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Phellinus linteus mushroom protects against tacrine-induced mitochondrial impairment and oxidative stress in HepG2 cells
Phytomedicine 20 (2013) 705–709
Contents lists available at SciVerse ScienceDirect
Phytomedicine journal homepage: www.elsevier.de/phymed
Phellinus linteus mushroom protects against tacrine-induced mitochondrial impairment and oxidative stress in HepG2 cells Chunpeng Gao a , Laifu Zhong b , Liping Jiang b , Chengyan Geng b , Xiaofeng Yao a , Jun Cao a,∗ a b
Occupational and Environmental Health Department, Dalian Medical University, No. 9, West Segment of South Lvshun Road, Dalian 116044, China China-Japanese Joint Institute for Medical and Pharmaceutical Science, Dalian Medical University, No. 9, West Segment of South Lvshun Road, Dalian 116044, China
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Keywords: Phellinus linteus mushroom Polysaccharides/glycoproteins Mitochondrial impairment Oxidative stress mtDNA damage
a b s t r a c t Tacrine (THA) was the first drug licensed for the treatment of Alzheimer’s disease. Unfortunately, reversible hepatotoxicity is evident in about 30% of patients and limits its clinical use. The intracellular mechanisms have not yet been elucidated. Phellinus linteus (PL) is a mushroom that has long been used as a folk medicine. In our previous study, we found that PL could decrease the reactive oxygen species (ROS) formation in HepG2 cells. Presently, protective effects of PL on tacrine-induced ROS formation and mitochondria dysfunction were evaluated. The results showed that PL significantly reduced tacrineinduced ROS production, disruption of m, 8-OHdG formation in mitochondrial DNA, and cytotoxicity in HepG2 cells. These data suggest that PL could attenuate the cytotoxicity and mitochondria dysfunction induced by tacrine in HepG2 cells. The protection is probably mediated by an antioxidant protective mechanism. Consumption of PL may be a plausible way to prevent tacrine-induced hepatotoxicity. © 2013 Elsevier GmbH. All rights reserved.
Introduction The liver is one of the first organs to be exposed to orally delivered compounds and is also the major site for xenobiotic metabolism, which can lead to the formation of toxic metabolites. Hepatotoxicity is one of the most common adverse drug reactions, as are more than half of marketed drugs with black box warnings (Hunt 2010). Tacrine, or 9-amino1,2,3,4-tetrahydroaminoacridine, is a competitive inhibitor of cholinesterase. It was the first drug licensed by the US Food and Drug Administration for the treatment of Alzheimer’s disease (Johnson et al. 2004). Unfortunately, reversible hepatotoxicity is evident by elevated alanine aminotransferase (ALT) levels in about 30% of patients and limits its clinical use. However, the intracellular mechanisms have not yet been elucidated. Many studies have shown an association with mitochondrial dysfunction (Robertson et al. 1998) and glutathione depletion in hepatocytes and liver cell necrosis (Watkins et al. 1994). Mehta et al. have shown that tacrine accumulated in mitochondria and increase the risk of mitochondrial toxicity (Mehta et al. 2008).
Mitochondria are the key cellular energy source, supplying more than 90% of cellular ATP (Modica-Napolitano and Singh 2004). Therefore, drug-induced mitochondrial impairment directly affects hepatocyte viability (Boelsterli and Lim 2007). Most drugs withdrawn from the market are mitochondrial toxicants. Mushrooms have attracted great interest due to their nutritional, medical and pharmacological properties. Phellinus linteus (PL) is a Basidomycota fungus which is rich in polysaccharides and aromatic compounds. It has been used in traditional oriental medicine for over 2000 years to treat various diseases, such as tumors, inflammation and lymphatic diseases (Han et al. 1999; Yang and Jong 1989). A large body of research about PL indicates that it possesses antitumor (Kim et al. 2004), immuno-modulating (Lim et al. 2004), and anti-oxidant activity etc. (Park et al. 2004). Moreover, hepatoprotective effects have been reported by Jeon et al. (Jeon et al. 2003). In our previous study, we found that PL decrease reactive oxygen species (ROS) in HepG2 cells. In this study, we investigated the protective effects of PL on tacrine-induced oxidative stress and mitochondrial impairment in HepG2 cells. Materials and methods
Abbreviations: ALT, alanine aminotransferase; DCFH-DA, 2,7dichlorofluorescein diacetate; GSH, glutathione; mtDNA, mitochondrial DNA; MTT, methylthiazol tetrazolium bromide; nDNA, nuclear DNA; PL, Phellinus linteus; ROS, reactive oxygen species. ∗ Corresponding author. Tel.: +86 411 86110330. E-mail addresses: [email protected], [email protected] (J. Cao). 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.02.014
Cell culture and reagents HepG2 cells (American Type Culture CollectionHB-8065) were obtained from Peking Union Medical College (Peking, China) and cultured in minimum essential Eagle’s medium containing 10% fetal
