Sex chromosome systems in Neotropical Primates: What have we learnt so far from cytogenetics and genomics?

Neotropical Primates (Platyrrhini) show great diversity in their life histories, ecology, behaviour and genetics. This diversity extends to their chromosome complements, both to autosomes and to sex chromosomes. In this contribution, we will review what is currently known about sex chromosomes in this group, both from cytogenetic and from genomic evidence. The X and Y chromosomes in Neotropical Primates, also known as New World Monkeys, have striking structural differences compared with Old World Monkeys when Catarrhini sex chromosomes are considered. The XY bivalent displays a different meiotic behaviour in prophase I, and their Y chromosome shows extensive genomic differences. Even though the most widespread sex chromosome system is the XX/XY and thus considered the ancestral one for Platyrrhini, modifications of this sexual system are observed within this group. Multiple sex chromosome systems originated from Y-autosome translocations were described in several genera (Aotus, Callimico and Alouatta). In the howler monkeys, genus Alouatta, an independent origin of the sexual systems in South American and Mesoamerican species was postulated. All the above-mentioned evidence suggests that the Y chromosome of Platyrrhini has a different evolutionary history compared with the Catarrhini Y. There is still much to understand regarding their sex chromosome systems.

Assessment of genetic variability in captive capuchin monkeys (Primates: Cebidae).

Capuchin monkeys (genera Cebus and Sapajus) show a wide range distribution, from Honduras to Argentina. The aim of this work was to evaluate the genetic and phenotypic variability of captive specimens putatively belonging to S. cay (SCY) and S. nigritus (SNI) at their southernmost distribution limit. Forty-four individuals held in five captive centers from Argentina were analyzed based on external morphology, karyology and DNA sequences of mitochondrial control region (mtDNA-CR). Three morphotypes associated with their probable geographical origin in SCY and a single morphotype in SNI were found. For SCY we could associate each morphotype with the most frequent karyotype. SNI showed a single phenotype and a homogenous karyotype. Heterochromatin showed geographical patterns within species. A 515-bp mtDNA-CR fragment was sequenced, defining fourteen haplotypes at 59 polymorphic sites. A network constructed with our 14 haplotypes and other 77 from S. apella, S. macrocephalus, S. cay and S. nigritus from bibliography revealed some phylogeographic signals. Our SCY and SNI samples rendered four groups that differed in multiple mutational steps, with SCY being more similar to S. apella than to S. macrocephalus. Also, we identified two genetic divergent SCY groups: samples from NOA and from NEA with high mitochondrial diversity. Our results highlight the relevance of using complementary genetic tools throughout the distribution ranges of SCY and SNI for a better assessment of their diversity.

Cytogenetic damage in peripheral blood cultures of Chaetophractus villosus exposed in vivo to a glyphosate formulation (Roundup).

Different concentrations of a glyphosate formulation, Roundup® Full II (66.2% glyphosate) were tested in culture peripheral blood of armadillo Chaetophractus villosus with cytogenetic biomarkers like mitotic index (MI), chromosomal aberrations (CA), sister chromatid exchange (SCE) and cell proliferation kinetics (CPK) by means of replication index. Adults animals of both sexes were exposed to RU at four concentrations ranging from 0.026 mL RU solution to 0.379 mL RU daily in oral treatment with the same volume (0.2 mL) during 7 days. We analyzed the induced damage at different times considering T0 as control value, one (T1), seven (T7) and 30 days (T30). One day after, only the higher concentration shows MI significant differences (p < 0.05), at T7 the frequency increases and at T30 it decreases reaching T0 values. The analysis of CA frequencies shows that only 0.106 mL RU/day exhibit significant differences vs T0 values. A great variability is expressed in the values of standard deviation (SD) and in the wide confidence intervals of the media. One day after treatments (T1) all four concentrations shows significant differences in SCE vs T0 values. Replication Index (RI) does not show significant differences. The dose-response behavior was not observed in either CA or SCE. The consistency of the findings obtained with the same biomarkers in vitro support the idea of expanding studies in order to characterize the risk doses for these mammals.

Genotoxic effects of Roundup Full II® on lymphocytes of Chaetophractus villosus (Xenarthra, Mammalia): In vitro studies.

