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Due to the large production and growing use of titanium dioxide nanoparticles (n-TiO2), their release in the marine environment and their potential interaction with existing toxic contaminants represent a growing concern for biota. Different end-points of genotoxicity were investigated in the European sea bass Dicentrarchus labrax exposed to n-TiO2 (1mgL(-1)) either alone and combined with CdCl2 (0.1mgL(-1)) for 7 days. DNA primary damage (comet assay), apoptotic cells (diffusion assay), occurrence of micronuclei and nuclear abnormalities (cytome assay) were assessed in peripheral erythrocytes and genomic stability (random amplified polymorphism DNA-PCR, RAPD assay) in muscle tissue. Results showed that genome template stability was reduced after CdCl2 and n-TiO2 exposure. Exposure to n-TiO2 alone was responsible for chromosomal alteration but ineffective in terms of DNA damage; while the opposite was observed in CdCl2 exposed specimens. Co-exposure apparently prevents the chromosomal damage and leads to a partial recovery of the genome template stability., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The increasing use of nanotechnologies will lead to significant releases of engineered nanoparticles into the aquatic environment, where their impact is still poorly characterized. In the present paper, the genotoxic and cytotoxic properties of CdS quantum dots (QDs) and silver sulphide (Ag2S) coated with methyl polyethylene glycol (M-PEG) were investigated in a rainbow trout cell line (RTG-2). The results showed that CdS QDs were highly cytotoxic at high concentrations (10 and 50μg/ml), and exhibited a concentration-dependent genotoxicity in the sub-toxic range (0.01-1μg/ml) after 24h exposure. Ag2S showed neither genotoxic nor cytotoxic effects., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We induced brief secondarily generalized seizures of limbic origin in Sprague-Dawley rats by bicuculline microinfusion into the anterior piriform cortex. After 1 h or 5 days we performed comet assay, a sensitive marker for DNA damage, within entorhinal cortex, hippocampus (limbic areas recruited by seizure spreading) and striatum (which is not recruited). DNA damage occurred selectively in the ipsilateral entorhinal cortex and hippocampus at 1 h, but not at 5 days. These data shed new light on molecular genetics as a marker during limbic seizures, the most common in epileptic patients.

In the present study, the genotoxic potential of nanosized TiO2 anatase and micro-sized rutile on bottlenose dolphin (Tursiops truncatus) fibroblasts and leukocytes was investigated. Human and mouse cells were also studied in order to compare susceptibility to TiO2 in different mammalian species. Cell lines were exposed for 4, 24, and 48 h to different concentrations of TiO2 (20, 50, 100, 150 μg/ml) and DNA damage was investigated by single cell gel electrophoresis (Comet assay). Both anatase and rutile induced increased DNA damage, even though statistically significant effects were scattered according to species and cell lines. Bottlenose dolphin leukocytes and murine fibroblasts exhibited increased DNA damage after rutile exposure at some doses/times, while human fibroblasts showed a significant dose-response effect after a 4 h exposure to anatase. Human leukocytes were tolerant to both anatase and rutile. Ultrastructural investigation showed that TiO2 particles entered the cell and were compartmentalized within membrane-bound vesicles., (Copyright © 2014. Published by Elsevier Ltd.)

Although amorphous silica is used in food products, cosmetics and paints and as vector for drug delivery, data on its potential health hazard are limited. The aim of this study was to investigate the cytotoxic and genotoxic potential of silica particles of different sizes (250 and 500nm) and structures (dense and mesoporous). Dense silica (DS) spheres were prepared by sol-gel synthesis, mesoporous silica particles (MCM-41) were prepared using hexadecyltrimethyl ammonium bromide as a structure-directing agent and tetraethylorthosilicate as silica source. Particles were accurately characterised by dynamic light scattering, nitrogen adsorption, X-ray diffraction and field emission scanning electron microscopy. Murine macrophages (RAW264.7) and human epithelial lung (A549) cell lines were selected for investigation. Genotoxicity was evaluated by Comet assay and micronucleus test. Cytotoxicity was tested by the trypan blue method. Cells were treated with 0, 5, 10, 20, 40 and 80 µg/cm(2) of different silica powders for 4 and 24 h. The intracellular localisation of silica was investigated by transmission electron microscopy. Amorphous particles penetrated into the cells, being compartmentalised within endocytic vacuoles. DS and MCM-41 particles induced cytotoxic and genotoxic effects in A549 and RAW264.7 although to different extent in the two cell lines. A549 were resistant in terms of cell viability, but showed a generalised induction of DNA strand breaks. RAW264.7 were susceptible to amorphous silica exposure, exhibiting both cytotoxic and genotoxic responses as DNA strand breaks and chromosomal alterations. The cytotoxic response of RAW264.7 was particularly relevant after MCM-41 exposure. The genotoxicity of amorphous silica highlights the need for a proper assessment of its potential hazard for human health.

