Mechanisms for epigallocatechin gallate induced inhibition of drug metabolizing enzymes in rat liver microsomes

Toxicology Letters 214 (2012) 328–338

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Mechanisms for epigallocatechin gallate induced inhibition of drug metabolizing enzymes in rat liver microsomes Zuquan Weng, James Greenhaw, William F. Salminen 1 , Qiang Shi ∗ Division of Systems Biology, National Center for Toxicological Research, Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA

h i g h l i g h t s    

Epigallocatechin gallate (EGCG) bound to a subset of protein in the microsomes. EGCG decreased the Western blot signals of a selective group of protein. EGCG inhibited microsomal glutathione transferase 1 but not cytosolic glutathione transferase. EGCG effects were partially abolished by glutathione.

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Article history: Received 24 August 2012 Received in revised form 14 September 2012 Accepted 14 September 2012 Available online 23 September 2012 Keywords: Epigallocatechin gallate Cytochrome P450 Rat Microsome Covalent-binding Glutathione

a b s t r a c t Epigallocatechin gallate (EGCG) inhibits drug metabolizing enzymes by unknown mechanisms. Here we examined if the inhibition is due to covalent-binding of EGCG to the enzymes or formation of protein aggregates. EGCG was incubated with rat liver microsomes at 1–100 ␮M for 30 min. The EGCG-binding proteins were affinity purified using m-aminophenylboronic acid agarose and probed with antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin, cytochrome P450 (CYP) 1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A, catechol-O-methyltransferase (COMT) and microsomal glutathione transferase 1 (MGST1). All but actin and soluble COMT were positively detected at ≥1 ␮M EGCG, indicating EGCG selectively bound to a subset of proteins including membrane-bound COMT. The binding correlated well with inhibition of CYP activities, except for CYP2E1 whose activity was unaffected despite evident binding. The antioxidant enzyme MGST1, but not cytosolic GSTs, was remarkably inhibited, providing novel evidence supporting the pro-oxidative effects of EGCG. When microsomes incubated with EGCG were probed on Western blots, all but the actin and CYP2E1 antibodies showed a significant reduction in binding at ≥1 ␮M EGCG, suggesting that a fraction of the indicated proteins formed aggregates that likely contributed to the inhibitory effects of EGCG but were not recognizable by antibodies against the intact proteins. This raised the possibility that previous reports on EGCG regulating protein expression using GAPDH as a reference should be revisited for accuracy. Remarkable protein aggregate formation in EGCG-treated microsomes was also observed by analyzing Coomassie Blue-stained SDS-PAGE gels. EGCG effects were partially abolished in the presence of 1 mM glutathione, suggesting they are particularly relevant to the in vivo conditions when glutathione is depleted by toxicant insults. Published by Elsevier Ireland Ltd.

1. Introduction Epigallocatechin gallate (EGCG) is the predominant green tea flavonoids with various claimed pharmacological effects (Singh et al., 2011) and is widely consumed as a major component of many dietary supplements, such as antioxidants and weight loss aides (Williamson et al., 2011). Clinical trials are ongoing to

∗ Corresponding author. Tel.: +1 870 543 7365; fax: +1 870 543 7736. E-mail address: [email protected] (Q. Shi). 1 Currently with PAREXEL International, 7216 Palm Beach Circle, Benton, AR 72019, USA. 0378-4274/$ – see front matter. Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.toxlet.2012.09.011

explore the possibility of developing EGCG into a novel anti-cancer drug (Shanafelt et al., 2012). Despite its claimed beneficial effects, EGCG alone (Galati et al., 2006; Lambert et al., 2010) or in combination with other drugs (Salminen et al., 2012) has been associated with various adverse effects including hepatotoxicity in both human and experimental animals (Mazzanti et al., 2009). EGCG was found to profoundly affect hepatic cytochrome P450s (CYPs), presumably leading to altered pharmacokinetics and toxicity of coadministrated drugs (Yang and Pan, 2012) such as acetaminophen (Salminen et al., 2012). Under in vitro conditions, EGCG inhibits both phase I and phase II drug metabolizing enzymes. For example, CYP1A activity was remarkably suppressed by EGCG in rat microsomes (Wang et al.,

