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.

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.

Identification and developmental expression of the ets gene family in the sea urchin (Strongylocentrotus purpuratus).

A systematic search in the available scaffolds of the Strongylocentrotus purpuratus genome has revealed that this sea urchin has 11 members of the ets gene family. A phylogenetic analysis of these genes showed that almost all vertebrate ets subfamilies, with the exception of one, so far found only in mammals, are each represented by one orthologous sea urchin gene. The temporal and spatial expression of the identified ETS factors was also analyzed during embryogenesis. Five ets genes (Sp-Ets1/2, Sp-Tel, Sp-Pea, Sp-Ets4, Sp-Erf) are also maternally expressed. Three genes (Sp-Elk, Sp-Elf, Sp-Erf) are ubiquitously expressed during embryogenesis, while two others (Sp-Gabp, Sp-Pu.1) are not transcribed until late larval stages. Remarkably, five of the nine sea urchin ets genes expressed during embryogenesis are exclusively (Sp-Ets1/2, Sp-Erg, Sp-Ese) or additionally (Sp-Tel, Sp-Pea) expressed in mesenchyme cells and/or their progenitors. Functional analysis of Sp-Ets1/2 has previously demonstrated an essential role of this gene in the specification of the skeletogenic mesenchyme lineage. The dynamic, and in some cases overlapping and/or unique, developmental expression pattern of the latter five genes suggests a complex, non-redundant function for ETS factors in sea urchin mesenchyme formation and differentiation.