Measuring Apoptosis and Necrosis in Cholestatic Liver Injury

Cholestasis can be induced by obstruction of bile ducts or intrahepatic toxicity of drugs and chemicals. However, the mode of cell death during cholestasis, i.e., apoptosis or necrosis, has been controversial. There are fundamental reasons for the controversies, both of which are discussed here, namely the design of experiments and the use of parameters with limited specificity for a certain mode of cell death. Based on the assumption that cholestatic liver injury is caused by accumulation of bile acids, rodent (mainly rat) hepatocytes have been exposed to hydrophobic, glycine-conjugated bile acids, which resulted in apoptotic cell death. The problems with this experimental design are that in rodents bile acids are predominantly taurine conjugated and rodent hepatocytes are never exposed to these levels of glycine-conjugated bile acids. In contrast, taurine-conjugated bile acids trigger inflammatory gene activation in rodent hepatocytes and a necro-inflammatory injury in vivo. On the other hand, human hepatocytes are more resistant to glycine-conjugated bile acids and die by necrosis when exposed to high biliary levels of these bile acids. In this chapter, we describe multiple assays including the caspase activity assay, which is specific for apoptosis, and the general cell death assays alanine aminotransferase or lactate dehydrogenase activities in cell culture medium or plasma. An increase in these enzyme activities without caspase activity indicates necrotic cell death. Thus, both the experimental design and the selection of cell death parameters are critical for the relevance of the experiments for the human pathophysiology.