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J. Agric. Food Chem. 2003, 51, 7617−7623
Effects of Garlic Powders with Varying Alliin Contents on
Hepatic Drug Metabolizing Enzymes in Rats
ANNE-MARIE LE BON,*,† MARIE-FRANCE VERNEVAUT,† LUCIEN GUENOT,† REMI KAHANE,‡ JACQUES AUGER,§ INGRID ARNAULT,§ THOMAS HAFFNER,| AND Unite´ Mixte de Recherche de Toxicologie Alimentaire, Institut National de la Recherche Agronomique, 17 rue Sully, 21065 Dijon Cedex, France, Coop d’Or R&D, INRA, Laboratoire de Physiologie et de Culture In vitro, 21100 Bretenie`res, France, Universite´ Franc¸ois Rabelais, IRBI, CNRS UMR 6035, Parc Grandmont, 37200 Tours, France, and Lichtwer Pharma AG, Wallenroder Strasse 8-10, D-13435 Berlin, Germany The anticarcinogenic effect of garlic has been demonstrated in both epidemiologic and experimentalstudies. In this study, possible mechanisms involved in the anticarcinogenic effect of garlic consumptionwere assessed by determining its capacity to alter drug metabolizing enzymes, in relation with itsalliin content. Rats were fed a diet for 2 weeks containing 5% garlic powders produced from bulbsgrown on soils with different levels of sulfate fertilization and therefore containing differing amountsof alliin. Activities of several hepatic enzymes, which are important in carcinogen metabolism suchcytochromes P450 (CYP) and phase II enzymes, were determined. Garlic consumption slightlyincreased ethoxyresorufin O-deethylase and CYP 1A2 levels. In contrast, garlic consumptiondecreased CYP 2E1 activity and the level of the corresponding isoform. UDP glucuronosyl transferaseand glutathion S-transferase activities were increased by garlic powders. The alliin content of thegarlic powders was positively correlated with UGT activity although not with other activities. Effectsproduced by garlic consumption were qualitatively similar to that of diallyl disulfide, a sulfur compoundthat has been extensively studied. These data could partially explain the chemoprotective effect ofgarlic.
KEYWORDS: Garlic; alliin; diallyl disulfide; liver; drug-metabolizing enzymes; rat
INTRODUCTION
carcinogens and thereby may influence the carcinogenicity ofchemicals (5). Several crude extracts of fruits, vegetables, and Fruit and vegetables, or their constituents, are considered good spices also have the ability to inhibit chemically induced candidates in the prevention of carcinogenesis (1, 2). Most carcinogenesis by modifying carcinogen metabolizing enzymes carcinogens are not reactive in themselves but require bio- activation to form ultimate metabolites that bind covalently to Garlic has long been considered a healthy food. More DNA and cause an alteration of the genome, which can further recently, the anticarcinogenic effects of garlic have been lead to mutagenesis and carcinogenesis. The toxicity of a demonstrated in both epidemiologic and experimental studies chemical carcinogen depends on the balance between detoxi- (6, 7). Among the possible mechanisms involved in the cation and metabolic activation. Compounds that are able to anticarcinogenic effects of garlic, its capacity to decrease modulate the activities of enzymes responsible for the metabo- activation and increase detoxication of carcinogens appears to lism of carcinogens are of interest when they reduce the level be of prime importance. Indeed, several organosulfur compounds of activation enzymes and enhance the activities of detoxication or garlic preparations such as garlic oil have been shown to be (3, 4). Many phytochemicals present in fruits and vegetables efficient inhibitors of CYP 2E1 and can therefore block the such as polyphenols, indoles, isothiocyanates, and organosulfur activation of nitrosamines and other compounds activated by compounds are known to affect the biotransformation of this CYP (8, 9). Induction of CYP 1A, 2B, and 3A has alsobeen observed with sulfides and polysulfides (10-12). The * To whom correspondence should be addressed. E-mail: lebon@ induction of phase II enzymes such as GST, QR, and UGT has † Institut National de la Recherche Agronomique.
also been demonstrated (13-17). These increased activities ‡ Present address: CIRAD-FLHOR, bd de la Lironde, 34398 Montpellier partially account for the protection provided by garlic sulfur compounds against mutagenesis and chemical carcinogenesis J. Agric. Food Chem., Vol. 51, No. 26, 2003 Table 1. Composition of the Experimental Diets
Figure 1. Chemical structures of alliin and DADS.
