Vincenzo Stingo

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.© 2011 Wiley Periodicals, Inc. Environ Toxicol 2012.

New developments in vertebrate cytotaxonomy II. the chromosomes of the cartilaginous fishes

To date, the evolutionary history of the Chondrichthyes, their origins, mutual relationships and those to the other Vertebrates, are far from being reliably defined. This is primarily due to the paucity of the fossil record, in that only rarely have the cartilaginous skeletons of these animals been preserved. Paleontologists have been forced to refer, for most of their data, to isolated teeth or dermal scales (denticles). The earliest selachian fossils are traceable back to the Upper Devonian (Romer, 1966) and belong to forms displaying characteristics similar to those of primitive Placoderms (cf. Stensi6, 1963). The most recent survey of selachian evolution is that of Schaeffer (1967), who has proposed a scheme in which fossil and extant selachian fishes are distinguished according to three subsequent organizational levels (Fig. 1), i.e. the cladodont level, comprizing selachians from the Paleozoic; the hybodont level, related to forms exhibiting more advanced morphologic characteristics, which became established in the Lower Trias, and the modern level, composed of presentday forms, which arose in the Jurassic. The study of the available fossil record suggests that Paleozoic selachian fishes had a strikingly widespread radiation, giving rise to a variety of forms with

Genotoxicity in Zebrafish (Danio rerio) Exposed to two Pharmacological Products from an Impacted Italian River

The aim of this study was to identify the pharmaceutical substances in the waters of the river Volturno in Campania and to identify the genetic alterations that they induce in fish. These vertebrates, living in water, are particularly sensitive to polluting substances dissolved in their water that interact with DNA leading to the formation of additions or breaks, as well as to the loss or chemical modifications of the bases, because they can conserve and concentrate the substances discharged into their natural habitat. A preventive chemical analysis was carried out on the waters of the river Volturno. The results showed the presence of various drugs, among which Carbamazepine, a known antiepileptic and Diclofenac, a non-steroid anti-inflammatory agent. The genotoxicity of these two compounds was evaluated in 155 individuals of Danio rerio using the Comet Test, the Diffusion Assay and RAPD-PCR. The alkaline version of the Comet Test was used for the erythrocytes of the zebrafish to evaluate the presence of DNA breaks of a single strand. Furthermore, the Diffusion Assay was used to estimate the number of apoptotic cells. The specimens were treated with Carbamazepine and Diclofenac at the same average concentrations found in the river Volturno and were killed after three, seven and fifteen days. The results of the Comet Test showed a statistically significant loss of DNA integrity after only three days of exposure to the drugs. Also the percentage of apoptotic cells reached its maximum values after three days of exposure to Carbamazepine and after one week to Diclofenac. By RAPD-PCR the genotoxicity was evaluated at the molecular level. The amplification products from the individuals exposed to the same concentrations of the environmental pollutants showed significant changes in their electrophoretic pattern with respect to the negative controls. The results of all three tests clearly show the genotoxicity of the compounds analyzed.

NOR and heterochromatin analysis in two cartilaginous fishes by C-, Ag- and RE (restriction endonuclease)-banding

Selachian chromosomes were investigated for the first time by C-, Ag- and RE (restriction endonuclease)-banding. A pair of NOR-bearing acrocentric chromosomes, as well as a third extra site, were demonstrated in two species of Torpedo. In situ digestion with restriction endonucleases revealed a peculiar arrangement of constitutive heterochromatin: in Torpedo ocellata the centromere never appeared C-banded, and was always digested by AluI, PstI, DdeI and HaeIII; in Torpedo marmorata, instead, heterochromatin was present in the centromere, and was not digested by the same enzymes. The occurrence of different types of centromeric heterochromatin suggests an involvement of highly repeated DNA fractions in the Robertsonian phenomena that occurred in chromosome rearrangements in the genus Torpedo.

