Diclofenac induces apoptosis in hepatocytes.
Hepatotoxicity is one of the side effects associated with the administration of diclofenac, a non-steroidal anti-inflammatory drug widely used clinically. The effect of diclofenac on the early events that trigger apoptosis cascade have been evaluated in rat hepatocytes. To do this, early and late apoptotic markers, associated with the pivotal steps of the execution phase, have been evaluated after incubation with the drug. The results show that the apoptotic effect of diclofenac occurs after exposure to sub-cytotoxic concentrations of the drug (maximal non toxic concentration, MNTC, after 24-h treatment was 450 microM), without overlapping with cell necrosis (LDH leakage evaluation). Flow cytometric analysis revealed a time- and dose-dependent increase of apoptotic nuclei with sub-diploid DNA content. Caspase 3 activation (3-5-fold control) was maximal after 12 h of exposure to 350 microM of the drug. The involvement of the mitochondrial permeability transition (MPT) in diclofenac-induced apoptosis was investigated. Cyclosporine A and decylubiquinone, MPT specific inhibitor, prevented the activation of caspase 3, thus showing that diclofenac opened the MPT pore. Treatment of hepatocytes with antioxidants (alpha-tocopherol, N,N-dimethylthiourea, superoxide dismutase) were able to prevent caspase cascade activation by diclofenac, revealing that oxidative stress at the mitochondrial level is involved in MPT induction. Finally, the differential cytotoxic and apoptotic effect produced in hepatocytes and non-metabolizing hepatoma cells suggest that CYP-mediated metabolism of diclofenac apoptosis may be related to the apoptotic effect of the drug.
Centro de Investigación, Hospital La Fe, Avda de Campanar 21, E-46009, Valencia, Spain. gomez_mjo@gva.es
Maria-José
MJ
Gómez-Lechón
Xavier
X
Ponsoda
Enrique
E
O'Connor
Teresa
T
Donato
R
R
Jover
José V
JV
Castell
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000633:toxicity*; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation; D004008:Diclofenac / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D018197:Hepatoblastoma; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008113:Liver Neoplasms; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009336:Necrosis; D018384:Oxidative Stress / Q000187:drug effects; D010539:Permeability / Q000187:drug effects
D000894:Anti-Inflammatory Agents, Non-Steroidal; D000975:Antioxidants; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D004008:Diclofenac; D007770:L-Lactate Dehydrogenase; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases