Stefania Gorbi

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.

Pollutants bioavailability and toxicological risk from microplastics to marine mussels.

Microplastics represent a growing environmental concern for the oceans due to their potential of adsorbing chemical pollutants, thus representing a still unexplored source of exposure for aquatic organisms. In this study polyethylene (PE) and polystyrene (PS) microplastics were shown to adsorb pyrene with a time and dose-dependent relationship. Results also indicated a marked capability of contaminated microplastics to transfer this model PAH to exposed mussels Mytilus galloprovincialis; tissue localization of microplastics occurred in haemolymph, gills and especially digestive tissues where a marked accumulation of pyrene was also observed. Cellular effects included alterations of immunological responses, lysosomal compartment, peroxisomal proliferation, antioxidant system, neurotoxic effects, onset of genotoxicity; changes in gene expression profile was also demonstrated through a new DNA microarray platform. The study provided the evidence that microplastics adsorb PAHs, emphasizing an elevated bioavailability of these chemicals after the ingestion, and the toxicological implications due to responsiveness of several molecular and cellular pathways to microplastics.

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.

Use of the Land Snail Helix aspersa as Sentinel Organism for Monitoring Ecotoxicologic Effects of Urban Pollution: An Integrated Approach

Atmospheric pollution from vehicular traffic is a matter of growing interest, often leading to temporary restrictions in urban areas. Although guidelines indicate limits for several parameters, the real toxicologic impacts remain largely unexplored in field conditions. In this study our aim was to validate an ecotoxicologic approach to evaluate both bioaccumulation and toxicologic effects caused by airborne pollutants. Specimens of the land snail Helix aspersa were caged in five sites in the urban area of Ancona, Italy. After 4 weeks, trace metals (cadmium, chromium, copper, iron, manganese, nickel, lead, and zinc) and polycyclic aromatic hydrocarbons (PAHs) were measured and these data integrated with the analyses of molecular and biochemical responses. Such biomarkers reflected the induction of detoxification pathways or the onset of cellular toxicity caused by pollutants. Biomarkers that correlated with contaminant accumulation included levels of metallothioneins, activity of biotransformation enzymes (ethoxyresorufin O-deethylase, ethoxycoumarin O-deethylase), and peroxisomal proliferation. More general responses were investigated as oxidative stress variations, including efficiency of antioxidant defenses (catalase, glutathione reductase, glutathione S-transferases, glutathione peroxidases, and total glutathione) and total oxyradical scavenging capacity toward peroxyl and hydroxyl radicals, onset of cellular damages (i.e., lysosomal destabilization), and loss of DNA integrity. Results revealed a marked accumulation of metals and PAHs in digestive tissues of organisms maintained in more traffic-congested sites. The contemporary appearance of several alterations confirmed the cellular reactivity of these chemicals with toxicologic effects of potential concern for human health. The overall results of this exploratory study suggest the utility of H. aspersa as a sentinel organism for biomonitoring the biologic impact of atmospheric pollution in urban areas.

Experimental development of a new protocol for extraction and characterization of microplastics in fish tissues: First observations in commercial species from Adriatic Sea.

The presence of microplastics in the marine environment has raised scientific interest during the last decade. Several organisms can ingest microplastics with potentially adverse effects on the digestive tract, respiratory system and locomotory appendages. However, a clear evidence of tissue accumulation and transfer of such microparticles in wild organisms is still lacking, partially hampered by technical difficulties in isolation and characterization protocols from biological samples. In this work, we compared the efficacy of some existing approaches and we optimized a new protocol allowing an extraction yield of microplastics from fish tissues ranging between 78% and 98%, depending on the polymer size. FT-IR analyses confirmed that the extraction procedure did not affect the particles characteristics. The method was further validated on the fish mullet, Mugil cephalus, exposed under laboratory conditions to polystyrene and polyethylene; the particles were isolated and quantified in stomach and liver, and their presence in the hepatic tissue was confirmed also by histological analyses. A preliminary characterization revealed the presence and distribution of microplastics in various fish species collected along the Adriatic Sea. FT-IR analyses indicated polyethylene as the predominant polymer (65%) in the stomach of fish. The overall results confirmed the newly developed method as a reliable approach to detect and quantify microplastics in the marine biota.