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bovine serum (Gibco, Grand Island, NY), penicillin (100 units/ml, Gibco), and strepto-mycin (100 mg/ml, Gibco). Tacrine was purchased from Sigma. PL was from Hun’chun lvdao medicinal Co. Ltd. (Hn’chun,China). The water extract was prepared as follows. The crushed dry mushroom (1.00 kg) was boiled for 4 h at 100 ◦ C, cooled to room temperature, and then filtered followed by ethanol precipitation. The extract was evaporated to dryness with a rotary vacuum evaporator and freeze-dried to give a powder (250 g). The polysaccharide concentration and protein contents were determined, and the monosaccharides and amino acids compositions were analyzed as described by Kim et al. (Kim et al. 2006). The stock solution of PL extract was prepared by dissolving the powder in distilled water (50 mg/ml). For each experiment, PL was diluted with cell culture medium to the concentrations indicated (0.0625, 0.125, and 0.25 mg/ml) freshly before the experiment. ROS assay The production of ROS was measured using the 2,7dichlorofluorescein diacetate (DCFH-DA) method (Cao et al. 2006). HepG2 cells (5 × 105 ) were suspended in 2 ml medium and were incubated with tacrine at different concentrations (0, 9.4, 18.8, 37.5 M) for 1 h at 37 ◦ C. Cells were washed twice with cold PBS and resuspended in PBS at 5 × 105 cells/ml, and loaded with DCFHDA at a final concentration of 5 M and incubated for 40 min at 37 ◦ C in the dark. To test the protective effect of PL, cells were pre-treated with or without PL at 37 ◦ C for 1 h. The fluorescent intensity of the cell suspensions was detected using a fluorescence spectrophotometer (HITACHI 650-60, Tokyo, Japan). All our ROS experiments were corrected with the appropriate control sample containing only PL. Excitation and emission wavelengths were 485 and 550 nm, respectively. The results were expressed as fluorescent intensity per 1 × 106 cells. Mitochondrial membrane potential assay The uptake of the cationic fluorescent dye rhodamine 123 has been used for the estimation of mitochondrial membrane potential (Cao et al. 2007). After pre-treated with PL for 1 h, HepG2 cells were challenged with tacrine at determined concentrations for 0.5 h. Control and treated cells were harvested and washed twice with PBS, and the cell pellet was then resuspended in 2 ml of fresh incubation medium containing 1.0 M rhodamine 123 and incubated at 37 ◦ C in a thermostatic bath for 10 min with gentle shaking. Hepatocytes were then separated by centrifugation, and the amount of rhodamine 123 remaining in the incubation medium was measured using a fluorescence spectrophotometer set (Hitachi 650-60; Tokyo, Japan) at 490 nm excitation and 520 nm emission wavelengths. Results were expressed as the fluorescence retained within the cells. Immunocytochemistry staining for 8-OHdG An immunoperoxidase method using a monoclonal 8-OHdG antibody has been developed for detection and quantitation of oxidative damage in single cells. Using this method we detected 8OHdG in mtDNA in situ, eliminating the need for isolation of DNA. Exponentially growing cells were seeded onto coverslips in 12-well tissue culture plates at 1 × 105 cells and cultured for 24 h. Cells were pre-treated with PL for 1 h at 37 ◦ C and then treated with tacrine for 2 h. After treatment, cells were rinsed twice with PBS and fixed with cold acetone for 10 min. To avoid detecting 8-OHdG incorporated into RNA, the fixed cultures were treated with RNase (100 mg/ml) for 1 h at 37 ◦ C. DNA was denatured for 5 min at 4 ◦ C and treated with 0.1% Triton X-100 for 5 min at 4 ◦ C. To block nonspecific antibody binding sites, 10% normal horse serum was used, and the
Table 1 Monosaccharide Components of PL extracts. Monosaccharide
Content (%)
Glucose Mannose Galactose Arabinose Rhamnose
68.7 12.4 7.6 4.3 3.1
cells were incubated with the primary antibody (JaICA, Fukuroi, Japan) in PBS (1:200) at 4 ◦ C overnight. Subsequently, using Ultrasensitive Streptavidin-peroxidase Kit (Maixin-Bio, Fujian, China), the cells were rinsed and biotin-conjugated secondary antibody was added for 30 min at room temperature, rinsed with PBS three times and streptavidin-peroxidase for 10 min at room temperature. Diaminobenzidene was applied as color presentation (3–10 min). The cells were counterstained for nuclei with hematoxylin. The images were taken by microscope (Olympus BX-51, Omachi, Japan), and multiparameter image analysis software Image-pro plus 4.5.1 was used to quantify the staining intensity from 50 randomly selected cells per group per experiment. Staining data represent the average absorbance multiplied by 1000. Cell viability assay To determine the protective effect of PL in tacrine-induced cytotoxicity, Cell viability was assessed using the methylthiazol tetrazolium bromide (MTT) assay (Cao et al. 2008). HepG2 cells were plated in a 96-well microtiterplate at a density of 1 × 104 cells per well in a final volume of 100 l modified Eagle’s medium (MEM). After pretreatment with PL (0.0625 mg/ml) for 2 h, the medium was replaced with fresh medium containing tacrine (final concentration ranging from 9.4 to 75 M). After incubation for 24 h, the cells were incubated with MTT solution (5 mg/ml) for 2 h at 37 ◦ C. The formazan crystals formed were dissolved in DMSO at 37 ◦ C for 1 h in the dark, and the absorbance was read at 595 nm in a microplate reader (BIO-RAD Model 3550). Statistical analysis Results are expressed as means and SDs. Statistical analyses were performed with one way ANOVA. Differences were considered statistically significant when p < 0.05. Results The chemical analysis revealed that polysaccharides are the major components of PL extract, consisting of about 86.7% of the mixture, and 12% protein confirmed that it also contained proteinbound polysaccharide. As shown in Table 1, the monosaccharide components of the PL extract were glucose and mannose at 68.7% and 9.4%, respectively. The amino acid analysis showed that the
Table 2 Amino Acid Components of PL extracts. Amino acid
Content (%)
Amino acid
Content (%)
Asp Thr Ser Glu Pro Gly Ala Cys
8.6 6.4 2.7 4.8 6.5 9.4 9.2 3.1
Val Met Leu Tyr Phe His Lys Arg
11.0 0.9 7.4 4.3 5.0 4.9 10.4 4.1
C. Gao et al. / Phytomedicine 20 (2013) 705–709
Fig. 1. ROS formation measured by DCFH-DA in HepG2 cells incubated with or without tacrine for 1 h. Data are mean fluorescent intensity ± SD calculated from three independent experiments (*p < 0.05 vs. control).
