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Genotoxicity is the ability of specific substances to cause DNA damage, affecting development, physiology, and reproduction. This is often mediated by induction of oxidative stress. This in vitro study aims to test the ability of two antioxidants, ellagic acid (EA, 100 µM) and curcumin (Cur, 40 µM) to protect zebrafish blood cells from the genotoxic action of benzene (10 µL/mL). Cells were treated for 30, 60, and 90 min with EA or Cur alone and in combination with benzene. The antigenotoxic role of antioxidants was evaluated in terms of cytotoxicity by trypan blue dye, genome stability by RAPD-PCR technique, DNA fragmentation and percentage of apoptotic cells using Comet and Diffusion assay, respectively. The results did not show statistical differences in terms of cell viability, genome stability, DNA damage and apoptosis between cells treated with antioxidants. When zebrafish blood cells were co-incubated with individual antioxidants and benzene, a significant improvement of these parameters was observed in comparison with cells incubated in benzene. Our results suggested that EA and Cur are able to protect zebrafish blood cells against DNA damage and apoptosis caused by mutagenic substance, and laid the foundation for future studies investigating their antigenotoxic potential in DNA oxidative damage therapy.

The increased titanium dioxide nanoparticles (TiO2-NPs) spread and their interaction with organic and inorganic pollutants arouses concern for the potential hazards for organisms and environment. This study tested in vitro the genotoxic effects of TiO2-NPs (1 μg/mL) and cadmium (Cd) (0.1 μg/mL) co-exposure using Dicentrarchus labrax embryonic cells (DLEC) as experimental model. The genotoxicity tests (Comet assay, Diffusion Assay and Random Amplification of Polymorphic DNA (RAPD-PCR) were conducted after 3, 24 and 48 hours of exposure to TiO2-NPs and Cd alone and in combination. The results showed that the percentage of DNA damage and apoptotic cells increases following 48 hours TiO2-NPs exposure, while DNA instability was detected for all the times tested. Cd induced genotoxic effects starting from 3 hour-exposure and for all the treatment times. Cd + TiO2-NPs co-exposure did not cause any genomic damage or apoptosis for all the exposure times. The possibility that Cd and TiO2-NPs form aggregates no longer able of penetrating the nucleus and damaging the genetic material is discussed. Graphical abstract: [Figure not available: see fulltext.].

Cryopreservation causes decreased sperm fertility potential due to reactive oxygen species (ROS) production and physical-chemical damage, resulting in reduced sperm viability and motility. The addition of antioxidants to freezing media could protect sperm from cryo-damage, counteracting the harmful effects of ROS. The aim of this study was to assess the effects of curcumin supplementation in freezing medium on preventing cryo-damage in human semen. Semen samples collected from fertile men were cryopreserved in freezing medium supplemented with different concentrations of curcumin (2.5, 5, 10, and 20 μM). After freezing-thawing, sperm parameters, DNA fragmentation, intracellular ROS, and glutathione peroxidase 4 (GPX4) gene expression were evaluated. Supplementation with 20 μM curcumin in freezing medium caused increases in progressive and nonprogressive motility and significant reductions in intracellular ROS and DNA fragmentation in frozen-thawed sperm cells. Following cryopreservation,GPX4 mRNA expression was significantly upregulated in thawed semen supplemented with 20 μM curcumin compared to the control. The results showed that curcumin supplementation in freezing medium was protective against human sperm parameters and sperm DNA, counteracting oxidative damage induced by the freeze-thaw process.

Oxidative stress (OS) plays a significant role in the etiology of male infertility, resulting in the impairment of male reproduction. This condition, characterized by an imbalance in the levels of oxidizing and antioxidant species in the seminal fluid, has a harmful impact on sperm functions and DNA integrity. The present study aimed to evaluate the anti-genotoxic action of ellagic acid, a polyphenolic molecule of natural origin having a powerful antigenotoxic, anti-inflammatory and antiproliferative role. An OS condition was induced in vitro by incubating normozoospermic human semen samples in benzene for 45, 60 and 90 min. DNA integrity was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay, RAPD-PCR was performed to calculate the genome template stability, while the percentage of intracellular reactive oxygen species (ROS) was assessed by the 2ʹ, 7ʹ-dichlorofluorescein assay. Our results showed that ellagic acid has a consistent protective effect on DNA integrity, as well as on sperm vitality and motility, by counteracting generation of intracellular ROS. The results of this study suggest ellagic acid as a suitable molecule to protect sperm DNA from oxidative stress, with a potentially significant translational impact on the management of the male infertility.

