Establishment of brain, umbilical cord, liver and muscle cell lines from Tursiops truncatus as models to assess Persistent Pollutants effects

Ocean Health is increasingly threatened by the widespread presence of Persistent organic Pollutants (POPs), as the per- and polyfluoroalkyl substances (PFAS), known for their persistence, bioaccumulation, and toxicity. Cetaceans are widely recognized as sentinels of marine pollution as they accumulate contaminants as a result of their long lifespans, coastal habitat, and high trophic positions. These characteristics make them valuable “indicators of the health” of marine ecosystems. Ethical and practical limitations prevent the in vivo testing on live cetaceans in toxicological studies. The establishment of cetacean-derived in vitro models provides a valuable and sustainable alternative to investigating pollutant impacts on alive cells. In this study, first we established four cell lines derived from brain, liver, muscle, and umbilical cord tissues of the bottlenose dolphin. Then, the cell lines were characterized morphologically, ultrastructurally, and immunocytochemically and the population doubling time was estimated. Finally, we assessed the cells responsiveness to the environmental contaminants (PFOA, PFOS, PFBS, PFBA, and C6O4) to increasing concentrations, assessing cell viability evaluated by MTT assay and the proliferative potential monitoring alterations in the cell cycle profile. By setting a semi-automated model based on the mathematical combination of nuclear parameters (Intensity, Length and Ratio), we evaluated the nuclear changes in cells classifying cells in six groups: G0+G1, S, G2+ early-Mitosis, Late-Mitosis respectively, Large-group and Small-group. Our results revealed that the umbilical cord cell line exhibits the higher population doubling time (23-58 h), followed by the liver cell line (29-40 h), the brain cell line (24-27 h) and the muscle cell line (22-23 h). The immunocytochemical results revealed that the brain cell line expressed β-actin, vimentin, Ki-67, and caspase-3, suggesting the fibroblast-like identity. The umbilical cord cell line expressed vimentin, β-actin, Ki-67, and caspase-3, indicating mesenchymal-like phenotype. The liver cell line and the muscle cell line expressed vimentin, β-actin, Ki-67, and caspase-3. According to the MTT assay results, in the brain cell line the C6O4 exhibited a modest decrease in cell viability. A similar trend was observed in the umbilical cord cell line, where PFAS displayed a decreased in cell viability. In the liver cell line C6O4 and PFOA revealed an increasing of cell viability. The exposure to PFAS on muscle cell line displayed a decreased cell viability. Dose response analyses showed that PFAS induced cell cycle dysregulation depending on the nature of the cell line. In the brain cell line, PFBA and C6O4 reduced viability and increased cells number in S and M phases. In the umbilical cord cell line, cells resulted sensitive to PFOA, PFOS, and PFBA, showing a decreasing in the number of cells in the S, G2+early-M and late-M groups. In the liver cell line, PFOA and PFOS increased the number of nuclei in S and G2+early-M phase, while PFBA and C6O4 mainly induced decrease in the S and mitotic phases. In the muscle cell line, cells resulted sensitive to PFOA and PFBA showing an increasing in the number of cells in the S group. PFOS and C6O4 reduced the S group, while no significant effects were observed for PFBS. This study highlights the complexity of PFAS-induced cellular responses, influenced by the PFAS chemical structure, especially functional groups and chain length. Our study, for the first time, provides evidence that PFAS induce cell cycle dysregulation in Tursiops truncatus cells, highlighting the importance of cetacean in vitro system to investigate pollutants effects in alive cells. The methodology we set provides a direct, rapid and objective tool for automatically classify the entire nuclear population.