ACETAMINOPHEN METABOLISM AND CYTOTOXICITY IN PC12 CELLS TRANSFECTED WITH CYTOCHROME P4502E1

Although a number of studies confirm the important role of metabolites in the cytotoxicity of acetaminophen, its precise mechanisms remain u nknown. Acetaminophen is metabolized by microsomal enzymes. Cytochrome P4502E1 (CYP2E1) mediated N-hydroxy-lation results in the formation o f N-acetyl-benzo-quinoneimine, a highly reactive intermediate. We exam ined biochemical parameters related to necrotic and apoptotic processe s in acetaminophen-exposed PC12 cells is and in a PC12 cell line genet ically engineered to express human CYP2E1. Both the [H-3]thymidine inc orporation test and the protein assay uniformly showed dose- and time- related significant growth retardation in both cell lines exposed to t he drug. This was more evident in CYP2E1-transfected cells. Moreover, the cytotoxic effect of acetaminophen was increased as evidenced by la ctate dehydrogenase activity in the culture medium. Both random oligon ucleotide primed synthesis assay and enzyme-linked immunosorbent assay revealed significant DNA fragmentation in both cell lines, which was greater in transfected cells, reaching about 11% of total cellular DNA . These results were confirmed by flow cytometry and microscopic exami nation of cell nuclei. Intracellular calcium levels were increased onl y in transfected cells, approximately threefold when 5 mM acetaminophe n was administered for 48 h. These results indicate the cytotoxic effe cts of acetaminophen via apoptosis, necrosis, and growth retardation. While the precise mechanism remains obscure, it seems that DNA fragmen tation and apoptotic cascade represent a preliminary biochemical event in acute cell death, and that acetaminophen biotransformation by CYP2 E1 stimulates this pathway.