The relationship between ATP depletion and the loss of cell integrity was examined in the killing of hepatocytes by anoxia and P388D1 macrophages by silica. ATP depletion is a feature of the reaction to either hazard. Treatment of hepatocytes, however, with antimycin, oligomycin, sodium azide, or N,N'-dicyclohexylcarbodiimide produced a rate and extent of ATP depletion comparable with anoxia without significant loss of viability. Treatment of P388D1 cells with 2-deoxyglucose plus antimycin, oligomycin, or sodium azide reproduced the loss of ATP accompanying silica particle intoxication. Again, there was no loss of viability. These data dissociate the loss of cellular ATP from the genesis of lethal injury in both cell types. ATP depletion was, however, associated with a loss of lysosomal integrity. With the metabolic inhibitors, loss of lysosomal integrity occurred in the absence of irreversible cell injury over the time course that anoxia and silica intoxication significantly damaged the cells. This implies that neither hazard produces lethal damage through mechanisms dependent on intracellular lysosomal enzyme release. While ATP depletion can cause lysosomal rupture in P388D1 macrophages, phagocytosis of silica particles in the absence of extracellular Ca2+ ions is associated with release of lysosomal contents without depletion of ATP or loss of cell integrity. Silica particles are concluded to interact directly with both the plasma and lysosomal membranes. The former leads to Ca2+ influx with resultant cell death and ATP depletion. The latter leads to release of lysosomal contents that is not followed by irreversible cell injury.

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Silica particles are toxic to primary cultures of macrophages or the P388D1 cell line in vitro. Loss of viability in these model systems is accompanied by depletion of ATP content within 3 to 6 hours. The mechanisms responsible for ATP depletion will be explored in this paper. After prelabeling for 1 hour with 3H-adenine, silica-treated cells released 60-80% of their labeled acid-soluble pool into the culture medium. This release did not occur after phagocytosis of nontoxic titanium dioxide particles and was specific for purines. ATP depletion was accompanied by purine catabolism: inosine, hypoxanthine, xanthine, and uric acid were detected in the culture medium using thin layer or high-performance liquid chromatography. The final xanthine oxidase step in purine catabolism generates reactive oxygen metabolites. Silica toxicity was not prevented by the xanthine oxidase inhibitor allopurinol nor exogenous purines. It is concluded that adenine nucleotide depletion and purine catabolism are not solely responsible for irreversible injury in silica toxicity. It is hypothesized that purine catabolism and release from injured macrophages may lead to generation of reactive oxygen species, injury to surrounding tissue, and fibrosis.

Rabbit polyribosomal globin messenger RNA (mRNA) and messenger ribonucleoprotein (mRNP) were labelled at the 3' poly(A) tail to high specific activity with T4 RNA ligase and [5'-(32)P]pCp without consequent loss of functional activity. Labelled message was translated in both micrococcal nuclease treated and untreated rabbit reticulocyte lysates, as shown by the formation of labelled polyribosomes. The utilisation of labelled messenger was abolished by T2 toxin or sodium fluoride which are known to inhibit protein synthesis.Images