The aquatic environment is involved in the pollutants spreading mechanisms, including nanomaterials and heavy metals. The aims of this study were to assess the in vivo genotoxicity of Cd (1 mg/L) and to investigate the genomic effects generated by its co-exposure with TiO2-NPs (10 µg/L). The study was performed using zebrafish as a model for 5, 7, 14, 21, and 28 days of exposure. The genotoxic potential was assessed by three experimental approaches: DNA integrity, degree of apoptosis, and molecular alterations at the genomic level by genomic template stability (% GTS) calculation. Results showed an increased in DNA damage after Cd exposure with a decrease in % GTS. The co-exposure (TiO2-NPs + Cd) induced a no statistically significant loss of DNA integrity, a reduction of the apoptotic cell percentage and the recovery of genome stability for prolonged exposure days. Characterization and analytical determinations data showed Cd adsorption to TiO2-NPs, which reduced free TiO2-NPs levels. The results of our study suggest that TiO2-NPs could be used for the development of controlled heavy metal bioremediation systems.

During its development, embryo is easily susceptible to reactive oxygen species (ROS). Evidence demonstrate protective role of the antioxidants, improving both cellular growth and embryonic development. Among these, ellagic acid (EA) is a natural antioxidant with anti-inflammatory and anti-carcinogen properties. The aim of this work was to assess in vitro the protective and anti-genotoxic role of EA during Danio rerio (zebrafish) embryonic development. For the study, zebrafish embryos were treated with H2O2 (15 μM, 30 μM and 45 μM) to simulate an oxidative damage, and with EA (2.5 mM, 5 mM and 10 mM) for 8, 20, 24, 48, 96 hpf (hours post fertilization). Vitality rate, alterations in the morphology and behavior of the larvae and the genomic stability were analyzed. The exposure to H2O2 caused genotoxicity for all exposure times. The incubation in 45 μM H2O2 and 30 μM H2O2 resulted in increased mortality rate of the larvae, as well as 10 mM EA. The co-exposure was performed using to 15 μM H2O2 and 2.5 mM and 5 mM EA and it demonstrated the EA capacity to protect the embryo DNA and development from the oxidative insult. Particularly, the co-exposure to 15 mM H2O2 and 5 mM EA showed an increase in the embryo survival rate and absence of alterations in morphology and behavior at 96 hpf. Interestingly, we observed a higher genomic stability at 8h and 20h co-exposure (15 mM H2O2 and 5 mM EA) time. The decline observed in ROS concentration for both exposure times confirmed the observation. In conclusion, EA protects the zebrafish embryonic development from DNA oxidative damage increasing the embryo survival rate and improving morphological parameters of the larvae.

Continuous cell lines could provide an important tool for studying epidemiology, toxicology, cellular physiology and the host-pathogen interactions. Random amplified polymorphic deoxyribonucleic acid analysis by PCR (RAPD-PCR) was used for the molecular characterization of Dicentrarchus labrax embryonic cells (DLEC) as a possible tool to detect DNA alterations in environmental genotoxic studies. We studied the DNA pattern of the DLEC fish cell line, a fibroblast-like cell line derived from European sea bass. From a total of 15 primers only six showed good discriminatory power for the amplification process on DNA samples collected from cells by three different methods (organic extraction, salting-out method and chelating agent extraction). The results obtained show that the cell line chosen for this study could be used as a possible tool for the detection of potential genotoxicity of numerous chemical compounds. © 2013 Springer Science+Business Media Dordrecht.

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.

We investigated the influence of titanium dioxide nanoparticles (nano-TiO2) on the response to cadmium in the gills of the marine mussel Mytilus galloprovincialis in terms of accumulation and toxicity. Mussels were in vivo exposed to nano-TiO2, CdCl2, alone and in combination. Several cellular biomarkers were investigated in gills: ABC transport proteins and metallothioneins at gene/protein (abcb1, abcc-like and mt-20) and functional level, GST activity, NO production and DNA damage (Comet assay). Accumulation of total Cd and titanium in gills as in whole soft tissue was also investigated. Significant responses to Cd exposure were observed in mussel gills as up-regulation of abcb1 and mt-20 gene transcription, increases in total MT content, P-gp efflux and GST activity, DNA damage and NO production. Nano-TiO2 alone increased P-gp efflux activity and NO production. When combined with Cd, nano-TiO2 reduced the metal-induced effects by significantly lowering abcb1 gene transcription, GST activity, and DNA damage, whereas, additive effects were observed on NO production. A lower concentration of Cd was observed in the gills upon co-exposure, whereas, Ti levels were unaffected. A competitive effect in uptake/accumulation of nano-TiO2 and Cd seems to occur in gills. A confirmation is given by the observed absence of adsorption of Cd onto nano-TiO2 in sea water media.