protein portion predominantly consisted of Val (11.0%), Lys (10.4%), Gly (9.4%), and Ala (9.2%) (Table 2). Effects of PL on ROS formation induced by tacrine in HepG2 cells In this study, we employed DCFH-DA to measure intra-cellular generation of ROS. There was no increase in intracellular level of ROS after the 1-h incubation with 9.4 M tacrine (p > 0.05) compared to untreated. When the concentrations of tacrine were raised to 18.8 and 37.5 M, the level of ROS was significantly elevated (p < 0.05). This dose response demonstrated that tacrine had a strong effect on ROS production (Fig. 1). To investigate the protective effects of PL, we employed high concentration of tacrine to observe the short-term effects on ROS formation. As shown in (Fig. 2), when the cells were pretreated with PL, the level of ROS was significantly decreased compared to that of only tacrine-treated (p < 0.05) cells. This demonstrates that PL acts as a scavenger of the ROS generated by tacrine in HepG2 cells. Effects of PL on tacrine-induced m collapse Measurement of m is essential for an integrated appraisal of mitochondrial function. We examined the state of the m after application of tacrine by measuring the relative differences in fluorescence of the cationic dye rhodamine 123 between control
Fig. 2. Effects of PL on tacrine-induced ROS formation measured by DCFH-DA in HepG2 cells. Each bar represents mean ± SD of three independent experiments (*p < 0.05 vs vehicle control; # p < 0.05 vs tacrine alone).
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Fig. 3. Effects of tacrine on mitochondrial membrane potential. HepG2 cells were incubated with or without tacrine for 0.5 h and stained with rhodamine 123. Data are mean fluorescent intensity ± SD calculated from three independent experiments (*p < 0.05 vs. control).
and tacrine-treated HepG2 cells. After exposure to tacrine for 0.5 h, there was a dose-dependent decline in the m (Fig. 3), indicating that exposure to tacrine results in a depolarization and m collapse. When HepG2 cells were pretreated with PL 1 h before tacrine treatment, a statistically significant elevation (p < 0.01) in the m was observed compared with only tacrine treated cells (Fig. 4). These results suggest that PL could attenuate the depolarization of mitochondria induced by tacrine in HepG2 cells. Effects of PL on tacrine-induced 8-OHdG formation in mtDNA 8-OHdG is the most widely measured oxidative damage to mtDNA as well as nDNA. In the present study, we employed an immunocytochemical approach using a monoclonal antibody against 8-OHdG to detect the 8-OHdG in situ in the cytoplasm for mtDNA damage. Fig. 5 shows representative immunocytochemical staining for 8-OHdG in HepG2 cells with or without tacrine treatment. These results indicated that 8-OHdG staining was positive in the mtDNA at all tested tacrine concentrations. As tacrine concentration increased, the staining intensity of 8-OHdG increased, giving a 5-fold increase in 8-OHdG staining in mtDNA between 37.5 M and control. When the cells were pretreated with PL for 1 h, the staining for 8-OHdG was evident, but the staining intensity was significantly decreased compared to that of only tacrine-treated cells (p < 0.05) (Fig. 5). These data show that PL could significantly decrease tacrine-induced oxidative damage to mtDNA in HepG2 cells.
Fig. 4. Effects of PL on tacrine-induced depolarization of mitochondria in HepG2 cells. Each bar represents mean ± SD of three independent experiments (*p < 0.05 vs vehicle control; # p < 0.05 vs tacrine alone).
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Fig. 5. Effects of PL on tacrine-induced 8-OHdG formation in HepG2 cells. Immunohistochemistry staining of 8-OHdG in HepG2 cells following a 2-h incubation with (a) 0 M, (b) 9.4 M, (c) 18.8 M, (d) 37.5 M tacrine and (e) 0.0625 mg/ml PL, (f) 0.0625 mg/ml PL + 9.4 M tacrine, (g) 0.0625 mg/ml PL + 18.8 M tacrine, (h) 0.0625 mg/ml PL + 37.5 M tacrine in the cytoplasm (A). The staining intensity of 8-OHdG was calculated in the cytoplasm (B) at all groups. Each figure is a typical representation of the 50 cells observed in at least three areas per group in three independent experiments; magnification: 3400 (*p < 0.05 vs vehicle control; # p < 0.05 vs tacrine alone).
Effects of PL on tacrine-induced growth inhibition in HepG2 Cells The growth inhibition by tacrine in HepG2 cells was observed after incubation for 24 h, and the inhibition was concentration dependent (Fig. 6). After the 1-h pre-incubation with 0.0625 mg/ml PL, there was significant increase in cell viability (p < 0.05) compared to only tacrine-treated. These data suggest that PL is effective on protection from cytotoxicity induced by tacrine in HepG2 cells. Discussion Liver is the important target for the adverse effects of tacrine. Tacrine exhibits a well-established hepatotoxicity with liver cell necrosis and an oxidative stress which was evidenced by enhanced ROS production and GSH depletion (Ezoulin et al. 2006). By accumulating within mitochondria, tacrine inhibits mtDNA synthesis inducing severe mtDNA depletion, enhancing p53, Bax induction, mitochondrial permeability transition and hepatocyte necrosis and/or apoptosis in vivo (Mansouri et al. 2003). In the present study, the HepG2 cell line was employed to investigate the protective effects of PL against tacrine-induced hepatoxicity, because this cell line is originally derived from a human hepatoblastoma which retains many characteristics of hepatocytes such as the activities of phase I and phase II enzymes. It reflects the metabolism of xenobiotics in the human body better than other
Fig. 6. Effects of PL on tacrine-induced growth inhibition in HepG2 cells determined by MTT assay. Each bar is a mean ± SD of 3 independent experiments (*p < 0.05 vs tacrine alone).
