Giada Frenzilli

The Comet assay for the evaluation of genotoxic impact in aquatic environments.

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.

Oxidative stress in ecotoxicology: from the analysis of individual antioxidants to a more integrated approach.

An integrate approach will be discussed for investigations on oxidative stress in xenobiotic toxicity. While the analysis of individual antioxidants is useful for their sensitivity and to understand the mode of action of a stressor, the integration with the analysis of the total antioxidant capacity provides a more holistic assessment of the overall biological significance of such variations. TOSC has a greater predictive value on the health condition of the organisms and allows to discriminate the different role of specific ROS in oxidative stress syndrome.

Integrating enzymatic responses to organic chemical exposure with total oxyradical absorbing capacity and DNA damage in the European eel Anguilla anguilla.

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.

Herbicide-induced DNA damage in human lymphocytes evaluated by the single-cell gel electrophoresis (SCGE) assay.

The genotoxicity of the herbicides, alachlor, atrazine, maleic hydrazide, paraquat and trifluralin has been evaluated in the single-cell gel electrophoresis (SCGE) assay by using human peripheral blood lymphocytes. All treatments were conducted with and without the presence of an external bioactivation source (S9 mix). The results indicate that all the herbicides tested are able to give positive results by increasing the comet tail length, which would confirm both the genotoxicity of the herbicides and the sensitivity of the assay in front of these chemicals. Alachlor and atrazine give similar results in treatments with and without S9, while when the S9 mix was not used paraquat and trifluralin genotoxicity was higher. On the other hand, although maleic hydrazide genotoxicity was higher when S9 mix was used at normal pH (7.4), our data show that its genotoxicity depends largely on the pH solution, increasing as the pH decreased.

DNA integrity and total oxyradical scavenging capacity in the Mediterranean mussel, Mytilus galloprovincialis: a field study in a highly eutrophicated coastal lagoon

In Mediterranean coastal lagoons, the combination of human impact and wide variability of natural environmental factors can lead to upsets in ecosystem homeostasis resulting in biodiversity decline. Oxidative damage has been causally linked to various kinds of environmental stress, both natural and artificial, the result being impairment of cellular functions. DNA damage and the efficiency of antioxidant defences in Mytilus galloprovincialis from the highly eutrophicated Orbetello Lagoon (Tuscany, Italy) were investigated, respectively by the single cell gel electrophoresis (or Comet test) and the total oxyradical scavenging capacity assay. Results showed significantly higher levels of DNA damage in mussels collected from the inner parts of the lagoon compared to specimens from more external sites. Specimens with the lower genetic integrity also exhibited a reduced efficiency in neutralizing three potent cellular oxidizing species, namely peroxyl radicals (ROO*), hydroxyl radicals (*OH) and peroxynitrite (HOONO), suggesting the involvement of reactive oxygen species in mediating the genetic damage. The analyzed biological parameters also showed a seasonal variability with a minimum of both DNA integrity and antioxidant scavenging efficiency during the warm months and an opposite trend in winter. The potential of analyzed techniques is discussed for the assessment of both anthropogenic and natural disturbance.

Common strategies and technologies for the ecosafety assessment and design of nanomaterials entering the marine environment.

The widespread use of engineered nanomaterials (ENMs) in a variety of technologies and consumer products inevitably causes their release into aquatic environments and final deposition into the oceans. In addition, a growing number of ENM products are being developed specifically for marine applications, such as antifouling coatings and environmental remediation systems, thus increasing the need to address any potential risks for marine organisms and ecosystems. To safeguard the marine environment, major scientific gaps related to assessing and designing ecosafe ENMs need to be filled. In this Nano Focus, we examine key issues related to the state-of-the-art models and analytical tools being developed to understand ecological risks and to design safeguards for marine organisms.

Evaluation of DNA damage in leukocytes of ex-smokers by single cell gel electrophoresis

Single cell gel electrophoresis (SCGE), or comet assay, appears to be a promising tool to estimate DNA damage at the single cell level and it provides information on the presence of damage among individual cells. A follow-up study of 90 smokers who ceased smoking was undertaken to determine the possible decrease of DNA damage in their leukocytes. Before beginning the trial, volunteers smoked on average 26.1 +/- 8.4 cigarettes/day. Comet length did not correlate with the number of cigarettes/day or with the condensate tar content. At the end of the study, 28 volunteers had abandoned the trial, 40 volunteers relapsed into smoking at different times, but with a reduced number of cigarettes/day, whereas 22 fully succeeded in smoking cessation. Throughout the 5 sampling times, a great variability of comet length at individual level was found. However, after 1 year of follow-up, comet length means were found to be significantly shorter (p < 0.0001) in those volunteers who completely quit smoking compared to those who relapsed into smoking (27.2 +/- 1.6 vs. 31.9 +/- 5.1 microns, respectively), irrespective of the amount of cigarettes previously smoked. No effect of age or sex was found. Six months later, these results were confirmed by a further study carried out on a reduced sample of volunteers. The present data strongly suggest that, in spite of the great variability observed, 1 year of smoking cessation is associated with a significant reduction of DNA damage in circulating leukocytes.

