C. Della Torre

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.

Time-dependent modulation of cyp1a gene transcription and EROD activity by musk xylene in PLHC-1 and RTG-2 fish cell lines

Musk xylene (MX) is a common synthetic nitromusk fragrance. Its high release in aquatic environments and evidence of bioaccumulation in biota suggest that it could have serious toxicological consequences for aquatic ecosystems. However, not much data is available on cellular pathways and mechanisms of toxicity in aquatic organisms. The aim of the present study was to investigate the interaction of MX with CYP1A by looking at gene transcription and EROD activity in two fish cell lines: PLHC-1 and RTG-2. Time-dependent (6 and 24 h) exposure experiments with three doses of MX (2, 4 and 20 μM) were performed also with co-exposure to B(a)P. Low cytotoxicity was observed in both cell lines. Reduction of cyp1a gene transcription was observed after 6 h with full dose-dependent recovery in 24 h in RTG-2 and partial recovery in PLHC-1. EROD activity was inhibited after 6 and 24 h of exposure except in PLHC-1 at 6 h at the two higher doses. MX did not alter CYP1A induction by B(a)P at gene transcription. A dose and time-dependent induction of GST activity was observed in PLHC-1 cells exposed to MX. These findings suggest that distinct signalling pathways not mediated by AhR and distinct regulatory mechanisms by CYP1A inducers are likely.

n-TiO2 and CdCl2 co-exposure to titanium dioxide nanoparticles and cadmium: Genomic, DNA and chromosomal damage evaluation in the marine fish European sea bass (Dicentrarchus labrax).

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.

Effects of 2,4,6-trinitrotoluene (TNT) on neurosteroidogenesis in the European eel (Anguilla anguilla; Linnaeus 1758)

The aim of the present study is to investigate the endocrine disrupting potential of the explosive 2,4,6-trinitrotoluene (TNT) on marine organisms using the European eel (Anguilla anguilla Linnaeus, 1758) as model fish species. Crucial brain steroidogenic parameters such as the estrogen receptors (ERs), P450 aromatase (CYP19), steroidogenic acute regulatory (StAR) protein and P450-mediated cholesterol side-chain cleavage (P450scc) enzyme were selected as potential biomarkers for the assessment of TNT endocrine disrupting potential. Juvenile European eels were exposed for 6 and 24 h to 0.5, 1 and 2.5 mg/l nominal concentration of TNT dissolved in dimethyl sulfoxide (DMSO). ERα, CYP19, StAR protein and P450scc genes expression were quantified using real-time PCR. Exposure to TNT altered the expression of ER gene at the lowest TNT concentration after 6 h of exposure, while after 24 h gene expression levels increased respect to controls only in eels at the maximum TNT dose (2.5 mg/l). A significant increase of CYP19 gene expression occurred after 6 h in all eels exposed to TNT compared to controls. On the opposite at 24 h, CYP19 expression seems reduced in eels exposed to 0.5 and 1mg TNT/l compared to 6 h, while no changes were observed at the highest dose (2.5 mg/l). Both StAR and P450scc gene expression were decreased at 6 h in eels exposed to 0.5 and 1mg TNT/l, while at 2.5 mg TNT/l, the gene expression levels were restored to background level. No further modulation was evident after 24 h for both genes. The overall results indicate the ability of TNT to affect neurosteroidogenic pathways.

The involvement of cytochrome P450 system in the fate of 2,4,6-trinitrotoluene (TNT) in European eel [Anguilla anguilla (Linnaeus, 1758)]

TNT (2,4,6-trinitrotoluene) was the most common nitro aromatic explosive available in World War II ammunitions. The presence of ordnance dumped at sea might represent a great concern for marine species living close to dumping sites and the toxicological properties of the chemicals released into the marine environments need to be evaluated. The aim of the present study is to investigate the involvement of CYP (cytochrome P450) system in the metabolism of TNT in marine organisms by using the European eel [Anguilla anguilla (Linnaeus, 1758)] as model species. In vivo exposure to sublethal concentration of TNT (0.5, 1 and 2.5 mg/l) leads to a significant decrease in the phase I CYP1A catalytic activities such as EROD (7-ethoxyresorufin-O-de-ethylase) and MROD (7-methoxyresorufin-O-de-ethylase). On the opposite, a significant increase in NADPH cytochrome c reductase activity as well as phase II UDP-glucuronosyltransferase activity is observed. An inhibition at enzyme level is hypothesized for both CYP1A enzymes, also confirmed by a similar decrease observed after in vitro exposure. An active role of NADPH cytochrome c reductase and phase II enzymes in the TNT metabolism may also be hypothesized.

Effects on CYP1A of the polycyclic musk tonalide (AHTN) in single and co-exposure with benzo(a)pyrene and 3,3′-4,4′,5-pentachlorobiphenyl in the PLHC-1 fish cell line

The aim of this study was to investigate the interaction of a common polycyclic musk fragrance, namely tonalide (AHTN), with the detoxification gene family CYP1A, by looking at both gene transcription (cyp1a) and enzyme activity (7-ethoxyresorufin-O-deethylase; EROD) in the PLHC-1 fish cell line. Time-dependent (6 and 24 h) exposure experiments with three doses of AHTN (1 nM, 10 nM and 2 μM) were performed. Co-exposure with known CYP1A inducers such as benzo[a]pyrene (B[a]P) and 3,3′-4,4′,5-pentachlorobiphenyl (PCB 126) was also investigated. A slight induction of cyp1a gene transcription was observed after 6 h, but not at 24 h. At 6 h, 40–45% of reduction of cyp1a transcription was observed in co-exposure with B[a]P and PCB 126 compared with single inducers. Complete recovery was observed after 24 h. No effect on EROD activity by AHTN was observed in either single exposure and co-exposure. AHTN seemed not to affect CYP1A at the gene or enzyme level, but in co-exposure with inducers ATHN seems able to reduce detoxification capability within a short time.

Acute Sublethal Effects Of 2,4,6-trinitrotoluene(TNT) On The European Eel Anguilla Anguilla(Linnaeus, 1758)

The aim of this study is to investigate the effects of the explosive 2,4,6-trinitrotoluene (TNT) on liver phase I and II biotransformation enzymes at gene (CYP1A1, GST and UDPGT) and catalytic levels by investigating EROD, GST and UDPGT activities, on crucial brain steroidogenic proteins (StAR and P450scc genes expression) and on gills histology in a model fish species such as the European eel (Anguilla anguilla, Linnaeus, 1758). Eels were exposed in vivo for 6 and 24 hours to 0.5mg/L, 1mg/L and 2.5mg/L nominal concentration of TNT by using 0.1‰ of DMSO as a carrier. The TNT produced a significant inhibition of EROD activity and an increase of UDPG T and GST genes expression and activities compared to controls. A decrease of StAR and P450scc genes was also observed in TNT exposed eels. Finally concerning gills, branchial lifting was evident at the lowest TNT concentration (0.5 mg/L) while lamellar aneurisms, vascular congestion, hypertrophy and hyperplasia of the epithelium were evident at 2.5 mg/L. Our results highlighted the concern related to the release of TNT on the seabed: the inhibition of EROD activity may result in an increased susceptibility of the organism to P450 inducers such as dioxins and PAH. TNT also seems to affect fish neurosteroidogenesis by downregulating key enzymes (StAR and P450scc genes). Gills seem to be a target organ for TNT in fish. The present research provided relevant information on TNT metabolism/toxicity and indicated sensitive targets of TNT toxicity in fish species.