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1988) and in over-expressed purified human CYPs (Muto et al., 2001). In human microsomes, UDP-glucuronosyltransferase (UGT) 1A1 and UGT1A4 activities were significantly decreased by EGCG (Mohamed and Frye, 2011; Mohamed et al., 2010). The underlying mechanisms of inhibition have not been explored. Recently, it was reported that EGCG may cause a functional change in its protein targets through covalent binding (Ishii et al., 2008; Tanaka et al., 2011). It was also discovered that EGCG may induce intermolecular cross-linking leading to protein aggregate formation in membrane proteins (Chen et al., 2011), although which protein is susceptible to this alteration remains unknown. Hepatic microsomes are derived from endoplasmic reticulum and contain mainly membrane proteins (Galeva and Altermann, 2002). This study was undertaken to test the hypothesis that EGCGinduced inhibition of drug metabolizing enzymes is due to covalent binding of EGCG to the enzymes and/or formation of enzyme aggregates. Assessing the interaction of EGCG with target proteins will help understand the mechanisms of possible EGCG-drug interactions as exemplified by our recent publication (Salminen et al., 2012).

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2. Materials and methods 2.1. Chemicals, reagents and antibodies EGCG was purchased from Blue California (Rancho Santa Margarita, CA). Its purity was determined to be 98.5% by high-performance liquid chromatography. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Sigma (St. Louis, MO; catalog number: G9545). Anti-actin (catalog number: sc-1616) and anti-microsomal glutathione transferase 1 (MGST1) (catalog number: sc17003) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-CYP1A1 (catalog number: AB1247), anti-CYP2B1/2 (catalog number: AB1283) and anti-CYP3A (catalog number: MAB10041) were from EMD Millipore Corporation (Billerica, MA). Anti-CYP2E1 (catalog number: AB28146) was from Abcam (Cambridge, MA). AntiCYP1A2 (catalog number: MA1-46106) was from Thermo Fisher Scientific Inc. (Rockford, IL). Anti-catechol-O-methyltransferase (COMT) was from BD Transduction Laboratories (San Jose, CA; catalog number: 611970). The secondary antibodies (IRDye® 680RD anti-goat, anti-mouse, and anti-rabbit IgG) were obtained from LI-COR Biosciences (Lincoln, NE). The fluorescent probe 7-benzyloxyquinoline was from BD Biosciences (San Jose, CA), and 7-ethoxyresorufin and 7pentoxyresorufin were from Invitrogen (Grand Island, NY). All other commonly used reagents including m-aminophenylboronic acid-agarose (PBA-agarose) and Nethylmaleimide (NEM) were either from Sigma (St. Louis, MO) or Bio-Rad (Hercules, CA).

Fig. 1. EGCG induced protein aggregate formation and selectively bound to a subset of microsomal proteins. EGCG-treated microsomes were either directly subjected to SDS-PAGE (A), lanes 2–10 correspond to 0–100 ␮M EGCG-treated samples) or used for purification of EGCG-binding proteins and subsequent analysis by SDS-PAGE (C), lanes 3–11 correspond to 0–100 ␮M EGCG treated samples and lane 2 represents the intact microsomes). The average density and standard deviation of protein bands represented in (A) are shown in (B), in which all the EGCG-treated samples (except 1–45 ␮M ECGC-treated samples for the zone 4 signals) had means that were statistically different from control (0 ␮M EGCG) sample (p < 0.05).

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2.2. Animal care Male Sprague-Dawley rats weighing 250–350 g were obtained from the US Food and Drug Administration National Center for Toxicological Research (NCTR) breeding colonies. Animal care and experimental procedures were performed in accordance with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” and were authorized by the NCTR Institutional Animal Care and Use Committee. 2.3. Isolation of rat hepatic sub-cellular fractions Rats were euthanized with carbon dioxide asphyxiation and the previously described method (Schenkman and Jansson, 1998) of isolating hepatic microsomes was followed in detail. The cytosolic and mitochondrial fraction was also collected either for the measurement of cytosolic glutathione transferase (cGST) activity or for the detection of specified proteins using Western blot. Protein concentrations were measured by the Bradford method (Bradford, 1976) using bovine serum albumin (BSA) as a standard. 2.4. Incubation of EGCG with microsomes The protein concentration of microsomes was adjusted to 1 mg/ml using PBS. EGCG stock solution was freshly prepared in PBS, and was added in small volumes to the microsomes to final concentrations of 0, 1, 7.5, 15, 30, 45, 60, 75 and 100 ␮M. After incubation at 37 ◦ C for 30 min, the microsomes were either centrifuged at 100,000 × g at 4 ◦ C for 1 h to remove excessive EGCG for subsequent purification of EGCG-binding proteins or used directly to measure the enzyme activity. To test the influence of glutathione on EGCG effects, additional microsomes were supplemented with 1 mM glutathione before incubation with EGCG. To test