Plant Material, Cultivation, and Preparation of Garlic Powders.
Garlic (Allium satiVum) was produced in a field experiment carried Most investigations on the effects of garlic on carcinogen out in Crest, Droˆme (France). Certified seed material, variety Printanor, metabolizing have dealt with isolated pure compounds. So far, was supplied by the Institut National de la Recherche Agronomique effects of the whole garlic bulb have been the subject of little d’Avignon (France). Seeds were planted early in the year 2000 and research. Park et al. (20) have demonstrated a decrease in CYP harvested 6 months later. Sulfur fertilization was provided by dehydrated 2E1 in rats fed garlic powders while Ip et al. showed an increase CaSO4 (50% CaSO4), which was applied before bulb formation. The in phase II enzymes by selenium-enriched garlic powder in rats experimental design included four treatments: 0, 50, 100, and 200 kg/ (21). The first objective of this study was to determine the effect ha CaSO4, with four replications for each level of fertilization. Each of garlic powder administered in the diet on a set of carcinogen plot comprised 100 plants. The bulbs were harvested when consideredmature (juice above 30° Brix of the juice). They were air-dried naturally metabolizing enzymes and to compare these effects with those and cured when completely dry (3-4 weeks later).
produced by a pure sulfur compound, DADS (Figure 1), whose
Afterward, the bulbs were processed as follows. Bulbs were properties are well-characterized (10, 12). DADS was described mechanically peeled in a four step process, which consisted of heating to be a breakdown product from allicin, which is formed from (3-5 h at 50 °C), cracking, cutting, and blowing with high air pressure.
sulfur compounds present in situ in the bulb. As it is possible Slicing produced pieces of garlic, 0.5 mm thick, that subsequently to enrich garlic bulbs in sulfur compounds by fertilizing the underwent a dehydrating process as follows: 2 h at 70 °C, followed by crop with sulfate (22), the second objective was to evaluate 65 °C overnight and 60 °C with 10% air renewal for the last 2 h.
whether the fertilization conditions of the crop could influence Samples were dehydrated separately to check the evolution of the dry the efficiency of garlic to modify drug metabolizing enzymes.
matter content. When stabilized, the dry matter content was registered, In the present study, rats were fed a diet for 2 weeks and the dry flakes were cooled before ground to a powder in a mill(<25 µm particles).
containing powdered garlic produced from bulbs grown in soils Analysis of Sulfur Compounds in the Powder. Garlic powder (1
with different levels of sulfate fertilization and therefore g) was extracted at room temperature with 10 mL of methanol/water containing different amounts of sulfur compounds such as alliin (80/20, v/v) and 0.05% formic acid (pH < 3). An aliquot was diluted (Figure 1). Alliin is a precursor of volatile sulfur compounds
five times and filtered (0.2 µm), and 10 µL was analyzed by HPLC.
