LIVER PARENCHYMAL CELL INJURY III. The Nature of Calcium-Associated Electron-Opaque Masses in Rat Liver Mitochondria Following Poisoning with Carbon Tetraehloride

Accumulation of calcium in the mitochondria of rat liver parenchymal cells at 16 and 24 hours after poisoning with carbon tetrachloride is associated with an increase in amount of liver inorganic phosphate, the persistence of mitochondrial adenosine triphosphatase activity, and the formation of electron-opaque intramitochondrial masses in cells with increased calcium contents. These masses, which form within the mitochondrial matrix adjacent to internal mitochondrial membranes, resemble those observed in isolated mitochondria which accumulate calcium and inorganic phosphate; are present in a locus similar to that of electron opacities which result from electron-histochemical determination of mitochondrial ATPase activity; and differ in both appearance and position from matrix granules of normal mitochondria. After poisoning, normal matrix granules disappear from mitochondria prior to their accumulation of calcium. As calcium-associated electron-opaque intramitochondrial masses increase in size, mitochondria degenerate in appearance. At the same time, cytoplasmic membrane systems of mid-zonal and centrilobular cells are disrupted by degranulation of the rough endoplasmic reticulum and the formation of labyrinthine tubular aggregates. The increase in amount of inorganic phosphate in rat liver following poisoning is balanced by a decreased amount of phosphoprorein. These chemical events do not appear to be related, however, as the inorganic phosphate accumulated is derived from serum inorganic phosphate. Mitochondria isolated from liver and kidney of normal animals sequester up to several hundredfold their normal calcium content when this alkaline earth is presented to them in vitro (4, 5). Inorganic phosphate accumulates concurrently, under these conditions, in a metabolic process which requires either adenosine triphosphate (ATP) or an electron transport-linked translocation of inorganic phosphate from the medium to the mitochondrial matrix (2, 6). The continued uptake of calcium and phosphate by mitochondria results in the appearance of electron-opaque masses within the mitochondrial matrix (2, 7). As a consequence, it has been postulated that mitochondria take part in the translocation of calcium and phosphate in normal cells (4, 5). Indeed, similar granules are observed in osteoclasts of normal bone (8), have been described in calcium phosphate-sequestering tumors (9), and appear in renal tubular epithelium following the administration of parathormone (10). Although calcium uptake by mitochondria has been most 53 on O cber 0, 2017 jcb.rress.org D ow nladed fom extensively studied, other metals such as barium and strontium are deposited within mitochondria of epithelial cells in vitro (7), and iron accumulates intramitochondrially within erythroblasts in vivo (ll). Thc scqucstering of calcium by livcr parenchyreal cells also occurs following poisoning by chemical agents such as carbon tetrachloridc (12), thioacetamide (13, 14), dimethylnitrosamine (12), and phosphorus (14). Intracellular calcium accumulation in carbon tetrachloride poisoning is localized to mitochondria (12, 15), is associated with continuing mitochondrial respiration in calcium-sequestering cells (12, 16), and is accompanied by an apparent activation of an alkaline earth~tependent mitochondrial adenosine triphosphatase (ATPase) (17). At the same time, leadstainable cation-binding sites appear within the cytoplasm of the cells involved (12). Continued accumulation of calcium by mitochondria results in failure of their respiration and ultimate disintegration of this organelle (12, 16). Electron-opaque calcium-containing masses also form within the matrix of mitochondria of midzonal and centrilobular parenchymal cells as a consequence of carbon tetrachloride poisoning. The present study examines in detail this cytochemical event, which occurs between 8 and 24 hours after the oral administration of hepatotoxin.