FASEB J 24(12): 4722-4732

PMC: PMC2992378

PMID: 20696856

Flavokawain B, the hepatotoxic constituent from kava root, induces GSH-sensitive oxidative stress through modulation of IKK/NF-κB and MAPK signaling pathways

MATERIALS AND METHODS

Compounds

Six kavalactones [kawain (K), dihydrokawain (DHK), methysticin (M), dihydromethysticin (DHM), yangonin (Y), and desmethoxyyangonin (DMY)] and 3 chalcones [flavokawain A (FKA), flavokawain B ((E)-2′-hydroxy-4′,6′-dimethoxychalcone; CAS no. 1775-97-9 (FKB), and flavokawain C (FKC)] were isolated and purified from an ethanolic extract from roots of Piper methysticum (kava). Briefly, a 95% EtOH extract of kava roots (150 g) (obtained from PureWorld; Naturex, South Hackensack, NJ, USA) was subjected to silica gel column chromatography (CC; 800 g, 63–200 mM) by elution with CH₂Cl₂ to obtain 3 fractions, followed by elution with MeOH to afford fraction F4. F1 (11.0 g) was subjected to CC (SiO₂, 130 g) by elution with n-hexane/acetone with increasing polarity to afford 9 further fractions. Flavokawain A (510 mg), flavokawain B (500 mg), and flavokawain C (25 mg) were obtained from Fr1–3 and Fr1–6 after crystallization. F4 (5 g) was subjected to CC (SiO₂, 80 g) by elution with n-hexane/acetone to afford 6 further fractions followed by reverse-phase C₁₈ MPLC [MeOH/H₂O (50:50), 1.7 ml/min] resulting in the separation of dihydromethysticin (60 mg) and yangonin (30 mg), methysticin (66 mg), kawain (38 mg), dihydrokawain (23 mg), and desmethoxyyangonin (12 mg). The purity of all tested compounds was shown to be >98% by HPLC. Their structures were confirmed by comparison of their mass and NMR spectral data with those in the literature (29). Spectral data of FKB were the following: LC/MS (ESI): m/z 285 [M+H]^{[MF : C}₁₇H₁₆O₄ (284)]; ^{H NMR (300 MHz, CD}₃OD): 3.84 (s, 3H), 3.94 (s, 3H), 6.10 (s, 2H), 7.41 (bs, 3H), 7.63 (m, 2H), 7.71 (d, 1H), 7.91 (d, 1H); ^{C NMR (300 MHz, CD}₃OD) δ: 56.3, 56.6, 92.3, 95.1, 128.9, 129.5, 130.2, 131.4, 137.0, 143.4, 164.3, 168.2, 168.9, 194.4. Glutathione was obtained from Sigma-Aldrich (St. Louis, MO, USA). Tumor necrosis factor α (TNF-α) was from R&D Systems (Minneapolis, MN, USA). The mitogen-activated protein/ERK kinase1 (MEK1) inhibitor (U0126), p38 MAPK inhibitor (SB202190), JNK inhibitor (SP600125), and IKK inhibitor (Bay11-7085) were purchased from Calbiochem (La Jolla, CA, USA).

Cell culture

The human hepatocyte line L-02 was obtained from the Tissue Culture Collection of the Chinese Academy of Sciences (TCCCAS; Beijing, China). HepG2 human hepatoma cells and HeLa cervical carcinoma cells were obtained from the American Tissue Culture Collection (ATCC; Manassas, VA, USA). HeLa and HepG2 cells were cultured in DMEM supplemented with 10% FBS, glutamine (2 mM), and penicillin/streptomycin (100 U/ml each). L-02 cells were maintained in a RPMI 1640 medium containing similar supplements as above. All medium components were from Invitrogen (Carlsbad, CA, USA). All cell cultures were grown at 37°C in a humidified atmosphere of 5% CO₂.

MTT viability assay

L-02 or HepG2 cells were plated in 96-well tissue culture plates at 1 × 10^{or 5 × 10}^{cells/well, respectively. Twenty-four hours later, the cells were exposed to test compounds at the indicated concentrations for 48 h or treated with vehicle only (DMSO). MTT assay was carried out in quadruplicates as described previously (}30) using MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] (Sigma-Aldrich). All experiments were repeated independently 2–3 times. Data are presented as means ± se.

Scanning electron microscope (SEM) analysis

L-02 cells were cultured on glass coverslips for 24 h at 37°C. Cells were exposed to the indicated concentrations of FKB for 6, 12, or 24 h. Following washing with cold PBS, cells were fixed (2.5% gluteraldehyde), dehydrated (ethanol), dried, and coated with a 20-nm gold layer according to standard procedures. Samples were examined by SEM (AKASHI SX-40, Seisakushu, Japan) at ×3000.

Western blot analysis

HepG2 cells were grown in full medium for 3 d and subsequently in serum-free DMEM for 24 h. Cells were than treated with FKB (30 μM) or vehicle only (DMSO) for 3 h and then stimulated with TNF-α (20 ng/ml) for the indicated periods. Western blot analysis was performed by using 30–40 μg total proteins as described previously (31). Antibodies for p38, phospho-p38, ERK, phospho-ERK, JNK, phospho-JNK, p-IκBα, and activated caspase 3 were from Cell Signaling Technology (Danvers, MA, USA). Antibodies for IκBα and p65 were from Santa Cruz Biotechnology, (Santa Cruz, CA, USA). Anti-β-actin antibody to validate equal loading was from Sigma-Aldrich.