Interactive effects of n-TiO2 and 2,3,7,8-TCDD on the marine bivalve Mytilus galloprovincialis

Despite the growing concern over the potential biological impact of nanoparticles (NPs) in the aquatic environment, little is known about their interactions with other pollutants. The bivalve Mytilus sp, largely utilized as a sentinel for marine contamination, has been shown to represent a significant target for different types of NP, including n-TiO2, one of the most widespread in use. In this work, the possible interactive effects of n-TiO2 and 2,3,7,8-TCDD, chosen as models of NP and organic contaminant, respectively, were investigated in Mytilus galloprovincialis. In vitro experiments with n-TiO2 and TCDD, alone and in combination, were carried out in different conditions (concentrations and times of exposure), depending on the target (hemocytes, gill cells and biopsies) and the endpoint measured. Mussels were also exposed in vivo to n-TiO2 (100 μg L(-1)) or to TCDD (0.25 μg L(-1)), alone and in combination, for 96 h. A wide range of biomarkers, from molecular to tissue level, were measured: lysosomal membrane stability and phagocytosis in hemocytes, ATP-binding cassette efflux transporters in gills (gene transcription and efflux activity), several biomarkers of genotoxicity in gill and digestive cells (DNA damage, random amplified polymorphic DNA-RAPD changes), lysosomal biomarkers and transcription of selected genes in the digestive gland. The results demonstrate that n-TiO2 and TCDD can exert synergistic or antagonistic effects, depending on experimental condition, cell/tissue and type of measured response. Some of these interactions may result from a significant increase in TCDD accumulation in whole mussel organisms in the presence of n-TiO2, indicating a Trojan horse effect. The results represent the most extensive data obtained so far on the sub-lethal effects of NPs and organic contaminants in aquatic organisms. Moreover, these data extend the knowledge on the molecular and cellular targets of NPs in bivalves.

Induction of DNA strand breakage and apoptosis in the eel Anguilla anguilla

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.

Individual sensitivity to cytogenetic effects of 1,2:3,4-diepoxybutane in cultured human lymphocytes: influence of glutathione S-transferase M1, P1 and T1 genotypes

Although some blood parameters have been suggested to modulate in-vitro induction of sister chromatid exchanges by 1,2:3,4-diepoxybutane (DEB), a metabolite of 1,3-butadiene, the increased sensitivity has largely been assigned to a homozygous deletion of glutathione S-transferase T1 gene (GSTT1 null genotype). However, some DEB-sensitive individuals have been shown to be GSTT1 positive (having at least one undeleted GSTT1 allele). To examine potential causes for this overlap, we evaluated the effect of GSTM1, GSTP1, and GSTT1 genotypes, together with various life-style and blood parameters, on the DEB induction of sister chromatid exchanges and cells with chromosomal aberrations (aberrant cells) in lymphocyte cultures of 115 and 62 human donors, respectively. Our results supported the important role of the GSTT1 genotype in DEB sensitivity; 76% of cultures from GSTT1 null donors but only 4% of those from GSTT1 positive donors were DEB-sensitive, as defined by sister chromatid exchange measurements. The GSTT1 genotype also clearly affected DEB-induced aberrant cells, 92% of GSTT1 null and 8% of GSTT1 positive donors being sensitive to DEB. All individuals showing a high response to DEB in both sister chromatid exchange and aberrant cell analyses were GSTT1 null. Baseline aberrant cell measurements but not sister chromatid exchange measurements were marginally higher among GSTT1 null donors compared with GSTT1 positive donors. GSTM1 and GSTP1 genotypes had no influence on these cytogenetic end-points. Blood transaminases, gamma-glutamyl transferase, urea, creatinine and white blood cell count showed a clear negative association with DEB-induced aberrant cells, whereas wine drinkers had more aberrant cells than non-drinkers. A higher sister chromatid exchange-response to DEB was observed in lymphocytes from women and smokers than from men and non-smokers, respectively. Erythrocyte count correlated negatively with DEB-induced sister chromatid exchanges. Thus, a variety of parameters seemed to modulate the individual DEB-sensitivity together with the GSTT1 genotype. Although the known contributing factors accounted for a considerable part of individual variability in sister chromatid exchanges (59.4%) and aberrant cells (46.7%) in DEB treatment, they did not, however, fully explain the overlap in cytogenetic response between GSTT1 positive and null individuals.