Ilaria Corsi

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.

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.

Accumulation and embryotoxicity of polystyrene nanoparticles at early stage of development of sea urchin embryos Paracentrotus lividus.

Nanoplastic debris, resulted from runoff and weathering breakdown of macro- and microplastics, represents an emerging concern for marine ecosystems. The aim of the present study was to investigate disposition and toxicity of polystyrene nanoparticles (NPs) in early development of sea urchin embryos (Paracentrotus lividus). NPs with two different surface charges where chosen, carboxylated (PS-COOH) and amine (PS-NH2) polystyrene, the latter being a less common variant, known to induce cell death in several in vitro cell systems. NPs stability in natural seawater (NSW) was measured while disposition and embryotoxicity were monitored within 48 h of postfertilization (hpf). Modulation of genes involved in cellular stress response (cas8, 14-3-3ε, p-38 MAPK, Abcb1, Abcc5) was investigated. PS-COOH forms microaggregates (PDI > 0.4) in NSW, whereas PS-NH2 results are better dispersed (89 ± 2 nm) initially, though they also aggregated partially with time. Their respectively anionic and cationic nature was confirmed by ζ-potential measurements. No embryotoxicity was observed for PS-COOH up to 50 μg mL(-1) whereas PS-NH2 caused severe developmental defects (EC50 3.85 μg mL(-1) 24 hpf and EC50 2.61 μg mL(-1) 48 hpf). PS-COOH accumulated inside embryos digestive tract while PS-NH2 were more dispersed. Abcb1 gene resulted up-regulated at 48 hpf by PS-COOH whereas PS-NH2 induced cas8 gene at 24 hpf, suggesting an apoptotic pathway. In line with the results obtained with the same PS NPs in several human cell lines, also in sea urchin embryos, differences in surface charges and aggregation in seawater strongly affect their embryotoxicity.

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.

Effects of nanomaterials on marine invertebrates.

The development of nanotechnology will inevitably lead to the release of consistent amounts of nanomaterials (NMs) and nanoparticles (NPs) into marine ecosystems. Ecotoxicological studies have been carried out to identify potential biological targets of NPs, and suitable models for predicting their impact on the health of the marine environment. Recent studies in invertebrates mainly focused on NP accumulation and sub-lethal effects, rather than acute toxicity. Among marine invertebrates, bivalves represent by large the most studied group, with polychaetes and echinoderms also emerging as significant targets of NPs. However, major scientific gaps still need to be filled. In this work, factors affecting the fate of NPs in the marine environment, and their consequent uptake/accumulation/toxicity in marine invertebrates will be summarized. The results show that in different model species, NP accumulation mainly occurs in digestive tract and gills. Data on sub-lethal effects and modes of action of different types of NPs (mainly metal oxides and metal based NPs) in marine invertebrates will be reviewed, in particular on immune function, oxidative stress and embryo development. Moreover, the possibility that such effects may be influenced by NP interactions with biomolecules in both external and internal environment will be introduced. In natural environmental media, NP interactions with polysaccharides, proteins and colloids may affect their agglomeration/aggregation and consequent bioavailability. Moreover, once within the organism, NPs are known to interact with plasma proteins, forming a protein corona that can affect particle uptake and toxicity in target cells in a physiological environment. These interactions, leading to the formation of eco-bio-coronas, may be crucial in determining particle behavior and effects also in marine biota. In order to classify NPs into groups and predict the implications of their release into the marine environment, information on their intrinsic properties is clearly insufficient, and a deeper understanding of NP eco/bio-interactions is required.

Nano-sized polystyrene affects feeding, behavior and physiology of brine shrimp Artemia franciscana larvae

Nano-sized polymers as polystyrene (PS) constitute one of the main challenges for marine ecosystems, since they can distribute along the whole water column affecting planktonic species and consequently disrupting the energy flow of marine ecosystems. Nowadays very little knowledge is available on the impact of nano-sized plastics on marine organisms. Therefore, the present study aims to evaluate the effects of 40nm anionic carboxylated (PS-COOH) and 50nm cationic amino (PS-NH2) polystyrene nanoparticles (PS NPs) on brine shrimp Artemia franciscana larvae. No signs of mortality were observed at 48h of exposure for both PS NPs at naplius stage but several sub-lethal effects were evident. PS-COOH (5-100μg/ml) resulted massively sequestered inside the gut lumen of larvae (48h) probably limiting food intake. Some of them were lately excreted as fecal pellets but not a full release was observed. Likewise, PS-NH2 (5-100µg/ml) accumulated in larvae (48h) but also adsorbed at the surface of sensorial antennules and appendages probably hampering larvae motility. In addition, larvae exposed to PS-NH2 undergo multiple molting events during 48h of exposure compared to controls. The activation of a defense mechanism based on a physiological process able to release toxic cationic NPs (PS-NH2) from the body can be hypothesized. The general observed accumulation of PS NPs within the gut during the 48h of exposure indicates a continuous bioavailability of nano-sized PS for planktonic species as well as a potential transfer along the trophic web. Therefore, nano-sized PS might be able to impair food uptake (feeding), behavior (motility) and physiology (multiple molting) of brine shrimp larvae with consequences not only at organism and population level but on the overall ecosystem based on the key role of zooplankton on marine food webs.