metabolically incompetent cells (Mersch-Sundermann et al. 2004). Moreover, it is a relevant in vitro model to evaluate the possible mitochondrial toxicity of newly developed drugs in liver cells and to examine a drug’s interaction with liver functions (Sassa et al. 1987). Our results confirm that tacrine induces oxidative stress by increasing levels of ROS. ROS is mainly produced by the mitochondria and when the ROS levels exceed the capacity of the cell in general and the mitochondria in particular to scavenge, the resulting oxidative stress may initiate mitochondrial permeability transition, which then in turn potentiates the oxidative stress (Yin et al. 2005). In this study, loss of m was detected when HepG2 cells were exposed to tacrine for 0.5 h, just before ROS production increasing with tacrine-treatment for 1 h. This result suggested that tacrine-induced depolarization of mitochondria initiated ROS formation. The underling mechanism may be that the weak base tacrine is protonated in the acidic intermembrane space of mitochondria and is electrophoretically transported into the mitochondrial matrix to release a proton in the alkaline matrix and uncouple mitochondrial respiration. In the present study, we failed to detect an increase in lipid peroxidation in HepG2 cells treated with tacrine (results not shown). However, oxidative damage to mtDNA was detected after tacrine-treatment for 2 h. It is known that mtDNA is more prone to oxidative damage because it is in closer contact to the ROS produced in the mitochondria. Presently, as a marker of oxidative DNA damage, 8-OHdG was detected using immunocytochemistry staining in mtDNA. 8-OHdG, constituting 10% of the total base adducts, is just one out of many lesions produced by ROS in DNA. However, it is the most widely used method to detect mtDNA oxidative modification because 8OHdG is usually more abundant in oxidative damage in mtDNA than in nuclear DNA (Hayakawa et al. 1992). The data of our study showed that 8-OHdG increased significantly in the mtDNA treated with tacrine. It suggested that tacrine could induce oxidative damage to mtDNA. Some studies have reported that tacrine is concentrated in mitochondria and particularly mtDNA is an important target in tacrine toxicity. The damage to mtDNA is potentially more important than the damage to nuclear DNA (nDNA) because the genes coded by the mitochondrial genome are all expressed while nDNA contains a large amount of nontranscribed sequence (Liang and Godley 2003). At the same time, mtDNA, unlike nDNA, is continuously replicated. Also, the mitochondrial genome encodes polypeptides that participate in the electron transport chain and oxidative phosphorylation, so damage to mtDNA will cause electron transport chain dysfunction, generate oxidative stress, impair the mitochondrial function, and finally trigger the cell death pathways (Santos et al. 2003).
C. Gao et al. / Phytomedicine 20 (2013) 705–709
In this study, we did find the cytotoxicity of tacrine imposed on HepG2 cells. Mitochondrial depolarization may be a prerequisite for tacrine-induced cytotoxicity and the presence of mtDNA damage may be a probable mechanism for tacrine to induce the cytotoxicity of HepG2 cells. Mitochondrial dysfunction is a consequence of oxidative damage caused by increased oxidant levels. Therefore, decreasing oxidant generation and oxidative damage should be an effective way to inhibit mitochondrial impairment. In the present study, PL showed a significant protective effect against tacrine-induced mitochondrial dysfunction and toxicity in HepG2 cells. PL is one of the most well-known traditional medicinal mushrooms, which was widely used in Asian countries. The main medicinal components of PL are polysaccharides and several aromatic compounds such as phydroxybenzaldehyde, caffeic acid and hispidin (Santos et al. 2003). Some of these components may afford protection against ROS. This effect can be attributed, at least partially, to their abilities of inducing antioxidant enzyme activities and increasing the glutathione (GSH) level (Park et al. 2004; Park et al. 2001; Song et al. 2003). In our previous study, we found that PL could decrease ROS in HepG2 cells. In this study, PL prevented tacrine-induced cytotoxicity and mitochondrial dysfunction, which correlates with the reduction in tacrine-induced ROS generation. Also, PL could attenuate tacrine-induced oxidative damage to mtDNA. These results suggest that PL not only serve as free radical scavengers but also prevent mitochondrial dysfunction and subsequent mtDNA damage and cytotoxicity. In conclusion, our results demonstrate that tacrine-induced depolarization of mitochondria initiates ROS formation and the excessive production of ROS creates a situation of oxidative bursts and thus causes mtDNA damage, further mitochondrial dysfunction and cytotoxicity. Supplementation of a PL-polysaccharide/glycoprotein mixture may reduce tacrinemediated mitochondrial dysfunction and cytotoxicity due to its direct ROS scavenging activity, and consumption of PL may be a plausible way to prevent tacrine-mediated hepatoxicity. It would be of interest to evaluate whether our results obtained with PL in HepG2 cells would be predictive for the human situation. Conflict of interest statement The authors declare no competing financial interest. Acknowledgement This work was supported by the National Natural Science Foundation of China (81172718). References Boelsterli, U.A., Lim, P.L., 2007. Mitochondrial abnormalities – a link to idiosyncratic drug hepatotoxicity? Toxicology and Applied Pharmacology 220, 92–107. Cao, J., Jia, L., Zhou, H.M., Liu, Y., Zhong, L.F., 2006. Mitochondrial and nuclear DNA damage induced by curcumin in human hepatoma G2 cells. Toxicological Sciences: An Official Journal of the Society of Toxicology 91, 476–483. Cao, J., Liu, Y., Jia, L., Jiang, L.P., Geng, C.Y., Yao, X.F., Kong, Y., Jiang, B.N., Zhong, L.F., 2008. Curcumin attenuates acrylamide-induced cytotoxicity and genotoxicity in HepG2 cells by ROS scavenging. Journal of Agricultural and Food Chemistry 56, 12059–12063. Cao, J., Liu, Y., Jia, L., Zhou, H.M., Kong, Y., Yang, G., Jiang, L.P., Li, Q.J., Zhong, L.F., 2007. Curcumin induces apoptosis through mitochondrial hyperpolarization and mtDNA damage in human hepatoma G2 cells. Free Radical Biology and Medicine 43, 968–975.