and can form allicin, which is considered a biologically active HPLC analysis was carried out using a Waters 616 pump and DAD compound (23). The sulfur compounds of the garlic powders 996, diode array detector (Waters, Milford, MA). Compounds were were analyzed by HPLC. Activities of several hepatic CYPs separated on a 150 mm × 3 mm I.D., 3 µm particle Hypurity Elite C18 enzymes, which are important in carcinogen metabolism, were column Thermo Quest at 38 °C (Thermo Hypersil, Keystone, Bellefonte, determined: EROD as a marker of CYP 1A1 and CYP 1A2, PA) and a UV detector operating at 208 nm. The column flow rate which are active toward polycyclic hydrocarbons and hetero- was 0.4 mL/min. The mobile phase consisted of (A) 20 mM NaH2PO4 cyclic amines; PROD as a marker of CYP 2B1/2, which is active 10 mM heptane sulfonic acid, pH 2.1; and (B) acetonitrile-20 mM + 10 mM heptane sulfonic acid, pH 2.1 (50:50 v/v). The 1 activation; PNPH as a marker of CYP 2E1, which gradient program was previously described (24). Data were processed is able to metabolize nitrosamines and numerous low molecular with Millenium software from Waters. Sulfur compounds were identi- weight chemicals; and NO as a marker of CYP 3A. Phase II fied by the comparison of their retention times and their spectra with enzyme activities, such as UGT, GST, and QR, involved in standard compounds. Synthetic reference compounds were characterized carcinogen detoxication, were also measured. In addition, by ion trap mass spectrometry (electrospray ionization) in the mutiple immunoblot analyses were performed to assess the microsomal MS mode as previously described (24).
levels of CYP isoenzymes (1A1, 1A2, 2B1/2, 2E1, and 3A2) Animals and Dietary Treatments. The experiment was performed
and the cytosolic levels of GST subunits (A1/A2, A5, M1, M2, with male SPF Wistar rats. Three week old rats purchased from Janvier (Le Genest Saint Isle, France) were housed in individual stainless wirecages, maintained at 22 °C with a 12 h light-dark cycle. They weremaintained in accordance with the French Ministry of Agriculture MATERIAL AND METHODS
guidelines for care and use of laboratory animals. They were fed a Chemicals. DADS (purity 80%) was obtained from Sigma-Aldrich
purified diet whose composition is reported in Table 1. Water was
Chimie (Saint Quentin Fallavier, France). Polyclonal antibodies raised added to the diet in the ratio of 50 g of water/100 g of dry matter.
against rat CYP 1A1, CYP 2B1, CYP 2 E1, and CYP 3A2 were At the start of the study, the animals were divided into six groups, purchased from Gentest (Woburn, MA). Those raised against rat each containing six rats. These groups were designated as C, S0, S50, GSTA1/A2, GSTM1, and GSTM2 were obtained from Biotrin Inter- S100, S200, and DADS. Group C was the control group and was given national (Dublin, Ireland), and the polyclonal antibody raised against the purified diet. Groups S0, S50, S100, and S200 were given the same GST P was obtained from Medical and Biological Laboratories Co.
diet containing 5% of the different powders issued from bulbs grown Ltd. (Nagoya, Japan). The polyclonal antibody raised against GSTA5 on soils fertilized with 0, 50, 100, and 200 SO4 kg/ha, respectively.
was kindly donated by Prof. D. J. Hayes (University of Dundee, U.K.).
The garlic powders were incorporated into the diet at the expense of Other chemicals were of the highest quality available.
sucrose and casein (Table 1). The group DADS received a diet
Garlic and Hepatic Drug Metabolizing Enzymes in Rat J. Agric. Food Chem., Vol. 51, No. 26, 2003 containing 0.05% DADS (3.4 mmol/kg diet). DADS was first dissolvedin corn oil and then mixed with the rest of the diet. Food intake wasrecorded daily, and rats were weighed twice a week during the 2 weekfeeding period. At the end of this period, rats were killed after 16 h offasting.
Preparation of Microsomal and Cytosolic Fractions. The animals
were killed, and the livers were immediately removed. Microsomesand cytosols were prepared by differential centrifugation and stored insmall aliquots at -80 °C (25). The protein levels of the microsomaland the cytosolic fractions were measured by the method of Bradford(26) adapted for the use of a Cobas Fara II centrifugal analyzer (RocheInstruments), using serum albumin as a standard.
Enzyme Assays. Total microsomal CYP content was quantified
Figure 2. HPLC profile of garlic powder. Key: 1, alliin; 2, γ-glutamyl-S-
according to Omura et al. (27). Determination of EROD and PROD allyl-L-cysteine; 3, γ-glutamyl-S-(trans-1-propenyl)-L-cysteine; and 4, activities was adapted from the method of Burke et al. (28). Reactions were carried out in a fluorimeter at 37 °C using a Cobas Fara IIcentrifugal analyzer (Roche Instruments, Switzerland). The concentra-tions of substrates ethoxyresorufin and pentoxyresorufin were 5 and10 µM, respectively.
