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Carcinogenesis Advance Access originally published online on July 8, 2006
Carcinogenesis 2006 27(12):2483-2490; doi:10.1093/carcin/bgl118
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Potent protective effect of isoimperatorin against aflatoxin B1-inducible cytotoxicity in H4IIE cells: bifunctional effects on glutathione S-transferase and CYP1A

Yuba Raj Pokharel, Eun Hee Han, Ji Young Kim, Soo Jin Oh1, Sang Kyum Kim1, Eun-Rhan Woo, Hye Gwang Jeong* and Keon Wook Kang*

College of Pharmacy, Chosun University Seosuk-dong, Dong-gu, Gwangju 501-759, South Korea
1 College of Pharmacy and Research Center for Transgenic Cloned Pigs, Chungnam National University Daejeon, 305-764, South Korea

*To whom correspondence should be addressed Email: kwkang{at}chosun.ac.kr or hgjeong{at}chosun.ac.kr


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Typically chemopreventive agents either induce phase II detoxifying enzymes or inhibit the cytochrome P450 enzymes (CYPs) that are required for the metabolism of carcinogens. In this study, we isolated a coumarin compound, isoimperatorin from Poncirus trifoliata Raf., and studied its protective effects against aflatoxin B1 (AFB1)-induced cytotoxicity in H4IIE cells. Isoimperatorin (>0.3 µM) significantly inhibited the cytotoxic effect of AFB1. CDNB [1-chloro-2,4-dinitrobenzene; glutathine S-transferase (GST) subtype-non-specific] and NBD (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; GST{alpha} type-specific) assays revealed that isoimperatorin (0.3–3 µM) increased GST activity in a concentration-dependent manner. Western blot analyses using subtype-specific antibodies confirmed that GST{alpha} protein, but not GSTµ or GST{pi}, was induced in cells treated with isoimperatorin. Reporter gene analysis using an antioxidant response element (ARE) containing construct and subcellular fractionation assays revealed that GST{alpha} induction by isoimperatorin is associated with Nrf2/ARE activation. Moreover, ethoxyresorufin-O-deethylase assays showed that isoimperatorin (2 µM) completely inhibited 3-methylchoranthrene-inducible CYP1A activity. These results indicate that isoimperatorin from Poncirus trifoliata Raf. possesses a potent hepatoprotective effect against AFB1, presumably through the induction of GST{alpha} and the direct inhibition of CYP1A, and suggest that isoimperatorin should be considered a potential chemopreventive.

Abbreviations: 3-MC, 3-methylcholanthrene; AFB1, aflatoxin B1; AhR, aryl hydrogen receptor; ARE, antioxidant response element; CDNB, 1-chloro-2,4-dinitrobenzene; C/EBP, CCAAT/enhancer binding protein; CYP, cytochrome P450; DRE, dioxin response element; EROD, ethoxyresorufin-O-deethylase; ETRF, ethoxyresorufin; GST, glutathione S-transferase; NBD, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; Nrf2, NF-E2 related factor2; XRE, xenobiotics response element


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Many chemotherapeutic agents have been synthesized and applied clinically. However, serious side effects and resistance to anticancer drugs frequently occur. Hence, much effort has been directed toward identifying the natural constituents of herbs that influence apoptosis and carcinogenesis. Cancer chemoprevention can be defined as the use of naturally occurring or synthetic agents to prevent, inhibit or reverse carcinogenesis (1). Diverse phytochemicals can act as chemopreventive agents, including diallyl sulfide from garlic and dithiolthiones from cruciferous plants, which are generally recognized to protect tissues and inhibit carcinogenesis (2–4).

