ToxSci Advance Access published online on April 28, 2004
Toxicological Sciences, doi:10.1093/toxsci/kfh151
Toxicological Sciences © Society of Toxicology 2004; all rights reserved
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1 Liver Research Institute, University of Arizona, College of Medicine, Tucson, AZ 85724
* To whom correspondence should be addressed. E-mail: jaeschke{at}email.arizona.edu.
The increase in cellular and mitochondrial glutathione disulfide (GSSG) levels and the GSSG-to-GSH-ratio after acetaminophen (AAP) overdose suggest the involvement of an oxidant stress in the pathophysiology. However, the initial severe depletion of hepatocellular glutathione makes the quantitative assessment of the oxidant stress difficult. Therefore, we tested the hypothesis that oxidant stress precedes the onset of cell injury in a cell culture model using 2',7'-dichlorofluorescein (DCF) fluorescence as a marker for intracellular oxidant stress. Cultured primary murine hepatocytes were exposed to 5 mM AAP. DCF fluorescence, XTT reduction, lactate dehydrogenase (LDH) release and trypan blue uptake were determined from 0-12h. After glutathione depletion at 3h, DCF fluorescence increased by 16-fold and was maintained at that level up to 12h. At 1.5h after AAP, a significant decrease of the cellular XTT reduction capacity was observed, which continued to decline until 9h. Cell necrosis (LDH release, trypan blue uptake) was detectable in 20% of cells at 6h with a significant further increase at later time points. Pretreatment with 20 mM N-acetylcysteine (NAC) 1h before AAP enhanced cellular glutathione content, prevented or attenuated the AAP-induced decrease of GSH levels and XTT reduction capacity, respectively, and reduced the loss of cell viability. Additionally, treatment with NAC 2h after AAP exposure prevented further deterioration of XTT reduction at 3h and later, and attenuated cell necrosis. Thus, AAP-induced oxidant stress precedes cell necrosis and, in cultured hepatocytes, the oxidant stress is involved in the propagation of cell injury.
Accepted April 7, 2004
Systems Evaluation
Acetaminophen-Induced Oxidant Stress and Cell Injury in Cultured Mouse Hepatocytes: Protection by N-Acetyl Cysteine
2 Liver Research Institute, University of Arizona, College of Medicine, Tucson, AZ 85724; Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205
3 Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC 27599
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