J Vet Sci.  2015 Mar;16(1):17-23. 10.4142/jvs.2015.16.1.17.

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

• Hwang GH
• Jeon YJ
• Han HJ
• Park SH
• Baek KM
• Chang W
• Kim JS
• Kim LK
• Lee YM
• Lee S
• Bae JS
• Jee JG
• Lee MY

• KMID: 2160816
• DOI: http://doi.org/10.4142/jvs.2015.16.1.17

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.

Keyword

apoptosis; butylated hydroxyanisole; primary mouse hepatocytes; reactive oxygen species

MeSH Terms

Animals
Apoptosis/*drug effects
Butylated Hydroxyanisole/chemistry/*pharmacology
Cell Survival/drug effects
Cells, Cultured
Hepatocytes/*drug effects
Hydrogen Peroxide/*toxicity
Male
Mice
Mice, Inbred ICR
Molecular Structure
Butylated Hydroxyanisole
Hydrogen Peroxide

Figure

• 

  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.

Reference

1. Aviram M. Review of human studies on oxidative damage and antioxidant protection related to cardiovascular diseases. Free Radic Res. 2000; 33:Suppl. S85–S97.

2. Cai Y, Luo Q, Sun M, Corke H. Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sci. 2004; 74:2157–2184.

3. Chen KC, Zhou Y, Zhang W, Lou MF. Control of PDGF-induced reactive oxygen species (ROS) generation and signal transduction in human lens epithelial cells. Mol Vis. 2007; 13:374–387.

4. Cole KK, Perez-Polo JR. Poly(ADP-ribose) polymerase inhibition prevents both apoptotic-like delayed neuronal death and necrosis after H₂O₂ injury. J Neurochem. 2002; 82:19–29.

5. Conde de la Rosa L, Schoemaker MH, Vrenken TE, Buist-Homan M, Havinga R, Jansen PLM, Moshage H. Superoxide anions and hydrogen peroxide induce hepatocyte death by different mechanisms: involvement of JNK and ERK MAP kinases. J Hepatol. 2006; 44:918–929.

6. Daniel H. Molecular and integrative physiology of intestinal peptide transport. Annu Rev Physiol. 2004; 66:361–384.

7. de Magalhães JP, Church GM. Cells discover fire: employing reactive oxygen species in development and consequences for aging. Exp Gerontol. 2006; 41:1–10.

8. Dumont A, Hehner SP, Hofmann TG, Ueffing M, Dröge W, Schmitz ML. Hydrogen peroxide-induced apoptosis is CD95-independent, requires the release of mitochondria-derived reactive oxygen species and the activation of NF-κ B. Oncogene. 1999; 18:747–757.

9. Englert RP, Shacter E. Distinct modes of cell death induced by different reactive oxygen species: amino acyl chloramines mediate hypochlorous acid-induced apoptosis. J Biol Chem. 2002; 277:20518–20526.

10. Evans ZP, Mandavilli BS, Ellett JD, Rodwell D, Fariss MW, Fiorini RN, Schnellmann RG, Schmidt MG, Chavin K. Vitamin E succinate enhances steatotic liver energy status and prevents oxidative damage following ischemia/reperfusion. Transplant Proc. 2009; 41:4094–4098.

11. Fang L, Gou S, Fang X, Cheng L, Fleck C. Current progresses of novel natural products and their derivatives/analogs as anti-Alzheimer candidates: an update. Mini Rev Med Chem. 2013; 13:870–887.

12. Halliwell B. Free radicals, antioxidants, and human disease: curiosity, cause, or consequence? Lancet. 1994; 344:721–724.

13. Ishikawa K, Takenaga K, Akimoto M, Koshikawa N, Yamaguchi A, Imanishi H, Nakada K, Honma Y, Hayashi J. ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis. Science. 2008; 320:661–664.

14. Iverson F. In vivo studies on butylated hydroxyanisole. Food Chem Toxicol. 1999; 37:993–997.

15. Jaeschke H. Reactive oxygen and mechanisms of inflammatory liver injury. J Gastroenterol Hepatol. 2000; 15:718–724.

16. Jiang J, Yu S, Jiang Z, Liang C, Yu W, Li J, Du X, Wang H, Gao X, Wang X. N-acetyl-serotonin protects HepG2 cells from oxidative stress injury induced by hydrogen peroxide. Oxid Med Cell Longev. 2014; 2014:310504.

17. Kähkönen MP, Hopia AI, Vuorela HJ, Rauha JP, Pihlaja K, Kujala TS, Heinonen M. Antioxidant activity of plant extracts containing phenolic compounds. J Agric Food Chem. 1999; 47:3954–3962.

18. Kim KC, Lee C. Curcumin induces downregulation of E2F4 expression and apoptotic cell death in HCT116 human colon cancer cells; involvement of reactive oxygen species. Korean J Physiol Pharmacol. 2010; 14:391–397.

