Erythrocytes of the Rhesus and Cynomolgus Monkeys.

The monograph Erythrocytes of the Rhesus and Cynomolgus Monkeys is devoted to the cytologic, quantitative, generative normal and abnormal presentations of the erythrocytes in these two species of primates...

Anti-genotoxic ability of α-tocopherol and Anthocyanin to counteract fish DNA damage induced by musk xylene.

Many compounds released into the environment are able to interact with genetic material. The main purpose of genetic toxicology is to investigate the adverse effects of genotoxic molecules such as reduced fitness, changes in gene frequencies and their impact on genetic diversity in populations following genotoxic exposure. However, the ecological effects of many genotoxic compounds remain poorly understood. The aim of this research was to evaluate the genotoxic activity of an artificial musk (musk xylene, MX) and the potential anti-genotoxicity against this chemical compound of two antioxidant substances (α-tocopherol and an anthocyanins enriched extract). The studies were performed both in vivo and in vitro, using the teleost Danio rerio and the DLEC (Dicentrarchus labrax embryonic cells) cell line. We carried out the exposure to these substances at different times. DNA and cell damage and their possible repair were detected by various experimental approaches: DNA strand breaks (Comet Assay), degree of apoptosis (Diffusion Assay) and molecular alterations at the genomic level (RAPD-PCR technique). Data were collected and analyzed for statistical significance using the Student's t test. The results of this study showed that MX exhibited a genotoxic activity even after short exposure times. The anti-genotoxicity experiments evidenced that both α-tocopherol and Anthocyanin were able to contrast the genotoxic effects induced by MX, both in vivo and in vitro.

Genotoxicity assessment of TiO2 nanoparticles in the teleost Danio rerio.

Titanium dioxide nanoparticles (TiO2 NPs), widely used in paints, pharmaceutical preparations and in many consumer products, have been shown to induce cytotoxicity, genotoxicity and carcinogenic responses both in vitro and in vivo. Numerous studies have shown the potential impact of nanoparticles on a series of aquatic organisms and their toxicity has been linked to their dissolution, surface properties and size. In vitro studies have raised concerns about the toxicity of TiO2 NPs, but there are very limited data on ecotoxicity to aquatic life. This in vivo study aimed to describe the genotoxicity of TiO2 NPs in the zebrafish Danio rerio. After 2 weeks of adaptation, groups of zebrafish were exposed to TiO2 NPs (1 and 10µg/L) for 5, 7, 14, 21 and 28 days. The genotoxic potential of TiO2 NPs was assessed by the Comet assay, the Diffusion assay and RAPD-PCR technique. The use of multi-biomarkers has become an important aspect of ecotoxicology to evaluate environmental quality through a wide panel of biological responses triggered by contaminants. The highest genotoxic effect was observed at the maximum concentrations of nanoparticles (10µg/L) with all three tests at 14 and 21 days of exposure. The results suggests the presence of mechanisms that can reduce the n-TiO2 genotoxicity. Future studies are necessary to analyze the DNA repairing capacity in zebrafish cells and so verify the role of the antioxidant defence system in modulating the response to exposure to n-TiO2 in fish.

Genotoxic effects in fish induced by pharmacological agents present in the sewage of some Italian water-treatment plants.

The presence of pharmaceutical substances in the municipal effluents is currently considered the principal source of bio-active molecule emissions into aquatic environments. This study analyzes the genotoxic damage caused by gemfibrozil and atorvastatin, two regulators of the hematic level of lipids, and sildenafil citrate, a vasodilator, on the teleost Danio rerio. The genotoxicity of these three compounds was evaluated using the comet assay, diffusion assay, and RAPD-PCR. The alkaline version (pH 12.1) of the comet assay was used for the erythrocytes of the zebrafish to evaluate the presence of single strand DNA breaks. Furthermore, the diffusion assay was used to estimate the number of apoptotic cells. The fish were treated with the three pharmacological agents at the average concentrations previously found at some Italian treatment plants and were then sacrificed from 5 to 35 days after exposure. The data of the comet assay showed a statistically significant loss of DNA integrity after 5 days of exposure to atorvastatin and after one week of exposure to gemfibrozil. This damage was, however, repaired after 14 days. Sildenafil citrate produced, instead, a statistically significant loss of DNA integrity at the concentrations found only after 35 days of exposure. The genotoxicity at the molecular level was tested by RAPD-PCR. The results from this investigation are in agreement with those from two other tests, confirming the efficacy of the use of the three experimental approaches for the complete evaluation of genotoxic damage.

Micronucleus test and comet assay for the evaluation of zebrafish genomic damage induced by erythromycin and lincomycin.

