¿Qué nos dicen los primates neotropicales bajo la mirada de la citogenética? / What do neotropical primates tell us under the look of cytogenetics?

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.

HEp-2 cell line as an experimental model to evaluate genotoxic effects of pentavalent inorganic arsenic / HEp-2 como modelo experimental para evaluar los efectos genotóxicos del arsénico inorgánico pentavalente

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.

Genome size of two cebus species (primates: platyrrhini) with a fertile hybrid and their quantitative genomic differences.

Genome size or C-value is defined as the total amount of DNA contained within a haploid chromosome set and is regarded as a species-specific constant. Speciation among neotropical primates seems to be accompanied by marked quantitative changes in DNA content. A direct correlation between genome size and the presence of heterochromatin has also been proposed. In this work, we analyzed the genome of a female fertile hybrid between Cebus libidinosus and C. nigritus using interspecies comparative genomic hybridization (iCGH), in order to detect quantitative differences between the hybrid and the parental genomes. We also estimated the genome sizes of C. libidinosus and C. nigritus. Both species, considered subspecies of C. apella until 2001, have a highly homologous karyotype but are easily distinguishable at the chromosomal level due to the noncentromeric heterochromatin block on C. libidinosus chromosome 11. Our findings on C-value quantification support the species status for C. libidinosus and C. nigritus, each having a different genome size. The iCGH analysis of the hybrid revealed quantitative differences in comparison to both parental species. The hybrid genome contains a greater amount of DNA in the heterochromatic blocks related to those in the genomes of both parental species. In view of observations in previous and the present work, some hypotheses about genome dynamics of neotropical primates are proposed and discussed.

Chromosome painting in two genera of South American monkeys: species identification, conservation, and management.

Cytogenetic studies showed that a number of New World primate taxa, particularly the genera Alouatta, Aotus, and Callicebus, have highly derived karyotypes. Cytogenetics in these primates, at every level of analysis, has contributed to the recognition of species and revealed that their number was certainly underestimated by researchers relying solely on traditional morphological data. Further attention was drawn to Alouatta and Aotus because they are characterized by translocations of the Y chromosome to autosomes, generating multiple sex chromosome systems. Here we present a report on the hybridization of human chromosome-specific paints on metaphases from 4 individuals originally assigned to Alouatta caraya and 1 individual of Aotuslemurinus. This is only the third karyotype studied with chromosome painting out of more than 10 known karyomorphs in Aotus. The banded chromosomes matched those of karyotype II as defined by Ma et al. [1976a], and we were able to more precisely assign the origin of the sample to A. l. griseimembra. Our results on the Argentinean Alouatta caraya samples were generally comparable to the banding and hybridization pattern of previous studies of A. caraya including the presence of an X(1)X(1)X(2)X(2)/X(1)X(2)Y(1)Y(2) sex chromosome system. The karyotype of the Brazilian Alouatta sample labeled as A. caraya differs from the 3 Argentinean samples by at least 10 chromosome rearrangements. The diploid number, G banding, and hybridization pattern of this female cell line was almost identical to previous painting results on Alouatta guariba guariba. Therefore we must conclude that this cell line is actually from an A. guariba guariba individual. The contribution of cytogenetic tools in identifying species or in this case assigning individuals or cell lines to their precise taxonomic allocation is stressed. Gathering further molecular cytogenetic data on New World primates should be conservation and management priorities.

Cebus paraguayanus and Cebus nigritus (Primates, Platyrrhini): a comparative genomic hybridization analysis.

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.

Genotoxicity of the herbicide formulation Roundup (glyphosate) in broad-snouted caiman (Caiman latirostris) evidenced by the Comet assay and the Micronucleus test.

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.

The karyotype of Alouattapigra (Primates: Platyrrhini): mitotic and meiotic analyses.

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.

Caiman latirostris (broad-snouted caiman) as a sentinel organism for genotoxic monitoring: basal values determination of micronucleus and comet assay.

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.

Karyological diagnosis of Cebus (Primates, Platyrrhini) in captivity: detection of hybrids and management program applications.

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.

Chromosomal localization of the telomeric (TTAGGG)n sequence in eight species of New World Primates (Neotropical Primates, Platyrrhini).

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.

Mitochondrial sequence diversity of the southernmost extant New World monkey, Alouatta caraya.

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.

Genotoxic and aneugenic properties of an imidazole derivative.

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).

Heterochromatin and chromosome evolution: a FISH probe of Cebus apella paraguayanus (Primate: Platyrrhini) developed by chromosome microdissection

Neotropical Primate karyotypes are highly variable, particularly in the heterochromatic regions, not only regarding the amount of heterochromatin, but also the composition. G and C banding and FISH techniques provide useful information to characterize interspecific relationships. We used chromosome microdissection to develop a FISH probe of the chromosome 11 heterochromatic block (11qHe+) of Cebus apella paraguayanus (CAPp). Fragments of the 11qHe+ microdissected from fibroblast cell culture were collected in a PCR tube, amplified by degenerate oligonucleotide primer-PCR and subsequently labeled. The specificity of the FISH probe was confirmed in metaphases of some Ceboidea species. Signals were located in the He+ of chromosomes 4, 11, 12, 13, and 19 of CAPp and in the He+ of chromosomes 4, 12 and 13 of C. a. nigritus (CAPn); no signals were observed when other Ceboidea species were analyzed. We propose that the heterochromatin observed in CAPp and CAPn is specific for these species. We consider this C. apella heterochromatin identity as a possible key for the interpretation of chromosomal evolution in these Ceboidea.

Teratogenic evaluation of metronidazole and ornidazole using Drosophila melanogaster as an experimental model.

BACKGROUND: Drosophila and vertebrates show similarities that suggest that the mechanisms involved in the induction of developmental defects may be similar in both. Therefore, Drosophila has been proposed as a useful, rapid, and economical model in the preliminary screening for teratology studies. The objective of the present study was to investigate the effect of metronidazole (MTZ) and ornidazole (ONZ) on the developmental stages of Drosophila melanogaster. METHODS: Samarkand wild-type females were allowed to lay eggs for 24 hr in media containing MTZ or ONZ at concentrations of 0, 500, 1000, and 2000 microg/ml. When larvae completed their development, the emerging flies were counted and examined for morphological abnormalities. RESULTS: After the analysis of 400-1000 flies for each concentration, ONZ-treated flies did not show an incidence of malformations above control values, although a significant high number of individuals with reduced body size was observed (p < 0.005, chi2 test). On the other hand, the 1000- and 2000-microg/ml MTZ-treated series presented higher frequencies of total abnormalities than did concurrent and historic controls (p < 0.05, chi2 test), indicating an MTZ effect during developmental morphogenesis. CONCLUSIONS: These findings contribute to the characterization of both nitroimidazoles, which are widely used, especially in underdeveloped countries. At the same time, this Drosophila bioassay is sensitive enough to detect differential effects of MTZ and ONZ (abnormalities vs. growth effects), showing specificity and selectivity.

Toxicogenetic evaluation of metronidazole in the treatment of women infected with Trichomonas vaginalis.

Samples of peripheral blood were collected once from non-smoking women who were healthy (controls) and twice (immediately before and immediately after 7 days of treatment with metronidazole at 500 mg/day) from non-smoking women infected with Trichomonas vaginalis. Lymphocyte cultures were prepared and used, in toxicogenetic studies, to determine the frequency of sister-chromatid exchange (SCE), the mitotic index (MI), and the replication index (RI) for each sample. MTZ treatment of the infected women led to an increase in the frequency of SCE (P <0.001), a decrease in the MI (P <0.003), and a modification in the kinetics of cell proliferation, with a decrease in the RI (P <0.0006). The differences seen between the results for the controls and those for the infected women, before and after MTZ treatment, may be attributed to the presence of the parasite, to the treatment itself, and/or to variation in the host's response to infection with T. vaginalis.

Cytogenetic evaluation of two nitroimidazole derivatives.

5-Nitroimidazoles are a well-established group of antiprotozoal and antibacterial agents. Thanks to their antimicrobial activity these chemotherapeutic agents inhibit the growth of both anaerobic bacteria and certain anaerobic protozoa such as Trichomonas vaginalis, Entamoeba histolytica and Giardia lamblia. The aim of the present study is to achieve a precise characterization of the genotoxic activity of these compounds and to establish the value of cytogenetic assays in order to determine the effect of these drugs, at therapeutic doses, to settle an improved risk assessment. Two nitroimidazole were studied, metronidazole and ornidazole, at four different concentrations (0.1, 1, 10 and 50 microg/ml of peripheral blood lymphocyte culture). Endpoints analyzed included: mitotic index (MI), replication index (RI), sister chromatid exchange (SCE) and chromosomal aberrations (CA). An analysis of variance test (ANOVA) was performed to evaluate the results. A significant decrease (P<0.0001) in MI as well as an increase in SCE (P<0.0001) and CA (0.0001) frequencies for both drugs was observed. No modifications in RI were found. The results suggest a genotoxic and cytotoxic effect of MTZ and ONZ in human peripheral blood cultures in vitro.