the involvement of thiol groups, microsomes were incubated with 0.5 mM NEM for 15 min before exposure to EGCG. In some experiments, the mitochondrial and cytosolic fractions were treated with EGCG in the same manner as for microsomes before using Western blot to detect GAPDH and actin. 2.5. Measurement of enzyme activities MGST1 and cGST activities were measured using 5 mM glutathione and 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) as substrates (Shi and Lou, 2005), respectively. The rate of p-nitrophenol hydroxylation was determined to reflect CYP2E1 activity (Chang et al., 2006). CYP1A and CYP2B activities were measured using 7-ethoxyresorufin and 7-pentoxyresorufin as substrates, respectively, as detailed previously (Dyck and Davis, 2001). The probe 7-benzyloxyquinoline was used to determine CYP3A activities following the published procedure (Stresser et al., 2002). 2.6. Purification of EGCG-binding proteins We slightly modified a previously described procedure (Ishii et al., 2008) to purify the EGCG-binding proteins in the microsomes. Briefly, the microsomes from 3 rats were isolated and pooled. EGCG was added at varying concentrations and incubated as described above. For each EGCG concentration, 5 mg protein was taken from each rat and pooled to a final concentration of 1 mg/ml. After incubation, the microsomes were centrifuged at 100,000 × g for 1 h. The supernatant was discarded and the microsomal pellets were washed briefly and re-suspended in 15 ml 100 mM Tris–HCl buffer (pH 8.6) with the aid of a Dounce type homogenizer. This step ensured that excessive EGCG was removed. The stock solution of Triton X-100 (20%) was then added to a final concentration of 0.2% to help solubilize the proteins. After shaking for 30 min, the samples were centrifuged at 20,000 × g for 20 min. No apparent pellets were observed, indicating essentially all proteins went into

Fig. 2. EGCG bound to and likely induced aggregate in GAPDH but showed no effects on actin. The EGCG-binding proteins were probed with anti-GAPDH (A) or anti-actin (B) antibody (lanes 3–11 correspond to 0–100 ␮M EGCG treated samples), with the intact microsome as a control (lane 2 in (A) and (B)). The EGCG-treated microsomes were probed by Western blot using anti-actin (C) or anti-GAPDH (E) antibodies, with quantitatively data presented in (D) and (F), respectively. The mitochondrial and cytosolic fractions treated with EGCG were also probed with anti-GAPDH (G) and (H). In (C), (E) and (G) and (H), lanes 2–10 correspond to 0–100 ␮M EGCG treated samples. The means of EGCG-treated samples were compared to control (0 ␮M EGCG) samples by one-way ANOVA followed by Dunnett’s test. *p < 0.05.

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Fig. 2. (Continued ).

solution. The samples were then added to 0.5 ml settled PBA-agarose and incubated with gentle shaking overnight. The PBA-agarose was then extensively washed with 12 ml buffer following this order: once with 100 mM Tris–HCl buffer (pH 8.6) containing 0.2% Triton X-100, once with the same buffer without Triton X-100, once with 1 mM Tris–HCl buffer (pH 8.6), twice with 250 mM Tris–HCl buffer (pH 6.5), and twice with 1 mM Tris–HCl buffer (pH 6.5). The EGCG-binding proteins were then eluted with 1.5 ml 50 mM glycine (pH 2.0) by gentle shaking for 30 min. The elution was repeated once and the samples were pooled and neutralized with about 8 ␮l 10 M NaOH.

calculated using the software GraphPad Prism 5 and presented in the figures. One-way or two-way analysis of variance (ANOVA) followed by Dunnett’s test or Bonferroni test were used to compare the means among groups. A p value of less than 0.05 was considered to be of statistical significance.