An enormous quantity of pharmacologically active principles are currently being introduced into the environment, with consequent escalation of environmental problems, but only a small number of studies are focusing on an assessment of their genotoxic effects. The aim of this article is to assess the genotoxic effects of erythromycin, lincomycin, and of a combination of these two antibiotics on the genome of the zebrafish. The genotoxicity of the two antibiotics was assessed by applying the micronucleus test to erythrocytes and performing a Comet assay on erythrocytes and hepatocytes. The fish were exposed to antibiotics at different concentrations and times of exposure, under standard laboratory conditions. Depending on the different experimental conditions, erythromycin and lincomycin induced a significant increase in DNA migration (tail moment) and a significant increase in micronuleus frequency. We also conducted an analysis on the activation of repair mechanisms when the genotoxic agent was removed. Only a few of the cells displayed a decrease in damage under these test conditions.

Cytogenetic analysis in Polypterus ornatipinnis (Actinopterygii, Cladistia, Polypteridae) and 5S rDNA.

Polypteridae is a family of archaic freshwater African fish that constitute an interesting subject for the study of the karyological evolution in vertebrates, on account of their primitive morphological characters and peculiar relationships with lower Osteichthyans. In this paper, a cytogenetic analysis on twenty specimens of both sexes of Polypterus ornatipinnis the ornate "bichir", coming from the Congo River basin, was performed by using both classical and molecular techniques. The karyotypic formula (2n=36; FN=72) was composed of 26 M+10 SM. The Alu I banding, performed to characterize heterochromatin in this species, was mainly centromeric. Both the chromosome location of the ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA(3) staining and FISH. CMA(3) marked all centromerical regions and showed the presence of two GC rich regions on the p arm of the chromosome pair n°1 and on the q arm of the pair n°14. Staining with Ag-NOR marked the only telomeric region of the chromosome n°1 p arm. After PCR, the 5S rDNA in this species was cloned, sequenced and analyzed. In the 665bp 5S rDNA sequence of P.ornatipinnis, a conserved 120bp gene region for the 5S rDNA was identified, followed by a non-transcribed variable spacer (NTS) which included simple repeats, microsatellites and a fragment of a non-LTR retrotransposon R-TEX. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair n°14, previously marked by CMA(3). FISH with 18S rDNA marked the telomeric region of the p arm of the pair n°1, previously marked both by Ag-NOR and CMA(3). The (GATA)(7) repeats marked the telomeric regions of all chromosome pairs, with the exclusion of the n°1, n°3 and n°14; hybridization with telomeric probes (TTAGGG)(n) showed signals at the end of all chromosomes. Karyotype evolution in Polypterus genus was finally discussed, including the new data obtained.

Evaluation of zebrafish DNA integrity after exposure to pharmacological agents present in aquatic environments.

Over the past few years, the increasing and uncontrolled use of pharmaceutical substances in agriculture, fish farming, human health and in veterinary medicine, together with an improper use of out-of-date medicines, has led to a consequent increase in the environmental problems linked to their disposal. In some Italian waste water treatment plants were found furosemide, a diuretic; ranitidine, an antiulcer drug; bezafibrate, a lipid regulator and ibuprofen, a painkiller. The present paper shows, by means of the synergic application of three tests (the Comet Test, the Diffusion Assay and the RAPD-PCR technique), how the DNA of zebrafish can be damaged after exposure to the above mentioned drugs. The data from the Comet Test, the Diffusion Assay and the RAPD-PCR technique were generally in agreement; these results show that all four drugs are genotoxic.

Polypteridae (Actinopterygii: Cladistia) and DANA-SINEs insertions.

SINE sequences are interspersed throughout virtually all eukaryotic genomes and greatly outnumber the other repetitive elements. These sequences are of increasing interest for phylogenetic studies because of their diagnostic power for establishing common ancestry among taxa, once properly characterized. We identified and characterized a peculiar family of composite tRNA-derived short interspersed SINEs, DANA-SINEs, associated with mutational activities in Danio rerio, in a group of species belonging to one of the most basal bony fish families, the Polypteridae, in order to investigate their own inner specific phylogenetic relationships. DANA sequences were identified, sequenced and then localized, by means of fluorescent in situ hybridization (FISH), in six Polypteridae species (Polypterus delhezi, P. ornatipinnis, P. palmas, P. buettikoferi P. senegalus and Erpetoichthys calabaricus) After cloning, the sequences obtained were aligned for phylogenetic analysis, comparing them with three Dipnoan lungfish species (Protopterus annectens, P. aethiopicus, Lepidosiren paradoxa), and Lethenteron reissneri (Petromyzontidae)was used as outgroup. The obtained overlapping MP, ML and NJ tree clustered together the species belonging to the two taxonomically different Osteichthyans groups: the Polypteridae, by one side, and the Protopteridae by the other, with the monotypic genus Erpetoichthys more distantly related to the Polypterus genus comprising three distinct groups: P. palmas and P. buettikoferi, P. delhezi and P. ornatipinnis and P. senegalus. In situ hybridization with DANA probes marked along the whole chromosome arms in the metaphases of all the Polypteridae species examined.

