Camilla Della Torre

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.

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.

Hepatic biotransformation genes and enzymes and PAH metabolites in bile of common sole (Solea solea, Linnaeus, 1758) from an oil-contaminated site in the Mediterranean Sea: A field study

The aim of the present field study was to evaluate the response of hepatic biotransformation genes and enzymes of the common sole collected from an area characterized by the presence of an oil-refinery. Based on ∑PAHs levels detected in sediments, three sites were identified: an high-impact site in front of the refinery, a moderate impact site and a reference site at increasing distances from the refinery. Transcription of cyp1a, udpgt and gst genes and related enzyme activities, such as EROD, BROD, MROD, B(a)PMO, UDPGT and GST, were assessed in sole liver. PAHs bile metabolites were measured. The link between phases I and II is discussed with regard to levels of PAHs measured in sediments and fillets. Results provide sequencing data on biotransformation genes essential for further studies on transcriptional responses in common sole and confirm phase I enzyme activities as useful tools for future biomonitoring studies in marine coastal areas.

Titanium dioxide nanoparticles modulate the toxicological response to cadmium in the gills of Mytilus galloprovincialis

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.

Interaction of ABC transport proteins with toxic metals at the level of gene and transport activity in the PLHC-1 fish cell line.

The aim of this study was to investigate the interaction of four toxic metals with ABC transport proteins in piscine cell line PLHC-1. Cells were exposed for 24 h to 0.01-1 μM of CdCl(2), HgCl(2), As(2)O(3), or K(2)Cr(2)O(7) and the expression of a series of ABC genes (abcb1, abcc1-4) was determined using qRT-PCR. Using the fluorescent model substrates calcein-AM and monochlorbimane we measured interaction of metals with the transport activity of ABC transporters. P-glycoprotein (P-gp) activity was measured in PLHC-1/dox (P-gp overexpressing cells) while activity and interactions of metals with MRPs was measured in PLHC-1/wt cells. After 24 h exposure, abcc2-4 genes were dose-dependently up-regulated by all metals, while abcb1 and abcc1 were less affected. Up-regulation of abcc2 was more pronounced, with up to 8-fold increase in expression. Abcc3 and abcc4 were moderately inducible by HgCl(2) with 3.3-fold and 2.2-fold, respectively. All metals caused a significant inhibition of both P-gp (2.9- to 4-fold vs. controls) and MRP (1.3- to 1.8-fold) transport activities. Modulation of ABC genes and transport activities was further investigated in PLHC-1/wt cells exposed to 1 μM HgCl(2) for 72 h and in Hg resistant cells selected by long term cultivation of PLHC-1/wt cells in increasing concentrations of HgCl(2). Exposure to HgCl(2) for 72 h induced MRP genes expression and efflux activity. The long term cultivation of PLHC-1/wt cells in HgCl(2), did not cause prolonged up-regulation of the tested abc genes but resulted in higher MRP transport activities as determined by the increased sensitivity of these cells to MK571 (MRP specific inhibitor). Results of the present study indicated specific interaction of metals with selected ABC transport proteins. Modulation of ABC transporters takes place at both transcriptional and functional level. An active involvement of efflux pumps in Hg clearance in fish is suggested.

Transcriptional and post-transcriptional response of drug-metabolizing enzymes to PAHs contamination in red mullet (Mullus barbatus, Linnaeus, 1758): a field study.

Aim of this study was to evaluate the responsiveness of red mullet (Mullus barbatus) liver detoxification enzymes to PAHs at transcriptional and post-transcriptional levels in the field. Fish were captured in the north-eastern Adriatic Sea, close to an oil refinery. Sixteen PAHs (EPA) were determined in sediments and fish fillets; transcription levels of cyp1a, cyp3a and abcc2 genes and EROD, BROD, B(a)PMO, BFCOD, GST and UDPGT enzymatic activities were measured. Levels of PAHs in sediments reflect the oil pollution gradient of the area, with weak correspondence in fish fillets. cyp1a gene transcription and EROD, B(a)PMO and BFCOD activities were significantly induced in the oil refinery site, and a slight up-regulation of cyp3a and abcc2 was also observed. GST and UDPGT remained unchanged. The present study provides the first data on detoxification responses at transcriptional levels in the liver of red mullet and confirms phase I enzymes as suitable biomarkers of exposure to PAHs in field studies.

DNA damage, severe organ lesions and high muscle levels of As and Hg in two benthic fish species from a chemical warfare agent dumping site in the Mediterranean Sea.