RT-PCR

Total RNA was isolated using RNeasy kits (Qiagen, Valencia, CA, USA). First strand cDNA was generated from 1 μg total RNA by using SuperScript^{First-Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer's instructions. One fifth of the cDNA product, 45 μl of PCR SuperMix (Invitrogen), and 0.5 μM primers for tested genes were amplified in a PCR Express Thermocycler (Thermo Scientific, Waltham, MA, USA). cDNA was denatured at 94°C for 4 min and subsequently submitted to various amplification cycles composed of 94°C for 40 s, 60°C for 45 s, and 72°C for 60 s. The following primers were used: IκBα: 5′-GCCTGGACTCCATGAAGGAC-3′ and 5′-CAAGTGGAGTGGAGTCTGCTGCAGGTTGTT-3′; GAPDH: 5′-ACTTTGTCAAGCTCATTTCC-3′ and 5′-TGCAGCGAACTTTATTGATG-3′.}

Determination of cellular GSH and GSSH

L-02 cells (7×10^{) were grown for 24 h and then incubated for another 36 h in the presence of FKB (44 μM) or vehicle only (DMSO). Cells were harvested, washed twice with ice-cold PBS, and suspended in double distilled water adjusted to pH 3.0 with HCl. Following several freeze-thaw cycles, cell lysates were centrifuged at 1000}g for 10 min at 4°C, and supernatants were filtered through a Microcon-3 membrane (Millipore, Billerica, MA, USA). Uridine (10 mM) were added to the samples and directly analyzed by high-performance capillary electrophoresis (HPCE) using a Beckman MDQ system (Beckman-Coulter, Fullerton, CA, USA) equipped with a diode array. Samples were injected under 0.05 PSI pressure for 5 s using buffer containing sodium borate (100 mM) and tricine (10 mM). The separation conditions (20 kV, normal polarity) were held at a constant voltage for 8 min. Separations were carried out with the detector set at 200 nm. GSH and GSSG for standards were obtained from Shanghai Shengxing Biochemical Technology Company (Shanghai, China).

Transfection of reporters and bioluminescent imaging

HeLa⁽32), HeLa⁽33, 34), parental HeLa cervical carcinoma, or HepG2 hepatoma cells were used for bioluminescence imaging. HeLa and HepG2 cells were transiently transfected with pκB₅→FLuc (Stratagene, La Jolla, CA, USA) or pCMV→FLuc (32). Forty-eight hours later, cells were washed and incubated with increasing concentrations of FKB (0–100 μM) for 3 h and subsequently challenged with TNF-α (20 ng/ml) or vehicle only (PBS) in the presence of D-luciferin (150 μg/ml; Biosynth, Naperville, IL, USA) and sequentially imaged for bioluminescence activity for 3 h using an IVIS-100 imaging system (Xenogen, Alameda, CA, USA; exposure, 60 s; binning, 4; FOV, 25 cm; image-image interval, 5 min). Total photon fluxes were normalized for cell viability and transfection efficiency by adjusting total photon counts with respect to pCMV→FLuc-expressing cells treated in a similar manner. Data are presented as fold-initial, fold-untreated, fold-CMV→FLuc control ± sem. For assessing cell viability, HeLa cells stably expressing FLuc were incubated with FKB (0–100 μM) and d-luciferin (150 μg/ml) and imaged for bioluminescence activity for 2 h at 5 min intervals. To analyze inhibition of total proteasomal activity, HeLa cells stably expressing tetraubiquitin-FLuc (Ub-FL) or FLuc were incubated with FKB (0–50 μM) or the proteasome inhibitor bortezomib (1 μM, positive control) and imaged for bioluminescence activity for 2 h at 5 min intervals. Data are presented as Ub-FLuc/FLuc (fold-untreated, fold-initial).

Real-time imaging of NF-κB activation in living mice

All animal experimentation was approved by the Animal Studies Committee of the Washington University School of Medicine. Six-week-old, male Balb/C mice were injected intravenously with 6 μg of κB₅→FLuc construct diluted in PBS (1 ml/10 g body weight) to induce stable expression of this NF-κB transcriptional reporter in liver hepatocytes in vivo (32). Three weeks later, mice were orally treated with FKB (25 mg/kg body weight) or vehicle (methyl cellulose, 0.5%, p.o.) daily for a week. Acute hepatic activation of NF-κB was induced by challenging the mice with LPS (4 mg/kg, i.p.). Bioluminescence imaging was carried out before FKB treatment as well as before and 3 h after LPS treatment using the IVIS-100 imaging system (exposure, 300 s; binning, 4; FOV, 25 cm; stop, 1/f; open filter). For each experimental group, n = 4.

Assessment of FKB-induced hepatotoxicity in vivo

Male ICR mice (18–22 g) were treated orally with FKB (25 mg/kg body weight) or vehicle (0.5% carboxymethyl cellulose) daily for 7 d. The left lobe of the liver was removed and immediately fixed in formaldehyde (4%, pH 7.0), embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin.

Compounds

Cell culture

MTT viability assay

Scanning electron microscope (SEM) analysis

Western blot analysis

RT-PCR

Determination of cellular GSH and GSSH

Transfection of reporters and bioluminescent imaging

Real-time imaging of NF-κB activation in living mice

Assessment of FKB-induced hepatotoxicity in vivo

RESULTS

Hepatocellular toxicity induced by chalocones

The major constituents of ethanolic kava root extract are kavalactones, including kawain, dihydrokawain, methysticin, dihydromethysticin, yangonin, and desmethoxyyangonin. Kava root extracts also contain chalcones, including flavokawain A, flavokawain B, and flavokawain C. We initially screened all 6 major kavalactones and 3 chalcones for cytotoxicity toward HepG2 hepatoma cells using MTT assays. None of the kavalactones, except yangonin, exhibited toxicity at concentrations up to 150 μM (Fig. 1A). Yagonin is a weak toxin with apparent LD₅₀ of 100 μM (Fig. 1A). Surprisingly, all 3 chalcones induced significant cell death in HepG2 cells at concentrations ranging from 10 to 50 μM (Fig. 1A). FKB is the most potent cytotoxin, exhibiting an apparent LD₅₀ value of 15.3 ± 0.2 μM. To confirm these findings, we next tested all 9 compounds against an immortalized nontumor origin human liver cell line, L-02. Again, FKB and FKC induced cell death in L-02 cells, with LD₅₀ values of 32 and 70 μM, respectively (data not shown). Interestingly, all other compounds tested, including FKA and yangonin, failed to induce cell death in L-02 cells (data not shown). FKB (Fig. 1B) was therefore chosen for further investigation, not only because it was a more potent cytotoxin in liver cells as compared to FKC, but also because FKB was >20-fold more abundant than FKC in acetone or ethanol extracts of kava (Table 1).

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Figure 1.

FKB is a potent hepatotoxin. A) Concentration-dependent cytotoxicity profiles of various kavalactones (dotted lines) and chalcones (solid lines) in HepG2 cells (at 48 h). Cell viability was assayed by MTT. B) Structure of FKB. C) Scanning electron micrographs of L-02 hepatocytes treated with FKB (44 μM) for 0, 6, 12, or 24 h. D) HepG2 cells were treated with FKA, FKB, or FKC (30 μM, 24 h). Cell lysates were immunoblotted with antibodies as indicated.

Table 1.

Contents of chalcones (FKB, FKC) and total kavalactones in kava root extracts (mg/g dried weight) by different extraction solvents

Extract solvent	FKB (mg/g)	FKC (mg/g)	Total kava lactones (mg/g)
Water	0.2	0	46.6
60% acetone	26.0	1.1	474.8
Acetone	33.7	1.5	570.0
95% ethanol	32.3	1.4	548.8

We next assessed the morphology of L-02 cells exposed to FKB at 44 μM for 24 h by SEM. FKB-treated cells showed loss of microvilli, cell rounding, and blebbing, suggesting that these cells were undergoing apoptosis (Fig. 1C). Since caspase 3 is cleaved and activated during apoptosis, we next investigated whether caspase 3 was activated on treatment of liver cells with FKB. FKB at 30 μM was shown to activate caspase 3 in HepG2 cells, whereas FKA and FKC at the same concentration failed to do so. Based on morphological changes (Fig. 1C) and caspase 3 cleavage (Fig. 1D) in response to treatment with FKB, we therefore suggest that this compound induced apoptotic cell death in cultured hepatocytes.

FKB blocks TNF-α-induced activation of NF-κB

NF-κB activity is essential to protect liver cells against TNF-α hepatotoxicity during development (24, 25, 35) and is also crucial for survival of hepatocytes in adult mice on ligand (i.e., TNF-α, ConA)-induced hepatic damage (27, 28). To determine the effect FKB on hepatocellular NF-κB activity, we first assessed the effects of FKB on TNF-α-induced nuclear translocation of the NF-κB subunit p65. As expected, HepG2 cells treated with 20 ng/ml TNF-α for 30 min exhibited nuclear localization of p65 (Fig. 2A), whereas vehicle-treated cells exhibited a diffuse cytosolic staining pattern of p65. On the other hand, pretreatment with FKB (30 μM, 3 h) completely abrogated the TNF-α-induced nuclear translocation of p65 (Fig. 2A). We next investigated the effects of FKB on NF-κB-dependent transcription of its own inhibitor IκBα. As determined by RT-PCR analysis (Fig. 2B), FKB inhibited TNF-α-induced transcription of IκBα, providing direct evidence that the transcriptional activity of NF-κB can be blocked by FKB treatment. To quantify the concentration-dependent effects of FKB on TNF-α-dependent NF-κB transcription, we next used a conventional NF-κB reporter gene assay. HepG2 or HeLa cells were transiently transfected with the NF-κB transcriptional reporter pκB₅→FLuc. As expected, TNF-α (20 ng/ml) robustly activated NF-κB transcriptional activity in both HeLa and HepG2 cells (Fig. 2C). FKB inhibited NF-κB transcriptional activation in a concentration-dependent manner, exhibiting apparent half maximal inhibition (IC₅₀) values of 10 and 25 μM for HepG2 and HeLa cells, respectively (Fig. 2C). These data suggested a mild cell-type selective effect of FKB to block NF-κB transcriptional activity preferentially in liver cells.

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Figure 2.

FKB blocks NF-κB activity. A) HepG2 cells were treated with or without 30 μM FKB for 3 h, followed by stimulation with TNF-α (20 ng/ml, 30 min) or vehicle. Cells were immunostained for p65 (top panels) and counterstained with DAPI (bottom panels). B) HepG2 cells were treated as in panel A. RNA was isolated, and levels of IκBα and GAPDH mRNA were determined by RT-PCR. C) HepG2 or HeLa cervical carcinoma cells were transiently transfected with the pκB₅→FLuc reporter. Forty-eight hours later, cells were incubated with increasing concentrations of FKB (0–100 μM) for 3 h and subsequently challenged with TNF-α (20 ng/ml) or vehicle only. Cell was imaged for bioluminescence activity. Readouts (+ sem) were normalized for cell viability and transfection efficiency by calculating the ratio of total photon counts with respect to pCMV→FLuc-expressing cells treated in a similar fashion. Significant difference was obtained when cells were treated with FKB at or greater than 6 μM for HepG2 and 12 μM for HeLa cells when compared to the control cells (P<0.005). Inset: Inhibition of NF-κB activity in HeLa (●) and HepG2 (○) cells as a function of FKB concentration, as calculated from data in panel C.

