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Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona (C.D.F., L.M.A., N.J.C.); Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas (J.M.M., C.D.K.); Department of Discovery Toxicology, Bristol-Myers Squibb, Princeton, New Jersey (D.M.N., L.D.L.-M.); and Department of Biomedical Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island (A.L.S.)
(Received December 13, 2006; accepted March 7, 2007)
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Abstract |
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; Ohaeri and Adoga, 2006). Epidemiological studies suggest that high garlic intake hinders development of certain human cancers and can decrease the risk of distal colon cancer by 50% (Steinmetz et al., 1994). Given these potential benefits of garlic, dietary supplementation with garlic oil (GO) or various types of garlic extracts has increased (Ross et al., 2006).
Gas chromatographic-mass spectral analysis has identified 47 compounds in GO, 18 of which are volatile linear sulfur-containing molecules that account for 94% of GO constituents (Calvo-Gomez et al., 2004). Among the most abundant of these linear sulfur-containing compounds are diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS). DAS can inhibit CYP2E1 activity in vivo and can induce hepatic mRNA levels of CYP1A, CYP2B, and CYP3A (Cherrington et al., 2003; Le Bon et al., 2003). Additionally, administration of DADS or DATS results in induction of phase II and antioxidant enzymes such as glutathione S-transferase (GST), NAD(P)H quinone oxidoreductase 1 (NQO1), UDP-glucuronosyl transferase (UGT), and epoxide hydrolase (Singh et al., 1998; Wu et al., 2002; Fukao et al., 2004). However, the mechanism(s) of drug-metabolizing enzyme induction by GO constituents remains unclear.
The induction of several drug-metabolizing enzymes has been shown to be regulated by the activation of specific transcription factors, including constitutive androstane receptor (CAR) and nuclear factor E2-related factor 2 (Nrf2). These transcription factors act as biosensors for endogenous and xenobiotic chemicals and respond by increasing drug-metabolizing enzyme levels (Zhang et al., 2004). CYP2B and NQO1 induction is the hallmark of CAR and Nrf2 activation, respectively, and both of these genes are induced by GO constituents.
CAR is best known for its ability to regulate induction of the CYP2B gene family following activation by phenobarbital (PB) and a family of PB-like inducers (Swales and Negishi, 2004). CAR plays a key role in the control of drug metabolism by mediating the induction of many phase I and II drug-metabolizing enzymes (such as CYP2B, CYP2C, CYP3A, UGT1A1, and GSTA1), as well as drug transporters, including Mrp2 and Oatp4 (Huang et al., 2003; Arnold et al., 2004).
Nrf2 regulates the gene expression of a battery of enzymes that serve to detoxify electrophiles and pro-oxidative stressors (Numazawa and Yoshida, 2004). Activation of Nrf2 results in transcriptional activation of several genes involved in the antioxidant response, including NQO1, NRH/quinone oxidoreductase 2, GSTA1, 
-glutamylcysteine synthetase, and heme oxygenase 1 (Hayes and McMahon, 2001; Chen and Kong, 2004; Jaiswal, 2004).
Because the intake of garlic and garlic supplements is prevalent, understanding the mechanisms governing the pharmacological actions of garlic is paramount to predict the potential for garlic and garlic supplements to alter drug metabolism. Previous studies reported that garlic alters the pharmacokinetics of several therapeutic drugs, including the HIV protease inhibitor saquinavir, the analgesic/antipyretic paracetamol, and the anticoagulant warfarin (Izzo and Ernst, 2001; James, 2001). Thus, the current study was conducted to determine whether GO and GO constituents, namely, DAS, DADS, and DATS, coordinately regulate drug-metabolizing enzymes by activation of the transcription factors CAR and Nrf2.
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Materials and Methods |
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Animals. Male and female Wistar-Kyoto (WKY) rats and male C57BL6/J wild-type (WT) mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Male mice homozygous for the targeted mutation of CAR were developed as described previously (Wei et al., 2000) and were obtained from Deltagen (San Carlos, CA). Nrf2-/- mice were generated as described previously (Itoh et al., 1997). CAR-/- mice used in the present studies were bred on a mixed SVJ129/C57BL/6 background, whereas Nrf2-/- mice were bred on a C57BL/6 background.