Many carcinogens are metabolized by cytochrome P450 (CYP) enzymes to chemically reactive species that covalently bind to DNA and promote carcinogenesis. These reactive metabolites may subsequently be involved in additional metabolic processes by phase II detoxifying enzymes and be converted to inactive products. Hence, the possible mechanisms of chemoprevention by plant compounds involve either the induction of phase II detoxifying enzymes, such as glutathione S-transferase (GST) (5), or the inhibition of the CYPs required for the metabolism of carcinogens, such as, the CYP1A family (6). The long-term exposure of animals to chemopreventive agents increases hepatic GST activity by upregulating transcription (7,8). Oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione], a dithiolethione, is under development as a chemopreventive for malignancies such as hepatic and colorectal cancers (4,9), and the mechanism underlying the effect of oltipraz involves the induction of GST (9,10). CYP1A1 and 1A2 are believed to activate carcinogens. In particular, CYP1A1, an inducible form, is mainly involved in the biotransformation of polycyclic aromatic hydrocarbons (PAHs) to carcinogenic metabolites (6). Thus, CYP1A1 expression is likely to be positively correlated with the risk of lung (11) or colorectal cancer (12).

Several phytochemicals have been shown to modulate the activities of drug metabolizing enzymes. These include the induction, activation and inhibition of these enzymes (13). We previously reported that chemical species containing the flavone moiety induce GST{alpha} protein (14). Oral flavone administration to Wistar rats increases GST activity (15). Moreover, dietary genistein or daidzein have been reported to elevate GST activities in rat kidney (16), and quercetin was found to increase CYP1A1 enzyme activity through aryl hydrogen receptor (AhR)-dependent CYP1A1 induction (17). Moreover, tangeretin, a flavone found in citrus fruits, inhibits CYP1A2 in rat and in human liver microsomes (18).

The dried immature fruit of Poncirus trifoliata Raf. (Rutaceae), Ponciri Fructus, is widely used as a traditional medicine in Eastern Asia, especially for treating inflammation, ulcers and hepatotoxicity, and it has been reported that crude extracts and a coumarin compound isolated from Ponciri Fructus have anti-inflammatory, anti-bacterial and anti-mucin releasing activities (19–21). Recently, Yi et al. (22) demonstrated that high concentrations (500 µg/ml) of Ponciri Fructus extracts cause cancer cell-specific apoptosis in HL-60 cells, a human leukemia cell line. During our on-going screening program to identify chemopreventive compounds in medicinal plants, we found that the coumarin, isoimperatorin, isolated from the dried immature fruits of Poncirus trifoliata Raf. potently inhibits aflatoxin B1 (AFB1)-induced cytotoxicity in H4IIE cells, a rat hepatocyte-derived cell line. In addition, we found that isoimperatorin selectively induces GST{alpha} by activating NF-E2 related factor2 (Nrf2)/antioxidant response element (ARE) and inhibits CYP1A enzyme activity irrespective of the enzyme expression.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials
Anti-GST{alpha}, µ and {pi} antibodies were obtained from Detroit R&D (Detroit, MI). Monoclonal antibodies against rat CYP1A1 (Mab 1-7-1) were a generous gift from Dr Sang Shin Park (Seoul, Korea, NCI, NIH). Anti-Nrf2 antibody (H-300) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA); alkaline phosphatase or horseradish peroxidase (HRP)-conjugated donkey anti-mouse, anti-rabbit and anti-goat IgGs were from Jackson ImmunoResearch (West Grove, PA); 5-bromo-4-chloro-3-indoylphosphate/nitroblue tetrazolium and pRL-SV40 plasmid were from Promega (Madison, WI); anti-actin antibody and general reagents used in the molecular studies were from Sigma Chemical (St Louis, MO); 7-ethoxyresorufin and resorufin from Pierce Chemical (Rockford, IL). Human Nrf2 overexpressing plasmid was obtained from 21C Frontier human gene bank (Daejon, South Korea).

Extraction and isolation of isoimperatorin
The dried immature fruit of Poncirus trifoliata Raf. (Rutaceae), Ponciri Fructus (600 g) was extracted with MeOH at room temperature to afford 140.0 g of residue. This extract was then suspended in water and partitioned sequentially against dichloromethane, ethyl acetate and n-butanol. Subsequently, 1 g of the methylene chloride soluble fraction was subjected to silica gel column chromatography and eluted using a hexane–EtOAc gradient system (20:1-> 15:1-> 10:1-> 8:1-> 6:1-> 4:1-> 2:1-> 1:1-> 1:2-> MeOH only). Fractions were combined based on TLC results to yield subfractions designated D1–D10. Subfraction D10 (19.2 mg) was purified by repeated RP silica gel column chromatography by eluting with MeOH:H2O (3:1, v/v) to afford isoimperatorin (4.5 mg). The physical and chemical data of the isolated product, which included UV, IR, 1H NMR, 13C NMR and HSQC data were identical with published results (23,24).

