Mitochondrial DNA damage and altered membrane potential (delta psi) in pancreatic acinar cells induced by reactive oxygen species.
BACKGROUND
Reactive oxygen species (ROS) have been implicated in the induction of acute pancreatitis. Mitochondria possess a distinct genome (mtDNA) that is more susceptible to ROS-induced damage than nuclear DNA (nDNA). The purpose of our study was to determine the effect of ROS on mitochondrial function and membrane potential (delta psi mt), to identify signal transduction mechanisms activated by ROS, and to quantify damage to mtDNA in an in vitro pancreatitis model.


METHODS
Pancreatic acinar cells, AR4-2J, were treated with saline solution (control) or hydrogen peroxide (H2O2), a representative ROS. Mitochondrial function was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; to determine delta psi mt, rhodamine-123 uptake was measured. Intracellular calcium levels and c-Jun N-terminal kinase activity was determined; gel mobility shift assays were performed to assess induction of the transcription factor NF-kappa B. To quantitate DNA damage, a novel polymerase chain reaction-based procedure was performed.


RESULTS
Mitochondrial function and delta psi mt were significantly decreased with oxidative damage. H2O2 treatment resulted in increased intracellular calcium levels, activation of c-Jun N-terminal kinase, and induction of NF-kappa B DNA binding. Treatment of AR4-2J cells with H2O2 resulted in selective mtDNA damage; nDNA was not affected.


CONCLUSIONS
Our data demonstrate that pancreatic mtDNA is more susceptible to oxidative damage than nDNA; this damage is associated with decreases in mitochondrial function and delta psi mt and activation of downstream signal transduction pathways. Mitochondrial damage mediated by ROS may play a central role in pancreatic cell injury associated with acute pancreatitis.
Ehlers
R A
RA
Department of Surgery, University of Texas Medical Branch, Galveston 77555-0533, USA.
Hernandez
A
A
Bloemendal
L S
LS
Ethridge
R T
RT
Farrow
B
B
Evers
B M
BM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D004249:DNA Damage; D004272:DNA, Mitochondrial / Q000378:metabolism*; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D008564:Membrane Potentials; D008928:Mitochondria / Q000502:physiology*; D020929:Mitogen-Activated Protein Kinase Kinases; D016328:NF-kappa B / Q000378:metabolism; D010179:Pancreas / Q000378:metabolism*; D011494:Protein Kinases / Q000502:physiology; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004272:DNA, Mitochondrial; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D011494:Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; D002118:Calcium