Male and female WKY (n = 5) rats were administered PB (80 mg/kg, i.p.), GO (300 mg/kg, p.o.), DAS (500 mg/kg, p.o.), DADS (200 mg/kg, p.o.), DATS (80 mg/kg, p.o.), or CO. Male WT, CAR-/-, and Nrf2-/- mice were administered GO (175 mg/kg, p.o.), DAS (500 mg/kg, p.o.), DADS (80 mg/kg, p.o.), DATS (80 mg/kg, p.o.), or CO. All the treatments were carried out for 4 days at a volume of 5 ml/kg, and total RNA was prepared from livers.
All the animals were acclimated for at least 1 week before the experiments and were allowed water and standard chow ad libitum. Housing and experimental procedures were in accordance with the Guide for the Care and Use of Laboratory Animals as determined by the U.S. National Institutes of Health.
Total RNA Isolation. Total RNA was isolated using RNAzol B reagent (Tel-Test Inc., Friendswood, TX) as per the manufacturer's protocol. RNA concentrations were determined by UV spectrophotometry, and integrity was examined by ethidium bromide staining after agarose gel electrophoresis.
Branched DNA Assay. Probe sets for rat CYP2B1/2, rat NQO1, mouse CAR, CYP2B10, mouse Nrf2, and mouse NQO1 were used as described previously (Hartley and Klaassen, 2000; Cherrington et al., 2002, 2003; Cheng et al., 2005). Specific oligonucleotide probes were diluted in lysis buffer supplied in the Quantigene HV Signal Amplification Kit (Panomics, Inc., Freemont, CA). All the reagents for analysis (i.e., lysis buffer, capture hybridization buffer, amplifier/label probe buffer, and substrate solution) were supplied in the Quantigene Discovery Kit. Total RNA (1 µg/µl; 10 µl) was added to each well of a 96-well plate containing capture hybridization buffer and 50 µl of each diluted probe set. Total RNA was allowed to hybridize to each probe set overnight at 53°C. Subsequent hybridization steps were carried out as per the manufacturer's protocol, and luminescence was quantified with a Quantiplex 320 branched DNA luminometer interfaced with Quantiplex Data Management Software Version 5.02 in 96-well plates.
In Vivo Luciferase Assay. A human CYP2B6 promoter construct containing a 1.7-kilobase fragment that maintains the core promoter (+39/-364) and the distal enhancer region (-1461/-2013), including the PB-responsive element cloned into pGL3-basic, was obtained from Dr. Richard Kim (Vanderbilt University, Nashville, TN). An AREx5 promoter construct was obtained from Dr. David Ross (University of Colorado Health Sciences Center, Denver, CO) and contains five consecutive copies of the human antioxidant response element (ARE) sequence found in the human NQO1 promoter cloned into an RSV180-luciferase reporter. C57BL6/J mice were matched for age and weight. Mice were given a rapid (5-s) tail vein injection of naked plasmid DNA, either 10 µg of human CYP2B6-luciferase or 3 µg of human AREx5-luciferase reporter constructs, in sterile saline at a volume equal to 10% body weight. Following an 18-h recovery, mice were anesthetized with ketamine/xylazine and injected with 0.07 µl of 25 mg/ml D-luciferin in saline vehicle (Molecular Imaging Products Company) 5 min before imaging. A VersArray 1300B camera (Princeton Instruments, Trenton, NJ) thermoelectrically cooled to -100°C was used to image mice. Images were obtained using Win View 32 software (Princeton Instruments) in gray scale, and pseudo-color maps were created with the Win View 32 program. Color maps were superimposed over the light image of the mouse using Adobe Photoshop CS2 software (Adobe Systems, Mountain View, CA). This image was considered time 0 h. Mice (n = 4) were then administered GO (300 mg/kg, p.o.), DAS (500 mg/kg, p.o.), DADS (200 mg/kg, p.o.), DATS (80 mg/kg, p.o.), or CO in a volume of 5 ml/kg. Images were repeated at 12 and 24 h for the human AREx5- or human CYP2B6-transfected mice, respectively. Quantification of promoter induction was determined by densitometry using SimplePCI software (Compix Inc., Sewickley, PA).
Statistics. Statistical significance was determined by one-way analysis of variance followed by a Newman-Keuls post hoc test between all the groups (p 
0.05).
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Results |
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Figure 1A shows CYP2B1/2 mRNA levels in male and female WKY livers following 4-day treatment with CO vehicle, PB (positive control for CAR activation), GO, and its constituents. PB increased CYP2B1/2 mRNA levels 5.6-fold higher in male WKY rats than in female WKY rats. Similarly, GO, DAS, and DADS induced CYP2B1/2 mRNA 8.5-, 1.3-, and 9.2-fold, respectively, higher in male WKY rats compared with WKY female rats. DATS treatment failed to induce CYP2B1/2 in either male or female WKY rats.
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CYP2B10 and NQO1 Induction in CAR-/- Mice. To definitively determine whether GO and its constituents induce CYP2B10 via CAR activation, levels of hepatic CYP2B10 mRNA were examined in WT and CAR-/- mice following treatment with GO, DAS, DADS, and DATS. GO tended to increase levels of CYP2B10 mRNA in WT and CAR-/- mice; however, this induction was not statistically significant (Fig. 2A). DAS induced CYP2B10 mRNA levels 530-fold in WT mice but not at all in CAR-/- mice. DADS and DATS did not affect hepatic CYP2B10 mRNA levels in WT or CAR-/- mice.
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Hepatic NQO1 levels were also determined in WT and CAR-/- mice administered GO, DAS, DADS, and DATS (Fig. 2B). GO tended to increase NQO1 mRNA levels in WT mice, although this induction did not reach statistical significance from control, whereas GO induced hepatic NQO1 mRNA levels approximately 10-fold in CAR-/- mice. DAS increased NQO1 mRNA levels similarly in WT and CAR-/- mice (31- and 32-fold, respectively). DADS tended to increase the levels of NQO1 mRNA in WT and CAR-/- mice (8- and 9-fold, respectively), but these increases were not statistically significant from control. Similarly, DATS tended to increase NQO1 mRNA levels by 9- and 6-fold in WT and CAR-/- mice, respectively, but neither group was statistically significant from control. To ensure that Nrf2 expression was not altered by the deletion of the CAR gene, Nrf2 mRNA levels were determined and found to be unchanged in WT and CAR-/- mice (data not shown).
CYP2B10 and NQO1 mRNA Induction in Nrf2-/- Mice. To obtain further evidence on whether GO or its constituents induce hepatic drug-metabolizing enzymes via Nrf2 and/or CAR, NQO1 and hepatic CYP2B10 mRNA levels were determined in WT and Nrf2-/- mice. Figure 3A indicates the induction of CYP2B10 in WT and Nrf2-/- mice following treatment with GO, DAS, DADS, and DATS. Surprisingly, GO failed to induce CYP2B10 in WT mice, whereas it produced a 37-fold induction of mRNA levels in Nrf2-/- mice. Mice treated with DAS displayed a robust induction of CYP2B10 that was not dependent on the presence or absence of Nrf2. DADS and DATS treatment did not induce hepatic CYP2B10 in WT or Nrf2-/- animals. To ensure that CAR expression was not altered by the deletion of the Nrf2 gene, CAR mRNA levels were determined, and there was no difference between WT and CAR-/- mice (data not shown).
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Figure 5 shows transcriptional activation of an AREx5-luciferase reporter construct by GO and its constituents. Because induction of mouse NQO1 is not as marked as CYP2B10, we used a human AREx5-luciferase reporter to increase the response of the promoter to Nrf2 binding. Furthermore, Nrf2 activation and nuclear translocation occurs more rapidly than does CAR. Preliminary experiments showed that optimal transcriptional activation occurs at 12 h following administration of the known Nrf2 inducer oltipraz (data not shown). DAS resulted in a robust increase in AREx5 transcriptional activation seen in images of mice treated by DAS. Quantification by densitometry revealed that DAS increased transcriptional activation of the human AREx5 8-fold. However, GO, DADS, and DATS administration resulted in little to no increase in transcriptional activation of the human AREx5-luciferase reporter construct when compared with 0-h images.
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Discussion |
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; Chen et al., 2004; Zhang et al., 2006). Because several of these enzymes fall into specific gene batteries regulated by CAR and Nrf2, the goal of the present study was to determine whether GO and its constituents induce CYP2B and NQO1 via activation of these transcription factors.