In Argentina, Chaetophractus villosus has a wide distribution that overlaps with agricultural areas where soybean is the predominant crop. In such areas the pesticide Roundup Full II® (RU) is widely applied. The genotoxic effect of its active ingredient glyphosate (RU is 66.2% glyphosate) on the peripheral blood lymphocytes of C. villosus was tested over a range of concentrations (280, 420, 560, 1120 µmol/L). Culture medium without glyphosate served as negative control, while medium containing mitomycin C served as positive control. Genetic damage was characterized in terms of the percentage of cells with chromosome aberrations (CA), the mean number of sister chromatid exchanges (SCE) per cell, and the modification of cell proliferation kinetics via the calculation of the replication index. Significant increases (p < 0.0001) were seen in the CA frequency and the mean number of SCEs per cell compared to negative controls at all the RU concentrations tested. Chromatid breaks, the only form of CA observed, under the 560 µmol/L RU conditions and in presence of mitomycin C were four to five times more common than at lower concentrations, while no viable cells were seen in the 1120 µmol/L treatment. The mean number of SCEs per cell was significantly higher under the 280 µmol/L RU conditions than the 420 or 560 µmol/L RU conditions; cells cultivated in the presence of MMC also showed significantly more SCEs. All the RU concentrations tested (except in the 1120 µmol/L RU treatment [no viable cells]) induced a significant reduction in the replication index (p < 0.0001). The present results confirm the genotoxic effects of RU on C. villosus lymphocytes in vitro, strongly suggesting that exposure to RU could induce DNA damage in C. villosus wildlife.

Chaetophractus villosus as a sentinel organism: Baseline values of mitotic index, chromosome aberrations and sister chromatid exchanges.

Sentinel species are useful tools for studying the deleterious effects of xenobiotics on wildlife. The large hairy armadillo (Chaetophractus villosus) is the most abundant and widely distributed mammal in Argentina. It is a long-lived, omnivorous, burrowing species, with fairly restricted home ranges. To evaluate the level of spontaneous genetic damage in this mammal, we determined the baseline values of several genotoxicity biomarkers. The study included 20 C. villosus adults of both sexes from eight pristine localities within its geographic distribution range. Genotoxicity analysis was performed on 72-h lymphocyte cultures, using mitomycin C as positive control. We obtained the baseline values of mitotic index (MI=10.52±0.30 metaphases/total cells, n=20), chromosome aberrations (CA=0.13±0.22, n=20), sister chromatid exchanges (SCE)=6.55±0.26, n=6) and replication index (RI=1.66, n=6). MI and CA did not show significant differences (P>0.05) among localities or between sexes. No significant differences in MI, CA, SCE, and RI (P>0.05) were found between values from the pristine localities and historical data. There were significant differences in CA, SCE, and RI (P<0.05) between lymphocyte cultures from pristine localities and those exposed to mitomycin C. We propose the large hairy armadillo as a sentinel organism for environmental biomonitoring of genotoxic chemicals due to its abundance, easy manipulation, well-known biology, the fact that it is usually exposed to different mixtures and concentrations of environmental contaminants, and the baseline values of genetic damage characterized by MI, CA, SCE and RI as biomarkers.

Etanol e isopropanol: genotoxicidad y teratogénesis evaluada aplicando el modelo de Drosophila melanogaster / Ethanol and isopropanol: genotoxicity and teratogenicity evaluated using Drosophila melanogaster as a model system

El etanol y el isopropanol son, de los alcoholes alifáticos de cadena corta, los más frecuentemente asociados a la actividad humana tanto a nivel industrial como en el entorno doméstico. En este trabajo se presentan los principales hallazgos reportados en la literatura para ensayos de genotoxicidad y teratogénesis en modelos experimentales de distinto nivel de complejidad, con especial énfasis en Drosophila melanogaster. El metabolismo de estos alcoholes es semejante en Drosophila y en humanos por lo cual la mosca es un buen modelo in vivo para la evaluación de sus potenciales efectos tóxicos, genotóxicos y teratogénicos.

Ethanol and isopropanol are two of the short chain aliphatic alcohols more frequently associated to the human environment, both in the industrial and domestic conditions. The aim of this work was to present the main findings reported in the literature about their genotoxicity and teratogenicity in experimental models of different level of complexity, with special emphasis in Drosophila melanogaster. Taking into account that the metabolism of both alcohols in Drosophila and humans is similar, the fly is a good model for the evaluation of their potentially toxic, genotoxic and teratogenic effects.

Multiple sex chromosome systems in howler monkeys (Platyrrhini, Alouatta).

In light of the multiple sex chromosome systems observed in howler monkeys (Alouatta Lacépède, 1799) a combined cladistic analysis using chromosomal and molecular characters was applied to discuss the possible origin of these systems. Mesoamerican and South American howlers were karyologically compared. FISH analysis using the chromosome painting probes for the #3 and #15 human chromosomes was applied to corroborate the homeology of the sexual systems. We found that the HSA3/15 syntenic association, present in the sex chromosome systems of South American Howlers, is not present in those of Mesoamerican ones. The autosomes involved in the translocation that formed the sexual systems in the Mesoamerican and South American species are different, thus suggesting an independent origin. Parsimony analysis resolved the phylogenetic relationships among howler species, demonstrating utility of the combined approach. A hypothesis for the origin of the multiple sex chromosome systems for the genus is proposed.

Metronidazole induced DNA damage in somatic cells of Drosophila melanogaster.