19. Lee MY, Jung SC, Lee JH, Han HJ. Estradiol-17β protects against hypoxia-induced hepatocyte injury through ER-mediated upregulation of Bcl-2 as well as ER-independent antioxidant effects. Cell Res. 2008; 18:491–499.

20. Li WM, Liu HT, Li XY, Wu JY, Xu G, Teng YZ, Ding ST, Yu C. The effect of tetramethylpyrazine on hydrogen peroxide-induced oxidative damage in human umbilical vein endothelial cells. Basic Clin Pharmacol Toxicol. 2010; 106:45–52.

21. Loguercio C, Federico A. Oxidative stress in viral and alcoholic hepatitis. Free Radic Biol Med. 2003; 34:1–10.

22. Lu Y, Zhang YY, Hu YC, Lu YH. Protective effects of 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone against hydrogen peroxide-induced oxidative stress in hepatic L02 cell. Arch Pharm Res. 2014; 37:1211–1218.

23. Martin D, Salinas M, Fujita N, Tsuruo T, Cuadrado A. Ceramide and reactive oxygen species generated by H₂O₂ induce caspase-3-independent degradation of Akt/protein kinase B. J Biol Chem. 2002; 277:42943–42952.

24. Menon SG, Goswami PC. A redox cycle within the cell cycle: ring in the old with the new. Oncogene. 2007; 26:1101–1109.

25. Moriya K, Nakagawa K, Santa T, Shintani Y, Fujie H, Miyoshi H, Tsutsumi T, Miyazawa T, Ishibashi K, Horie T, Imai K, Todoroki T, Kimura S, Koike K. Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis. Cancer Res. 2001; 61:4365–4370.

26. Murakami Y, Shoji M, Ogiwara T, Tanaka S, Yokoe I, Fujisawa S. Preventive effect of ortho dimer of butylated hydroxyanisole on activator protein-1 activation and cyclooxygenase-2 expression in macrophages stimulated by fimbriae of Porphyromonas gingivalis, an oral anaerobe. Anticancer Res. 2006; 26:2915–2920.

27. Pham-Huy LA, He H, Pham-Huy C. Free radicals, antioxidants in disease and health. Int J Biomed Sci. 2008; 4:89–96.

28. Rajendran P, Nandakumar N, Rengarajan T, Palaniswami R, Gnanadhas EN, Lakshminarasaiah U, Gopas J, Nishigaki I. Antioxidants and human diseases. Clin Chim Acta. 2014; 436:332–347.

29. Ruiz C, Casarejos MJ, Gomez A, Solano R, de Yebenes JG, Mena MA. Protection by glia-conditioned medium in a cell model of Huntington disease. PLoS Curr. 2012; 4:e4fbca54a2028b.

30. Saito M, Sakagami H, Fujisawa S. Cytotoxicity and apoptosis induction by butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Anticancer Res. 2003; 23:4693–4701.

31. Sandoval-Acuña C, Ferreira J, Speisky H. Polyphenols and mitochondria: an update on their increasingly emerging ROS-scavenging independent actions. Arch Biochem Biophys. 2014; 559:75–90.

32. Sarada SK, Himadri P, Ruma D, Sharma SK, Pauline T, Mrinalini . Selenium protects the hypoxia induced apoptosis in neuroblastoma cells through upregulation of Bcl-2. Brain Res. 2008; 1209:29–39.

33. Tribble DL, Aw TY, Jones DP. The pathophysiological significance of lipid peroxidation in oxidative cell injury. Hepatology. 1987; 7:377–386.

34. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol. 2007; 39:44–84.

35. Vandghanooni S, Forouharmehr A, Eskandani M, Barzegari A, Kafil V, Kashanian S, Ezzati Nazhad Dolatabadi J. Cytotoxicity and DNA fragmentation properties of butylated hydroxyanisole. DNA Cell Biol. 2013; 32:98–103.

36. Wang CC, Fang KM, Yang CS, Tzeng SF. Reactive oxygen species-induced cell death of rat primary astrocytes through mitochondria-mediated mechanism. J Cell Biochem. 2009; 107:933–943.

37. Wang X. The expanding role of mitochondria in apoptosis. Genes Dev. 2001; 15:2922–2933.

38. Weng D, Lu Y, Wei Y, Liu Y, Shen P. The role of ROS in microcystin-LR-induced hepatocyte apoptosis and liver injury in mice. Toxicology. 2007; 232:15–23.

39. Xu S, Chen F, Deng M, Sui Y. Simple simultaneous determination of butylated hydroquinone (TBHQ) and butylated hydroxyanisole (BHA) antioxidants in oil using high-performance liquid chromatography with chemiluminescence detection. Luminescence. 2014; 29:1027–1032.

40. Xue T, Luo P, Zhu H, Zhao Y, Wu H, Gai R, Wu Y, Yang B, Yang X, He Q. Oxidative stress is involved in Dasatinib-induced apoptosis in rat primary hepatocytes. Toxicol Appl Pharmacol. 2012; 261:280–291.