Molecular characterization and chromosomal localization of spermatogenesis related sequences in Torpedo torpedo (Chondrichthyes, Torpediniformes).

Cytogenetic data in cartilaginous fishes are currently very inconsistent, considering that the karyotype morphology of only about seventy living species is actually known. Only in the last few years different molecular approaches, first of all physical mapping on metaphase chromosomes, have been used to investigate the cytotaxonomic relationship existing inside this interesting group of vertebrates. The aim of the work was to characterize new molecular chromosomal markers both to discriminate the different chromosome pairs and to distinguish the probable sex chromosomes in the species Torpedo torpedo, since its karyotype does not seem to exhibit heterochromosomes. Evolution of the SRY gene has received considerable attention, mainly because it has been shown to be the sex-determining locus in mammals. The gene is located in the Y chromosome where it normally occurs as a single copy. Using primers taken from the conserved SRY sequences, we characterized these regions at the molecular level and localized them on metaphase chromosomes. The PCR products revealed similar patterns in specimens of both sexes of T. torpedo, but only one fragment of the male amplification product showed a high percentage of identity with human spermatogenesis related genes, SPATA 16, SPATA 18 and UTY. Fluorescent in situ hybridization with these sequences showed the presence of spots at the subtelomeric level of two chromosome pairs in the male and of one pair in the female. Finally, these sequences are particularly useful as chromosome markers to differentiate between the male and the female karyotypes in this species.

Karyotypic characterization and genomic organization of the 5S rDNA in Polypterus senegalus (Osteichthyes, Polypteridae).

Polypteridae (Cladistia) is a family of archaic fishes, confined to African freshwaters. On account of their primitiveness in anatomical and morphological characters and mosaic relationships among lower Osteichthyans fishes, they constitute an important subject for the study of evolution in vertebrates. Very little is known about the karyological structure of these species. In this article, a cytogenetic analysis on twenty specimens of Polypterus senegalus (Cuvier, 1829) was performed using both classical and molecular techniques. Karyotype (2n=36; FN=72), chromosome location of telomeric sequences (TTAGGG)(n), (GATA)(7) repeats and ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA(3) staining and FISH. Staining with Ag-NOR showed the presence of two GC rich NORs on the p arm of the chromosome pair no. 1. CMA(3) marked all centromerical and some (no. 1 and no. 14) telomeric regions. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair no. 14. FISH with 18S rDNA marked the telomeric region of the p arm of the chromosome pair no. 1, previously marked by Ag-NOR. (GATA)(7) repeats marked the subtelomeric regions of all chromosome pairs, with the exclusion of the no. 1, 3 and 14. Hybridization with telomeric probes (TTAGGG)(n) showed bright signals at the end of all chromosomes. After cloning, the 5SrDNA alignment revealed an organization of sequences made up of two different classes of tandem arrays (5S type I and 5S type II) of different lengths.

Molecular and karyological aspects of Batoidea (Chondrichthyes, Elasmobranchi) phylogeny.

Although considerable progress has been made in elucidating the relationships within the Chondrichthyes, there is no agreement as it concerns the systematics of Batoidea, the most derived superorder among cartilaginous fishes, and many different interpretations exist. Our investigation provides the first assessment of relationships among the described batoid species using sequences from both mtDNA and nuclear genes as well as karyological morphology. Our work consists primarily in reconstructing the phylogenetic relationships of Batoidea by examining the mtDNA (16S) and nuclear gene (18S) sequences from 11 batoid species. The three analytical methods (NJ, MP and Bayesian analysis) grouped Rajiformes, Myliobatiformes and Rhinobatiformes. In these trees the two torpedoes diverge from the other batoid fishes. We also compare the molecular data with the available karyological evidence, which consist of the diploid number and the karyotype morphology of eight species belonging to the four orders examined. The results show that the karyological structure in the different species is generally consistent with the various phylogenetical trees, and that Torpediniformes confirm their unique genome organization.

Karyotypic characterization and genomic organization of the 5S rDNA in Erpetoichthys calabaricus (Osteichthyes, Polypteridae).