Since phosphorylation, polyubiquitinylation, and degradation of IκBα precedes NF-κB nuclear translocation, we next assessed whether these processes were impaired by FKB. HepG2 cells were treated with 30 μM FKB or vehicle and then stimulated with TNF-α for 5 or 15 min. Immunoblotting with antibodies specifically recognizing phosphorylated-IκBα (p-IκBα S32/36) and total IκBα indicated that FKB blocks TNF-α-induced phosphorylation and degradation of IκBα (Fig. 3A). To assess the effects of FKB on the dynamics of TNF-α-induced IκBα processing, HeLa cells stably expressing an IκBα-luciferase fusion reporter (IκBα-FLuc) or the control unfused reportor (FLuc) were treated with increasing concentrations of FKB for 3 h and then stimulated with TNF-α (20 ng/ml) followed by bioluminescence imaging for 2 h (Fig. 3B) using established methods (32). In the absence of FKB, TNF-α induced a rapid decrease in net IκBα-FLuc bioluminescence, followed by a gradual increase in bioluminescence, representing IKK-dependent degradation of the reporter, followed by its resynthesis and post-translational stabilization (32, 36, 37). FKB inhibited the TNF-α-induced decrease of IκBα-FLuc bioluminescence in a concentration-dependent manner, exhibiting an apparent IC₅₀ value of 25 μM (Fig. 3B). Interestingly, in HepG2 cells, transiently transfected with pIκBα-FLuc, FKB inhibited TNF-α-induced IκBα degradation with an apparent IC₅₀ value of ∼12 μM (Fig. 3C). These data were in good agreement with the observation that FKB was more potent in inhibiting NF-κB transcriptional activity in HepG2 cells than in HeLa cells.

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Figure 3.

FKB inhibits IKK but not the 26S proteasome. A) HepG2 cells were treated with or without 30 μM FKB for 3 h, followed by stimulation with TNF-α (20 ng/ml) for the indicated times. Cell lysates were immunoblotted with antibodies against total IκBα, phosphorylated IκBα (p-IκBα), and β-actin. B) HeLa cells stably expressing IκBα-FLuc were incubated for 3 h with FKB (0–100 μM), subsequently challenged with TNF-α (20 ng/ml), and sequentially imaged for bioluminescence for 2 h at 5 min intervals. Data are presented as fold of TNF-α-untreated. Significant differences were found when FKB concentrations were ≥25 μM (P<0.005). C) Concentration-dependent effects of FKB on IκBα-FLuc degradation (calculated as in panel B) as recorded from HeLa or HepG2 cells (stably or transiently expressing the reporter, respectively). Data are presented as mean ± se percentage degradation (fold-FKB untreated). D) HeLa cells stably expressing tetraubiquitin-FLuc (Ub-FLuc) or unfused FLuc were incubated with FKB (0–50 μM) and imaged as in panel B. The proteasome inhibitor bortezomib (1 μM) served as a positive control. Data are presented as Ub-FLuc/FLuc (fold-vehicle). E) HeLa cells stably expressing unfused FLuc were incubated with FKB (0–100 μM) and imaged for 2 h at 5 min intervals. Significant differences were found only when FKB concentrations were at 100 μM (P<0.05).

To test if FKB directly inhibited global 26S proteasomal degradation and thereby prevented IκBα degradation, we analyzed the effects of FKB on HeLa cells stably expressing a tetraubiquitin-FLuc reporter (Ub-FLuc) (33). Treatment of these cells with increasing concentrations of FKB exhibited no significant effect on Ub-FLuc turnover (Fig. 3D), while bortezomib (used as a positive control) induced in the same time frame (3 h) a more than 150-fold increase in bioluminescence and hence in the stability of the reporter. Furthermore, FKB at the tested concentrations had only minor effects on control FLuc bioluminescence (Fig. 3E; ∼20% decrease in signal after 3 h with 100 μM), suggesting that FKB treatment for short periods (3 h) does not significantly affect viability of the cells. These data strongly supported the hypothesis that FKB did not interrupt global 26S proteasomal activity, but instead acted on or upstream (indirectly) of IKK.

FKB constitutively activates MAPKs signaling pathways

Besides activation of NF-κB, TNF-α also transiently activates MAPKs including JNK, p38, and ERK. Although transient activation of MAPKs leads to proliferation, prolonged activation of JNK has been linked to hepatocytic death (38,–42). To test whether FKB affects TNF-α-induced MAPK signaling, HepG2 cells were pretreated with FKB (30 μM, 3 h) and then stimulated (for 0, 5 or 15 min) with TNF-α (20 ng/ml) or vehicle (PBS) only. Western blot analysis revealed that in the absence of FKB, TNF-α induces minimal, transient phosphorylation of JNK, p38, and ERK in these cells. However, pretreatment with FKB induced constitutive and significantly higher levels of phosphorylation of these proteins (Fig. 4A). We therefore sought to investigate whether FKB by itself can activate these MAPKs and characterize the dynamics of this process. HepG2 cells were treated with FKB (30 μM), and whole-cell lysates were prepared at the indicated time points over 6 h. Western blot analysis revealed that FKB induced prolonged phosphorylation of JNK, p38, and ERK in a time-dependent manner (Fig. 4B) even without TNF-α stimulation.

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Figure 4.

MAPKs are constitutively activated by FKB. A) HepG2 cells were treated with FKB (30 μM, 3 h) or vehicle only and then stimulated with TNF-α (20 ng/ml) for the indicated times. Cell lysates were immunoblotted for ERK, p-ERK, p38, p-p38, JNK, and p-JNK. B) HepG2 cells were exposed to 30 μM FKB for the indicated time periods. Cell lysates were immunoblotted as in panel A.