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Contents lists available at SciVerse ScienceDirect
Phytomedicine journal homepage: www.elsevier.de/phymed
Phellinus linteus mushroom protects against tacrine-induced mitochondrial impairment and oxidative stress in HepG2 cells Chunpeng Gao a , Laifu Zhong b , Liping Jiang b , Chengyan Geng b , Xiaofeng Yao a , Jun Cao a,∗ a b
Occupational and Environmental Health Department, Dalian Medical University, No. 9, West Segment of South Lvshun Road, Dalian 116044, China China-Japanese Joint Institute for Medical and Pharmaceutical Science, Dalian Medical University, No. 9, West Segment of South Lvshun Road, Dalian 116044, China
a r t i c l e
i n f o
Keywords: Phellinus linteus mushroom Polysaccharides/glycoproteins Mitochondrial impairment Oxidative stress mtDNA damage
a b s t r a c t Tacrine (THA) was the first drug licensed for the treatment of Alzheimer’s disease. Unfortunately, reversible hepatotoxicity is evident in about 30% of patients and limits its clinical use. The intracellular mechanisms have not yet been elucidated. Phellinus linteus (PL) is a mushroom that has long been used as a folk medicine. In our previous study, we found that PL could decrease the reactive oxygen species (ROS) formation in HepG2 cells. Presently, protective effects of PL on tacrine-induced ROS formation and mitochondria dysfunction were evaluated. The results showed that PL significantly reduced tacrineinduced ROS production, disruption of m, 8-OHdG formation in mitochondrial DNA, and cytotoxicity in HepG2 cells. These data suggest that PL could attenuate the cytotoxicity and mitochondria dysfunction induced by tacrine in HepG2 cells. The protection is probably mediated by an antioxidant protective mechanism. Consumption of PL may be a plausible way to prevent tacrine-induced hepatotoxicity. © 2013 Elsevier GmbH. All rights reserved.
Introduction The liver is one of the first organs to be exposed to orally delivered compounds and is also the major site for xenobiotic metabolism, which can lead to the formation of toxic metabolites. Hepatotoxicity is one of the most common adverse drug reactions, as are more than half of marketed drugs with black box warnings (Hunt 2010). Tacrine, or 9-amino1,2,3,4-tetrahydroaminoacridine, is a competitive inhibitor of cholinesterase. It was the first drug licensed by the US Food and Drug Administration for the treatment of Alzheimer’s disease (Johnson et al. 2004). Unfortunately, reversible hepatotoxicity is evident by elevated alanine aminotransferase (ALT) levels in about 30% of patients and limits its clinical use. However, the intracellular mechanisms have not yet been elucidated. Many studies have shown an association with mitochondrial dysfunction (Robertson et al. 1998) and glutathione depletion in hepatocytes and liver cell necrosis (Watkins et al. 1994). Mehta et al. have shown that tacrine accumulated in mitochondria and increase the risk of mitochondrial toxicity (Mehta et al. 2008).
Mitochondria are the key cellular energy source, supplying more than 90% of cellular ATP (Modica-Napolitano and Singh 2004). Therefore, drug-induced mitochondrial impairment directly affects hepatocyte viability (Boelsterli and Lim 2007). Most drugs withdrawn from the market are mitochondrial toxicants. Mushrooms have attracted great interest due to their nutritional, medical and pharmacological properties. Phellinus linteus (PL) is a Basidomycota fungus which is rich in polysaccharides and aromatic compounds. It has been used in traditional oriental medicine for over 2000 years to treat various diseases, such as tumors, inflammation and lymphatic diseases (Han et al. 1999; Yang and Jong 1989). A large body of research about PL indicates that it possesses antitumor (Kim et al. 2004), immuno-modulating (Lim et al. 2004), and anti-oxidant activity etc. (Park et al. 2004). Moreover, hepatoprotective effects have been reported by Jeon et al. (Jeon et al. 2003). In our previous study, we found that PL decrease reactive oxygen species (ROS) in HepG2 cells. In this study, we investigated the protective effects of PL on tacrine-induced oxidative stress and mitochondrial impairment in HepG2 cells. Materials and methods
Abbreviations: ALT, alanine aminotransferase; DCFH-DA, 2,7dichlorofluorescein diacetate; GSH, glutathione; mtDNA, mitochondrial DNA; MTT, methylthiazol tetrazolium bromide; nDNA, nuclear DNA; PL, Phellinus linteus; ROS, reactive oxygen species. ∗ Corresponding author. Tel.: +86 411 86110330. E-mail addresses: [email protected], [email protected] (J. Cao). 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.02.014
Cell culture and reagents HepG2 cells (American Type Culture CollectionHB-8065) were obtained from Peking Union Medical College (Peking, China) and cultured in minimum essential Eagle’s medium containing 10% fetal