Polypterids are a group of Osteichthyan fish whose evolutionary relationships with closer basal ray-finned and lobe-finned fish have been disputed since their discovery. Very little is known about the evolutive karyology in the whole Polypteriformes group. In order to fill this gap, a cytogenetic analysis of Erpetoichthys calabaricus species was performed, using both classical and molecular techniques. Karyotype structure (2n = 36; FN = 72), chromosome location of telomeric sequences (TTAGGG)n and ribosomal 5S and 18S rRNA genes were examined in twenty specimens of E. calabaricus by using Ag-NOR, classical C-banding, sequential CMA3/4',6-diaminidino-2-phenylindole (DAPI) staining and fluorescent in situ hybridization (FISH). CMA3 marked all centromerical and some (no. 1 and no. 15) telomeric regions. Staining with Ag-NOR and CMA3 showed the presence of two NORs on the p arm of the chromosome pair no. 1. Hybridization with telomeric probes (TTAGGG)n showed signals at the end of all chromosomes. 5S rDNA was cloned and sequenced. After the alignment, the 5S rRNA sequences revealed an organization made up of two different classes of tandem arrays (type I and type II). FISH with 5S rDNA marked the telomeric regions of the small chromosome pair no. 15, while FISH with 18S rDNA marked the telomeric region of the pair no. 1. The results obtained were compared with cariological data on closer species now available in literature.

Assessment of environmental stress by the micronucleus test and the Comet assay on the genome of teleost populations from two natural environments.

The aim of the present paper was to assess the biological damage caused by exposure of the test organism (Gambusia holbrooki: Cyprinodontiformes, Poecilidae) to various mutagenic agents present in the polluted waters of the Sarno River. For this purpose, we performed a micronuclei (MN) test and single cell gel electrophoresis (the Comet assay), testing DNA migration in an electrophoretic field using erythrocytes of G. holbrooki specimens both from the Sarno River and from the waters of the crater of the Astroni natural reserve as negative controls. The results indicate statistically higher values for both MN and DNA migration in the samples from the Sarno River compared with those from Astroni and point to a strong genotoxic action of the mixture of pollutants present in the Sarno River. These data were compared with the values found in the G. holbrooki specimens from the Sarno River kept under laboratory conditions for 100 days in clean water.

Cytogenetic and molecular studies in a lungfish, Protopterus annectens (Osteichthyes, Dipnoi).

A neontological approach to the problem of the origin of tetrapods consists in the examination of the available cytological and molecular data on the genome of these vertebrates. Dipnoans are a group of osteichthyian fishes, the evolutionary relationships of which with tetrapods have been disputed since their discovery. In the past, they were variously considered as being related to actinistians, tetrapods, and lower actinopterygians, though nowadays they are considered a monophyletic group, the sister group of crossopterygians. Dipnoans first appeared in the geologic record in the Early Devonian with 50 extinct genera, surviving up to date, with only three genera: Lepidosiren, Neoceratodus and Protopterus, including only six recognized species. Nothing is known of the genome of the early tetrapods, except that they and the Choanoichthyes exhibited a remarkable interspecific variability of the karyotype and of DNA content. These characteristics are often found in dipnoans and in the extant lissamphibians. Very little is known about the evolutionary karyology in the four Protopterus species and in the dipnoan clade in general. In this paper, we karyotyped ten male and female specimens of P. annectens (2n=34) from Nigeria. Moreover, we localized heterochromatin and nucleolar organizer regions by using base-specific fluorochromes and detected the human telomeric (TTAGGG)(n) sequences on all the telomeric sites of P. annectens chromosomes. DNA was also extracted and digested with seven restriction enzymes, which revealed the probable presence of almost three different families of satellite DNA. Nuclear DNA content was identified from blood samples by flow cytometry. New genomic and karyological data were compared and discussed with those on closer genera and taxa available in literature.

Karyotype and genome characterization in four cartilaginous fishes.

Different approaches can be used to elucidate the unsolved questions concerning taxonomic evolution in cartilaginous fish. The study of the karyological characteristics of these vertebrates by combining molecular and traditional techniques of chromosome preparation and banding has been demonstrated to be a very effective method. In this paper we studied the localization and the composition of the constitutive heterochromatin by using C- and restriction endonuclease-banding in four selachian species, belonging to two of the four superorders. We also characterized two different types of repetitive genomic sequences in these species: satellite DNA and (TTAGGG)(n) telomeric sequences. Finally, we analysed the nuclear ribosomal gene to determine the number of the nucleolar organizers and their position on chromosomes by using silver staining, chromomycin A(3), and FISH (fluorescent in situ hybridization). The results showed a prevailingly telomeric localization of constitutive heterochromatin in the Galeomorphii, the presence of additional nucleolar organizer sites in Raja asterias, an exclusively telomeric localization of the (TTAGGG)(n) sequences in Scyliorhinus stellaris and both telomeric and interstitial in Taeniura lymma. These data, together with those concerning the conservation of the satellite DNA, seem to support the hypothesis that Chondrichthyes have an evolutionary history leading them to the acquisition of large genomes rich in highly repeated sequences and subjected to some selective pressures favoring the conservation of this DNA fraction.