3. Results 3.1. EGCG-induced protein aggregate formation and selective binding to a subset of microsomal proteins

2.7. Western blot detection of target proteins The purified EGCG-binding proteins were combined with an equal volume of 2× reducing Laemmli buffer and boiled in 95 ◦ C water for 5 min before SDS-PAGE. To each lane were loaded 15 ␮l of sample. As a control, the same volume of microsomal protein (0.5 mg/ml final concentration) was loaded in the parallel lane. The resolved proteins were transferred to nitrocellulose membranes. After blocking for 1 h with 5% non-fat dry milk in Tris-buffered saline (TBS), the primary antibody was added at a 1:1000 dilution in TBS with 5% BSA and incubated with the membrane at 4 ◦ C for about 16 h. The membrane was washed and then the corresponding secondary antibody was added at a dilution of 1:10,000 in the same buffer. The Western blot signal was obtained on an Odyssey® Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). The Image Studio Software from LI-COR Biosciences (Lincoln, NE) was used to quantitatively analyze the images. In cases where indicated, the membranes were stripped by the restore Western blot stripping buffer from Thermo Fisher Scientific Inc. and re-probed with anti-actin antibody for the purpose of normalization. 2.8. Statistical analysis All the experiments were repeated at least three times using different batches of microsomes from different rats. The mean and the standard deviation were

The global molecular weight changes of EGCG-treated microsomes were first detected by Coomassie Blue stained SDS-PAGE gels as in a previous report (Chen et al., 2011). As shown in Fig. 1A, when we arbitrarily divided each protein lane into four zones according the molecular weight, we observed a notable increase of protein intensity in zones 1 and 2 (highest molecular weights) and a significant decrease of protein intensity in zones 3 and 4 (lowest molecular weights) in EGCG-treated samples (lanes 3–10) as compared to the un-treated samples (lane 2). These results indicate that some low molecular weight proteins in zones 3 and 4 formed protein aggregates that migrated slower in the gels causing an increased signal in zones 1 and 2. Quantitative results derived from Fig. 1A are shown in Fig. 1B. A well-established procedure (Ishii et al., 2008) was adopted to observe the EGCG-binding protein. As shown in Fig. 1C, while we saw no protein bands in an un-treated sample (lane 3) or a 1 ␮M

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EGCG-treated sample (lane 4), significant protein signals began to appear in 7.5–100 ␮M EGCG-treated samples (lanes 5–11), indicating that EGCG bound to the proteins that could be selectively enriched. Not all the original microsomal protein bands (lane 2) were present in the EGCG-binding proteins (lanes 5–11), such as those indicated by arrows in lane 2 of Fig. 1C, suggesting that the binding to EGCG was protein selective. Among the EGCG-binding proteins, we consistently saw at >150 kDa the “smearing” of protein bands (lanes 7–11 in Fig. 1C), a characteristic feature of protein aggregates (Sagne et al., 1996). 3.2. EGCG bound to and likely induced aggregates in GAPDH but showed no effects on actin To identify the specific proteins that bound to EGCG, we used the antibodies against GAPDH and actin to probe the purified EGCG-binding protein on Western blots. GAPDH was chosen as it is the best-characterized protein target for EGCG-binding (Ishii et al., 2008). Actin was selected as a theoretical target for EGCGbinding based on the hypothesis that the binding mainly occurs via cysteines (Ishii et al., 2008) and the cysteines of actin are susceptible to other forms of modifications such as S-nitrosylation (Shi

et al., 2008). As illustrated in Fig. 2, GAPDH (Fig. 2A) but not actin (Fig. 2B) was positively detected among the EGCG-binding proteins on Western blots, demonstrating that the binding to EGCG was protein selective. The fact that actin was readily detectable in the microsomes (lane 2 in Fig. 2B) confirmed that our Western blot procedure was reliable. The formation of GAPDH and actin aggregates was then assessed. When the microsomal proteins were probed with these two antibodies, the Western blot signal of GAPDH (Fig. 2E and F) but not actin (Fig. 2C and D) was decreased by EGCG starting at 1 ␮M. Three normalization methods were used to quantitatively analyze the GAPDH Western blot signal. First, the signal was normalized to the total protein signal on a Coomassie-Blue stained SDS-PAGE gel that was run in parallel (data not shown). Second, the signal was normalized to the total protein signal on blot membranes that were Ponceau S-stained before blocking with BSA (data not shown). Third, the signal was normalized to the actin Western blot signal, which was obtained by stripping the membrane for GAPDH and re-probing it with anti-actin antibody (Fig. 2F). All methods showed similar results. We did not see other notable changes in the higher molecular weight range that may represent the aggregates of GAPDH. The negative actin results (Fig. 2C and D) suggest

Fig. 3. EGCG significantly inhibited the activity of CYP1A and bound to and triggered aggregate in CYP1A1 and CYP1A2. The CYP1A activity was measured using 7ethoxyresorufin as a probe (A). The EGCG-binding proteins were purified and probed with anti-CYP1A1 (B) or anti-CYP1A2 (C) on Western blots. The EGCG-treated microsomes were also directly probed with anti-CYP1A1 (D) and anti-CYP1A2 (F) on Western blots, with the quantitative results presented in (E) and (G), respectively. The lane layout was the same as in Fig. 2. The means of EGCG treated samples were compared to control (0 ␮M EGCG) samples by one-way ANOVA followed by Dunnett’s test. *p < 0.05. The CYP1A activity in the control microsomes was 38.92 ± 5.53 pmol/min/mg protein.