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bovine serum (Gibco, Grand Island, NY), penicillin (100 units/ml, Gibco), and strepto-mycin (100 mg/ml, Gibco). Tacrine was purchased from Sigma. PL was from Hun’chun lvdao medicinal Co. Ltd. (Hn’chun,China). The water extract was prepared as follows. The crushed dry mushroom (1.00 kg) was boiled for 4 h at 100 ◦ C, cooled to room temperature, and then filtered followed by ethanol precipitation. The extract was evaporated to dryness with a rotary vacuum evaporator and freeze-dried to give a powder (250 g). The polysaccharide concentration and protein contents were determined, and the monosaccharides and amino acids compositions were analyzed as described by Kim et al. (Kim et al. 2006). The stock solution of PL extract was prepared by dissolving the powder in distilled water (50 mg/ml). For each experiment, PL was diluted with cell culture medium to the concentrations indicated (0.0625, 0.125, and 0.25 mg/ml) freshly before the experiment. ROS assay The production of ROS was measured using the 2,7dichlorofluorescein diacetate (DCFH-DA) method (Cao et al. 2006). HepG2 cells (5 × 105 ) were suspended in 2 ml medium and were incubated with tacrine at different concentrations (0, 9.4, 18.8, 37.5 M) for 1 h at 37 ◦ C. Cells were washed twice with cold PBS and resuspended in PBS at 5 × 105 cells/ml, and loaded with DCFHDA at a final concentration of 5 M and incubated for 40 min at 37 ◦ C in the dark. To test the protective effect of PL, cells were pre-treated with or without PL at 37 ◦ C for 1 h. The fluorescent intensity of the cell suspensions was detected using a fluorescence spectrophotometer (HITACHI 650-60, Tokyo, Japan). All our ROS experiments were corrected with the appropriate control sample containing only PL. Excitation and emission wavelengths were 485 and 550 nm, respectively. The results were expressed as fluorescent intensity per 1 × 106 cells. Mitochondrial membrane potential assay The uptake of the cationic fluorescent dye rhodamine 123 has been used for the estimation of mitochondrial membrane potential (Cao et al. 2007). After pre-treated with PL for 1 h, HepG2 cells were challenged with tacrine at determined concentrations for 0.5 h. Control and treated cells were harvested and washed twice with PBS, and the cell pellet was then resuspended in 2 ml of fresh incubation medium containing 1.0 M rhodamine 123 and incubated at 37 ◦ C in a thermostatic bath for 10 min with gentle shaking. Hepatocytes were then separated by centrifugation, and the amount of rhodamine 123 remaining in the incubation medium was measured using a fluorescence spectrophotometer set (Hitachi 650-60; Tokyo, Japan) at 490 nm excitation and 520 nm emission wavelengths. Results were expressed as the fluorescence retained within the cells. Immunocytochemistry staining for 8-OHdG An immunoperoxidase method using a monoclonal 8-OHdG antibody has been developed for detection and quantitation of oxidative damage in single cells. Using this method we detected 8OHdG in mtDNA in situ, eliminating the need for isolation of DNA. Exponentially growing cells were seeded onto coverslips in 12-well tissue culture plates at 1 × 105 cells and cultured for 24 h. Cells were pre-treated with PL for 1 h at 37 ◦ C and then treated with tacrine for 2 h. After treatment, cells were rinsed twice with PBS and fixed with cold acetone for 10 min. To avoid detecting 8-OHdG incorporated into RNA, the fixed cultures were treated with RNase (100 mg/ml) for 1 h at 37 ◦ C. DNA was denatured for 5 min at 4 ◦ C and treated with 0.1% Triton X-100 for 5 min at 4 ◦ C. To block nonspecific antibody binding sites, 10% normal horse serum was used, and the
Table 1 Monosaccharide Components of PL extracts. Monosaccharide
Content (%)
Glucose Mannose Galactose Arabinose Rhamnose
68.7 12.4 7.6 4.3 3.1
cells were incubated with the primary antibody (JaICA, Fukuroi, Japan) in PBS (1:200) at 4 ◦ C overnight. Subsequently, using Ultrasensitive Streptavidin-peroxidase Kit (Maixin-Bio, Fujian, China), the cells were rinsed and biotin-conjugated secondary antibody was added for 30 min at room temperature, rinsed with PBS three times and streptavidin-peroxidase for 10 min at room temperature. Diaminobenzidene was applied as color presentation (3–10 min). The cells were counterstained for nuclei with hematoxylin. The images were taken by microscope (Olympus BX-51, Omachi, Japan), and multiparameter image analysis software Image-pro plus 4.5.1 was used to quantify the staining intensity from 50 randomly selected cells per group per experiment. Staining data represent the average absorbance multiplied by 1000. Cell viability assay To determine the protective effect of PL in tacrine-induced cytotoxicity, Cell viability was assessed using the methylthiazol tetrazolium bromide (MTT) assay (Cao et al. 2008). HepG2 cells were plated in a 96-well microtiterplate at a density of 1 × 104 cells per well in a final volume of 100 l modified Eagle’s medium (MEM). After pretreatment with PL (0.0625 mg/ml) for 2 h, the medium was replaced with fresh medium containing tacrine (final concentration ranging from 9.4 to 75 M). After incubation for 24 h, the cells were incubated with MTT solution (5 mg/ml) for 2 h at 37 ◦ C. The formazan crystals formed were dissolved in DMSO at 37 ◦ C for 1 h in the dark, and the absorbance was read at 595 nm in a microplate reader (BIO-RAD Model 3550). Statistical analysis Results are expressed as means and SDs. Statistical analyses were performed with one way ANOVA. Differences were considered statistically significant when p < 0.05. Results The chemical analysis revealed that polysaccharides are the major components of PL extract, consisting of about 86.7% of the mixture, and 12% protein confirmed that it also contained proteinbound polysaccharide. As shown in Table 1, the monosaccharide components of the PL extract were glucose and mannose at 68.7% and 9.4%, respectively. The amino acid analysis showed that the
Table 2 Amino Acid Components of PL extracts. Amino acid
Content (%)
Amino acid
Content (%)
Asp Thr Ser Glu Pro Gly Ala Cys
8.6 6.4 2.7 4.8 6.5 9.4 9.2 3.1
Val Met Leu Tyr Phe His Lys Arg
11.0 0.9 7.4 4.3 5.0 4.9 10.4 4.1
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Fig. 1. ROS formation measured by DCFH-DA in HepG2 cells incubated with or without tacrine for 1 h. Data are mean fluorescent intensity ± SD calculated from three independent experiments (*p < 0.05 vs. control).
