Cell cycle alterations due to perfluoroalkyl substances PFOS, PFOA, PFBS, PFBA and the new PFAS C6O4 on bottlenose dolphin (Tursiops truncatus) skin cell.

Per- and polyfluoroalkyl substances (PFAS) have become ubiquitous environmental contaminants in aquatic ecosystems worldwide. Marine mammals, as top predators, are constantly exposed to several PFAS compounds that accumulate in different tissues. As a proxy to assess cytotoxicity of PFAS in the bottlenose dolphin (Tursiops truncatus), we generated a new immortalized cell line derived from skin samples of bottlenose dolphin. Using high content imaging, we assessed the effects of increasing concentrations of PFOS, PFOA, PFBS, PFBA and C6O4 on cell viability and cell cycle phases. In particular, we classified all cells based on multiple morphometric differences of the nucleus in three populations, named respectively "Normal" (nuclei in G0, S and M phase); "Large" (nuclei showing characteristics of senescence) and "Small" (nuclei with fragmentation and condensed chromatin). Combining this approach with cell cycle analysis we determined which phases of the cell cycle were influenced by PFAS. The results revealed that the presence of PFOS, PFBS and PFBA could increase the number of cells in G0+G1 phase and decrease the number of those in the S phase. Moreover, PFOS and PFBS lowered the fraction of cells in the M phase. Interestingly PFOS, PFBS and PFBA reduced the prevalence of the senescence phenotype ("large" nuclei), suggesting a potential tumorigenic effect. Besides, the presence of PFOS and PFBS correlated also with a significant decrease in the number of "small" nuclei. The C6O4 exposure did not highlighted morphometric alteration or cell cycle modification bottlenose dolphin skin cell nuclei. While the effects of PFAS on cell cycle was clear, no significant change was detected either in term of cell proliferation or of viability. This study fosters the overall knowledge on the cellular effects of perfluoroalkyl substances in marine mammals.

Microscopic anatomical, immunohistochemical, and morphometric characterization of the terminal airways of the lung in cetaceans.

The lungs of cetaceans undergo anatomical and physiological adaptations that facilitate extended breath-holding during dives. Here, we present new insights on the ontogeny of the microscopic anatomy of the terminal portion of the airways of the lungs in five cetacean species: the fin whale (Balaenoptera physalus); the sperm whale (Physeter macrocephalus), the Cuvier's beaked whale (Ziphius cavirostris); the bottlenose dolphin (Tursiops truncatus); and the striped dolphin (Stenella coeruleoalba). We (a) studied the histology of the terminal portion of the airways; (b) used immunohistochemistry (IHC) to characterize the muscle fibers with antibodies against smooth muscle (sm-) actin, sm-myosin, and desmin; (c) the innervation of myoelastic sphincters (MESs) with an antibody against neurofilament protein; and (d) defined the diameter of the terminal bronchioles, the diameter and length of the alveoli, the thickness of the septa, the major and minor axis, perimeter and section area of the cartilaginous rings by quantitative morphometric analyses in partially inflated lung tissue. As already reported in the literature, in bottlenose and striped dolphins, a system of MESs was observed in the terminal bronchioles. Immunohistochemistry confirmed the presence of smooth muscle in the terminal bronchioles, alveolar ducts, and alveolar septa in all the examined species. Some neurofilaments were observed close to the MESs in both bottlenose and striped dolphins. In fin, sperm, and Cuvier's beaked whales, we noted a layer of longitudinal smooth muscle going from the terminal bronchioles to the alveolar sacs. The morphometric analysis allowed to quantify the structural differences among cetacean species by ranking them into groups according to the adjusted mean values of the morphometric parameters measured. Our results contribute to the current understanding of the anatomy of the terminal airways of the cetacean lung and the role of the smooth muscle in the alveolar collapse reflex, crucial for prolonged breath-holding diving.