FKB depletes hepatocellular GSH, and replenishment of GSH, but not MAPK inhibition, protects cells from FKB-induced cell death

Reduced GSH plays a key role in detoxification and is a major regulator of thiol-disulfide redox state (43). Profound GSH depletion is known to be lethal by impairing defenses against endogenous reactive oxygen species (ROS) produced by mitochondria (44). GSH depletion also sensitizes hepatocytes to TNF-α-induced cell death and leads to sustained JNK activation (45,–47). Our finding that FKB induced sustained activation of JNK and cell death prompted us to investigate the impact of FKB on cellular GSH. Incubation of L-02 cells with FKB (30 μM) resulted in progressive decrease of cellular GSH from 6 to 12 h (Fig. 5A). FKB at this concentration leads to only minimal cell death up to 12 h. At the same time, GSSG, the oxidized disulfide form of GSH, increased from 51.18 ± 4.24 (0 h) to 181.18 ± 7.32 μM/mg cells (12 h). Therefore, our data suggest that FKB led to conversion of GSH to GSSH, thus dramatically affecting hepatocellular redox state.

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Figure 5.

Role of GSH in FKB-induced cell death. A) HPCE analysis of GSH in L-02 cells treated with 30 μM FKB at indicated time points. *P < 0.05 vs. 0 h. B) HepG2 cells were treated with FKB (30 μM) with or without exogenous GSH (2 mM) for 3 h and then stimulated with TNF-α (20 ng/ml) for the indicated time periods. Cell lysates were immunoblotted as indicated. C) HepG2 cells were treated with FKB (30 μM) or vehicle only in the presence or absence of Bay11–7085, U0126, SP, SB, or GSH (at 20, 10, 5, 10, or 2000 μM, respectively). Cytotoxicity was monitored 48 h later by MTT assay. D) HepG2 cells were incubated with FKB (30 μM) and increasing concentrations of GSH (0–4 mM) for 48 h. Cytotoxicity was assessed by MTT. *P < 0.001 vs. vehicle.

We next aimed at determining the effects of exogenous GSH on TNF-α-induced activation of NF-κB and MAPK signaling pathways in the absence or presence of FKB. HepG2 cells were incubated for 3 h with FKB (30 μM) in the absence or presence of reduced GSH (2 mM). Cells were than stimulated with TNF-α (20 ng/ml) for 5 or 15 min. Consistent with our preceding data, FKB abrogated TNF-α-induced IκBα degradation and activated all 3 MAPKs constitutively (Fig. 5B). Surprisingly, supplementation with exogenous GSH normalized the effects of FKB on TNF-α-induced activation of both IKK (as reflected by IκBα degradation) and MAPK signaling pathways (as reflected by phosphorylation of JNK, p38, and ERK; also compare Figs. 3A, ,44A, and and55B).

Since GSH was shown to normalize the effects of FKB on NF-κB and MAPK signaling, one would expect that GSH may rescue cells from FKB-induced death. Furthermore, since prolonged activation of MAPK signaling and especially JNK was shown to have a role in hepatocellular apoptosis (38,–42), one may also expect that MAPK inhibition could reverse the hepatotoxic effects of FKB. To test these hypotheses, we analyzed FKB-induced hepatocellular toxicity in the absence or presence of exogenous GSH, U0126 (MEK1 inhibitor, blocks ERK phosphorylation), SB2023580 (p38 inhibitor), or SP600125 (JNK inhibitor). We found that none of the MAPK inhibitors by themselves or in combination were capable of rescuing HepG2 cells from FKB-induced cell death (Fig. 5C). Surprisingly, exogenous GSH not only counterbalanced the effects of FKB on NF-κB and MAPK signaling (Fig. 5B), but also blocked FKB-induced hepatotoxicity (Fig. 5C) in a concentration-dependent manner (Fig. 5D). Interestingly, GSH supplementation also rescued HepG2 cells that were treated with Bay 11-7085, a small molecule that was previously shown to specifically inhibit IKK activity (Fig. 5C) (48). These data implied that exogenous GSH supplementation had the potential to reverse hepatotoxicity induced by prolonged activation of MAPKs (as provoked by FKB) or by IKK/NF-κB inhibition (as induced by FKB or Bay 11-7085).

FKB induces hepatotoxicity and inhibits NF-κB transcriptional activity in vivo

To evaluate the hepatotoxic effects of FKB in vivo, mice were orally administered FKB (25 mg/kg body weight) or vehicle (0.5% methyl-cellulose) daily for 1 wk. Histological analysis of liver specimens revealed that FKB induced massive liver damage, as demonstrated by diffuse cloudy hepatocellular swelling and vesiculated cytoplasm (Fig. 6A, white arrow). Inflammatory infiltration was also evident predominantly in the periportal area (black arrow). Consistent with histologically observed liver damage, serum AST and AKP levels were also increased in mice treated with FKB (data not shown).

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Figure 6.

FKB inhibits hepatic NF-κB activity and induces liver damage in vivo. A) Mice were administered FKB (p.o., 25 mg/kg) or vehicle only (0.5% methyl-cellulose) daily for 7 d. Liver specimens were sectioned (5 μm) and stained with hematoxylin and eosin. Open and solid arrows indicate regions of cell death and macrophage infiltration, respectively. B, C) Schematic representation of the experimental timeline for assessing real-time NF-κB activity (B) and corresponding images (C). Briefly, liver hepatocytes were transduced by somatic gene transfer using high-volume i.v. injection of the reporter construct κB₅→FLuc (8 μg/mouse, n = 8). Three weeks later, mice were imaged (C, top row) and randomly divided to 2 groups (n=4/group). Mice were treated daily with FKB (p.o., 25 mg/kg; C, right panels) or vehicle (methyl cellulose, 0.5%, p.o.; C, left panels) and imaged after 7 d of treatment (C, middle row). Three hours later, all mice were challenged by a single i.v. dose of LPS (4 mg/kg) to activate NF-κB signaling and reimaged 2 postchallenge (C, bottom row). D) Quantitative representation of the changes in mean ± se total photon flux over the experimental timeline of vehicle- or FKB-pretreated mice. Inset: net LPS-induced increase in NF-κB activity in vehicle- or FKB-pretreated animals (presented as fold-LPS-prechallenged).