protein portion predominantly consisted of Val (11.0%), Lys (10.4%), Gly (9.4%), and Ala (9.2%) (Table 2). Effects of PL on ROS formation induced by tacrine in HepG2 cells In this study, we employed DCFH-DA to measure intra-cellular generation of ROS. There was no increase in intracellular level of ROS after the 1-h incubation with 9.4 M tacrine (p > 0.05) compared to untreated. When the concentrations of tacrine were raised to 18.8 and 37.5 M, the level of ROS was significantly elevated (p < 0.05). This dose response demonstrated that tacrine had a strong effect on ROS production (Fig. 1). To investigate the protective effects of PL, we employed high concentration of tacrine to observe the short-term effects on ROS formation. As shown in (Fig. 2), when the cells were pretreated with PL, the level of ROS was significantly decreased compared to that of only tacrine-treated (p < 0.05) cells. This demonstrates that PL acts as a scavenger of the ROS generated by tacrine in HepG2 cells. Effects of PL on tacrine-induced m collapse Measurement of m is essential for an integrated appraisal of mitochondrial function. We examined the state of the m after application of tacrine by measuring the relative differences in fluorescence of the cationic dye rhodamine 123 between control
Fig. 2. Effects of PL on tacrine-induced ROS formation measured by DCFH-DA in HepG2 cells. Each bar represents mean ± SD of three independent experiments (*p < 0.05 vs vehicle control; # p < 0.05 vs tacrine alone).
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Fig. 3. Effects of tacrine on mitochondrial membrane potential. HepG2 cells were incubated with or without tacrine for 0.5 h and stained with rhodamine 123. Data are mean fluorescent intensity ± SD calculated from three independent experiments (*p < 0.05 vs. control).
and tacrine-treated HepG2 cells. After exposure to tacrine for 0.5 h, there was a dose-dependent decline in the m (Fig. 3), indicating that exposure to tacrine results in a depolarization and m collapse. When HepG2 cells were pretreated with PL 1 h before tacrine treatment, a statistically significant elevation (p < 0.01) in the m was observed compared with only tacrine treated cells (Fig. 4). These results suggest that PL could attenuate the depolarization of mitochondria induced by tacrine in HepG2 cells. Effects of PL on tacrine-induced 8-OHdG formation in mtDNA 8-OHdG is the most widely measured oxidative damage to mtDNA as well as nDNA. In the present study, we employed an immunocytochemical approach using a monoclonal antibody against 8-OHdG to detect the 8-OHdG in situ in the cytoplasm for mtDNA damage. Fig. 5 shows representative immunocytochemical staining for 8-OHdG in HepG2 cells with or without tacrine treatment. These results indicated that 8-OHdG staining was positive in the mtDNA at all tested tacrine concentrations. As tacrine concentration increased, the staining intensity of 8-OHdG increased, giving a 5-fold increase in 8-OHdG staining in mtDNA between 37.5 M and control. When the cells were pretreated with PL for 1 h, the staining for 8-OHdG was evident, but the staining intensity was significantly decreased compared to that of only tacrine-treated cells (p < 0.05) (Fig. 5). These data show that PL could significantly decrease tacrine-induced oxidative damage to mtDNA in HepG2 cells.
Fig. 4. Effects of PL on tacrine-induced depolarization of mitochondria in HepG2 cells. Each bar represents mean ± SD of three independent experiments (*p < 0.05 vs vehicle control; # p < 0.05 vs tacrine alone).
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Fig. 5. Effects of PL on tacrine-induced 8-OHdG formation in HepG2 cells. Immunohistochemistry staining of 8-OHdG in HepG2 cells following a 2-h incubation with (a) 0 M, (b) 9.4 M, (c) 18.8 M, (d) 37.5 M tacrine and (e) 0.0625 mg/ml PL, (f) 0.0625 mg/ml PL + 9.4 M tacrine, (g) 0.0625 mg/ml PL + 18.8 M tacrine, (h) 0.0625 mg/ml PL + 37.5 M tacrine in the cytoplasm (A). The staining intensity of 8-OHdG was calculated in the cytoplasm (B) at all groups. Each figure is a typical representation of the 50 cells observed in at least three areas per group in three independent experiments; magnification: 3400 (*p < 0.05 vs vehicle control; # p < 0.05 vs tacrine alone).
Effects of PL on tacrine-induced growth inhibition in HepG2 Cells The growth inhibition by tacrine in HepG2 cells was observed after incubation for 24 h, and the inhibition was concentration dependent (Fig. 6). After the 1-h pre-incubation with 0.0625 mg/ml PL, there was significant increase in cell viability (p < 0.05) compared to only tacrine-treated. These data suggest that PL is effective on protection from cytotoxicity induced by tacrine in HepG2 cells. Discussion Liver is the important target for the adverse effects of tacrine. Tacrine exhibits a well-established hepatotoxicity with liver cell necrosis and an oxidative stress which was evidenced by enhanced ROS production and GSH depletion (Ezoulin et al. 2006). By accumulating within mitochondria, tacrine inhibits mtDNA synthesis inducing severe mtDNA depletion, enhancing p53, Bax induction, mitochondrial permeability transition and hepatocyte necrosis and/or apoptosis in vivo (Mansouri et al. 2003). In the present study, the HepG2 cell line was employed to investigate the protective effects of PL against tacrine-induced hepatoxicity, because this cell line is originally derived from a human hepatoblastoma which retains many characteristics of hepatocytes such as the activities of phase I and phase II enzymes. It reflects the metabolism of xenobiotics in the human body better than other
Fig. 6. Effects of PL on tacrine-induced growth inhibition in HepG2 cells determined by MTT assay. Each bar is a mean ± SD of 3 independent experiments (*p < 0.05 vs tacrine alone).