Cell culture
H4IIE cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 50 U/ml penicillin and 50 µg/ml streptomycin at 37°C in humidified 5% CO2 atmosphere.

Preparation of nuclear extracts
Nuclear extracts were prepared essentially according to our previously published method (25). Briefly, cells in dishes were washed with ice-cold phosphate-buffered saline (PBS), removed by scraping, transferred to microtubes, treated with 100 µl hypotonic buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.5% Nonidet P-40, 1 mM dithiothreitol and 0.5 mM phenylmethylsulfonylfluoride (PMSF). Lysates were then incubated for 10 min in ice and centrifuged at 7200 g for 5 min at 4°C. Pellets containing crude nuclei, were resuspended in 50 µl of extraction buffer containing 20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 10 mM dithiothreitol and 1 mM PMSF and then incubated for 30 min in ice. Samples were then centrifuged at 12 000 g for 10 min to obtain supernatants containing nuclear fractions. Nuclear fractions were stored at –70°C until required.

Immunoblot analysis
SDS–PAGE and immunoblot analysis were performed as described previously (25). The cells were lysed in a buffer containing 20 mM Tris–HCl (pH 7.5), 1% Triton X-100, 137 mM sodium chloride, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 25 mM ß-glycerophosphate, 2 mM sodium pyrophosphate, 1 mM PMSF and 1 µg/ml leupeptin, and cell lysates were centrifuged at 10 000 g for 10 min to remove cell debris. Proteins were fractionated using a 10% separating gel, and electrophoretically transferred on to nitrocellulose paper, which was then incubated with form-specific anti-GST{alpha}, µ and {pi} antibodies or anti-CYP1A1 antibody, treated with alkaline phosphatase or HRP-conjugated secondary antibody, and developed using 5-bromo-4-chloro-3-indoylphosphate and nitroblue tetrazolium or using a West-Zol chemiluminescence detection kit (Intron Biotech, Seongnam, Korea).

Construction of GSTA2 promoter-luciferase constructs and luciferase assays
Firefly-luciferase reporter gene constructs pGL-1651 (–1.65 promoter region of the rat GSTA2 gene) and pGL-797 (797 bp promoter region of the GSTA2 gene) were generated, as described previously (25). To determine promoter activity we used a dual-luciferase reporter assay system (Promega, Madison, WI). Briefly, cells (3 x 105 cells/well) were replated in 12-well plates overnight and transiently transfected with each GSTA2 promoter-luciferase construct and pRL-SV40 plasmid (Renilla-luciferase expression was used for normalization) (Promega, Madison, WI) using Genejuice Reagent (Novagen, Madison, WI). Transfected cells were exposed to isoimperatorin for 18 h, and firefly- and Renilla-luciferase activities in cell lysates were measured using a luminometer (Turner Designs; TD-20, CA). Relative luciferase activities were calculated by normalizing GSTA2 promoter-driven firefly-luciferase activity versus that of Renilla-luciferase. pCYP1A1-DRE reporter plasmid (482 bp fragment of the murine CYP1A1 gene upstream region: –1306 to 824) (26) containing four dioxin response elements (DRE; TTGCGTGAGA) was also used to assess specific activation of xenobiotics response element (XRE) by isoimperatorin.

EROD activity
Ethoxyresorufin-O-deethylase (EROD) activity is a specific assay for the bioactivation of CYP1A and was determined in intact cells, as described by Kennedy and Jones (27) at 37°C using 5 µM ethoxyresorufin (ETRF) in growth medium as a substrate in the presence of 1.5 mM salicylamide to inhibit conjugating enzymes (28). Resorufin fluorescence generated by ETRF conversion by CYP1A was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm for 30 min in a FL-600 multi-plate fluorescence reader (BIO-TEK, USA).

