Subcellular distribution of lead in cultured rat hepatocytes

A clear understanding of the sequence and molecular mechanism of the events involved in lead toxicity is hampered by a lack of information about lead compartmentation within the cell. As part of a continuing effort to identify the mechanism by which lead affects cellular functions, we examined the subcellular distribution of 210Pb in cultured hepatocytes. The cells were isolated, labeled, homogenized in sucrose-N-[(2-hydroxyethyl)piperazine]-N'-2-ethanesulfonic acid buffer, and fractionated into mitochondrial, microsomal, and cytosolic components by differential centrifugation. Complete fractionation of the cells revealed that 71% of the cellular 210Pb was associated with the mitochondria, 5% with the microsomes, and 24% with the cytosol. A modified, rapid fractionation procedure indicated that 45% of the cellular lead was associated with both the mitochondria and the cytosol and 10% with the microsomes. When the cells were separated into total particulates and cytosol with a single centrifugation, 22% of the 210Pb was associated with the soluble fraction. The process of homogenization and fractionation of the isolated hepatocytes altered the intracellular distribution of 210Pb. This experimental approach to studying the localization of lead may be compromised by the redistribution of 210Pb during the extensive centrifugations and resuspensions required for subcellular fractionation and suggests that the subcellular distribution patterns of 210Pb obtained by the fractionation of cells reflects the distribution of lead in the homogenate rather than the distribution of 210Pb in the intact cell.