Chromosome location of major (18S, 5.8S and 28S) and 5S ribosomal RNA genes (rDNAs) was examined in Lebias fasciata collected from different Italian blackish-waters, using silver (Ag)- and chromomycin A3 (CMA 3)-staining and/or fluorescence in situ hybridization (FISH). Both 18S and 5S rDNA probes for FISH were obtained with polymerase chain reaction-directed cloning from genomic DNA of the examined species. Nucleolar organizer regions (NORs) containing the major rDNAs showed intraspecific polymorphism in number as detected by Ag-and CMA3-staining and FISH with the 18S rDNA probe. On the other hand, 5S rDNA loci constantly occurred on one chromosome pair and co-localized with a pair of the major rDNA loci as evidenced by two-color FISH using the 5S and 18S rDNA probes. Sequential CMA3- and Ag-NOR staining and FISH revealed apparent inactivation of some NORs. The cloned 5S rDNA was found to contain some TATA-like sequences that might play an important role in the regulation of gene expression.

The distribution of telomeric sequence (TTAGGG)n in the genomes of Chondrichthian species at different stages of evolution was investigated both by DNA genomic hybridization, and by fluorescence in-situ hybridization (FISH) of metaphase chromosomes. The sequence is highly conserved in all the species examined. FISH revealed a label uptake only by the telomeres in Raja asterias. However, in Torpedo ocellata, we revealed pericentromeric and interstitial sequence localization on some chromosome pairs in addition to the hybridization signal on telomeres. These findings confirm that the karyotype evolution of these fish began by Robertsonian fusion.

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 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.

Among vertebrates, chondrichthyans exhibit peculiar karyotypes and total amount and composition of DNA very different from those of tetrapods and teleosteans. Selachians have relatively large genome sizes (more than 30 pg/N), which are inversely related to the fraction rich in adenine‐thymine. Moreover, they show a high chromosome number (2n = 60–100), decreasing in the most specialized species. The karyotypes of Scyliorhinus stellaris, Torpedo ocellata, and T. marmorata have been investigated by several techniques in order to distinguish particular genome fractions along the chromosome arms. C‐banding appears to be positive in most telomeric regions in Scyliorhinus and at the interstitial level in the two torpedo species. Studies with restriction enzymes (RE) have just been started in selachians, employing ALU I and HIND III in Scyliorhinus stellaris. The former digests the whole chromosome except the telomeric regions, revealing patterns similar to the C‐bands. The latter cuts the chromosomes into several interstitial regions, producing G‐bands. Other RE are being studied, which will allow identification in situ of qualitative differences in the various DNAs. Copyright © 1989 Wiley‐Liss, Inc., A Wiley Company

The nuclear DNA content of 23 selachian species (10 Batoidea, 11 Galeomorphii, and 2 Squalomorphii) was histophotometrically studied. Their genome sizes range from 7.5 pg/N in Raja fillae (Batoidea) to 34.1 pg/N in Oxynotus centrina (Squalomorphii). Results show slight differences in the pattern of quantitative variations between the superorders Batoidea and Galeomorphii; Squalomorphii preserve their peculiar wide interspecific variability at the intrafamilial level, with values sited between 13.1 and 34.1 pg/N. In 21 species also the DNA base composition was determined by means of DAPI. The study shows that in the species examined the DAPI positive fraction varies from a minimum of 27.7% in Oxynotus centrina, which possesses the largest genome size among all the Selachians studied, to a maximum of 72.5% in Carcharhinus limbatus. As a whole the data show an inverse correlation between the DNA content and the DAPI positive fraction, a condition common to all cold-blooded vertebrates. The low percentage of DAPI positive DNA found in Oxynotus centrina could be attributable to a lower stainability by the fluorochrome caused by a higher chromatin condensation in the erythrocytes. The validity of the DAPI method was verified by comparison with the biochemical assay according to the thermal denaturation method in 6 selachian species. © 1989 Kluwer Academic Publishers.