The assay for PNPH activity was determined by HPLC (29). The concentration of the substrate p-nitrophenol was 0.1 mM, and themicrosomal protein was 0.5 mg/mL. The reaction product, p-nitrocat-echol, was monitored at 340 nm.
NO activity was determined by HPLC (30). The concentration of the substrate nifedipine was 0.2 mM, and the microsomal protein was1 mg/mL. UV detection of the product dihydronifedipine was performedat 254 nm.
Total GST activity was measured with 1-chloro-2,4-dinitrobenzene as the substrate (31). The reaction mixture contained 1 mM GSH and1 mM substrate. The formation of the conjugate was continuouslymonitored at 340 nm.
Figure 3. Alliin concentration of four garlic powders (S0, S50, S100, and
UGT activity was determined with 0.15 mM p-nitrophenol as a S200) grown with different levels of sulfate fertilization. Garlic powders substrate and with 3 mM UDP glucuronic acid (32). Microsomes were were produced from bulbs grown in a field. Sulfur fertilization was provided activated with Triton X-100, so that the ratio of Triton to protein by four levels of CaSO4 (0, 50, 100, and 200 kg/ha). Values are means concentration was 0.2. The measurement of GST and UGT activities was adapted for the use of a Cobas Fara II centrifugal analyzer.
Immunoblot Analyses. Immunoblot procedures were performed as
Table 2. Effects of Garlic Powders (S0, S50, S100, and S200) and
previously described (25). For the detection of CYP 1A1, CYP 1A2, DADS on Body Weights, Liver Weights, and Relative Liver Weights of CYP 2B1, CYP 2E1, and CYP 3A2, hepatic microsomes from rats treated, respectively, with methylcholanthrene, phenobarbital, pyrazole,and dexamethasone were used as positive controls. Quantification of individual bands was done by comparing blot density between treated and control rats using an image analyzer (Bioscan Optimetric, Edmonds, Statistical Analyses. Enzymatic activity data were treated by analysis
of variance followed by a Dunnett’s test to assess the difference between treatments and control. The level of significance was P e 0.05. Results of enzyme activities were correlated with the alliin content of the powder using linear regression analysis. The statistical significance wasanalyzed using the parametric test of Pearson. Correlations were a Values are means ± SEM (n ) 6). b Significantly different from control mean considered to be statistically significant when P was e0.05. Calculations were made with StatBoxPro v5. (Grimmer-soft, Paris, France).
significant (Table 2). The liver weights were also reduced by
treatment with garlic powders although the differences were not Sulfur Analysis of Garlic Powders. The following com-
significant. In contrast, DADS treatment significantly increased pounds were identified in the garlic powders: alliin, γ-glutamyl- S-allyl-L-cysteine, γ-glutamyl-S-(trans-1-propenyl)-L-cysteine, Enzyme Activities. Rats fed with the garlic powders and
and γ-glutamyl-phenylalanine (Figure 2). Alliin, a precursor
DADS had a lower content of hepatic CYP content (76-83% of biologically active compounds of garlic, was quantified in of the control) (Figure 4). PNPH activity was also decreased
the garlic powder (Figure 3). A strong relationship was found
by 50-60% in rats consuming diets containing garlic powders between the level of sulfate fertilization and the alliin content or DADS. EROD activity was increased by 30-40% by garlic of the garlic powders (correlation coefficient ) 0.998).
powders S50, S100, and S200. The increase was greater with Food Consumption, Body Weights, and Liver Weights.
