Responses of an American eel brain endothelial-like cell line to selenium deprivation and to selenite, selenate, and selenomethionine additions in different exposure media.

The effect of selenium deprivation and addition on the American eel brain endothelial cell line (eelB) was studied in three exposure media: complete growth medium (L15/FBS), serum-free medium (L15), and minimal medium (L15/ex). L15/ex contains only galactose and pyruvate and allowed the deprivation of selenium on cells to be studied. In L15/ex, without any obvious source of selenium, eelB cells survived for at least 7 d, formed capillary-like structures (CLS) on Matrigel, and migrated to heal wounds. Three selenium compounds were added to cultures: selenite, selenate, and selenomethionine (SeMet). Adding selenite or selenate to eelB cell cultures for 24 h caused dose-dependent declines in cell viability, regardless of the exposure media. Although varying with exposure media and viability end point, selenite was approximately 70-fold more cytotoxic than selenate. By contrast, 24 h exposures to either DL- or L-SeMet in the three media caused little or no cytotoxicity. However for 7 d exposures in L15/ex, DL- and L-SeMet were very cytotoxic, even at the lowest tested concentration of 31 µM. By contrast in L15 and L15/FBS, cytotoxicity was only observed with 500 and 1000 µM L-SeMet. In L15/FBS, eelB continued to migrate and form CLS in the presence of SeMet but at 500 µM, cell migration appeared stimulated. As judged from a colony-forming assay over 14 d in L15/FBS, 500 and 1000 µM DL- and L-SeMet inhibited cell proliferation. Overall, the responses of eel cells to selenium depended on the selenium form, concentration, and exposure media, with responses to SeMet being most dependent on exposure media.

Development of a cell line from the American eel brain expressing endothelial cell properties.

A cell line (eelB) was developed from the outgrowth of adherent cells from brain explants of the American eel, Anguilla rostrata (Lesueur). EelB cells have been grown routinely in L-15 with 10% fetal bovine serum (FBS), undergone over 100 passages, and cryopreserved successfully. The cells from late-passage cultures (>45) were polygonal, formed capillary-like structures (CLS) on Matrigel, and stained immunocytochemically for von Willebrand factor (vWF) and for three tight junction proteins, zonula occludens-1 (ZO-1), claudin 3, and claudin 5. These results suggest that eelB is an endothelial cell line, one of the few from fish and the first from the brain. Despite this, eelB did not respond to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with the induction of CYP1A protein. The cells from early-passage cultures (<20) had more varied shapes and did not form CLS on Matrigel. Only cells from early-passage cultures formed in suspension three-dimensional aggregates that had some cells expressing alkaline phosphatase and nestin. These cells are thought to be neural stem cells and the aggregates neurospheres. The emergence of endothelial-like cells upon the continued subcultivation of cells from early-passage cultures that had neural stem cells has been described previously for mammals, but this is a first for teleosts. Remarkably, cells from all passage levels were stained strongly for senescence-associated ß-galactosidase (SA ß-Gal) activity.