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Fig. 3. (Continued ).

that EGCG-induced aggregate formation was also protein selective. In addition, the Western blot signal of GAPDH in the mitochondrial (Fig. 2G) and cytosolic fraction (Fig. 2H) was also significantly decreased by EGCG treatment. The most remarkable change was observed with the mitochondrial fraction, in which the 100 ␮M EGCG treatment almost eliminated the GAPDH signal (Fig. 2G). In contrast, no changes of actin Western blot signals were observed (data not shown).

different pattern. As shown in Fig. 4A, although binding to EGCG was clearly observed, the reduction in its Western blot signal that is indicative of possible aggregate formation was not detected (Fig. 4B and C), and the enzyme activity was not changed after EGCG treatment (Fig. 4D). Among the CYPs tested, CYP3A appeared to be the most sensitive to EGCG- induced alterations in terms of binding, as it was positively detected in 1 ␮M EGCG treated samples (Supplementary Fig. 2A).

3.3. EGCG selectively inhibited the activity of several CYP isoenzymes but bound to and triggered aggregates in all of them

3.4. EGCG selectively inhibited the activity of MGST1 but not cGSTs and bound to and triggered aggregates of MGST1

Major CYP isoenzymes were investigated in the next step. Fig. 3A shows that CYP1A activity was significantly inhibited by EGCG at the concentrations of ≥30 ␮M. Using anti-CYP1A1 and CYP1A2 antibodies, we found that both CYP1A1 and CYP1A2 were among the EGCG-binding proteins (Fig. 3B and C). Fig. 3D–G illustrate that the Western blot signals of both CYP1A1 and CYP1A2 were slightly but statistically significantly reduced after EGCG treatment, indicating that they possibly formed protein aggregates that were not recognizable by the antibodies against the intact proteins. The binding to EGCG and possible aggregate formation correlated well with the inhibitory effects of EGCG on CYP1A activity (Fig. 3A). Similar results were obtained for CYP2B and 3A, as shown in the Supplementary Figs. 1 and 2. However, CYP2E1 displayed a

The abundant microsomal protein MGST1 (Shi and Lou, 2005) was explored in the next step. Fig. 5A shows that MGST1 was remarkably inhibited by EGCG. We also found that EGCG not only bound to MGST1 (Fig. 5B) but also likely induced aggregate formation in MGST1 (Fig. 5C and D). In Fig. 5C, very faint signals were observed at about 34 kDa as shown by the arrow, indicating the formation of MGST1 dimmers, a form of protein aggregates which had been previously observed in peroxynitritetreated MGST1 (Ji et al., 2006). To further study the mechanism of inhibition, the unique feature of MGST1 being activated by NEM via irreversible binding to its cysteine 49 (Ji et al., 2002) was exploited. As shown in Fig. 5A and reported in the legend for Fig. 5, the NEMactivated MGST1, whose activity was about 3-fold higher than in

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Fig. 4. EGCG bound to CYP2E1 but did not induced protein aggregates formation or inhibit its catalytic activity. The enriched EGCG-binding proteins were probed with anti-CYP2E1 (A) antibody, with the original microsomal protein as a control (lane 2). In B, the EGCG-treated microsomes were separated on SDS-PAGE gels followed by Western blot detection of CYP2E1, with the quantitative data presented in C. The lane layout was the same as in Fig. 2. The (D) shows the CYP2E1 activity. The means of EGCG treated samples were compared to control (0 ␮M EGCG) samples by one-way ANOVA followed by Dunnett’s test. No statistical differences were observed across all treatment groups. The CYP2E1 activity in the control microsomes was 1.18 ± 0.12 nmol/min/mg protein.

the control microsomes, was still inhibited by EGCG, but the extent of inhibition was significantly less than in the non-NEM-treated microsomes. To test if EGCG inhibition of MGST1 could be expanded to cGSTs, we measured the cGST activities in the liver cytosolic proteins. As illustrated in Fig. 5A, the activity of cGSTs was not affected by EGCG, suggesting that the inhibition is protein isoform selective.