DADS (76% of control). No significant variation of PROD and The presence of garlic powders or DADS in the diet modified NO activities was observed (Figure 4).
the food intake of the rats for few days. The growth of the treated UGT activity was significantly increased in the groups S100 rats declined during the first days due to lower food consumption and S200 and in the DADS group (50-100% of control value) and then recovered. At the end of the feeding period, weights (Figure 5). GST activity was slightly increased by all of the
of the rats fed with garlic powders or DADS were lower than treatments, although the increase was only significant for the those of the control group, although these differences were not S200 group and the DADS group. QR activity was increased J. Agric. Food Chem., Vol. 51, No. 26, 2003 Figure 4. Effects of garlic powders (S0, S50, S100, and S200) and DADS on hepatic CYP-dependent activities. Results are presented as means ± SEM
(n ) 6). Values with an asterisk differ significantly from the control value C (Dunnett’s test P e 0.05).
by 25-47% by rats consuming garlic powders, although these was observed in the S100 and S200 groups. CYP 1A1 was not differences were not significant. The QR activity was signifi- cantly doubled by rats consuming DADS.
GST levels of different classes R, µ, and π were also analyzed Correlation Between the Alliin Content of Garlic Powders
by immunodetection (Table 5). GST A1/A2 was increased by
and Their Effects on Enzyme Activities. As the garlic powders
35-50% in S0 and S50 groups and was doubled in the DADS contain different amounts of alliin, we assessed the relationship group. GST A5 was not detected in the groups treated with garlic between the alliin content of garlic and its efficiency as powders but was visible in the DADS group. GST M1 was not modulators of enzyme activities. The correlation coefficients modified by any treatment whereas GST M2 was increased by between the alliin content of the garlic powder and its efficiency S50, S100, S200, and DADS groups. GST P was detected only on enzyme activities are presented in the Table 3. Among all
of the enzyme activities, only UGT activity was linearly relatedto the alliin content of garlic, showing statistical significance.
DISCUSSION
Immunoblot Analyses. Immunoblot analyses were carried
out to ascertain whether the observed increased or decreased In this study, we have demonstrated that feeding rats with CYP activities were accompanied by elevation or repression of diets supplemented with garlic powders for 2 weeks modulates specific CYP isoenzymes. Table 4 shows that levels of CYP
drug metabolizing enzymes. The response elicited by garlic 1A2 apoprotein were increased by 40-100% by the garlic consumption was qualitatively similar to that produced by powders and by DADS, while CYP E1 levels were decreased DADS fed alone. The alliin content of the powder was positively by the same treatments. The level of CYP 2B1 isoform was correlated with the increase of UGT activity although not with increased by 57% only in the DADS group. In the other groups, the other drug metabolizing enzyme activities.
there was no elevation of this isoform. In the same manner, Feeding rats with garlic for 2 weeks increased some enzyme there was no elevation of CYP 3A2 although a slight decrease activities depending on the particular form of CYP. Garlic Garlic and Hepatic Drug Metabolizing Enzymes in Rat J. Agric. Food Chem., Vol. 51, No. 26, 2003 garlic. Because the CYP1A2 subfamily is closely associatedwith the activation of carcinogens such as heterocyclic amines,it is suggested that garlic might enhance their activation.
Actually, it has been shown previously that DADS treatmentincreased hepatic CYP1A2 levels with a parallel increase ofthe mutagenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (8). Garlic powder also caused a weak increase ofPROD and NO activities although the increases were notsignificant. Moreover, no increase of the correponding isoformsCYP 2B1 and CYP 3A2 was observed. The weak modificationsof CYP 2B1- and CYP 3A-dependent activities suggest thatgarlic consumption would have no effect on the activation ofcarcinogens, such as aflatoxin B1, which are activated by theseisoforms. Conversely, garlic consumption decreased total CYPconcentration in the liver, the level of CYP 2E1 isoform, andPNPH activity, suggesting a possible action of garlic on reducingthe activation of low molecular weight carcinogens or nitro-samines, which are metabolized by this CYP. The mutagenicityof dimethylnitrosamine, a nitrosamine selectively activated byCYP 2E1, has been demonstrated to be strongly inhibited byDADS (8). In addition, the carcinogenicity of diethylnitrosamineand dimethylhydrazine was also demonstrated to be reducedby administration of polysulfides from garlic (34-36). Inhibitionof CYP 2E1 is considered to be a major mechanism by whichgarlic consumption exerts its chemopreventive effect againstnitrosamine-induced cancer (20).
