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

Abstract

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 12 h). 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

“Test Tube Cetaceans”: From the Evaluation of Susceptibility to the Study of Genotoxic Effects of Different Environmental Contaminants Using Cetacean Fibroblast Cell Cultures

“…From Table 8 it appears evident that the carrier of OC mixture (DMSO) confounds the evaluation of the responses of the cells to the treatments, as previously demonstrated in other studies [15,43]. Only the sperm whale demonstrated a clear inductive effect on MICA due to OCs, compared to DMSO ( Figure 8A).…”

“…Immunofluorescence was quantified with a specially designed Olympus Soft Imaging Systems macro, DetectIntZ, which works with the image acquisition, processing and analysis system, analySIS^B (Olympus) [15]. The image analysis procedure has the objective of quantifying, with an adimensional index generated for this purpose, the amount of Alexa …”

“…But how is it possible to discriminate the effects of a specific toxic in a mixture of many pollutants and assess the susceptibility of a particular cetacean species to just one class of contaminants? The aim of the present study is to use cetacean fibroblast cell cultures, obtained from skin biopsy of free-ranging animals and from skin tissue of stranded animals dead within 12 h [14,15], as an "in vitro" method, called "Test Tube Cetaceans", to investigate the effects of environmental contaminants. In particular we use Test Tube Cetaceans to explore the susceptibility to genotoxic effects of different environmental contaminants in these marine mammals.…”

Toxicity assessment of 2,4-D and MCPA herbicides in primary culture of fish hepatic cells

“…Fish cell cultures provide an important tool for different research areas, including: fish immunology; [24] virology; [25] endocrinology; [26] gene expression; [27] oxidative stress; [28] toxicology and ecotoxicology. [29][30][31][32][33] In addition, cell cultures have been successfully employed as an alternative to the use of whole animal models. [34,35] In this study, we show that primary cultures of M. roosevelti hepatic cells provide a good model for the evaluation of changes in respiratory activity caused by 2,4-D and MCPA.…”

Toxicology of Marine Mammals: New Developments and Opportunities

“…Fossi et al 2006Fossi et al , 2008Marsili et al 2008;Spinsanti et al 2008) and on the evaluation of immunotoxicity and metabolic activity for pollutant breakdown or biotransformation (e.g. Boon et al 1998;De Guise et al 1998;Kim et al 1998;White et al 2000;Nakata et al 2002;Levin et al 2004Levin et al , 2007Mori et al 2006;Wilson et al 2007;Camara Pellissò et al 2008).…”