Effects of N-acetylcysteine and 3-aminotriazole on H2O2-induced apoptosis in HepG2 cells

The effects of N-acetylcysteine (NAC), an antioxidative amino acid, and 3-amino-1,2,4-triazole (3-AT), a potential catalase inhibitor, on hydrogen peroxide (H 2 O 2 )-induced apoptosis were studied using a human hepatoma cell line (HepG 2 ). The leakage of lactate dehydrogenase (LDH) into the culture medium from the cells, which was regarded as an indicator of the degree of cell death, was significantly increased after treatment with H 2 O 2 in a dose- or time-dependent manner. When NAC was added to the culture medium before the H 2 O 2 treatment, the leakage of LDH from the H 2 O 2 -induced HepG 2 cells was effectively suppressed by the NAC, dose-dependently. In contrast, 3-AT significantly increased the LDH leakage into the culture medium from the H 2 O 2 -induced HepG 2 cells. DNA ladder formation was demonstrated in the culture medium of H 2 O 2 -treated HepG 2 cells, which was suppressed by NAC and stimulated by 3-AT. TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay was employed to estimate the apoptosis in situ. H 2 O 2 -induced HepG 2 cells were stained by the TUNEL method, indicating nuclear fragmentation. Such nuclear degeneration was inhibited by NAC and increased by 3-AT. These findings suggest that NAC exerts antioxidative effects on H 2 O 2 -induced apoptosis in HepG 2 as estimated from apoptosis, DNA ladder formation and TUNEL staining.