Some phase II enzymes were modified by garlic powder consumption. GST activity was slightly increased. This activitywas significantly higher when rats were fed with garlic powdercontaining the highest amount of alliin (S200). There arenumerous studies showing the increasing effects of isolatedgarlic sulfur compounds or garlic oil on GST activity (13, 37,38). With regard to the effects of garlic consumption, there arecontradictory results. Schaffer et al. (39) reported that consump-tion of garlic powder (2% in the diet) had no effect on GSTactivity whereas Polasa et al. (40) showed a significantstimulation of GST activity in rats fed with 1% garlic powder.
In our study, we have studied this more deeply. We examined Figure 5. Effects of garlic powders (S0, S50, S100, and S200) and DADS
variations in the relative proportions of several GST subunits on phase II enzyme activities. Results are presented as means ± SEM and observed an increase of only GST M2, while GST A5 and (n ) 6). Values with an asterisk differ significantly from the control value P were not detected. The induction of the GST M2 subunit is C (Dunnett’s test P e 0.05).
interesting in terms of chemoprevention because it representsone of the major GST subunits in the rat liver and it is involved Table 3. Correlation of Enzyme Activities with the Alliin Content of the
in the detoxication of mutagenic metabolites of carcinogens such as benzo[a]pyrene 4,5-oxide and styrene oxide (41). Therefore,it seems reasonable to postulate that a protective effect of garlic powder against the carcinogenic action of these compounds could be expected. In fact, garlic powder consumption reduced dimethylbenzanthracene-induced mammary tumor in rats (42, 43). UGT measured with p-nitrophenol as substrate was significantly increased by almost all treatments. This kind of enzyme is actively involved in the detoxication of carcinogens such as polycyclic hydrocarbons and heterocyclic aromatic amines (44). All of the modifications of detoxication enzymessuch as GST and UGT cannot fully explain the anticarcinogenic a The statistical significance was analyzed using the parametric test of Pearson.
effects of garlic powder, but their increase may be a contributing Correlations were considered to be statistically significant when P was e0.05.
In the present study, we compared the effects of garlic with powder caused a modest, yet statistically significant increase those of DADS and observed that the pattern of enzyme in EROD activity. This increase was accompanied by an modifications was qualitatively similar as that of DADS, namely, elevation of CYP 1A2 levels while CYP 1A1 was not detected decrease of CYP 2E1 and increase of UGT and GST activities.
in any treated groups. Both CYP 1A1 and 1A2 are responsible In our extraction and analysis conditions, DADS was not for EROD activity (33). Therefore, the EROD increase is detected in the garlic powder. Therefore, two hypotheses have suggested to be mainly due to an up-regulation of CYP 1A2 by been put forward to explain this similarity: (i) sulfur compounds J. Agric. Food Chem., Vol. 51, No. 26, 2003 Table 4. Effect of Garlic Powders (S0, S50, S100, and S200) and DADS Consumption on the Expression of Hepatic CYP Isoenzymes
a Western blots analysis was performed with 20 µg of microsomal proteins. Quantification of individual bands was done by comparing blot density between treated and control rats with an image analyzer. Values are means of two rats and represent percentage of control, which was arbitrarily attributed to the value 100. b Antibody raisedagainst CYP 1A1, diluted 1:1000; this antibody cross-reacts with CYP 1A2; nd, not detected. c Antibody raised against CYP 2B1, diluted 1:2000. d Antibody raised againstCYP 2E1, diluted 1:4000. e Antibody raised against CYP 3A2, diluted 1:2000.
diallyl disulfide; EROD, ethoxyresorufin O-deethylase; PROD, Table 5. Effects of Garlic Powders (S0, S50, S100, and S200) and
DADS on Hepatic GST Subunits Expression
pentoxyresorufin O-dealkylase; PNPH, p-nitrophenol hydroxy-lase; NO, nifedipine oxidase; HPLC, high-performance liquid LITERATURE CITED
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