3.5. EGCG selectively bound to and triggered aggregate formation in membrane-bound but not soluble COMT COMT, an abundant liver enzyme with soluble and membrane bound isoforms (Karhunen et al., 1994), was examined in further experiments. As shown in lane 2 of Fig. 6A–B, anti-COMT antibody produced two bands. The band with the higher molecular weight, that is, about 28 kDa, represents the membrane-bound isoenzyme of COMT, while the one with lower molecular weight, that is, about 24 kDa, represents the soluble isoenzyme of COMT. In Fig. 6A, it is clearly demonstrated that the membrane-bound, but not soluble, COMT specifically bound to EGCG. Fig. 6B–D shows that both isoenzymes of COMT possibly formed protein aggregates after EGCG treatment, as indicated by the finding that their Western blot signals were decreased at the position of normal molecular weight,

with the membrane-bound form more profoundly affected than the soluble one.

3.6. EGCG induced protein aggregates were completely prevented but the binding to proteins was only partially abolished in the presence of 1 mM glutathione In the last set of experiments, we examined if the physiological anti-oxidant glutathione, which was absent in the previous tests, had any influence on EGCG effects. As shown in Fig. 7A, 1 mM glutathione prevented the formation of EGCG-induced protein aggregates. This was also confirmed by detecting specific proteins using the antibodies such an anti-CYP1A1, anti-CYP3A, and anti-MGST1 to probe the microsomal proteins on Western blots. No reduction in the signals was observed (data not shown). However, EGCG-binding to proteins was only partially abolished by 1 mM glutathione (Fig. 7B). Of note, in the high molecular weight range, that is, >150 kDa, no apparent “smearing” of bands, as appeared in glutathione-free microsomes (Fig. 1C), were observed, confirming that the protein aggregates were entirely prevented by glutathione. Among the specific proteins tested, binding to MGST1 and inhibition of MGST1 activity by 7.5–45 ␮M EGCG were completely eliminated by 1 mM glutathione, but the effects of 60–100 ␮M

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Fig. 5. EGCG selectively inhibited the activity MGST1 but not cGSTs and bound to and triggered aggregates of MGST1. (A) shows the GST activities. In (B), the EGCG-binding proteins were purified and probed with anti-MGST1 on Western blots. The EGCG-treated microsomes were also directly probed with anti-MGST1 (C) on Western blots, with the signals normalized to anti-actin antibody signal obtained from stripped membranes (D). The lane layout was the same as in Fig. 2. The means of EGCG treated samples were compared to control (0 ␮M EGCG) samples by one-way ANOVA followed by Dunnett’s test. *p < 0.05; # p < 0.05. The means of 0.5 mM NEM-treated samples were compared to 0 mM NEM-treated samples by two-way ANOVA followed by Bonferroni test. The MGST1 activity in control and NEM-treated microsomes were 44.79 ± 5.21 and 114.47 ± 3.83 nmol/min/mg protein, respectively. The cGST activity in the cytosolic fraction was 904.30 ± 29.03 nmol/min/mg protein.

EGCG were only partially abolished. Similar results were obtained in CYP isoenzymes (data not shown). 4. Discussions The EGCG concentrations we tested here are relevant to the in vivo conditions. After a single oral administration in healthy humans of purified EGCG or a green tea extract containing a mixture of flavonoids, the average maximum plasma concentration (Cmax ) of EGCG was determined to be 0.3–7.4 ␮M depending on the dose (Chow et al., 2005; Ullmann et al., 2003) and EGCG was predominantly found in the free form in plasma (Chow et al., 2005). Though the average Cmax of EGCG was 0.3–7.4 ␮M, some individuals had as high as 13.2 ␮M EGCG in the blood (Chow et al., 2005; Ullmann et al., 2003). We observed EGCG binding to proteins and protein aggregate formation starting as low as 1 ␮M EGCG, implying that these findings are likely physiologically or pathophysiologically relevant, at least in some individuals. It should be pointed out that glutathione is present at millimolar concentration in the liver under physiological conditions (Wu et al., 2004). We found that 1 mM glutathione completely prevented protein aggregate formation and partially abolished EGCG binding to protein. It seems that our findings, particularly protein aggregate formation, may not be applicable to healthy individuals that have sufficient reserves of glutathione. However, glutathione is easily