Chondrichthyan cytogenetics : a comparison with teleosteans

SummaryCytogenetic studies on cartilaginous fish conducted in recent years have shown that these vertebrates have peculiarities associated both with the karyotypes and the size and composition of their DNAs. Although the data for this group, which includes about 1000 extant species, are still fragmentary, there appear to be more differences than similarities with teleosts; e.g., chromosome sets are characterized by a high diploid number (2n=up to 106) and are often rich in acrocentric elements and in microchromosomes. From the quantitative standpoint, chondrichthyan genomes are relatively large (2C=up to 34 pg DNA/n), exhibiting sometimes wide interspecific variability (Squalidae).The few studies on genome composition for these species have revealed marked difference between chondrichthyans and teleosteans in the ratio of the amount of GC-rich DNA to the total increase in genome. Moreover, thermal denaturation of the genomes of six selachians revealed derived curves that are characteristic of heterogeneity in nucleotide distribution, which has not been evidenced in most of the teleosteans investigated thus far.Finally, for the first time in selachians, an investigation was conducted using restriction enzymes, the results of which showed a pattern of chromosome labeling that was in some cases (Alu I) similar to and in others (Hae III, Hind III) different from that of teleosteans.

Cloning and characterization of a repetitive DNA detected by HindIII in the genome of Raja montagui (Batoidea, Chondrichthyes)

A repetitive HindIII fragment of DNA from Raja montagui (Rajiformes) was cloned and sequenced for the first time in cartilaginous fishes. This element, which comprises approximately 5% of the whole genome of the spotted ray, is absent in long tandem arrays, being typical of satellite DNA. It appeared constituted by 311 AT-rich bp (61%). The clone was hybridized to the genomic DNA of species with varying phyletic distances, revealing a high degree of conservation.

Characterisation of 5S rDNA in Gasterosteus aculeatus (Teleostei, Gasterosteidae)

Abstract The 5S rRNA genes of Gasterosteus aculeatus were characterised. Genomic DNA was amplified by PCR using two primers constructed in different salmonid species in order both to extract the total 5S rDNA (coding region + nontranscribed spacer region) and to generate amplified DNA products only if 5S rDNAs were tandemly arrayed. Amplification revealed four bands (A, B, C, and D) of about 250, 370, 580, and 660 base pairs, respectively. The electrophoretic pattern yielded by PCR products appeared very similar in the various species examined, it was also present in a selachian, an amphibian and a reptile. However, differences were observed in the size of the bands. Band A was cloned and se‐quenced: it consisted of 225 base pairs (A+T = 48.6%; G+C = 51.4%) and showed a high sequence homology to 5S rDNA in many vertebrate species. Hybridization on nylon filters of genomic DNA digested with several restriction enzymes showed the typical tandem array of repeated DNAs.

A cytogenetic analysis of Gambusia holbrooki (Cyprinodontiformes, Poecilidae) from the River Sarno

Abstract The mosquitofish, Gambusia holbrooki, is one of the few tele‐ostean fish still present in the polluted waters of the River Sarno. The specimens taken in this river had 48 chromosomes, all acro‐centric, gradually decreasing in length in both sexes. However, several (12%) female plates showed a chromosome with a unilateral gap often accompanied by a chromosome number reduced to 47, due to the presence of a biarmed element derived by centric fusion of two uniarmed elements. The nuclear organizer regions (NORs) were localized on two of the smallest chromosome pairs by Ag NOR banding. C banding + DAPI revealed centromer‐ic AT‐rich heterochromatin on all elements. The results also suggest a possible mutagenic and toxic activity of the substances present in the polluted waters of the River Sarno.

Genomic Damage in Human Sperm Cells Exposed In Vitro to Environmental Pollutants

Environmental pollution of waters caused by the release of chemical substances is becoming a threat for both man and the environment. The active pharmacological agents discharged into the waters, can interact with the cell DNA and induce genetic damage. The aim of the present work was to evaluate the potential genotoxic effects of nine pharmacological agents, Atorvastatin, Sildenafil citrate, Gemfibrozil, Ibuprofen, Atenolol, Ofloxacin, Carbamazepine, Bezafibrate, and Diclofenac present in the waste waters of some Italian treatment plants. Genotoxic effects of the nine pharmacological compounds were evaluated by using four tests, the Comet Assay, the Diffusion Assay, the TUNEL test, and RAPD-PCR technique. The sperm cells were exposed in vitro for three different times: 15, 30 and 45 min at the mean concentrations detected in the waste waters. All drugs tested induced a statistically significant reduction in the integrity of sperm DNA and high fragmentation values. The drugs found in the wastewater were then able to induce damage to sperm DNA, highlighted in a very early stage of cell suffering, even before they are capable of inducing other metabolic disorders.