Statistical analysis
Scanning densitometry was performed using a Microcomputer Imaging Device, Model M1 (Imaging Research, Ontario, Canada). One way analysis of variance (ANOVA) was used to assess significant differences between treatment groups. For each significant treatment effect, the Newman–Keuls test was used to compare multiple group means. Criteria for statistical significance were set at P < 0.05 or P <0.01, as indicated. All statistical tests were two-sided.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Potent protective effect of isoimperatorin on AFB1-inducible cytotoxicity
We isolated the coumarin, isoimperatorin, from the immature fruits of Poncirus trifoliata Raf. The structure of isoimperatorin is illustrated in Figure 1A. Initially, we examined the effect of isoimperatorin on AFB1-induced cytotoxicity in H4IIE cells. AFB1 is a mycotoxin produced by the fungus Aspergillus flavus and a potent hepatotoxin and hepatocarcinogen (29). Pretreating cells with isoimperatorin (0.1–3 µM, 12 h) was found to significantly inhibit the cell death induced by 100 or 300 µM AFB1 (Figure 1B) and the IC50 value of isoimperatorin against the AFB1-cytotoxicity (100 µM) was 189 nM. However, other coumarins (heraclenol 3'-methyl ester, heraclenin, oxypeucedanin methanolate, isomerazin and poncimarin) also isolated from Poncirus trifoliata Raf., did not protect against the cytotoxicity by AFB1 (Figure 1C).


Figure 1
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  		Fig. 1 (A) Structure of isoimperatorin isolated from the dried immature fruits of Poncirus trifoliata Rafin. (B) Concentration-dependent effect of isoimperatorin on AFB1-induced cytotoxicity. Cells were serum-starved and incubated in the presence or absence of various concentrations of isoimperatorin (0.1–3 µM) for 12 h, and then treated with 100 or 300 µM AFB1 for additional 12 h. Cells viabilities were assessed by MTT assay. Data represent the means ± SD of 8–16 different samples (significant versus the untreated control: **P < 0.01; significant versus the AFB1-treated group: ##P < 0.01). (C) Effects of five coumarins (10 µM) isolated from Poncirus trifoliata Rafin. on AFB1-induced cytotoxicity. MTT assays were performed as described in (B). Data represent the means ± SD of eight different samples (significant as versus the untreated control: **P < 0.01; significant versus the AFB1-treated group: #P < 0.05; ##P < 0.01).

 
Selective induction of GST{alpha} by isoimperatorin
AFB1 is biotransformed to the reactive metabolite AFB1 8,9-epoxide, which reacts with and damages DNA. The operative major detoxification pathway involves glutathione (GSH) conjugation of AFB1 8,9-epoxide by GST. Thus, an increase in GST activity is believed to be an important protective mechanism in the detoxification of AFB1 (30). In order to determine whether the coumarin affects the enzyme activities of GST, we performed CDNB (1-chloro-2,4-dinitrobenzene) and NBD (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) assays using cell lysates obtained from H4IIE cells pretreated with isoimperatorin (0.1–10 µM, 24 h), and isoimperatorin was found to significantly increase CDNB (GST subtype non-specific) enzyme activity (Figure 2A). In addition, coumarin enhanced NBD enzyme activity (Figure 2A), which reflects specific GST{alpha} subtype catalytic activity (31).


Figure 2
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  		Fig. 2 (A) Effect of isoimperatorin–GST activity. Cells were serum-starved and incubated in the presence or absence of isoimperatorin (1–10 µM) for 24 h; cell lysates were used to determine GST activities. CDNB (1-chloro-2,4-dinitrobenzene) assays (left panel) determine subtype-non-specific GST activity and NBD (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) assays (right panel) determine GST{alpha} subtype-specific enzyme activities. Data represent means ± SD of six different samples (significant versus the untreated control: *P < 0.05; **P < 0.01). (B) Effect of isoimperatorin on GST protein levels. The protein expressions of GST subunits were monitored 18 h after treating cells with isoimperatorin (0.1–10 µM). Lanes were loaded with 10 µg of cytosolic protein. Data represent the means ± SD of three separate experiments (significant versus the untreated control: *P < 0.05; **P < 0.01; control level = 1).

