First detection of CYP1A1 and CYP2B induction in Mediterranean cetacean skin biopsies and cultured fibroblasts by Western blot analysis.

The need to develop powerful tools to detect exposure and effects of POPs and emerging contaminants in Mediterranean cetaceans led us to develop a suite of sensitive non-lethal biomarkers in integument biopsies of free-ranging animals. In order to propose induction of CYP1A1 and CYP2B, detected by Western blot analysis, as biomarkers of exposure to OCs, PAHs and PBDEs, a three-phase experimental protocol (in vitro experiments, calibration experiments and field applications) was followed using fibroblast cell cultures and biopsies of Mediterranean Stenella coeruleoalba and Tursiops truncatus. This methodology was confirmed to be sensitive and stable in comparison to previous methods used to detect CYP1A1 in biopsies, enabling analysis of several inducible proteins in non-lethal samples and analysis of material from stranded animals.

Use of immunofluorescence technique in cultured fibroblasts from Mediterranean cetaceans as new "in vitro" tool to investigate effects of environmental contaminants.

The aim of the present study was to propose the immunofluorescence technique in cultured fibroblasts from Mediterranean cetaceans as a new "in vitro" tool to explore the susceptibility of these marine mammals to different xenobiotic compounds. The cell lines were cultured from integument biopsies of free-ranging and stranded cetaceans (dead within 12h). Using the indirect immunofluorescence assay, we detected endogenous proteins induced by different contaminants. Here we present the method used for qualitative and quantitative evaluation of cytochromes P450 (CYP1A1 and CYP2B) induced by some POPs (DDTs and PCBs) and emerging contaminants (PBDEs) in fibroblast cell cultures of striped dolphin (Stenella coeruleoalba) and bottlenose dolphin (Tursiops truncatus). Immunofluorescence was quantified with a specially designed Olympus macro, DetectIntZ. A major result was the possibility of using this "in vitro" assay to quantify induction of endogenous proteins.

Selection of reliable reference genes for qRT-PCR studies on cetacean fibroblast cultures exposed to OCs, PBDEs, and 17beta-estradiol.

Quantitative real-time PCR (qRT-PCR) represents an effective molecular technique for the detection of mRNA expression in biological samples. Its sensitivity allows the quantification of slight changes in the regulation of gene transcription but is strictly dependent upon the method followed during the normalization procedure. Relative quantification determines changes in the steady-state mRNA levels of genes across multiple samples and it is assessed by comparison with the levels of one or more internal control RNA. In this context, the choice of constitutively expressed control genes, whose transcription is not affected by the contaminants, appears to be fundamental for the reliability of this technique. During this study, fibroblast cell cultures originated from integumentum biopsies, sampled in the cetacean species Stenella coeruleoalba, have been exposed for 6h to increasing concentrations of different mixtures of compounds with endocrine disruptor capacities (EDCs): organochlorines (OCs), polybrominated diphenyl ethers (PBDEs), and 17beta-estradiol. Ten common housekeeping genes have been tested for the expression of their transcripts in exposed cell cultures using qRT-PCR assays and raw data were analyzed with the two Excel applets geNorm and NormFinder. The genes encoding for SDHA, GAPDH and YWHAZ appear to be the most reliable controls, respectively, for the OC, PBDE and 17beta-estradiol treatments. These results clearly show that the transcription of even widely diffused control genes can be regulated by different treatments and underlie the importance of a careful selection of the optimal housekeeping genes in toxicological studies.

Application of a suite of biomarkers in Posidonia oceanica (L.) delile to assess the ecotoxicological impact on the coastal environment.

The main aim of this study was to develop and validate a suite of sensitive responses (biomarkers) for monitoring conservation status and ecotoxicological impact in Posidonia oceanica meadows. Analytical methods were developed for NADPH cytochrome c reductase, ethoxycoumarin-o-deethylase (ECOD), guaiacol peroxidase (GPOX) and superoxide dismutase (SOD) assays. A preliminary proteomic approach using 2-D electrophoresis was also proposed as a biomarker. These techniques were initially tested on samples of posidonia exposed experimentally to various contaminants. Once validated, this approach was applied to posidonia in a field study. Samples of the seagrass were collected at four sites with potentially different environmental impact along the northern Tyrrhenian coast. The results showed that reductase activity was significantly induced in the various sampling areas with respect to the reference site. GPOX and SOD showed a similar trend; the highest activities were found in samples collected off a chlor-alkali plant and near a river estuary. Analysis of trace elements, polycyclic aromatic hydrocarbons (PAHs) and organochlorine compounds (OCs) in posidonia leaves showed differences between sites. A significant correlation was found between Hg concentrations and GPOX activity and between Cr, Al and As concentrations and reductase activity. The results validated these biomarkers in posidonia for the assessment of ecotoxicological impact on the coastal ecosystem.

Development of new-tools to investigate toxicological hazard due to endocrine disruptor organochlorines and emerging contaminants in Mediterranean cetaceans.

The possibility that certain Mediterranean cetaceans are subject to toxicological hazard due to organochlorines and emerging contaminants, such as polybrominated diphenyl ethers (PBDEs) with endocrine disrupting capacity, was investigated using non-lethal methods. The need for new biomarkers for EDCs and for a "cell model" to explore the different susceptibilities to several classes of ECDs, including emerging contaminants, led us to culture fibroblasts of different cetacean species ("dolphins in test tubes"). We then explored interspecies and gender susceptibility to OC-EDCs and PBDEs using qualitative and semi-quantitative evaluation of target proteins, such as CYP1A and CYP2B in cultured cetacean fibroblasts (Stenella coeruleoalba, Tursiops truncatus and Balaenoptera physalus), by western blot and immunofluorescence techniques.