The lysosomal permability test modified for toxicity testing with cultured heart endothelioid cells

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Abstract
A modified lysosomal fragility test is described which is suitable for use with cultured cells. The permeability (fragility) of the lysosomal membranes of the cells to the substrate β-glycerophosphate is measured by assessing the degree of particulate lysosomal staining seen after exposing the cells to the Gomori acid phosphatase staining reaction under carefully controlled conditions. Monolayer cultures of endothelioid cells from the hearts of neonatal rats have been used in all experiments. The time-course of lysosomal staining for cells exposed to various treatments (normal saline, isotonic sucrose, 0.25 M sucrose, distilled water, acetate buffer pH 5.0, cold acetone, neutral formalin, acetic-ethanol, Triton X-100, hydrocortisone, chloroquine and vitamin A) was compared with that of control cells stained under identical conditions. Statistical differences in staining between the test and control cells were determined by the Wilcoxon Signed Rank Test and also by regression analysis following a transformation designed to allow for the saturation character of the reaction. The success of the modified technique depends upon meticulous methodology. It is capable of demonstrating both lysosomal membrane labilization and stabilization, second- and third-stage lysosomal activation, and apparent lysosomal enzyme loss or destructionin situ. The technique also allows the degree of reversible or first-stage lysosomal activation to be subdivided on an almost continuous basis and is suitable for investigating the effects of drugs and other agents on the integrity of the lysosomein situ.