Cytochrome P450-generated reactive metabolites cause mitochondrial permeability transition, caspase activation, and apoptosis in rat hepatocytes

Although cytochrome P-450 (CYP)-generated reactive metabolites can cause he patocyte apoptosis, the mechanism of this effect is incompletely understood . In the present study, we assessed the hepatotoxicity of skullcap, a diter penoid-containing herbal remedy. Male rat hepatocytes were incubated for 2 hours with skullcap diterpenoids (100 mu g/mL). This treatment decreased ce ll glutathione and protein thiols and increased cell [Ca2+]. This activated Ca2+-dependent tissue transglutaminase, forming a cross-linked protein sca ffold, and also opened the mitochondrial permeability transition pore, caus ing outer mitochondrial membrane rupture, increased cytosolic cytochrome c, activation of procaspase 3, internucleosomal DNA fragmentation, and ultras tructural features of apoptosis, Cell death was increased by a CYP3A induce r (dexamethasone) or a sulfur amino acid-deficient diet increasing glutathi one depiction. In contrast, cell death was prevented by decreasing CYP3A ac tivity (with troleandomycin), preventing glutathione depletion (with cystei ne or cystine), blocking Ca2+-modulated events (with calmidazolium), preven ting mitochondrial permeability transition (with cyclosporin A), or inhibit ing caspase 3 (with acetyl-Asp-G u-Va-Asp-a dehyde). Both calmidazolium and cyclosporin A also prevented the increase in cytosolic cytochrome c and pr ocaspase 3 activation. In conclusion, CYP3A activates skullcap diterpenoids into reactive metabolites that deplete cellular thiols and increase cell [ Ca2+]. This activates Ca2+-dependent transglutaminase and also opens the mi tochondrial permeability transition pore, causing outer mitochondrial membr ane rupture, cytochrome c release, and caspase activation, Preventing mitoc hondrial permeability transition pore opening and/or caspase activity block s apoptosis, showing the fundamental role of these final events in metaboli te-mediated hepatotoxicity.