depleted by toxicant insults, such as acetaminophen overdose (Yang et al., 2012). Our results would be particularly meaningful in these settings. In addition, EGCG itself may deplete hepatic glutathione (Galati et al., 2006), and we speculate that once glutathione is depleted below a protective level by EGCG, protein binding and aggregate formation would occur. Our findings are therefore helpful to understand the toxicity, especially hepatotoxicity (Galati et al., 2006; Lambert et al., 2010), triggered by EGCG alone. A key finding in this study was that MGST1 might be a novel protein target for EGCG. MGST1 has both GST and glutathione peroxidase activities and serves as an important antioxidant enzyme (Shi et al., 2012). The unique feature of MGST1 is that its single cysteine 49 can be modified to enhance the catalytic activities and thus help protect cells from oxidative damage (Shi et al., 2012). Here we found that EGCG significantly inhibited, instead of activated, MGST1, possibly leading to its decreased anti-oxidative capability. The findings that the inhibitory effect of 7.5–45 ␮M EGCG was completely abolished by the presence of 1 mM GSH and was significantly reduced in NEM-treated microsomes strongly suggest both the involvement of cysteine 49 and non-cysteine related modification(s). This could in part explain why EGCG, generally considered an anti-oxidant due to it polyphenolic structure, showed prooxidant effects under certain circumstances (Yang et al., 2004). Similar to MGST1, COMT also has two isoenzymes (Karhunen et al., 1994) and we show here that only the membrane-bound, but

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Fig. 6. EGCG selectively bound to and triggered aggregate formation in membrane-bound but not soluble COMT. The EGCG-binding proteins were purified and probed with anti-COMT antibody on Western blots (A). The EGCG-treated microsomes were also directly probed with anti-COMT antibody on Western blots (B), with the signals normalized to anti-actin antibody signal obtained from stripped membranes (C) and (D). (C) and (D) show the quantitative data of membrane-bound and soluble COMT, respectively. The lane layout was the same as in Fig. 2. The means of EGCG treated samples were compared to control (0 ␮M EGCG) samples by one-way ANOVA followed by Dunnett’s test. *p < 0.05.

Fig. 7. EGCG-induced protein aggregates were completely prevented but the binding to proteins was only partially abolished in the presence of 1 mM glutathione. The microsomes were supplemented with 1 mM glutathione and then incubated with 0–100 ␮M EGCG for 30 min. The proteins were either directly resolved in SDS-PAGE and visualized by Coomassie Blue staining (A) or subjected to purification of EGCG-binding proteins, which were then analyzed by SDS-PAGE (B). The lane layout was the same as in Fig. 1A and C, respectively.

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not soluble, form was susceptible to binding to EGCG. It has been well-documented that EGCG inhibits the soluble COMT in both rat (Lu et al., 2003) and human livers (Chen et al., 2005; Nagai et al., 2004), and such inhibition has been suggested to be an important mechanism for the beneficial effects of EGCG on Parkinson’s disease (Kang et al., 2010). Our data clearly exclude, in rat liver, the possibility that the inhibition is due to binding of EGCG to soluble COMT and highlight the importance to explore alternative mechanism(s). We additionally found that EGCG did bind to membrane-bound COMT. Whether the binding has functional significance represents an interesting future direction. The finding that CYP2E1 activity was not affected by EGCG in spite of significant binding to EGCG implies that the binding could be a possible protective mechanism against excessive EGCG, similar to that of protein S-glutathionylation against excessive oxidized glutathione (Dalle-Donne et al., 2009). It is also likely that the CYP2E1-EGCG complex is more resistant than CYP2E1 itself to other detrimental injuries such as inhibition by the anti-alcohol drug disulfiram via thiol modifications (Frye and Branch, 2002). The possible EGCG-disulfiram interaction represents an interesting topic, as both of them appear to cause a change in the thiol groups of CYP2E1, with the latter causing a decrease in activity (Frye and Branch, 2002) and the former being ineffective (Fig. 4D). To determine the relative expression levels of proteins by Western blot, GAPDH and actin are two widely used house-keeping proteins, that is, their expression levels are assumed to be constant and not to be affected by various treatments. Our finding that EGCG caused a decrease of antibody binding to GAPDH in a Western blot raises the possibility that previous reports on EGCG regulating protein expression using GAPDH as a control (Park et al., 2011) may not be sufficiently accurate, particularly those that used high concentrations of EGCG in in vitro systems (Park et al., 2011). In contrast, the reports using actin as a control (Yang and Raner, 2005) should be deemed more reliable and actin is strongly recommended for future studies. The reduction in GAPDH Western blot signal (Fig. 2C and D) was partially against our expectations, as we anticipated that a decrease of signal in the original GAPDH position, that is, around 36 kDa, would be accompanied by the appearance of additional bands in the higher molecular weight range that represent the aggregates of GAPDH. We speculate that significant structure changes occurred in the GAPDH aggregates, making them not recognizable by the antibody against the intact protein. Specifically, the epitopes recognized by antibody against the intact protein might be masked in the aggregates, as has been reported in other proteins (Choukhi et al., 1999). It is also likely that the aggregates have poor solubility and therefore did not enter into the gels and thus escaped the detection by Western blot. The possibility that EGCG-binding itself caused the decrease of GAPDH signal was dismissed, as EGCGbound GAPDH was readily detectable by its antibody (Fig. 2A). The above speculations may also explain the reduction of Western blot signals in other proteins tested. It was previously noted that EGCG enhanced the protein expression of some CYP isoenzymes but suppressed their corresponding catalytic activities (Yang and Raner, 2005). This puzzling issue might be partially explained by the present study. It is likely that EGCG not only induced the protein expression of these CYP isoenzymes, but also covalently bound to them causing the decreased activities. This is not uncommon for other irreversible (mechanismbased) CYP inhibitors since short-term decreases in CYP activity are often accompanied by longer term induction of CYP expression, most likely a result of a feedback loop (Phillip and Williams, 2000). Data presented here could also help explain our recent findings that green tea extract with 48.4% EGCG prevented acetaminophen hepatotoxicity when given 3 h prior to acetaminophen challenge, but enhanced the toxicity if administrated 6 h after acetaminophen

