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. 2010 Dec;24(12):4722-32.
doi: 10.1096/fj.10-163311. Epub 2010 Aug 9.

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

Ping Zhou  1 , Shimon Gross, Ji-Hua Liu, Bo-Yang Yu, Ling-Ling Feng, Jan Nolta, Vijay Sharma, David Piwnica-Worms, Samuel X Qiu
Affiliations

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

Ping Zhou et al. FASEB J. 2010 Dec.

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 (LD(50)=15.3 ± 0.2 μM) and L-02 (LD(50)=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.

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Figures

Figure 1.
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.
Figure 2.
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.
Figure 3.
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).
Figure 4.
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.
Figure 5.
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.
Figure 6.
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).
Figure 7.
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.
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