The basis for the cellular damage induced by ethacrynic acid in liver slices in vitro. Comparison of structure and...

The development of cellular damage in response to ethacrynic acid and to amytal has been studied in slices of rat liver. After a preincubation at 1 degree C, the slices were incubated with or without the agents at 38 degrees C. Control slices showed a net extrusion of water, Na+, Cl-, and Ca2+, and a net reaccumulation of K+, during incubation at 38 degrees C. Ethacrynic acid (3 mM) reduced the extrusion of water, Na+ and Cl- during the first 5 minutes at 38 degrees C, but Ca2+ extrusion was not affected. Morphological indications of damage to mitochondria were apparent already at 5 minutes, although unaccompanied by a change of tissue ATP content. The mitochondrial damage was more marked at 15 minutes, when signs of early necrosis were also evident in some cells and ATP content started to fall. At longer times with ethacrynate, water and Ca2+ started to re-enter slices and there were indications of a loss of selective permeability of the plasma membranes; no significant uptake of K+ occurred at any time. Electron-dense particles, apparently of Ca2+, were deposited in the mitochondria and cisternae of the endoplasmic reticulum. The morphological appearance rapidly progressed to a marked disorganization. Slices incubated with 4 mM amytal showed greater and earlier loss of ATP than slices with ethacrynate and complete inhibition of water extrusion; other biochemical and morphological effects were generally less marked than with ethacrynate. Some of the morphological and biochemical effects of the two agents after 15 minutes at 38 degrees C could be reversed upon removal of the drugs. However, Ca2+ contents showed a late rise which started 20 minutes after removal of the drugs, indicating that an early, nonreversible damage of the calcium regulating mechanism had occurred. The results suggest that early effects of ethacrynate are on cell volume regulation and mitochondrial structure and function; there follow more general changes of membrane permeability and loss of control of cell Ca2+. A variety of biochemical and morphological markers of cell damage gave different indications of the level of damage in the early phases of treatment with each of the drugs used here. At longer times (e.g., 60 min) there was a greater degree of uniformity in the indications of cell damage.