The use of immunofluorescence technique in the evaluation of presence of CYp450 and ER receptor in cetaceans fibroblast cell cultures

Various studies of Mediterranean cetaceans have revealed bioaccumulation of contaminants such as organochlorines (OCs) and polycyclic aromatic hydrocarbons (PAHs). With the aim of investigating interspecies susceptibility to lipophilic contaminants, we developed an investigative method based on fibroblast cell cultures used as an "in vitro" surrogate of the different species. In this paper we propose the indirect immunofluorescence technique in fibroblast cell cultures for a qualitative and semi-quantitative evaluation of the target proteins CYP450 1A1-1A2, CYP4502B4 and estrogen receptor (ER). In a pilot project, an experiment was performed with potent CYP450 inducers in fibroblast cell cultures (third generation) of common bottlenose dolphin (Tursiops truncatus) and striped dolphin (Stenella coeruleoalba), sampled using a biopsy dart in the Mediterranean Whale Sanctuary (Italy). In this experiment fibroblasts were exposed for 48 h to a mixture of Arochlor 1260, pp'DDT e pp'DDE solubilysed in DMSO (0.05%) at three different doses: 1μg/ml, 5μg/ml and 25μg/ml, plus a DMSO (0.05%) control. Immunofluorescence showed a good cross-reaction of the primary antibodies used, the presence of the cytochromes 1A1-1A2 and 2B4 and of the estrogen receptor in bottlenose dolphin and striped dolphin fibroblast cells, and an increasing intensity of fluorescence in relation to the treatment doses of contaminants in the male striped dolphin.