 
Next, we determined GST subtype levels after treating H4IIE cells with the coumarin. Cells exposed to isoimperatorin for 18 h showed significantly increased levels of GST{alpha}, but the protein levels of other GST subtypes, i.e. GSTµ and {pi}, were unaffected by isoimperatorin (Figure 2B). In particular, the expression of the GST{alpha} subtype in cells treated with 3 or 10 µM isoimperatorin increased 2.3- and 2.2-fold, respectively (Figure 2B). Moreover, this result is consistent with our NBD enzyme activity assay result. Hence, our results show that the selective induction of GST{alpha} by isoimperatorin seems to be associated with increased GST activity.

Nrf2/ARE activation by isoimperatorin
The expression of the GSTA2 gene depends on ARE and/or XRE (32). Nrf2 is a key transcription factor that binds to ARE sequences, which is implicated in the regulation of GST{alpha} expression (33–36). Whereas, the activations of C/EBPß and AhR were associated with the induction of GST{alpha}, via their binding to the XRE sequence (14,25,37). To determine whether the induction of GST{alpha} by isoimperatorin is mediated via the activation of Nrf2/ARE or XRE, reporter gene analyses were performed using H4IIE cells transfected with the mammalian expression vectors pGL-1651 or pGL-797, which both contained the luciferase structural gene and the GSTA2 promoter (25). Exposure of H4IIE cells, transiently transfected with pGL-1651 containing both ARE and XRE sequences, to isoimperatorin (3 µM) caused a 3.1-fold increase in luciferase activity (Figure 3A). In the case of pGL-797 reporter plasmid, the XRE sequence was deleted from the promoter sequence, and relative inducible luciferase activity by isoimperatorin (1 or 3 µM) in cells transfected with pGL-797 was also significantly increased and the increase ratio (5.4-fold by 3 µM isoimperatorin) was slightly higher than that obtained by transfecting with pGL-1651 (Figure 3B). These results provide evidence that isoimperatorin induces GST{alpha} via ARE activation.


Figure 3
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  		Fig. 3 (A) Induction of luciferase activity by isoimperatorin in H4IIE cells transiently transfected with the GSTA2 chimeric gene construct pGL-1651 containing both the ARE and XRE elements. Dual-luciferase reporter assays were performed on lysed H4IIE cells co-transfected with pGL-1651 (firefly-luciferase) and pRL-SV (Renilla-luciferase) (in the ratio 100:1) after exposure to isoimperatorin (0.3–3 µM) for 18 h. Reporter gene activations (firefly-luciferase activity) were expressed as ratios relative to Renilla-luciferase activity. Data represent the means ± SD of four separate experiments (significant as compared to the control: *P < 0.05). (B) Induction of luciferase activity by isoimperatorin in H4IIE cells transiently transfected with the GSTA2 chimeric gene construct, pGL-797, containing the ARE element. Dual-luciferase reporter assays were performed as described in (A). Data represent the means ± SD of four separate experiments (significant versus the control: **P < 0.01). (C) Nuclear translocation of Nrf2 by isoimperatorin. The nuclear localizations of Nrf2 were assessed immunochemically by using Nrf2 specific antibody in cells treated with isoimperatorin (3 µM). All lanes contained 20 µg of nuclear extracts. Nuclear extracts (hNrf2) obtained from H4IIE cells transfected with human Nrf2 overexpressing plasmid were used as controls. Equal protein loadings were verified by Ponceau-S staining.

 
To confirm this finding, we also examined whether isoimperatorin stimulates the translocation of Nrf2 to the nucleus, which is essentially required for Nrf2 binding to the ARE consensus sequence (34,38). Subcellular fractionation and immunoblot analyses revealed that isoimperatorin increased the level of Nrf2 in nuclear fractions at 3–6 h (Figure 3C). These data suggest that the induction of GST{alpha} by isoimperatorin is closely associated with Nrf2-mediated ARE activation.

