Biological effects of contaminants: measurement of lysosomal membrane stability.

Abstract

Lysosomes are ubiquitous cellular organelles that provide a waste disposal and macromolecular recycling system (autophagy) and also a membrane-bound compartment for intracellular digestion of food ingested by the cells. They accumulate many toxic metals and organic chemical contaminants, providing an evolutionarily primitive detoxication capacity, which if overloaded results in lysosomal damage leading to cell injury, tissue dysfunction, and reduction in animal “health status”. Major reactions of lysosomes to pollutants include loss of membrane integrity, enlargement associated with autophagy, and accumulation of lipid and lipofuscin (agepigment). These types of responses have been widely used to test for the effects of toxic contaminants in both experimental investigations and environmental impact assessments. Several methods are available to measure lysosomal functional status: these include measurement of lysosomal membrane stability in both frozen tissue sections and live cells. Protocols for the implementation of these methods are described here in practical detail for mussel/molluscan digestive gland or hepatopancreas and flatfish liver. Cytochemically determined latency of selected lysosomal marker enzymes is used as the measure of stability in frozen sections, and retention time of the chromogenic dye neutral red, as the measure of lysosomal integrity in live cells. Guidelines are included for sample handling, data analysis, and interpretation of results