The aim of the present study was to evaluate the environmental threat to benthic species from chemical weapons dumped in the southern Adriatic Sea. An ecotoxicological approach using chemical analysis and biological responses was applied, in two sentinel species: the Blackbelly rosefish Helicolenus dactylopterus and European conger Conger conger. Specimen were collected in a stretch of sea, where had been dumped war materials and from a reference site free of ordnance. Residues of yperite, Hg and As were measured in fish fillets. Skin, liver, kidney and spleen were examined for histopathological and macroscopical lesions. Liver detoxifying capacities (EROD and UDPGT) and genotoxicity (comet assay) were also investigated. As and Hg levels were three-four times higher than those from the reference site in both species (p<0.001). Both species captured in dumping site showed clear signs of chronic illness according to the health assessment index (HAI). Deep ulcers and nodules were observed on skin and external organs. Histological lesions such as periportal and bile duct fibrosis, pericholangitis, steatosis, granuloma and elevated splenic MMCs were detected in liver and spleen. Significantly higher EROD activities were also found in both species from dumping site (p<0.01). Comet assay revealed genotoxicty in gills of C. conger from dumping site, indicating uptake of chemical warfare agents through fish gills. European conger was found to be a more sensitive bioindicator of this type of contamination than the Blackbelly rosefish.

Effect of bioemulsificant exopolysaccharide (EPS2003) on microbial community dynamics during assays of oil spill bioremediation: A microcosm study

Microcosms experiments were carried out to evaluate the effect of bioemulsificant exopolysaccharide (EPS₂₀₀₃) on microbial community dynamics. An experimental seawater microcosm, supplemented with crude oil and EPS₂₀₀₃ (SW+OIL+EPS₂₀₀₃), was monitored for 15 days and compared to control microcosm (only oil-polluted seawater, SW+OIL). Determination of bacterial abundance, heterotrophic cultivable and hydrocarbon-degrading bacteria were carried out during all experimentation period. The microbial community dynamic was monitored by isolation of total RNA, RT-PCR amplification of 16S rRNA, cloning and sequencing. Oil degradation was monitored by GC-MS analysis. Bioemulsificant addition stimulated an increase of the total bacterial abundance, change in the community structure and activity. The bioemulsificant also increased of 5 times the oil biodegradation rate. The data obtained from microcosm experiment indicated that EPS₂₀₀₃ could be used for the dispersion of oil slicks and could stimulate the selection of marine hydrocarbon degraders thus increasing bioremediation process.

Environmental hazard of yperite released at sea: sublethal toxic effects on fish

The aim of this study was to evaluate the potential toxicological effects on fish related to the leakage of yperite from rusted bomb shells dumped at sea. Both in vivo and field studies have been performed. As for the in vivo experiment, specimen of European eel were subcutaneously injected with 0.015, 0.15 and 1.5mg/kg of yperite and sacrificed after 24 and 48 h. In the field study, specimen of Conger eel were collected from a dumping site in the Southern Adriatic Sea. The presence/absence of yperite in tissues, genotoxicity, detoxification enzymes, histological alterations and gross abnormalities were investigated. Results of the in vivo experiment showed a significant increase of EROD activity at both 24h and 48 h. UGT activity increased significantly at 48 h post injection. An acute inflammatory response after 24h in skin layers and muscle was observed, associated to cell degeneration and necrosis after 48 h at the highest dose. On field, comet assay revealed genotoxicity in gills of fish from the dumping site. Specimen from the dumping site showed significantly higher EROD activities compared to controls, deep ulcers and papules on skin together with liver and spleen histopathological lesions.

Effects of 2,4,6-trinitrotoluene (TNT) on phase I and phase II biotransformation enzymes in European eel Anguilla anguilla (Linnaeus, 1758)

The aim of this study was to investigate effects of the explosive 2,4,6-trinitrotoluene (TNT) on liver drug metabolizing genes and enzymes in the European eel Anguilla anguilla as a model fish species. Eels were exposed in vivo for 6h and 24h to 0.5, 1 and 2.5mg/L nominal concentrations of TNT. Expression of CYP1A, glutathione-S-transferase (pi-class; GST) and uridine-diphosphate glucuronosyltransferase (1-family) (UDPGT) genes was investigated by RT-PCR, and 7-ethoxy- and 7-methoxyresorufin-O-dealkylases (EROD, MROD), NADPH cyt c reductase (NADPH red), UDPGT and GST enzyme activities were measured by biochemical assays. An in vitro study was also performed, measuring only EROD activity. TNT exposure produced no modulation of CYP1A transcript expression while a significant inhibition of EROD enzyme activity was observed and confirmed in vitro. UDPGT transcript increased dose-dependently only at 6h while the UDPGT activity tended to increase dose-dependently at 24h. GST gene expression increased after 24h and significant increases of GST activity were observed both at 6 and 24h only at the highest TNT concentration. An increase of NADPH red activity was observed at 24h. Our results seem to indicate an inhibitory effect of TNT on CYP1A-dependent catalytic activities and a possible involvement of phase II enzymes as well as NADPH red in TNT metabolism in eels.