Our results show for the first time that GO and DADS significantly induce CYP2B1/2 in rats and confirm DAS induction of CYP2B1/2 observed in previous reports (Lii et al., 2006). Because CAR protein expression in WKY rats is much lower in female than in male livers (Yoshinari et al., 2001), the poor induction of CYP2B1/2 after GO, DAS, and DADS in female rats when compared with male rats strongly suggests that CAR is involved in the hepatic induction of this drug-metabolizing enzyme. Our laboratory has previously shown that DAS treatment results in a greater induction of CYP2B1/2 mRNA levels in male WKY rats than in female rats (Cherrington et al., 2003), similar to the current study. The observation that the sex difference in CYP2B1/2 induction by DAS is smaller than that observed by GO and DADS is curious and may be indicative of the multiple components involved in the CAR-mediated induction of CYP2B1/2. How CAR levels affect either Nrf2 or other transcription factors is the subject of ongoing research.
DAS also induces CYP2B10 in mice (Cheng et al., 2005). Furthermore, the CAR-RXR
heterodimer is important in basal CYP2B10 transcription, as shown by studies using hepatic RXR-/- mice (Cherrington et al., 2003). We have previously shown that DAS induction of CYP2B10 is significantly reduced in RXR-/- mice, suggesting that CYP2B10 induction following DAS administration is CAR-dependent because of the loss of the obligate heterodimerizing partner (Cherrington et al., 2003). The robust induction of CYP2B10 caused by DAS in WT mice was completely absent in CAR-/- mice and indicates that CYP2B10 induction is CAR-dependent. In addition, DAS was shown to activate a human CYP2B6 promoter-reporter construct containing the NR1 CAR-binding element in transiently transfected mice. These results suggest that DAS activation of CAR is a mechanism of CYP2B induction conserved between rats and mice.
DAS significantly increased NQO1 mRNA levels in both male and female WKY rats (Fig. 1B), as well as WT and CAR-/- mice (Fig. 2B). Together these results suggest that DAS activates NQO1 via a CAR-independent mechanism. Previous studies have noted that DAS causes a 3-fold increase in hepatic GST activity in mice, another antioxidant gene regulated by Nrf2 (Srivastava et al., 1997). Additionally, they reported 3.2- and 4.4-fold inductions of hepatic GST mRNA following DADS and DATS treatment, respectively. In contrast, Wu et al. (2004) reported that whereas DADS and DATS increased levels of NQO1 in vitro, DAS failed to induce these enzymes. In the current study, definitive evidence that Nrf2 is involved in the induction of NQO1 by GO and its constituents was determined using Nrf2-/- mice. DAS produced a 6-fold increase in NQO1 mRNA levels in WT mice, which was almost completely prevented in Nrf2-/- mice. In addition, DAS administration to transiently transfected mice was shown to activate a human AREx5-luciferase reporter, suggesting the possibility that DAS might also induce NQO1 in humans.
DAS has been shown to cause nuclear accumulation of CAR and binding to the CAR-specific NR1 element in the promoter of CYP2B1/2 in rats (Zhang et al., 2006). However, DAS activation of CAR has not been examined in mice, and Nrf2 activation has only been implied in vitro with varying results. Two studies have examined the possibility that DAS activates Nrf2 and thus induces antioxidant genes in HepG2 cells. One study showed that DATS was the most potent ARE inducer among the three garlic constituents examined in this study, whereas DAS had no effect on ARE transcriptional activity (Chen et al., 2004). Contrary to the current findings, another study noted significant increases in protein expression, nuclear translocation, and DNA binding of Nrf2, respectively, in HepG2 cells following treatment with DAS (Gong et al., 2004).
Several xenobiotics specifically activate either CAR (PB, chlorpromazine, and phenytoin) or Nrf2 (sulforaphane and butylated hydroxyanisole). The prototypical CAR activator, PB, has been shown to induce Nrf2-regulated genes, including NQO1 in addition to CYP2B10 in the mouse (Slitt et al., 2006). We have further shown that the Nrf2 activator ethoxyquin also increases mRNA levels of CYP2B1/2 in rats, suggesting that this xenobiotic may likewise activate CAR in addition to Nrf2 (Cherrington et al., 2003). Recently, trans-stilbene oxide has been shown to activate both CAR and Nrf2 (Slitt et al., 2006). These results led Slitt et al. (2006) to hypothesize that cross-talk between the CAR and Nrf2 activation pathways could occur with trans-stilbene oxide. The fact that DAS can activate these same transcription factors in both mice and rats suggests the possibility of cross-talk between CAR and Nrf2. Importantly, this may explain the unexpected induction of CYP2B10 by GO in Nrf2-/- mice where induction was not seen in WT mice. Additionally, the observed induction of NQO1 in both CAR-/- and Nrf2-/- mice by GO underscores the complexity of the several components that make up GO. Whereas the concept of cross-talk between CAR and Nrf2 activation pathways has been hypothesized (Slitt et al., 2006), further studies are necessary to determine the nature and biochemical consequences of this potential mechanism.
