Marta D. Mudry

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.

DNA single strand breaks in peripheral blood lymphocytes induced by three nitroimidazole derivatives

Tinidazole (TNZ), ornidazole (ONZ) and metronidazole (MTZ) are antiparasitic drugs (nitroimidazole derivatives) that have proven to be effective against Trichomonas vaginalis, Entoamoeba histolytica, Giardia lamblia and Helicobacter pylori. The reduction of the nitro group and the generation of short-lived reactive intermediates are the basis of its parasiticidal activity. This reduction is associated with its mutagenic activity in bacteria, although in mammalian cells DNA damage seems to be related to the production of reactive oxygen species (ROS). Using alkaline single cell electrophoresis, a significant increase in single strand breaks and alkali labile sites in human peripheral blood lymphocytes (PBL) exposed to MTZ, ONZ and TNZ at 10, 100 and 500 microg/ml is observed. MTZ causes less damage, especially at higher concentrations, when compared with TNZ, the most harmful of the drugs tested. These findings suggest that primary damage is induced under aerobic conditions and confirms that these nitroimidazoles are DNA damaging agents.

Genetic, enzymatic and developmental alterations observed in Caiman latirostris exposed in ovo to pesticide formulations and mixtures in an experiment simulating environmental exposure

In South America, economic interests in last years have produced a constant increase in transgenic soybean cropping, with the corresponding rise in pesticide formulated products. The aim of this study was to determine the effects of pesticides formulations and mixtures on a South American caiman, Caiman latirostris, after in ovo exposure. We conducted a field-like experiment which simulates the environmental exposure that a caiman nest can receive in neighbouring croplands habitats. Experimental groups were Control group, Treatment 1: sprayed with a glyphosate herbicide formulation, and Treatment 2: sprayed with a pesticide mixture of glyphosate, endosulfan and cypermethrin formulations. Results demonstrated genotoxicity, enzymatic and metabolic alterations, as well as growth delay in caimans exposed in ovo to Treatments 1 and 2, showing a higher toxicity for the mixture. Integral evaluation through biomarkers of different biological meaning is highly informative as early indicators of contamination with pesticides and mixtures in this wildlife species.

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.

Genetic variability in four Alouatta species measured by means of nine DNA microsatellite markers : Genetic structure and recent bottlenecks

We used microsatellite DNA to study the population genetics of 4 Alouatta species from Central and South America. Our main findings include the following: (1) A. seniculus had the highest level of microsatellite variability while A. caraya and A. palliata had the lowest mean number of alleles per locus and the lowest expected heterozygosity, respectively; (2) the samples of A. seniculus and A. palliata came from different regions and were not in Hardy-Weinberg equilibrium (HWE) which may indicate a Wahlund effect and differentiated gene pools – in contrast, A. macconnelli and A. caraya were in HWE; (3) the microsatellite genetic heterogeneity of the 4 Alouatta species was similar to the karyotype divergence found among these Alouatta species; the species pair with the lowest level of heterogeneity (genetic differentiation) was A. seniculus/A. caraya, while the Central American species, A. palliata, was highly differentiated from the other 3 South American species; (4) we recommend the establishment of a conservation plan to help protect A. caraya because the Cornuet and Luikart procedure demonstrated a recent bottleneck for this 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 X1 and acrocentric X2, Y1, and Y2. 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.

COII: a useful tool for inferring phylogenetic relationships among New World monkeys (Primates, Platyrrhini)

In this study we evaluated the performance of the cytochrome c oxidase subunit II (COII) mitochondrial gene as a tool for inferring phylogenetic relationships among platyrrhines. Twenty‐nine COII sequences were examined in seven platyrrhine genera (Alouatta, Ateles, Lagothrix, Brachyteles, Cebus, Saimiri, and Aotus) employing parsimony and distance methods. Phylogenetic signal (g1) was present in all codon positions, despite the transitional saturation detected at the third position. In tree reconstructions bootstrap support values decreased abruptly above the generic level. Parsimony trees based on weighted transversions (tv : ts, 10 : 1) at the third position showed similar topologies. The utility of COII in phylogenetic studies among platyrrhines seems to be limited, due to its low rate of replacement substitutions and a relatively fast saturation of silent substitutions at third codon positions. Our data suggest that its main utility in platyrrhine systematics lies at the intrageneric level.