Loud noise is generally considered an environmental stressor causing negative effects on acoustic, cardiovascular, nervous, and endocrine systems. In this study, we investigated the effects of noise exposure on DNA integrity in rat adrenal gland evaluated by the comet assay. The exposure to loud noise (100 dBA) for 12 hr caused a significant increase of DNA damage in the adrenal gland. Genetic alterations did not decrease 24 hr after the cessation of the stimulus. We hypothesize that an imbalance of redox cell status is responsible for the induction and persistence of noise-induced cellular damage.

The relationship between DNA damage and the exposure of marine organisms to environmental contaminants was examined in the Göteborg harbour area. This research is part of a wider ecotoxicological study planned to evaluate the biological impact of chemical contamination in the River Göta estuary, following a bunker oil (10-100 tonnes) spill occurred in June 2003. Here we present data on the DNA strand breaks derived using the comet assay and the presence of apoptotic cells using the diffusion assay in nucleated erythrocytes of the eelpout (Zoarces viviparus) from the study area and at a clean reference site. Polycyclic aromatic hydrocarbon metabolites were also analyzed in the bile of exposed fish. The results showed a high level of damaged DNA, paralleled by a peak in bile PAH metabolites, in fish from the most impacted site, 3 weeks after the oil spill. A significant recovery was observed in specimens from the spill site, 5 months later, but not in fish caught in the middle part of Göteborg harbour, which is chronically subjected to heavy chemical pollution. The levels of apoptic cells did not show any marked variations, but a significant recovery was observed in fish from the oil impacted site 5 months after the spill.

Harbours can be considered as model environments for developing and validating field monitoring procedures and to investigate mechanistic relationships between different biological responses. In this study, several biomarkers were investigated in marine mussels caged for 4 weeks into an industrialised harbour of north-west Italy. Organisms were collected at different time intervals to better characterise the sensitivity, temporal variations and interactions of analysed responses. Besides single antioxidants (catalase, glutathione S-transferases, glutathione reductase, total glutathione), the total oxyradical scavenging capacity (TOSC) assay was used to analyse the capability of the whole antioxidant system to neutralise specific forms of radicals: these data were further integrated by measurement of DNA integrity, oxidised bases and the impairment of lysosomal membrane stability in haemocytes. Results showed a biphasic trend for single antioxidants and TOSC, with no variation or increase during the first 2 weeks of exposure to the polluted site followed by a progressive decrease up to a severe depletion in the final part of the experiment. These findings suggest an initial counteractive response of mussels toward the enhanced prooxidant challenge, while antioxidants appeared overwhelmed at longer exposure periods. The hypothesis of reactive oxygen species (ROS) mediated toxicity is supported by the appearance of cell damages (DNA integrity and lysosome membrane stability), which exhibited a progressive enhancement during the course of the experiment with a maximum impairment after 30 days of exposure.

Rationale: 3,4-Methylenedioxymethamphetamine (MDMA) is an amphetamine derivative, which is neurotoxic to both serotonin (5HT) and dopamine (DA) nerve terminals. Previous reports, carried out in rodents and non-human primates, demonstrated neurotoxicity to monoamine axon terminals, although no study has analyzed nigral and striatal cell bodies at the sub-cellular level., Objective: In this study, we examined intrinsic nigral and striatal cells, and PC12 cell cultures to evaluate whether, in mice, MDMA might affect nigral and striatal cell bodies., Methods: After administering MDMA, we analyzed effects induced in vivo and in vitro using high-performance liquid chromatography (HPLC) analysis, light- and electron microscopy with immunocytochemistry, and DNA comet assay., Results: We found that MDMA (5 mg/kg x4, 2 h apart), besides a decrease of nigrostriatal DA innervation and 5HT loss, produces neuronal inclusions within nigral and intrinsic striatal neurons consisting of multi-layer ubiquitin-positive whorls extending to the nucleus of the cell. These fine morphological changes are associated with clustering of heat shock protein (HSP)-70 in the nucleus, very close to chromatin filaments. In the same experimental conditions, we could detect oxidation of DNA bases followed by DNA damage. The nature of inclusions was further investigated using PC12 cell cultures., Conclusions: The present findings lead to re-consideration of the neurotoxic consequences of MDMA administration. In fact, occurrence of ubiquitin-positive neuronal inclusions and DNA damage both in nigral and striatal cells sheds new light into the fine alterations induced by MDMA, also suggesting the involvement of nuclear and cytoplasmic components of the ubiquitin-proteasome pathway in MDMA toxicity.