Inhibition of CYP1A activity by isoimperatorin
Human and rodent AFB1 metabolisms are believed to be mainly catalyzed by CYP1A and CYP3A (29,39). A previous study also showed that the chemical inhibition of CYP1A blocked the formation of major metabolites (AFM1 and AFB1-dihydrodiol) in bovine hepatocytes (40). In order to test whether isoimperatorin affects the enzyme activity of CYP1A, we performed EROD assays. Isoimperatorin alone did not affect EROD activities, whereas marked increases in EROD activities were observed in H4IIE cells treated with 3-methylcholanthrene (3-MC), thus reflecting a specific increase in CYP1A enzyme activity (41). The induction of 7-ETSF was 17-fold that of untreated control in cells incubated with 1 µM of 3-MC for 18 h (Figure 4A). Moreover, pretreatment of cells with isoimperatorin significantly inhibited 3-MC induced EROD activity in a concentration-dependent manner (Figure 4A). In particular, the inhibitory effect of 2 µM isoimperatorin was >67% (Figure 4A). A variety of phytochemicals inhibit CYP1A1 activity by blocking CYP1A1 gene expression at the transcription level (42,43). Thus, we further evaluated whether isoimperatorin affects CYP1A1 expression. Western blot analysis revealed that isoimperatorin (2 µM) itself did not inhibit CYP1A1 protein (Figure 4B, upper panel), but the compound rather enhanced CYP1A1 protein levels in response to 3-MC (Figure 4B, lower panel). Although, potentiation of 3-MC-inducible CYP1A1 induction by isoimperatorin was found, the enzyme activity was potently blocked in cells treated with isoimperatorin. This indicates that CYP1A inhibiting effect of isoimperatorin may be mediated through direct enzyme inhibition.


Figure 4
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  		Fig. 4 (A) Effect of isoimperatorin on EROD activity. EROD activities were measured in H4IIE cells incubated with 3-MC (1 µM) in the presence or absence of isoimperatorin (0.1–2 µM) for 18 h. Data represent the means ± SD of three separate experiments (significant versus untreated control: **P < 0.01; significant versus the 3-MC-treated group: ##P < 0.01). (B) Immunoblot analysis of CYP1A1 (inducible CYP1A form) protein in H4IIE cells. Cells were treated with 3-MC (1 µM) with or without isoimperatorin (0.5–2 µM), and harvested 18 h after adding chemicals. {alpha}-tubulin levels were measured as loading controls. (C) Immunoblot analysis of nuclear AhR. Cells were incubated with 3-MC (1 µM) and/or isoimperatorin (2 µM) for 90 min. (D) Induction of luciferase activity by isoimperatorin or 3-MC in H4IIE cells transiently transfected with pCYP1A1-DRE plasmid. Dual-luciferase reporter assays were performed as described in (A). Data represent the means ± SD of three separate experiments (significant versus the control: **P < 0.01).

 
To clarify whether isoimperatorin causes transcriptional activation of CYP1A1 gene through AhR activation, we further determined nuclear levels of AhR in cells exposed to the coumarin and 3-MC. Isoimperatorin alone (2 µM) did not cause nuclear translocation of AhR (Figure 4C). Furthermore, 3-MC-induced AhR accumulation in nucleus was not enhanced by isoimperatorin addition (Figure 4C). We also assessed the effect of isoimperatorin on the transactivation of DRE reporter gene (pCYP1A1-DRE). 2 µM isoimperatorin slightly (2-fold) increased DRE reporter activity, but the induction ratio was not comparable to that by 1 µM of 3-MC (15-fold) (Figure 4D). Moreover, isoimperatorin did not potentiate 3-MC-inducible DRE reporter activity (Figure 4D). Hence, isoimperatorin may minimally affect AhR-dependent transcriptional activation of CYP1A1 gene. It would be possible that the coumarin changes CYP1A1 protein stability or regulates other transcription factor(s) (e.g. PPAR-{alpha} and nuclear factor-1) dominating the transcription of CYP1A1 gene (44,45).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Imperatorin and isoimperatorin have anti-tumor functions. In cell cultures, imperatorin was found to inhibit the proliferations of several cancer cell lines (46), and isoimperatorin has been reported to potently protect human hepatoma cells against tacrine-induced cell death (47). Moreover, the coumarins have also been reported to block benzo(a)pyrene or 7,12-dimethylbenz[a]anthracene-induced skin tumor formation (48).