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exposure (Salminen et al., 2012). When administered 3 h prior to the acetaminophen challenge, green tea extract essentially eliminated the production of toxic acetaminophen metabolite Nacetyl-p-benzoquinone imine (NAPQI), as measured by covalent binding to protein using Western blots (Salminen et al., 2012), presumably due to CYP inhibition. Our in vitro results provide mechanistic insight into this inhibition and indicate that EGCG in the green tea extract may covalently bind to various CYPs and inhibited their activity in vivo as we saw in the study referenced above. This in turn prevented the metabolism of acetaminophen to the reactive metabolite NAPQI and decreased acetaminophen hepatotoxicity. In contrast, when green tea extract was administered 6 h after the acetaminophen exposure, cellular damage had already occurred (Yang et al., 2012) and this would have allowed the administrated EGCG to effectively bind to and, possibly trigger the aggregation of MGST1, which might have already been activated by binding to NAPQI via its cysteine 49 (Yonamine et al., 1996). This could have led to remarkably decreased anti-oxidant capability of MGST1 and therefore enhanced liver toxicity in green tea extract co-treated animals as compared to the controls that were dosed with only acetaminophen. Further studies are needed to test this interesting possibility. Taken together, this study provided novel insights into the mechanisms of EGCG induced inhibition of drug metabolizing enzymes in hepatic microsomes. EGCG binding to the enzymes and protein aggregate formation were the key events at the molecular level that might contribute to the inhibitory effects of EGCG on various enzymes. These functional and structural changes are protein selective and protein isoform dependent, are partially preventable by glutathione, and do not always correlate well with each other. We also identified MGST1 and membrane-bound COMT as new protein targets for EGCG. These findings will aid in the understanding of the mechanisms of EGCG actions, particularly possible EGCG-drug interactions that lead to toxicity. Conflict of interest statement None declared. Acknowledgements Dr. Zuquan Weng is supported by the Research Participation Program at the National Center for Toxicological Research administrated by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and the US Food and Drug Administration. Disclaimer: This article is not an official guidance or policy statement of the US Food and Drug Administration (FDA). No official support or endorsement by the FDA is intended or should be inferred. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.toxlet.2012.09.011. References Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Chang, T.K., Crespi, C.L., Waxman, D.J., 2006. Spectrophotometric analysis of human CYP2E1-catalyzed p-nitrophenol hydroxylation. Methods in Molecular Biology 320, 127–131. Chen, D., Wang, C.Y., Lambert, J.D., Ai, N., Welsh, W.J., Yang, C.S., 2005. Inhibition of human liver catechol-O-methyltransferase by tea catechins and their metabolites: structure-activity relationship and molecular-modeling studies. Biochemical Pharmacology 69, 1523–1531.

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