We next sought to assess if FKB blocks IKK/NF-κB activity in intact animals on oral administration of FKB. We therefore used real-time bioluminescence imaging of NF-κB transcriptional activity in living mice to monitor the effects of FKB on the pathway in vivo. Using hydrodynamic somatic gene transfer, a cohort of mice (n=8) stably expressing an κB₅→FLuc reporter (see also Fig. 2C) in their liver was established (32). Three weeks later, mice were orally administered FKB (25 mg/kg body weight, n=4) or vehicle only (0.5% methyl-cellulose, n=4), daily for 1 wk. Because LPS not only induces TNF-α production but also activates NF-κB through TLR4/MyD88-dependent signaling, independently of TNF-α induction (49), LPS was then injected intravenously into all mice to induce acute hepatocellular activation of NF-κB. As expected, LPS potently activated NF-κB in vehicle-treated mice (Fig. 6C, D). However, LPS-induced activation of NF-κB was greatly reduced in FKB-treated mice (>60%). These data suggested that oral consumption of FKB can impair hepatic NF-κB transcriptional activity in vivo, thereby blocking prosurvival signals mediated by NF-κB.

Hepatocellular toxicity induced by chalocones

Figure 1.

Table 1.

Contents of chalcones (FKB, FKC) and total kavalactones in kava root extracts (mg/g dried weight) by different extraction solvents

Extract solvent	FKB (mg/g)	FKC (mg/g)	Total kava lactones (mg/g)
Water	0.2	0	46.6
60% acetone	26.0	1.1	474.8
Acetone	33.7	1.5	570.0
95% ethanol	32.3	1.4	548.8

FKB blocks TNF-α-induced activation of NF-κB

Figure 2.

Figure 3.

FKB constitutively activates MAPKs signaling pathways

Figure 4.

FKB depletes hepatocellular GSH, and replenishment of GSH, but not MAPK inhibition, protects cells from FKB-induced cell death

Figure 5.

FKB induces hepatotoxicity and inhibits NF-κB transcriptional activity in vivo

Figure 6.

DISCUSSION

The beneficial psychoactive effects of kavalactones for treating anxiety, stress, insomnia, restlessness, and muscle fatigue are well documented in the literature (1, 50,–52). Traditionally, extraction of these compounds is performed using aqueous solutions, yielding relatively low levels of kavalactones (∼4.6%, Table 1). Modern extraction techniques using organic solvents (e.g., acetone, ethanol) yield significantly higher levels of kavalactones (∼45–55%, Table 1), and dramatically higher levels of lipophilic chalcones in the extract (∼160-fold for FKB, Table 1). Recently organic extracts of kava root rhizomes, sold as over-the-counter herbal supplements, were reported to induce severe hepatotoxicity. Kavalactones have been proposed to account for kava-induced liver toxicity (14,–16, 53), but no noticeable toxicity was observed in rats fed with kavalactones (>500 mg/kg, daily for 4 wk) (17). In agreement with this in vivo observation, our data (Fig. 1A) showed that indeed kavalactones had no significant effects on the viability of selected liver cell lines. On the other hand, we show here that chalcones, compounds that are dramatically enriched in organic solvent-based extractions (Table 1), were accountable for the observed hepatotoxicity (Fig. 1). FKA exhibited toxicity toward HepG2 hepatoma cells (LD₅₀∼75 μM), but to a much lesser extent toward L-02 hepatocytes (data not shown), suggesting that this chalcone was not likely responsible for hepatotoxicity of kava extracts. Both FKB and FKC exhibited significant toxicity in both cell lines; however, FKB was shown to be a more potent hepatocellular toxin than FKC (LD₅₀∼15 vs. 70 μM, respectively). Also, FKB is 20-fold more abundant than FKC in organic kava extracts (Table 1). We further demonstrated that FKB was capable of activating caspase 3 and inducing apoptosis in cultured hepatocytes. Oral administration of FKB to mice induced significant liver damage. Although we do not exclude the possibility that other constituents can contribute to the hepatoxicity of kava extract, our data support that FKB is the major hepatocellular toxin found in kava root organic extracts.

In hepatocytes, proapoptotic signals mediated by TNF-α are counterbalanced by NF-κB via transcription of several antiapoptotic proteins, including cIAPs, c-FLIP, A1, A20, TRAF2, and Bcl-xl (54), which block either death receptors or mitochondrial apoptotic pathways. We found that FKB blocked TNF-α-induced phosphorylation and degradation of IκBα, as well as NF-κB nuclear translocation and transcriptional activation. Blockage of IκBα degradation could not be attributed to global inhibition of proteasomal activity, but rather to inhibition of IKK activity. At this point, however, we cannot determine if FKB inhibits NF-κB signaling via direct or indirect inhibition of IKK activity. Interestingly, inhibition of both IKK and NF-κB transcriptional activities was more potent in hepatocytes than in HeLa cervical carcinoma cells, suggesting a certain level of selectivity toward hepatocytes. This is in good agreement with a recently reported study demonstrating that FKB and FKA inhibit TNF-α-induced activation of NF-κB in a leukemia cell line at concentrations of 175 and 320 μM for FKB and FKA, respectively (55). These concentrations are significantly higher than those we used here to inhibit NF-κB signaling in cultured hepatocytes. We further demonstrated that oral administration of FKB to mice significantly attenuated LPS-dependent NF-κB hepatic activity, suggesting that FKB blocks NF-κB activity both in vitro and in vivo.

