Acetaminophen (
AAP) hepatotoxicity, resulting in centrilobular
necrosis, is frequently encountered following suicidal attempts, especially by adolescents, but also after its excessive use in infants. The subcellular and molecular sequences leading to hepatocellular cell death are not yet clear. We therefore investigated
AAP hepatotoxicity by using cultured
hepatoma-derived cells (Hep3B) exposed to
AAP and
N-acetylcysteine (NAC), used as a
protective agent. Specifically, we studied the role of apoptosis and oxidative damage as putative mechanisms of
AAP-associated cytotoxicity. Hep3B cells were exposed to
AAP (5-25 mM) and NAC (5 mM) for different time periods. Cell viability was assessed by the
Alamar Blue Reduction Test and LDH. Oxidative damage was evaluated by measuring
reactive oxygen species (ROS) and
glutathione.
AAP-induced apoptosis was investigated by flow cytometry and transmission electron microscopy. We found that: 1. In Hep3B cells,
AAP causes a time- and concentration-dependent cytotoxic effect, leading to oxidative stress,
mitochondrial dysfunction, alterations of membrane permeability and apoptosis; 2. In the course of
AAP cytotoxicity, the generation of ROS appears as an early event which precedes decrease of viability, LDH leakage,
glutathione depletion and apoptosis; 3. NAC protects Hep3B cells from
AAP-induced oxidative injury, but does not prevent apoptosis.