Protective effect of butylated hydroxylanisole against hydrogen peroxide-induced apoptosis in primary cultured mouse hepatocytes

Abstract

Butylated hydroxyanisole (BHA) is a synthetic phenolic compound consisting of a mixture of two isomeric organic compounds: 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole. We examined the effect of BHA against hydrogen peroxide (H2O2)-induced apoptosis in primary cultured mouse hepatocytes. Cell viability was significantly decreased by H2O2 in a dose-dependent manner. Additionally, H2O2 treatment increased Bax, decreased Bcl-2, and promoted PARP-1 cleavage in a dose-dependent manner. Pretreatment with BHA before exposure to H2O2 significantly attenuated the H2O2-induced decrease of cell viability. H2O2 exposure resulted in an increase of intracellular reactive oxygen species (ROS) generation that was significantly inhibited by pretreatment with BHA or N-acetyl-cysteine (NAC, an ROS scavenger). H2O2-induced decrease of cell viability was also attenuated by pretreatment with BHA and NAC. Furthermore, H2O2-induced increase of Bax, decrease of Bcl-2, and PARP-1 cleavage was also inhibited by BHA. Taken together, results of this investigation demonstrated that BHA protects primary cultured mouse hepatocytes against H2O2-induced apoptosis by inhibiting ROS generation.

Keywords: apoptosis; butylated hydroxyanisole; primary mouse hepatocytes; reactive oxygen species.

Fig. 1. Effect of hydrogen peroxide (H₂O₂) on the viability of primary cultured mouse hepatocytes. (A) Mouse hepatocytes were incubated with H₂O₂ at the indicated concentrations for 24 h. Cell viability was then measured by a CCK-8 assay. Values are expressed as the mean ± standard error (SE) of three independent experiments with triplicate dishes. ^*p < 0.05 vs. the control. (B) Cells were incubated with H₂O₂ at the indicated concentrations for 24 h. Cell lysates were subjected to Western blotting to measure the levels of Bax, Bcl-2, and PARP-1 expression. (C) The densities of bands corresponding to Bax and Bcl-2 protein were measured and the Bax/Bcl-2 ratio was calculated. Values are expressed as the mean ± SE of three independent experiments. ^*p < 0.05 vs. the control.

Fig. 2. Effect of butylated hydroxylanisole (BHA) on H₂O₂-induced cytotoxicity. (A) Chemical structure of BHA. (B) Mouse hepatocytes were incubated with 1,000 µM H₂O₂ for 24 h with or without BHA pretreatment at the indicated concentrations for 30 min. Cell viability was measured with a CCK-8 assay. The values are expressed as the mean ± SE of three independent experiments. ^*p < 0.05 vs. the control; ^**p < 0.05 vs. H₂O₂ alone.

Fig. 3. Effect of BHA on H₂O₂-induced ROS generation. (A~F) Dichlorofluorescein (DCF)-sensitive cellular ROS generation was assessed. (A) Control. (B) Treatment with 1,000 µM H₂O₂ for 2 h. (C) Pretreatment with 5 µM BHA for 30 min before exposure to 1,000 µM H₂O₂ for 2 h. (D) Incubtion with with BHA for 2 h. (E) Pretreatment with 1,000 µM N-acetyl-cysteine (NAC) for 30 min before exposure to 1,000 µM H₂O₂ for 2 h. (F) Incubation with NAC for 2h. (G) DCF-DA fluorescence was measured and quantified with a fluorometer. Values are expressed as the mean ± SE of three independent experiments. ^*p < 0.05 vs. the control, ^**p < 0.05 vs. H₂O₂ alone. Scale bars = 50 µm (A~F).

Fig. 4. Effect of BHA on H₂O₂-induced apoptosis. (A) Mouse hepatocytes were pretreated with 5 µM BHA or 1,000 µM NAC for 30 min and then incubated with or without 1,000 µM H₂O₂ for 24 h. Cell viability was measured with a CCK-8 assay. Values are expressed as the mean ± SE of three independent experiments with triplicate dishes. ^*p < 0.05 vs. control; ^**p < 0.05 vs. H₂O₂ alone. (B) Cells were pretreated with 5 µM BHA followed by treatment with 1,000 µM H₂O₂ for 24 h. Scale bar = 50 µm. (C) Cells were pretreated with 5 µM BHA and then incubated with 1,000 µM H₂O₂ for 24 h. Cell lysates were subjected to Western blotting to measure the levels of Bax, Bcl-2, and PARP-1 expression. (D) Densities of bands corresponding to Bax and Bcl-2 protein were measured, and the Bax/Bcl-2 ratio was calculated. Values are expressed as the mean ± SE of three independent experiments. ^*p < 0.05 vs. the control , ^**p < 0.05 vs. H₂O₂ alone.