The standard version of the wing somatic mutation and recombination test (SMART) in Drosophila melanogaster was employed in order to evaluate the genotoxic potential of metronidazole (MTZ) as a function of exposure concentration. MTZ was administered by chronic feeding of 3-day-old larvae with the parenteral solution at 0, 500, 1000 and 2000 µg/ml until pupation. The marker-heterozygous progeny (mwh+/+flr3) with phenotypically wild-type wings was analyzed. Non significant differences were found between control and each MTZ concentration tested for single small spots (SSS) frequencies. Large single spots (LSS) and twin spots (TS) were significantly increased with the higher dose. MTZ treatments with 1000 and 2000 µg/ml also significantly increased the frequency of Total spots. These findings suggest that MTZ is genotoxic in the present experimental conditions and induces recombinagenesis and/or gene conversion, two major mechanisms that cause loss of heterocigosity and could play an important role in tumorigenesis and carcinogenesis processes.

Genotoxic damage induced by isopropanol in germinal and somatic cells of Drosophila melanogaster.

Isopropanol (isopropyl alcohol, 2-propanol, IPA) is a volatile solvent widely used in domestic or industrial environments and reported as innocuous in various test systems. The aim of this work was to search for in vivo genotoxic effects of IPA in Drosophila melanogaster, studying its ability to induce nondisjunction (ND) in females, sex linked recessive lethals (SLRL) in males, and somatic mutation and/or recombination (SMART) in larvae. Treatments were acute (60min) and were administered via inhalation. IPA had low toxicity in adult flies (75% IPA mortality index, MI=12.7% (females) and 2.6% (males)) and larvae (MI=14.3%, 75% IPA). Female fertility was severely affected during the first 24h (brood I, BI) after treatment, but, afterwards, control values were recovered. IPA induced a 50-fold increase of ND (%) in 24h old females, and a six-fold rise in 4-5 d old BI offspring. Nondisjunction frequencies (%) in the offspring of broods II to V (24h in each case) were similar to control values. IPA doses of 25% and 50% (v/v), tested in 24h old females, showed a significant dose-dependent increase of ND(%)in BI only, with control values in subsequent broods. Flies gave normal offspring when kept in regular media for 24h before mating. The eye spot test (SMART) showed a significant increase at 50% IPA (p<0.05, m=2), but the response was not dose-dependent. IPA failed to induce SLRL in any of the spermatogenesis stages tested. These findings suggest that the main effect of IPA is to induce chromosomal malsegregation; IPA must be present at the resumption of M-phase I after fertilization, to exert these effects. The alcohol does not affect DNA directly, but perturbations of the nuclear membrane may be responsible for induction of ND.

Male and female reproductive toxicity induced by sub-chronic ethanol exposure in CF-1 mice.

Since genetic damage induced by ethanol exposure is controversial and incomplete and because germ and somatic cells constitute bioindicators for monitoring reproductive toxicity and genotoxic actions of ethanol consumption, the purpose of the present investigation was to evaluate morphological sperm, oocyte alterations and parental genotoxic effects after sub-chronic ethanol intake in the CF-1 outbred mouse strain. Ethanol 10% was administered to CF-1 adult male (treated males, TM) and female (treated females, TF) mice for 27 days, whereas water was given to controls from both sexes too (CM and CF). Post-treatment micronucleus frequency (MN-PCE/1,000/mouse) and gamete morphology were evaluated. To test whether change of female reproductive status results in maternal genotoxicity, CF-1 females received ethanol 10% (exposed group, periconceptionally treated females (PTF)) or water (control group, pregnant control females (PCF)) in drinking water for 17 days previous and up to 10 days of gestation. TM had a high percentage of abnormal spermatozoa vs CM (p < 0.001) and elevated parthenogenetic activated oocyte frequency appeared in TF vs CF (p < 0.001). Sub-chronic ethanol ingestion induced increased MN frequency in TM and TF (p < 0.01). In PTF, where blood alcohol concentrations were between 19-28 mg/dl, very significantly increased MN frequency was found vs PCF (p < 0.01), whereas MN values were similar to TF. These results show that sub-chronic alcohol ingestion in CF-1 mice produces sperm head dysmorphogenesis and oocyte nuclear anomalies, suggesting that morphological abnormalities in germ cells are probably related to parental genotoxicity after ethanol consumption.

Biomarkers of genotoxicity and genomic instability in a non-human primate, Cebus libidinosus (Cebidae, Platyrrhini), exposed to nitroimidazole derivatives.

