The mechanism by which apoptosis is induced by local anesthetic bupivacaine, a potent uncoupler of mitochondrial oxidative phosphorylation, was investigated. In promyelocytic leukemia cells HL-60, bupivacaine induced formation of apoptotic bodies and DNA fragmentation in a time- and dose-dependent manner similar to typical apoptosis inducers. Caspase-3, -8 and -9, which play a pivotal role in the initiation and execution of receptor- or mitochondria-mediated apoptosis, were all clearly activated by bupivacaine in good correlation with the degree of DNA fragmentation. However, bupivacaine did not induce either mitochondrial permeability transition (PT) or release of cytochrome c in experiments with isolated mitochondria. These results suggest that an indirect action of bupivacaine on mitochondria occurs and that other mechanisms may be involved in bupivacaine-induced apoptosis. To obtain additional information concerning the mechanism of action involved in bupivacaine-induced apoptosis, a microarray analysis of gene expression in bupivacaine-treated HL-60 cells was carried out. Several apoptosis-related genes were found to be transcriptionally regulated by bupivacaine using a high-density cDNA microarray. The expression levels of heat shock protein 70 (HSP70), c-jun and c-fos genes were remarkably up-regulated and those of c-myc and poly (ADP ribose) polymerase (PARP) were down-regulated in bupivacaine-treated cells. These results are of value in developing a better understanding of the molecular mechanism of bupivacaine-induced apoptosis leading to neuro- or myotoxicity.

Previously, we found that the cationic cyanine dye tri-S-C 4 (5) uncoupled mitochondrial oxidative phosphorylation by acting as an inducer of the mitochondrial permeability transition (PT). In the present study, the actions of cyanine dyes such as tri-S-C 4 (5) and tri-S-C 7 (5) on the mitochondrial structures and functions were further characterized. In the presence of inorganic phosphate (Pi), cyanine dyes were found to accelerate mitochondrial oxygen consumption that was partially sensitive to the PT inhibitor cyclosporin A (CsA). However, not only the CsAsensitive but also CsA-insensitive acceleration of mitochondrial respiration was induced by cyanine dyes; and both of them were found to be associated with the release of mitochondrial cytochrome c. On the contrary, in the absence of Pi, moderate acceleration of respiration was induced by cyanine dyes, but this respiratory effect was not associated with the induction of swelling or the release of mitochondrial cytochrome c. Thus, cyanine dyes were concluded to have 3 different effects on the mitochondrial functions depending on the Pi status.