DAS, DADS, and DATS have been identified as three major constituents in GO (Wu et al., 2004). Because these chemicals have all been documented to affect transcriptional regulation of hepatic phase I and phase II drug-metabolizing enzymes, it is reasonable to expect that GO would have similar effects. Although GO produced significant induction of CYP2B1/2 in WKY rats (Fig. 1A), induction of CYP2B10 was not observed in WT mice following a 4-day induction study. This is almost certainly because of the dose-limiting toxicity observed in mice treated with GO in the current study. Whereas rats were able to tolerate 4 consecutive days of GO (300 mg/kg, p.o.), this dose was not tolerated in mice. Therefore, the GO dose was decreased to 175 mg/kg to complete the 4-day induction studies in mice. A similar situation was observed in mice treated with DADS. Because of toxicity observed with 200 mg/kg DADS in mice, a dose of 80 mg/kg DADS was used to complete the 4-day induction studies. The apparent lack of mouse CYP2B10 induction following GO and DADS could be associated with this decrease in dose concentration. It is also noteworthy that Nrf2-/- mice were particularly susceptible to DATS-induced lethality, an observation not noted in WT or CAR-/- mice. Consistent with previous studies, it is likely that Nrf2 plays a role in preventing xenobiotic toxicity of compounds via induction of detoxification and antioxidant enzymes (Jaiswal, 2004). Unlike the 4-day dosing studies, designed to determine maximal induction of mRNA levels following administration of an inducer, the in vivo transcription assay is designed to measure activation of transcription. We have previously shown that a single dose of trans-stilbene oxide (Slitt et al., 2006) results in a robust transcriptional activation of the human CYP2B-luciferase reporter in this assay. Single doses of GO (200 mg/kg, p.o.) and DADS (200 mg/kg, p.o.) used in the in vivo transcription assay studies were better tolerated than in the 4-day induction studies.
Preclinical evidence continues to elucidate the antibacterial, antithrombotic, and chemotherapeutic properties of fresh garlic extracts, aged garlic, GO, and a number of specific organosulfur compounds generated by processing garlic (Ariga and Seki, 2006; Milner, 2006; Sengupta et al., 2006). As usage of garlic supplements increases, it is important to understand the biological effects of such intake. The present data indicate that a specific constituent of GO, DAS, activates CAR and Nrf2, thereby altering drug metabolism. Thus, the potential for herb-drug interactions with garlic and its organosulfur constituents exists, and garlic intake may need to be taken into consideration in the clinical setting.
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Acknowledgments |
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This work was supported by National Institutes of Health Grants ES007091 (C.D.F.), ES011646 (N.J.C.), DK068039 (N.J.C.), and ES09716 (C.D.K.). This work has been presented at the Mountain West Society of Toxicology meeting, Santa Fe, NM, 2005; the International Society for the Study of Xenobiotics meeting, Maui, HI, 2005; and the National Society of Toxicology, San Diego, CA, 2006.
ABBREVIATIONS: GO, garlic oil; DAS, diallyl sulfide; DADS, diallyl disulfide; DATS, diallyl trisulfide; GST, glutathione-S-transferase; NQO1, NAD(P)H quinone oxidoreductase 1; UGT, UDP-glucuronosyl transferase; CAR, constitutive androstane receptor; Nrf2, nuclear factor E2-related factor 2; PB, phenobarbital; CO, corn oil; WKY, Wistar-Kyoto; WT, wild-type; ARE, antioxidant response element; RLU, relative light unit(s).
Address correspondence to: Nathan J. Cherrington, Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, 1703 East Mabel, Tucson, AZ 85721. E-mail: cherrington{at}pharmacy.arizona.edu
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