Cytogenetic and microtubule array effects of the zineb-containing commercial fungicide formulation Azzurro© on meristematic root cells of Allium cepa L.

Zineb [ethylene bis(dithiocarbamate) zinc] is a widely employed foliar fungicide for agricultural and industrial applications. Allium cepa L. is a reliable model for the assessment of xenobiotic genotoxicity and cytotoxicity. We evaluated the effects of the zineb-containing commercial formulation Azzurro(®) (70% zineb) in cell cycle stages of the meristem root cells of A. cepa. The mitotic index (MI), chromosomal aberrations at anaphase/telophase (CAs), micronuclei (MN), and abnormalities in immunodetected microtubule structures, e.g., preprophasic band (PPB), mitotic spindle (MS), and phragmoplast (Phrag), were used as end-points. Azzurro(®) (1 and 10μg/ml) induced a significant increase in the frequency of CAs (P<0.05), and the higher concentration inhibited the MI (P<0.05) compared to control values. The frequency of MN did not differ from control values at any concentration. Treatment with 1μg/ml Azzurro(®) induced a significant increase in the frequency of abnormal PPB (P<0.01), MS (P<0.001), and Phrag (P<0.01) and, at 10μg/ml, enhancements in the frequencies of abnormal MS (P<0.05) and Phrag (P<0.05) were seen. A tubulin immunodetection assay showed that exposure to Azzurro(®) interferes with normal assembly of microtubule structures during mitosis.

A comparative study of the recombination pattern in three species of Platyrrhini monkeys (primates)

Homologous chromosomes exchange genetic information through recombination during meiotic synapsis, a process that increases genetic diversity and is fundamental to sexual reproduction. Meiotic studies in mammalian species are scarce and mainly focused on human and mouse. Here, the meiotic recombination events were determined in three species of Platyrrhini monkeys (Cebus libidinosus, Cebus nigritus and Alouatta caraya) by analysing the distribution of MLH1 foci at the stage of pachytene. Moreover, the combination of immunofluorescence and fluorescent in situ hybridisation has enabled us to construct recombination maps of primate chromosomes that are homologous to human chromosomes 13 and 21. Our results show that (a) the overall number of MLH1 foci varies among all three species, (b) the presence of heterochromatin blocks does not have a major influence on the distribution of MLH1 foci and (c) the distribution of crossovers in the homologous chromosomes to human chromosomes 13 and 21 are conserved between species of the same genus (C. libidinosus and C. nigritus) but are significantly different between Cebus and Alouatta. This heterogeneity in recombination behaviour among Ceboidea species may reflect differences in genetic diversity and genome composition.

Exposure of Bovine Oocytes to the Endogenous Metabolite 2-Methoxyestradiol During In Vitro Maturation Inhibits Early Embryonic Development

Abstract Catecholestrogens are endogenous metabolites that have been shown to modulate granulosa, theca, and luteal cell function in some species. The present study was aimed at determining the possible role of these steroids on oocyte maturation. Cumulus-enclosed bovine oocytes were matured for 24 h, fertilized, and then cultured for 8 days. Whereas estradiol was without effect, addition of catecholestrogens (2-hydroxyestradiol, 4-hydroxyestradiol, and 2-methoxyestradiol [2-MOE2]) to the maturation medium did not affect the cleavage rate but was associated with a decrease in blastocyst production on Day 8. Although 2-MOE2 was also able to inhibit blastocyst formation when added during embryo culture, the effects were less pronounced than those seen when the steroid was added only during maturation. In agreement with the known ability of 2-MOE2 to bind tubulin at the colchicine site, marked alterations were observed in the spindle assembly of oocytes exposed to 2-MOE2 during maturation, which lead to gross chromosomal aberrations after fertilization and consequent developmental arrest at the morula stage. Moreover, that the blastocyst rate was not affected when meiosis was blocked with roscovitine during 2-MOE2 exposure is consistent with the idea that altered nuclear maturation is the cause of the low developmental competence. Because 2-MOE2 could be increased in follicular fluid in response to aryl hydrocarbon-receptor ligands, such as some environmental contaminants, our results show that abnormally high intraovarian levels of catecholestrogens could have a deleterious effect on oocyte maturation and early embryonic development arising from the alterations in the meiotic spindle.