The present study shows that isoimperatorin potently blocks AFB1 toxicity in hepatocyte-derived cell line and that this may be associated with its differential effects on GST and CYP1A. Isoimperatorin was found to selectively induce GST{alpha}, a process associated with AFB1-detoxification. Moreover, an increase in the activity of GST could reduce AFB1-induced cytotoxicity. In general, conjugation between xenobiotics and GSH catalyzed by hepatic phase II enzymes is a critical detoxifying event, and is viewed as an efficient chemoprotective mechanism. Moreover, GSH-conjugation with xenobiotics can be accelerated by GST, and elevated GST activity can potentiate this conjugative reaction and lead to the detoxification of many xenobiotics (4).

The role of Nrf2/ARE in the inducible expression of phase II detoxifying enzymes by several antioxidants and chemopreventive agents has been extensively studied (25,33). Genetic defects in Nrf2 ultimately reduce the expressions of phase II enzymes including GST, and increase the incidence of carcinoma formation in response to carcinogen treatment (49,50). Hence, the activation of Nrf2, which controls the constitutive and inducible expression of GST{alpha}, is believed to be a protective mechanism against carcinogens. The findings of the present study show that isoimperatorin selectively increases the reporter activity of GSTA2 promoter containing ARE and causes the nuclear translocation of Nrf2. Both of these events might induce GST{alpha} and subsequently increase its enzyme activity. GST induction is also mediated through AhR-dependent XRE activation, which frequently observed in cells exposed to planar aromatic compounds (51). In the present study, we found that isoimperatorin increased the pGL-797 reporter activity reflecting ARE and the pGL-1651 reporter activity reflecting both ARE and XRE at the same levels. In addition, isoimperatorin did not increase nuclear level of AhR in H4IIE cells. Monofunctional inducers are known to transcriptionally activate GST{alpha} gene expression through ARE, whereas bifunctional inducers act through XRE and ARE (52,53). Hence, isoimperatorin can be classified as a monofunctional inducer of GST.

CYPs, including CYP1A and 3A4, metabolizes AFB1 to AFB1 epoxide (54) and AFB1-dihydrodiol, the hydrolysis product of AFB1 epoxide, then binds cellular protein via the dialdehydic phenolate ion, which has a cytotoxic effect (55). It was previously reported that AFB1-dihydrodiol formation is inhibited in hepatocytes exposed to AFB1 in the presence of {alpha}-naphthoflavone, which suggests that CYP1A catalyzes the epoxide opening reaction (40). Thus, the protective effects of isoimperatorin on AFB1-induced toxicity might be due to a reduction in CYP1A enzyme activity. Isoimperatorin was found to significantly reduce CYP1A enzyme activity as evidenced by EROD assay results, but levels of CYP1A1 were not decreased by the coumarin. It has been recently reported that phytochemicals containing the coumarin moiety competitively inhibit CYP1B1 enzyme activity in a yeast-microsome expressed enzyme system (56). Thus, our results in combination with previous report suggest that inhibition of CYP1A enzyme activity by isoimperatorin is due to direct inhibition of CYP1A enzyme subtypes, and not to transcriptional inactivation.

In summary, the results of this study demonstrate the protective effect of isoimperatorin on AFB1-induced cytotoxicity in H4IIE cells. GST{alpha} and CYP1A were found to respond differentially to isoimperatorin treatment. Both increased GST{alpha} activity via Nrf2/ARE activation and decreased CYP1A activity via direct enzyme inhibition in hepatocytes treated with isoimperatorin, could contribute to its inhibition of AFB1-induced cell death. Our results suggest that imperatorin isolated from Poncirus trifoliata Raf. potently protects cells from AFB1 and hepatocarcinogenesis.


   Acknowledgments
 
This work was supported by a grant from the Korean Research Foundation (KRF; Basic Science Research Program E00082 [GenBank] , 2005; K.W.K.) and by a grant from Research Center for Transgenic Cloned Pigs (KOSEF grant R11-2002-100-00000-0; S.K.K.).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 3, 2006; revised June 1, 2006; accepted June 23, 2006.


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