Meiosis and chromosome painting of sex chromosome systems in Ceboidea.

The identity of the chromosomes involved in the multiple sex system of Alouatta caraya (Aca) and the possible distribution of this system among other Ceboidea were investigated by chromosome painting of mitotic cells from five species and by analysis of meiosis at pachytene in two species. The identity of the autosome #7 (X2) involved in the multiple system of Aca and its breakage points were demonstrated by both meiosis and chromosome painting. These features are identical to those described by Consigliere et al. [1996] in Alouatta seniculus sara (Assa) and Alouatta seniculus arctoidea (Asar). This multiple system was absent in the other four Ceboidea species studied here. However, data from the literature strongly suggest the presence of this multiple in other members of this genus. The presence of this multiple system among several species and subspecies that show high levels of chromosome rearrangements may suggest a special selective value of this multiple. The meiotic features of the sex systems of Aca and Cebus apella paraguayanus (Cap) are strikingly different at pachytene, as the latter system is similar to the sex pair of man and other primates. The relatively large genetic distances between species presently showing this multiple system suggest that its origin is not recent. Other members of the same genus should be investigated at meiosis and by chromosome painting in order to know the extent and distribution of this complex sex-chromosome system.

Embryolethality induced by metronidazole (MTZ) in Rattus norvegicus.

Parasitic illnesses is increasing all over the world, especially in developing countries, and metronidazole (MTZ) is the therapeutic agent usually administered to children as well as adults at the reproductive age. In this work, we propose an evaluation of MTZ in order to analyze the potential reproductive damage in females by using Rattus norvegicus (Sprague-Dawley) as an animal model. Adult female rats were mated after MTZ treatments, and they were sacrificed at 21 days of gestation. Different types of damage were evaluated by using mortality, phenotypic abnormalities and reproductive capacity as parameters, and were studied and scored in 70 adult specimens (450 g/bw). They were divided into five groups: a) untreated females as a control group; females treated with b) DMSO as a solvent control group or c) 500 mg/kg/bw of MTZ per day for 7 days as therapeutic dose (TD); d) a half therapeutic dose (HD); and e) a double therapeutic dose (DD). Pre-implantation death in MTZ-treated groups was not significantly different from controls. However, drug treatments significantly increased the frequency of post-implantation deaths and the dominant lethals were ranged between 12.0 % and 17.8 %.

Revised karyotype of Alouatta caraya (Primates: Platyrrhini) based on synaptonemal complex and banding analyses.

Most primates studied have the usual XX/XY sex-chromosome system. However, exceptions to this rule among howler monkeys have been suggested by several authors. Recently a quadrivalent was discovered in male meiosis of Alouatta caraya and it was established that this species has an X1X2Y1Y2 sex chromosome system. On that basis, a cytogenetic analysis of 25 males of this species is described, showing the corrected karyotype of this species. Each chromosome involved in the particular sex-chromosome system of this species is identified on the basis of mitotic chromosome measurements, G and C-banding patterns as well as on the relative measurements of synaptonemal complexes. It is now established that A. caraya has a karyotype with 2n = 52 in both sexes, and that the male one shows a single autosome #7 (X2) besides the X (X1) and the two products of the reciprocal translocation between the second autosome #7 and the Y chromosome (Y1 and Y2), while females show a homomorphic pair #7 (X2) and a pair of X1. The evolutionary implications of the exceptional primate species having composite sex-chromosome systems are discussed.

Mutagenic bioassay of certain pharmacological drugs: III. Metronidazole (MTZ).

The genotoxic activity of MTZ was evaluated in vitro with the anaphase-telophase test in a CHO cell line, chromosomal aberration and micronucleus test in lymphocyte cultures, and in vivo using the micronucleus test in mouse bone marrow cells. The In vivo test was performed using clinical trial doses (23, 70 and 160 mg/kg). A significant increase in micronucleated cells (p < 0.02) was observed in the three assayed doses with a linear dose response (r = 0.91). In vitro studies showed a significant increase in the percentage of abnormal anaphases (p < 0.05), in chromosome aberrations (p < 0.01) and in the frequency of micronuclei (p < 0.02) at all the concentrations assayed (0.1, 1 and 10 micrograms/ml). These findings demonstrate the clastogenic effect of this drug which should be taken into account considering its wide human consumption.