Plastics and microplastics in the oceans: From emerging pollutants to emerged threat

Plastic production has increased dramatically worldwide over the last 60 years and it is nowadays recognized as a serious threat to the marine environment. Plastic pollution is ubiquitous, but quantitative estimates on the global abundance and weight of floating plastics are still limited, particularly for the Southern Hemisphere and the more remote regions. Some large-scale convergence zones of plastic debris have been identified, but there is the urgency to standardize common methodologies to measure and quantify plastics in seawater and sediments. Investigations on temporal trends, geographical distribution and global cycle of plastics have management implications when defining the origin, possible drifting tracks and ecological consequences of such pollution. An elevated number of marine species is known to be affected by plastic contamination, and a more integrated ecological risk assessment of these materials has become a research priority. Beside entanglement and ingestion of macro debris by large vertebrates, microplastics are accumulated by planktonic and invertebrate organisms, being transferred along food chains. Negative consequences include loss of nutritional value of diet, physical damages, exposure to pathogens and transport of alien species. In addition, plastics contain chemical additives and efficiently adsorb several environmental contaminants, thus representing a potential source of exposure to such compounds after ingestion. Complex ecotoxicological effects are increasingly reported, but the fate and impact of microplastics in the marine environment are still far to be fully clarified.

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.

Interactions between metabolism of trace metals and xenobiotic agonists of the aryl hydrocarbon receptor in the antarctic fish Trematomus bernacchii: Environmental perspectives

Although Antarctica is a pristine environment, organisms are challenged with contaminants either released locally or transported from industrialized regions through atmospheric circulation and marine food webs. Organisms from Terra Nova Bay also are exposed to a natural enrichment of cadmium, but to our knowledge, whether such environmental conditions influence biological responses to anthropogenic pollutants has never been considered. In the present study, the Antarctic rock cod (Trematomus bernacchii) was exposed to model chemicals, including polycyclic aromatic hydrocarbons (benzo[a]pyrene), persistent organic pollutants (2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]), cadmium, and a combination of cadmium and TCDD. Analyzed parameters included chemical bioaccumulation, activity, and levels of biotransformation enzymes (cytochrome P4501A); metallothioneins and the efficiency of the antioxidant system measured as individual defenses (catalase, glutathione, glutathione reductase, glutathione S-transferases, and glutathione peroxidases); and total scavenging capacity toward peroxyl and hydroxyl radicals. Reciprocal interactions between metabolism of inorganic and organic pollutants were demonstrated. Dioxin enhanced the accumulation of cadmium, probably stored within proliferating endoplasmic reticulum, and cadmium suppressed the inducibility of cytochrome P4501A, allowing us to hypothesize a posttranscriptional mechanism as the depletion of heme group availability. Clear evidence of oxidative perturbation was provided by the inhibition of antioxidants and enhanced sensitivity to oxyradical toxicity in fish exposed to organic chemicals. Exposure to cadmium revealed counteracting responses of glutathione metabolism; however, these responses did not prevent a certain loss of antioxidant capacity toward peroxyl radicals. The pattern of antioxidant responses exhibited by fish coexposed to cadmium and TCDD was more similar to that observed for cadmium than to that observed for TCDD. The overall results suggest that elevated natural levels of cadmium in Antarctic organisms from Terra Nova Bay can limit biotransformation capability of polycyclic (halogenated) hydrocarbons, thus influencing the bioaccumulation and biological effects of these chemicals in key sentinel species.

Oxidative stress defense in human-skin-derived mesenchymal stem cells versus human keratinocytes: Different mechanisms of protection and cell selection

Stem cells are undifferentiated cells with the capacity for self-renewal and differentiation. Here we have determined the susceptibility to oxidative stress of isolated mesenchymal stem cells from human skin (S-MSCs) in comparison with keratinocytes, which are differentiated cells of the same lineage. To induce pro-oxidant conditions, S-MSCs and keratinocytes were exposed to 0.5mM H(2)O(2) for 2 h, with oxidative effects analyzed after 4, 12, 24, and 48 h of recovery, in terms of cell growth, vitality, apoptosis, DNA damage, variations in individual antioxidant defense and total oxyradical scavenging capacity toward peroxyl and hydroxyl radicals. The data indicate different abilities across these two cell types to counteract this oxidative stress, which reflects stress that would normally be experienced by these cells under basal conditions. Human keratinocytes seem to have much greater antioxidant defense to counteract the oxidative injury to which they are continuously exposed in the skin. The S-MSCs are surrounded by a complex microenvironment that protects them from external insults, and so they do not have a particularly efficient defense system, and they were generally less responsive to enhanced pro-oxidant challenge. S-MSCs seem particularly prone to apoptotic events, which might thus represent their primary defense mechanism against stress.

Effect of biologic therapies targeting tumour necrosis factor-α on cutaneous mesenchymal stem cells in psoriasis

Summary Background  Psoriasis is a Th1 immune‐mediated, inflammatory disease, in which skin lesions appear many years before the related metabolic and cardiovascular comorbidities, according to the theory of the ‘psoriatic march’. Inducible nitric oxide synthetase (iNOS), tumour necrosis factor (TNF)‐α and vascular endothelial growth factor (VEGF) are directly implicated in determining both skin lesions and systemic involvement in psoriasis. Reactive oxygen species actively promote the secretion of inflammatory Th1 cytokines directly involved in the pathogenesis of psoriasis.