Sustained activation of JNK had been shown to participate in promoting apoptosis induced by TNF-α (for a review see ref. 56). It has been also reported that transcription of the antioxident enzyme SOD2 is dependent on NF-κB activation (57, 58), and therefore blockade of NF-κB activation may lead to oxidative stress and increases in cellular ROS. Since ROS can deactivate MAPK phosphates (MKPs), concurrent oxidative stress and inhibition of NF-κB transcriptional activity may lead to sustained activation of JNK (59). We found that activation of JNK, p38, and to a lesser extent ERK was markedly increased in FKB-treated cells. The sustained activation of JNK is likely due to increased ROS induced by FKB because, under similar conditions, FKB induced GSH depletion (Fig. 5A) and NF-κB inhibition (Fig. 2). However, pharmacological blockade of each MAPK independently or in combination failed to protect liver cells from FKB-induced cell death, which suggests that sustained activation of JNK alone may not be the primary cause for FKB-induced cell death. Nonetheless, our results do not exclude the possibility that sustained activation of JNK acts additively on other signaling pathways to mediate FKB-induced cell death.

Depletion of GSH by hepatotoxins (e.g., alcohol, acetaminophen) has been shown to contribute to sensitization of liver to hepatotoxin-induced injury through TNF-α-induced death (60, 61). GSH is a critical ROS scavenger and a major regulator of thiol-disulfide redox state. The balance of GSH and GSSG represents a key redox buffer for protein thiol-disulfides. We demonstrated that FKB depleted hepatocellular GSH, and strikingly, addition of exogenous GSH rescued liver cells from FKB-induced death, as well as normalizing IKK/NF-κB and MAPK signaling. This detoxification of FKB by GSH may also explain why liver damage occurs only in a small population of kava users that potentially exhibit a preexisting altered hepatic redox state.

In summary, we showed that in organic solvent-extracted kava root extracts, chalcones, and especially FKB are dramatically enriched. We also demonstrated that FKB is a potent hepatotoxin that induces hepatocellular apoptosis. These detrimental effects of FKB are mediated by affecting the intracellular redox state and, at least in part, by altering the balance of NF-κB and MAPK signaling (Fig. 7). Furthermore, pretreating hepatocytes with exogenous GSH normalizes NF-κB and MAPK signaling and rescues hepatocytes from FKB-induced toxicity. Finally, we demonstrated that FKB inhibits hepatic NF-κB transcriptional activity and induces liver damage in vivo. Kava is still widely used as a dietary supplement to relieve anxiety and stress in the United States. Controlling the levels of FKB in kava products by modifying existent extraction methods or possibly by genetically modifying FKB biosynthesis should in principle reduce, if not eliminate, those rare hepatotoxic effects observed in consumers of kava root extracts.

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Object name is z380121080400007.jpg

Figure 7.

Suggested mechanism for FKB-induced hepatocellular toxicity. FKB inhibits IKK activity (directly or indirectly), leading to down-regulation of NF-κB transcriptional activity, which is crucial for hepatocellular survival. FKB also alters intracellular redox levels and induces oxidative stress that is likely to be further enhanced by blockade of NF-κB-mediated transcription and down-regulation of SOD2. Proapoptotic signals, mediated by constitutive activation of MAPKs (mainly p38 and JNK), are induced by this oxidative stress possibly through inhibition of MKPs' toxicity.

^{Division of Oncology,}

^{Department of Developmental Biology, and}

^{Molecular Imaging Center, Mallinckrodt Institute of Radiology, Washington University, School of Medicine, St. Louis, MO, USA;}

^{Department of Internal Medicine, Stem Cell Program, University of California Davis, Sacramento, CA, USA;}

^{School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, China; and}

^{Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China}

^{These authors contributed equally to this work.}

^{Correspondence: Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China., E-mail:}nc.ca.gbcs@uiqxs

Received 2010 May 5; Accepted 2010 Jul 22.

Abstract

Kava (Piper methysticum Foster, Piperaceae) organic solvent-extract has been used to treat mild to moderate anxiety, insomnia, and muscle fatigue in Western countries, leading to its emergence as one of the 10 best-selling herbal preparations. However, several reports of severe hepatotoxicity in kava consumers led the U.S. Food and Drug Administration and authorities in Europe to restrict sales of kava-containing products. Herein we demonstrate that flavokawain B (FKB), a chalcone from kava root, is a potent hepatocellular toxin, inducing cell death in HepG2 (LD₅₀=15.3±0.2 μM) and L-02 (LD₅₀=32 μM) cells. Hepatocellular toxicity of FKB is mediated by induction of oxidative stress, depletion of reduced glutathione (GSH), inhibition of IKK activity leading to NF-κB transcriptional blockade, and constitutive TNF-α-independent activation of mitogen-activated protein kinase (MAPK) signaling pathways, namely, ERK, p38, and JNK. We further demonstrate by noninvasive bioluminescence imaging that oral consumption of FKB leads to inhibition of hepatic NF-κB transcriptional activity in vivo and severe liver damage. Surprisingly, replenishment with exogenous GSH normalizes both TNF-α-dependent NF-κB as well as MAPK signaling and rescues hepatocytes from FKB-induced death. Our data identify FKB as a potent GSH-sensitive hepatotoxin, levels of which should be specifically monitored and controlled in kava-containing herb products.—Zhou, P., Gross, S., Liu, J.-H., Yu, B.-Y., Feng, L.-L., Nolta, J., Sharma, V., Piwnica-Worms, D., Qiu, S. X. Flavokawain B, the hepatotoxic constituent from kava root, induces GSH-sensitive oxidative stress through modulation of IKK/NF-κB and MAPK signaling pathways.