In this work, susceptibility to oxidative stress was analyzed under laboratory conditions in the European eel Anguilla anguilla. Eels were treated with increasing concentrations of benchmark environmental pollutants, namely, benzo[a]pyrene ([BaP], at 0, 0.1, 1, 10, and 50 mg/kg), beta-naphthoflavone ([BNF], at 0, 0.1, 1, 10, and 50 mg/kg), Arochlor 1254 (at 0, 0.1, 1, 10, and 50 mg/kg), and 2,3,7,8-tetrachlorodibenzo p-dioxin ([TCDD], at 0, 0.01, 0.1, 1, and 2 microg/kg). The integral relationships were analyzed between induction of ethoxyresorufin O-deethylase (EROD) activity, its involvement in perturbing oxyradical metabolism, and the role of cytochrome P450 and/or oxidative stress in mediating genotoxic effects. To reveal whether the oxidative status in exposed organisms was altered as a result of chemical exposure, measurements of the main endogenous antioxidant defenses were integrated with the measurement of total oxyradical scavenging capacity (TOSC) toward peroxyl radicals and hydroxyl radicals (*OH). This approach permits discriminating the resistance of a tissue toward different forms of oxyradicals, thereby indicating a differential role for specific reactive oxygen species (ROS) in perturbing the balance between prooxidant and antioxidant mechanisms. All the analyzed chemicals promoted EROD induction (reflective of CYP1A) and altered either the levels or the activities of the antioxidants studied, which might be anticipated to exert alterations in oxyradical metabolism. Analysis of TOSC suggested the prevalence of metabolic oxidative pathways leading to the more reactive *OH on exposure to the chemicals studied. Of these chemicals, enhanced EROD activity correlated with genotoxic damage only in the cases of the nonhalogenated hydrocarbons BaP and BNF. The highest degree of genotoxic damage was consistently observed in organisms in which the capacity to absorb or scavenge OH was lowest. These data suggest a general relationship between oxidative stress and loss of DNA integrity in juvenile eels exposed to the chemicals studied herein.

Noise exposure causes changes at different levels in human organs, particularly the cardiovascular system, where it is responsible for increasing heart rate, peripheral vascular resistance, and blood pressure. In this study, we evaluated the effect of noise exposure on DNA integrity and ultrastructure of rat cardiomyocytes. The exposure to loud noise (100 dBA) for 12 hr caused a significant increase of DNA damage, accompanied by swelling of mitochondrial membranes, dilution of the matrix, and cristolysis. These alterations were concomitant with increased in situ noradrenaline levels and utilization. Genetic and ultrastructural alterations did not decrease 24 hr after the cessation of the stimulus. An elevated oxyradical generation, possibly related to altered sympathetic innervation, is hypothesized as responsible for the induction and persistence of noise-induced cellular damage.

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.

The ability of benzo[a]pyrene, Aroclor 1254, 2-3-7-8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone to induce DNA strand breaks (SB) and apoptosis in erythrocytes of the European eel (Anguilla anguilla) was investigated following by in vivo exposure. DNA damage was evaluated by the Comet assay, while the diffusion assay was used to investigate the induction of apoptosis 7 days after a single intraperitoneal administration. 2-3-7-8-Tetrachlorodibenzo-p-dioxin induced the highest genotoxic effect, followed by benzo[a]pyrene, while the other two substances had limited effects. A significant induction of apoptosis was observed at the highest doses after exposure to benzo[a]pyrene, when DNA damage was also elevated. The occurrence of apoptotic cells after exposure to Aroclor, 2-3-7-8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone was quite variable and did not show clear dose-related responses. The role of oxidative stress in mediating DNA damage was also discussed.

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.

Single cell gel electrophoresis (or Comet Assay) was used for evaluation of the in vitro genotoxicity of hydrogen peroxide (used as a positive control), polychlorinated biphenyls (PCBs) (Aroclor 1254) and methyl mercury chloride, in isolated bottlenose dolphin leukocytes. Results showed that hydrogen peroxide and methyl mercury induced DNA strand breakage in a dose-dependent manner, while PCBs did not induce a clear dose-effect response at the low doses investigated. Efficiency in repairing DNA breakage induced by methyl mercury was also evaluated. Findings demonstrated that dolphin cells are characterized by higher efficiency in DNA repair when compared to human leukocytes. The observed resistance to methyl mercury toxicity in dolphins was hypothesized to be a defence strategy developed to combat high dietary exposure and compensate for limited capacity to excrete persistent pollutants.