metabolically incompetent cells (Mersch-Sundermann et al. 2004). Moreover, it is a relevant in vitro model to evaluate the possible mitochondrial toxicity of newly developed drugs in liver cells and to examine a drug’s interaction with liver functions (Sassa et al. 1987). Our results confirm that tacrine induces oxidative stress by increasing levels of ROS. ROS is mainly produced by the mitochondria and when the ROS levels exceed the capacity of the cell in general and the mitochondria in particular to scavenge, the resulting oxidative stress may initiate mitochondrial permeability transition, which then in turn potentiates the oxidative stress (Yin et al. 2005). In this study, loss of m was detected when HepG2 cells were exposed to tacrine for 0.5 h, just before ROS production increasing with tacrine-treatment for 1 h. This result suggested that tacrine-induced depolarization of mitochondria initiated ROS formation. The underling mechanism may be that the weak base tacrine is protonated in the acidic intermembrane space of mitochondria and is electrophoretically transported into the mitochondrial matrix to release a proton in the alkaline matrix and uncouple mitochondrial respiration. In the present study, we failed to detect an increase in lipid peroxidation in HepG2 cells treated with tacrine (results not shown). However, oxidative damage to mtDNA was detected after tacrine-treatment for 2 h. It is known that mtDNA is more prone to oxidative damage because it is in closer contact to the ROS produced in the mitochondria. Presently, as a marker of oxidative DNA damage, 8-OHdG was detected using immunocytochemistry staining in mtDNA. 8-OHdG, constituting 10% of the total base adducts, is just one out of many lesions produced by ROS in DNA. However, it is the most widely used method to detect mtDNA oxidative modification because 8OHdG is usually more abundant in oxidative damage in mtDNA than in nuclear DNA (Hayakawa et al. 1992). The data of our study showed that 8-OHdG increased significantly in the mtDNA treated with tacrine. It suggested that tacrine could induce oxidative damage to mtDNA. Some studies have reported that tacrine is concentrated in mitochondria and particularly mtDNA is an important target in tacrine toxicity. The damage to mtDNA is potentially more important than the damage to nuclear DNA (nDNA) because the genes coded by the mitochondrial genome are all expressed while nDNA contains a large amount of nontranscribed sequence (Liang and Godley 2003). At the same time, mtDNA, unlike nDNA, is continuously replicated. Also, the mitochondrial genome encodes polypeptides that participate in the electron transport chain and oxidative phosphorylation, so damage to mtDNA will cause electron transport chain dysfunction, generate oxidative stress, impair the mitochondrial function, and finally trigger the cell death pathways (Santos et al. 2003).
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In this study, we did find the cytotoxicity of tacrine imposed on HepG2 cells. Mitochondrial depolarization may be a prerequisite for tacrine-induced cytotoxicity and the presence of mtDNA damage may be a probable mechanism for tacrine to induce the cytotoxicity of HepG2 cells. Mitochondrial dysfunction is a consequence of oxidative damage caused by increased oxidant levels. Therefore, decreasing oxidant generation and oxidative damage should be an effective way to inhibit mitochondrial impairment. In the present study, PL showed a significant protective effect against tacrine-induced mitochondrial dysfunction and toxicity in HepG2 cells. PL is one of the most well-known traditional medicinal mushrooms, which was widely used in Asian countries. The main medicinal components of PL are polysaccharides and several aromatic compounds such as phydroxybenzaldehyde, caffeic acid and hispidin (Santos et al. 2003). Some of these components may afford protection against ROS. This effect can be attributed, at least partially, to their abilities of inducing antioxidant enzyme activities and increasing the glutathione (GSH) level (Park et al. 2004; Park et al. 2001; Song et al. 2003). In our previous study, we found that PL could decrease ROS in HepG2 cells. In this study, PL prevented tacrine-induced cytotoxicity and mitochondrial dysfunction, which correlates with the reduction in tacrine-induced ROS generation. Also, PL could attenuate tacrine-induced oxidative damage to mtDNA. These results suggest that PL not only serve as free radical scavengers but also prevent mitochondrial dysfunction and subsequent mtDNA damage and cytotoxicity. In conclusion, our results demonstrate that tacrine-induced depolarization of mitochondria initiates ROS formation and the excessive production of ROS creates a situation of oxidative bursts and thus causes mtDNA damage, further mitochondrial dysfunction and cytotoxicity. Supplementation of a PL-polysaccharide/glycoprotein mixture may reduce tacrinemediated mitochondrial dysfunction and cytotoxicity due to its direct ROS scavenging activity, and consumption of PL may be a plausible way to prevent tacrine-mediated hepatoxicity. It would be of interest to evaluate whether our results obtained with PL in HepG2 cells would be predictive for the human situation. Conflict of interest statement The authors declare no competing financial interest. Acknowledgement This work was supported by the National Natural Science Foundation of China (81172718). References Boelsterli, U.A., Lim, P.L., 2007. Mitochondrial abnormalities – a link to idiosyncratic drug hepatotoxicity? Toxicology and Applied Pharmacology 220, 92–107. Cao, J., Jia, L., Zhou, H.M., Liu, Y., Zhong, L.F., 2006. Mitochondrial and nuclear DNA damage induced by curcumin in human hepatoma G2 cells. Toxicological Sciences: An Official Journal of the Society of Toxicology 91, 476–483. Cao, J., Liu, Y., Jia, L., Jiang, L.P., Geng, C.Y., Yao, X.F., Kong, Y., Jiang, B.N., Zhong, L.F., 2008. Curcumin attenuates acrylamide-induced cytotoxicity and genotoxicity in HepG2 cells by ROS scavenging. Journal of Agricultural and Food Chemistry 56, 12059–12063. Cao, J., Liu, Y., Jia, L., Zhou, H.M., Kong, Y., Yang, G., Jiang, L.P., Li, Q.J., Zhong, L.F., 2007. Curcumin induces apoptosis through mitochondrial hyperpolarization and mtDNA damage in human hepatoma G2 cells. Free Radical Biology and Medicine 43, 968–975.
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