Cellular cultures setup and ecotoxicological exposure to bisphenols in Caretta caretta

Population of various marine species are threatened. In addition to natural threats such as viruses and bacteria, there are also numerous anthropogenic threats such as contaminants, litter, climate change, food depletion from over-fishing, bycatch, noise, shipping and collision. Moreover, the incidence of some diseases is closely related to the level of environmental pollution. For this reason, it is difficult to discern the effects of one threat from the others, since multiple threats act simultaneously. On top of these, a clinical veterinary approach as well as experimental studies are unfeasible for wild marine species such as C.caretta. Ethical and legal constraints on marine wild species, lead to the development and use of the in vitro test-system as cell-lines. According to the 3R Principal and in respect of the European regulations, in vitro methods represent a cost-effective methodology and a great opportunity to study and conduct researches about endangered species. In this study, four primary cell-lines were prepared from loggerhead sea turtles. For the first time in C. caretta, a protocol to obtain cell cultures from hearth, kidney, spleen and thymus was defined, in addition to a protocol for the exposure (for 24-48 h) of the hearth cell-cultures to Bisphenols A, B, F and S at 5 different concentrations each (100 μg/ml, 10 μg/ml, 1 μg/ml, 0,1 μg/ml, 0,01 μg/ml). The different effects of bisphenols on the cell lineage were evaluated using a multidisciplinary approach including molecular biology and immunohistochemistry. In addition, spectroscopy techniques were used to highlight macromolecular changes in the treated cells such as FTIR and RAMAN. This study is key in getting a deeper understanding of the contaminants' effects on the loggerhead sea turtles’ health status. Moreover, the approach and protocols described could be used for other marine wild species.