Crystalline silica inhaled from occupational sources has been classified by IARC as carcinogenic to humans; in contrast, for amorphous silica, epidemiological and experimental evidence remains insufficient. The genotoxicity of crystalline silica is still debated because of the inconsistency of experimental results (“variability of silica hazard”), often related to the features of the particle surfaces. We have assessed the role of crystal habit in the genotoxicity of silica powders. Pure quartz (crystalline) and vitreous silica (amorphous), sharing the same surface features, were used in an in vitro study with human pulmonary epithelial (A549) and murine macrophage (RAW264.7) cell lines, representative of occupational and environmental exposures. Genotoxicity was evaluated by the comet and micronucleus assays, and cytotoxicity by the trypan blue method. Cells were treated with silica powders for 4 and 24 h. Quartz but not vitreous silica caused cell death and DNA damage in RAW264.7 cells. A549 cells were relatively resistant to both powders. Our results support the view that crystal habit per se plays a pivotal role in modulating the biological responses to silica particles. [ABSTRACT FROM AUTHOR]

Abstract: This review considers the potential of the Comet assay (or Single Cell Gel Electrophoresis, SCGE) to evaluate the environmental impact of genotoxins in aquatic environments. It focuses on in vivo and in situ studies that have been carried out in various marine and freshwater sentinel species, published in the last 5 years. A large number of the studies reviewed report that the Comet assay is more sensitive when compared with other biomarkers commonly used in genetic ecotoxicology, such as sister chromatid exchanges or micronucleus test. Due to its high sensitivity, the Comet assay is widely influenced by laboratory procedures suggesting that standard protocols are required for both fish and mussel cells. However, there are still a wide variety of personalised Comet procedures evident in the literature reviewed, making comparison between published results often very difficult. Standardization and inter-laboratory calibration of the Comet assay as applied to aquatic species will be required if the Comet assay is to be used routinely by national bodies charged with monitoring water quality. [Copyright &y& Elsevier]

Abstract: In developed countries, estuarine environments are often subjected to chemical pollution, whose biological impact is profitably evaluated by the use of multi-biomarker approaches on sentinel species. In this paper, we investigate genotoxicity and lysosomal alterations in the Mediterranean mussel (Mytilus galloprovincialis), from the estuary of the River Cecina (Tuscany, Italy), selected as “pilot basin” within the Water Frame Directive (2000/60 European Community). Both native and 1 month transplanted mussels were used in order to compare these two approaches in terms of sensitiveness of specific biomarker responses. Genotoxic effects were evaluated as strand breaks, by single cell gel electrophoresis (or Comet assay), and as chromosomal alterations, by the micronucleus test in gill cells. Lysosomal alterations were assessed by the neutral red retention time (in haemocytes), lipofuscin accumulation and ultrastructure (in digestive cells). Heavy metal bioaccumulation was also analysed. Mussels from the River Cecina showed a general alteration of all the biomarkers investigated, accompanied by an elevation of tissue metal levels. However, some differences in specific responses occurred between transplanted and native mussels. Early biomarkers, such as those based on DNA and lysosomal membrane integrity, were induced at similar degree in native and transplanted mussels; while alterations resulting from cumulative events, as the increase of micronuclei frequency were much more elevated in native specimens (23.1±7.6) than in transplanted (9.3±4.7) and reference ones (5.8±5.2). Similarly, the comparison between lipofuscin accumulation and mean lysosomal diameter in impacted and control sites, gave significant differences exclusively with transplanted mussels. These results suggest that the parallel use of caged and native mussels in environmental biomonitoring can improve the characterization of the study area. [Copyright &y& Elsevier]

Single cell gel electrophoresis (or Comet Assay) was used for evaluation of the in vitro genotoxicity of hydrogen peroxide (used as a positive control), polychlorinated biphenyls (PCBs) (Aroclor 1254) and methyl mercury chloride, in isolated bottlenose dolphin leukocytes. Results showed that hydrogen peroxide and methyl mercury induced DNA strand breakage in a dose-dependent manner, while PCBs did not induce aclear dose-effect response at the low doses investigated. Efficiencyin repairing DNA breakage induced by methyl mercury was also evaluated. Findings demonstrated that dolphin cells are characterized by higher efficiency in DNA repair when compared to human leukocytes. The observed resistance to methyl mercury toxicity in dolphins was hypothesized to be a defence strategy developed to combat high dietary exposure and compensate for limited capacity to excrete persistent pollutants. [ABSTRACT FROM AUTHOR]