Acetaminophen toxicity results in site-specific mitochondrial damage in isolated mouse hepatocytes.
Exposure of isolated mouse hepatocytes to a toxic concentration of acetaminophen (5 mM) resulted in damage to the mitochondrial respiratory apparatus. The nature of this damage was investigated by measuring respiration stimulated by site-specific substrates in digitonin-permeabilized hepatocytes after acetaminophen exposure. Respiration stimulated by succinate at energy-coupling site 2 was most sensitive to inhibition and was decreased by 47% after 1 h. Respiration supported by NADH-linked substrates (site 1) was also decreased but to a lesser extent, while there was no decrease in the rate of ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD)-supported respiration (site 3). The loss of mitochondrial respiratory function was accompanied by a decrease in ATP levels and ATP/ADP ratios in the cytosolic compartment and was preceded by a loss of reduced glutathione in both the cytosol and mitochondria. All these effects occurred well before the loss of cell membrane integrity. The putative toxic metabolite of acetaminophen, N-acetyl-p-benzoquinonimine (NAPQI), produced a similar pattern of respiratory dysfunction in isolated hepatic mitochondria. Respiration stimulated by succinate- and NADH-linked substrates was very sensitive to 50 microM NAPQI, while ascorbate + TMPD-supported respiration was unaffected. The interaction between NAPQI and the respiratory chain was further investigated using submitochondrial particles. Succinate dehydrogenase (associated with respiratory complex II) was found to be very sensitive to NAPQI, while NADH dehydrogenase (respiratory complex I) was inhibited to a lesser extent. Our results indicate that a loss of the ability to utilize succinate- and NADH-linked substrates due to attack of the respiratory chain by NAPQI causes a disruption of energy homeostasis in acetaminophen hepatotoxicity.
Burcham
P C
PC
Department of Pharmacology, Queen Elizabeth II Medical Centre, University of Western Australia, Nedlands.
Harman
A W
AW
D000082:Acetaminophen / Q000633:toxicity*; D000818:Animals; D002458:Cell Fractionation; D005978:Glutathione / Q000032:analysis; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D051379:Mice; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D010085:Oxidative Phosphorylation; D010100:Oxygen / Q000378:metabolism; D013367:Submitochondrial Particles / Q000187:drug effects / Q000378:metabolism; D013379:Substrate Specificity
D000082:Acetaminophen; D007770:L-Lactate Dehydrogenase; D005978:Glutathione; D010100:Oxygen