Biomarker responses at different levels of biological organisation in crabs (Carcinus aestuarii) experimentally exposed to benzo(α)pyrene

The aim of this study was to validate a multi-trial biomarker approach for the evaluation of toxicological risk due to benzo(alpha)pyrene. Carcinus aestuarii, exposed to increasing concentrations of B(alpha)P in the water, was used as the bioindicator organism. A set of biomarkers were tested in order to: identify biological materials for biomarker and residue analysis; determine a group of sensitive techniques for the assessment of PAH contamination; investigate correlation between responses at different levels of biological organisation. The results underlined that BPMO activities in hepatopancreas and gills were a good biomarker of exposure to PAH-type compounds. B esterases activities in hemolymph and porphyrin patterns in excreta could be proposed as a non-destructive approach for evaluating chemical exposure in this species.

Cytochrome P450, acetylcholinesterase and gonadal histology for evaluating contaminant exposure levels in fishes from a highly eutrophic brackish ecosystem: the Orbetello Lagoon, Italy

Biochemical markers and ovarian histology were investigated in prespawning females of grass goby (Zosterisessor ophiocephalus) and grey mullet (Mugil cephalus) collected, respectively, in late spring and summer 2000 in four sites of a highly eutrophic brackish ecosystem of central Italy, the Orbetello Lagoon. Exposure to chlorinated and aromatic hydrocarbons was evaluated in fish livers by the somatic liver index (SLI) and by measuring 7-ethoxyresorufin-O-deethylase (EROD) and benzo(a)pyrene monooxygenase (BaPMO) activities. Acetylcholinesterase (AChE) activity was measured in brain and gills to evaluate exposure to organophosphates (OPs) and carbamates (CBs). The gonad somatic index (GSI) was used to confirm ovarian maturation and ovarian histology was investigated as a potential biomarker for environmental effects. Samples from the Western Basin, near a sewage treatment plant (STP) off the town of Orbetello, showed higher SLI values and higher EROD and BaPMO activities than those collected from the Ansedonia Canal (AC) in the Eastern Basin (p<0.05) and respect to those from reference sites: the Albegna River (AR) Delta for grass goby and the Nassa Canal (NC), connected with the sea, for grey mullet both located in the Western Basin as well. Low brain AChE activity was observed in both species from the reference sites (AR and NC) in association with the presence of anomalies in developing oocytes: unexpectedly small in grass goby and irregular disintegrated cytoplasm in grey mullet. The results indicate that the Western Basin is more polluted than the Eastern Basin particularly in the Orbetello where the sewage treatment plant may be a source of aromatic and chlorinated compounds while the Albegna River and the Nassa Canal may be sources of OPs and CBs.

Environmental Levels of para-Nonylphenol Are Able to Affect Cytokine Secretion in Human Placenta

Background para-Nonylphenol (p-NP) is a metabolite of alkylphenols widely used in the chemical industry and manufacturing. It accumulates in the environment, where it acts with estrogen-like activity. We previously showed that p-NP acts on human placenta by inducing trophoblast differentiation and apoptosis. Objective The aim of the present study was to investigate the effect of p-NP on cytokine secretion in human placenta. Methods In vitro cultures of chorionic villous explants from human placenta in the first trimester of pregnancy were treated with p-NP (10−13, 10−11, and 10−9 M) in 0.1% ethanol as vehicle. Culture medium was collected after 24 hr and assayed by specific immunoassays for the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, and tumor necrosis factor-α (TNF-α). Results p-NP modulated cytokine secretion by inducing the release of GM-CSF, IFN-γ, IL-1β, IL-4, and IL-10, with a maximum effect at 10−11 M. It reduced the release of TNF-α at 10−13 M, whereas levels of IL-2 and IL-5 remained below the detection limit. IL-6 and IL-8 levels were 100–1,000 times higher than those of other cytokines, and they were not affected by p-NP. We observed significant differences from controls (ethanol alone) only for GM-CSF and IL-10. Conclusion An unbalanced cytokine network at the maternal–fetal interface may result in implantation failure, pregnancy loss, or other complications. The effects of extremely low doses of p-NP on the placental release of cytokines raise considerable concerns about maternal exposure to this endocrine disruptor during pregnancy.