Keywords: hepatocellular toxin, apoptosis, TNF-α, herb products, bioluminescence imaging

Abstract

Kava (Piper methysticum Foster, Piperaceae), also known as kava-kava, is a herbal shrub that has been used for centuries in the South Pacific as a social beverage and in traditional ceremonial rituals (1, 2). In the past 20 years, organic solvent (ethanol and/or acetone) extracts from kava roots and rhizomes have been used in Western industrialized countries for treating mild and moderate anxiety, stress, insomnia, restlessness, and muscle fatigue (1), leading to its emergence as one of the 10 best-selling botanical dietary supplements. Despite the apparent safety of traditional kava drinking in the South Pacific island states (3), severe side effects of liver damage resulting in several cases of mortality or liver transplantation were recently reported in both Europe and the United States (3, 4). In some patients, the use of certain kava supplements was shown to induce hepatic failure, severe acute hepatitis, panacinar necrosis, collapse of hepatic lobules, and hepatocellular apoptosis associated with increases in bilirubin, aspartate aminotransferase (AST), and alanine aminotransferases (5,–8). As a consequence, kava-containing products have represented a significant public health concern and are banned in a number of countries, including most European countries, Canada, Australia, and New Zealand (9,–11), with advisories issued in the United States by the Food and Drug Administration (10, 11). It is important to note that although Western “industrial” kava preparations are mainly extracted with organic solvents (e.g., ethanol, acetone), traditional kava drinks are prepared by dipping the kava roots/rhizomes in water or coconut juice with an apparently safe history. However, the contribution of different extraction methods to liver toxicity remains elusive.

Kavalactones are the major components of kava and are generally believed to be the psychoactive compounds accountable for kava's sedative and hypnotic activities (12, 13). In vitro studies demonstrated that kavalactones inhibit P450 enzymes, responsible for metabolism of more than 90% of pharmaceuticals in humans, and therefore are proposed to cause drug-drug interactions and liver toxicity in cases of concomitant use of kava preparations with conventional therapeutic antidepressants (14). Furthermore, kavalactones can form electrophilic quinone metabolites, potentially leading to glutathione depletion and oxidative stress (15, 16). However, these in vitro data were not supported by the in vivo observation that rats fed with aqueous kava root extracts containing as much as 500 mg kavalactones/kg body weight for 4 wk exhibited no noticeable toxicity (17). Recently it was reported that a piperidine alkaloid, pipermethystine (PM), induces apoptosis in human hepotoma HepG2 cells (18, 19) but fails to induce hepatic toxicity in vivo (20). However, PM is almost exclusively present in the aerial parts of kava but virtually absent in the roots and rhizomes, which are used in traditional drinks and herbal supplements. This raises doubts as to whether PM is responsible for the hepatotoxicity of kava extracts. The proinflammatory cytokine tumor necrosis factor α (TNF-α) has been associated with hepatocellular apoptosis and inflammatory liver injury (21). This cytokine activates parallel signaling pathways including mitogen-activated protein kinases (MAPKs), nuclear factor-κB (NF-κB), as well as caspase-dependent proapoptotic pathways. All 3 types of MAPKs, namely, ERK, JNK, and p38, can be activated by TNF-α, leading to either proliferation or cell death depending on the cell type. Under normal conditions, however, TNF-α does not induce apoptosis owing to a balanced activation of prosurvival NF-κB signaling (22). The NF-κB family of transcription factors is composed of dimers containing different combinations of Rel-domain-containing proteins (i.e., p65, Rel B, c-Rel, p50, and p52). In resting cells, NF-κB is sequestered in the cytosol by members of the IκB protein family (23). TNF-α indirectly activates the heterotrimeric IκB kinase (IKK, consisting of IKKα, IKKβ, and IKKγ) leading to phosphorylation, polyubiquitinylation, and proteasomal degradation of IκBα. As a consequence, NF-κB freely translocates into the nucleus, where it activates transcription of proinflammatory, proproliferative, and survival genes. Mice lacking IKKβ (24), p65 (25), or IKKγ display severe liver degeneration that results in embryonic lethality and that can be rescued by simultaneous knockout of TNF-α (26) or the receptor of TNF-α (24). NF-κB activity was also suggested to play a key role in hepatocellular survival of adult mice because animals deficient in hepatic IKKβ or IKKγ exhibited increased susceptibility to hepatoxic effects associated with injection of Con A or TNF-α (27, 28). Interestingly, these studies also implied a crucial role for MAPK signaling and especially prolonged activation of JNK in promoting TNF-α-induced hepatotoxicity.

In this study, we aimed at identifying the hepatotoxic constituent from kava roots/rhizomes and deciphering its effects on signaling pathways associated with hepatocellular survival. We report herein that organic solvent-extracted kava root extracts contain high levels of FKB that induce severe hepatocellular toxicity through inducing oxidative stress, modulating IKK/NF-κB and MAPK signaling pathways. It is therefore concluded that FKB is the major, and perhaps the exclusive, chalcone contributing to the hepatotoxicity of kava extracts, and its levels should be tightly monitored and controlled during preparation of kava root extracts if organic solvents are used for extraction.

Acknowledgments

This project was jointly funded by U.S. National Institutes of Health grant P50 CA94056, National Natural Science Foundation of China grants (30572320;, 30973635), a Chinese Academy of Sciences 100 Talents Program Endowment award to S.X.Q., and a grant from the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-YW-R-217).

Acknowledgments

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