The genotoxicity of two nitroimidazole derivatives, ornidazole (ONZ) and metronidazole (MTZ) in the peripheral blood lymphocytes of Cebus libidinosus (CLI) (Primates, Cebidae) was assessed. Endpoints measured included sister chromatid exchange (SCE) frequency, cell proliferation kinetics (CPK), replication index (RI), mitotic index (MI), and damage incidence in or near CLI heterochromatin regions. MI and SCE values following ONZ or MTZ treatments were significantly different (p<0.001) from control. SCE frequency per chromosome was not proportional to chromosome length. The chromosomes most affected for SCE were 1, 2, 4, 6, 11-13, 17, and 18, many of which possess interstitial or terminal heterochromatin. In the CLI genome, chromosomes 11 and 17 showed higher susceptibility to damage RI was the only biomarker that did not show statistically significant differences between control and treated cultures. C. libidinosus bands 11q1.4 and 11q1.5 may be hot-spots in the context of nitroimidazole exposure.

Nitroimidazole derivatives: non-randomness sister chromatid exchanges in human peripheral blood lymphocytes.

Nitroimidazole derivatives exhibited genotoxic effect in different experimental conditions. This study focuses on an evaluation of possible genomic targets, at a chromosomal level, of two 5-nitroimidazoles (ornidazole and metronidazole) using the in vitro human peripheral blood culture as experimental system. We observed that both derivatives showed a decrease in mitotic index (MI) (P < 0.001), an increase in sister chromatid exchanges (SCE) frequency (P < 0.001) and no modifications in cellular proliferation kinetics (CPK). As a null hypothesis we considered the assumption that larger chromosomes should harbor more SCE, which was viewed using a novel sequential G-band (400 band resolution)/SCE technique. The analysis showed highly significant chi square values (P < 0.001), indicating that SCE frequency per chromosome is not proportional to chromosome length. SCE could be considered an instability indicator due to the high correlation between SCEs in certain chromosomal bands and the exposure to nitroimidazole derivatives.

Phylogenetic relationships among some Ateles species: the use of chromosomic and molecular characters.

As with most platyrrhines, the systematics of Ateles is under discussion. In order to help clarify its systematic, we employed chromosomic and molecular characters to analyze the phylogenetic relationship among some species of the genus Ateles. Chromosomic studies were conducted on 14 atelid specimens: eight Ateles from A. paniscus, A. chamek, A. belzebuth and A. geoffroyi, and six Alouatta caraya. Ateles paniscus showed 2N=32, whereas A. chamek, A. belzebuth and A. geoffroyi presented 2N=34, XX/XY (with a submetacentric X and a variable Y) corroborated by male meiosis. Nucleotide sequence variation at the mitochondrial cytochrome c oxidase subunit II gene (COII) was analyzed in ten New World monkey specimens. Parsimony trees showed consistent phylogenetic relationships using both chromosomic forms and mitochondrial COII gene sequences as characters. Particularly, chromosomic phylogenies showed A. hybridus as a divergent taxon from the remaining group, whereas A. chamek, A. belzebuth and A. marginatus form an unresolved clade with A. geoffroyi as sister group.

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.

Mutagenicidad quínica y evaluación de daño potencial mediante ensayos de corto plazo (ECT) / Chemical mutagenesis and evaluatial of potential damage by short term test (STT)

Los estudios del daño al DNA por agentes químicos se realizan en distintos niveles según el sistema experimental que se utilice. Es así que los ensayos propuestos para el monitoreo biológico se presentan en cuatro niveles diferentes: a) primer nivel: ensayos moleculares o en bacterias; b) segundo nivel: pruebas in vitro en células de cultivo; c) tercer nivel: análisis in vivo ; d) estudios en poblaciones expuestas. Se pueden evaluar tanto agentes químicos o físicos, como muestras de suelo, agua o aire (mezclas complejas). Se trabajó con ensayos de corto plazo (ECPs) para evaluar el daño genético potencial de diferentes agente químocos, productos naturales o contaminantes ambientales. En la actualidad, el interés particular consiste en caracterizar de modo exhaustivo diferentes derivados nitroimidazólicos como mebendazol (MBZ), tiabendazol (TBZ), metronidazol (MTZ) y ornidazol (ONZ), por su uso muy difundido como drogas antiparasitarias. El trabajo fue desarrollado con un amplio espectro de biomarcadores: aberraciones cromosónicas, cinética de proliferación celular, intercambio de cromátidas hermanas, retraso en anafase, micronúcleos, mutaciones de punto, recombinación somática, etcétera. Los estudios se han realizado en niveles crecientes de complejidad: nivel II (estudios in vitro en cultivos de célñulas de roedores y primates), nivel III (estudios in vivo en células de discos imaginales de insectos, células de médula ósea de roedores y linfocitos de primates) y nivel IV (estudios epidemiológicos en poblaciones humanas expuestas). Estos estudios que conllevan un enfoque multidisciplinario sobre un problema mediante el uso de distintos modelos animales son herramientas muy valiosas y pueden aplicarse al estudio de distintos contaminantes ambientales o a mezclas complejas.