Acetaminophen induces apoptosis of C6 glioma cells by activating the c-Jun NH(2)-terminal protein kinase-related cell death pathway.

Acetaminophen (AAP), a widely used analgesic drug, can damage various organs when taken in large doses. In this study, we investigate whether AAP causes cell damage by altering the early signaling pathways associated with cell death and survival. AAP caused time- and concentration-dependent apoptosis and DNA fragmentation of C6 glioma cells used as a model. AAP activated c-Jun N-terminal protein kinase (JNK) by 5.3-fold within 15 min. The elevated JNK activity persisted for up to 4 h before it returned to the basal level at 8 h. In contrast, activities of other mitogen-activated protein (MAP) kinases and the level of Akt phosphorylation in the cell survival pathway remained unchanged throughout the treatment. Wortmannin, an inhibitor of phosphatidylinositol-3 kinase, or SB203580, an inhibitor of p38 MAP kinase, did not reduce AAP-induced toxicity, indicating that these enzymes do not play a major role in cell toxicity. AAP-induced apoptosis was preceded by the sequential elevation of the pro-apoptotic Bax protein, cytochrome c release, and caspase-3 activity. Treatment with caspase inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (Z-DEVD-FMK) significantly reduced AAP-induced caspase-3 activation and cytotoxicity. Transfection of cDNA for the dominant-negative mutant JNK-KR or stress-activated protein kinase kinase-1 Lys-->Arg mutant (SEK1-KR), an immediate upstream kinase of JNK, significantly reduced AAP-induced JNK activation and cell death rate. The noncytotoxic analog of AAP, 3-hydroxyacetanilide, neither increased JNK activity nor caused apoptosis. Pretreatment with YH439, an inhibitor of CYP2E1 gene transcription, markedly reduced CYP2E1 mRNA, protein content, and activity, as well as the rate of AAP-induced JNK activation and cell death. These data indicate that AAP can cause cell damage by activating the JNK-related cell death pathway, providing a new mechanism for AAP-induced cytotoxicity.