MORPHOLOGY OF RICIN AND ABRIN EXPOSED ENDOTHELIAL-CELLS IS CONSISTENTWITH APOPTOTIC CELL-DEATH

Cultures of bovine pulmonary endothelial (BPE) cells were exposed to L C(50) doses of ricin or abrin (15.5 and 4.5 pM respectively) over a pe riod of up to 40 h, The viability of the cultures (as determined by th e neutral red (NR) dye retention assay) declined after 6 h exposure to the toxins, From 15 h onwards, cellular material in toxin exposed cul tures became detached from the substratum of the culture vessels. Hoff man modulation contrast photomicrography showed that this process was due to ricin and abrin exposed cells collapsing into membrane bound ve sicles which retained the NR dye, became detached and floated into the medium, These apoptotic-like structural changes were further investig ated by transmission electron microscopy (TEM) and by agarose gel elec trophoresis of DNA from control and exposed cultures, Many of the char acteristic changes associated with apoptotic cell death were seen usin g TEM, including heterochromatin condensation at the nuclear periphery , crenulation of the nuclear membrane and progressive degeneration of residual nuclear and cytoplasmic structures. The plasma membrane of ma ny cells remained intact, and contained nuclear and cytoplasmic debris . Agarose gel electrophoresis of DNA extracted from toxin-treated cell s revealed oligonucleosome sized DNA fragments, characteristic of apop tosis, from adherent cells at 7 h and both adherent and floating popul ations when harvested from 15 h; DNA from unexposed control cells did not show this fragmentation. The identification of apoptosis as being a significant additional mechanism of toxicity following exposure to r icin and abrin holotoxins raises the possibility of developing new the rapeutic strategies against poisoning by these phytotoxins.