Durante mucho tiempo no había principios uniformes para la Atribución de nombres a los antibióticos https://antibioticos-wiki.es . Más a menudo se les llama por el nombre genérico o especie del producto, con menos frecuencia-de acuerdo con la estructura química. Algunos antibióticos se nombran de acuerdo con el lugar donde se asignó el producto.
No job name
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
(1) Dragsted, L. O.; Strube, M.; Larsen, J. C. Cancer-protective
factors in fruits and vegetables: biochemical and biological
background. Pharmacol. Toxicol. 1993, 72, 116-135.
(2) Huang, M. T.; Ferraro, T.; Ho, C. T. Cancer chemoprevention
by phytochemicals in fruits and vegetables. An overview. In Fooda Quantification of individual bands was done by comparing blot density between
Phytochemicals for Cancer PreVention I: Fruits and Vegetables;
treated and control rats with an image analyzer. Values are means of two rats
Ho, C.-T., Osawa, T., Huang, M.-T., Rosen, R. T., Eds.; ACS
and represent percentage of control, which was arbitrarily attributed to the value
Symposium Series 547; American Chemical Society: Washing-
100. b Western blot analysis performed with 10 µg of cytosolic proteins. Antibodies
raised against GST A1/A2 diluted 1:300. c Western blot analysis performed with
(3) Guengerich, F. P. Forging the links between metabolism and
20 µg of cytosolic proteins. Antibodies raised against GST A5 diluted 1:300; nd,
carcinogenesis. Mutat. Res. 2001, 488, 195-209.
not detected; +, visible band. d Western blot analysis performed with 10 µg of
(4) Wattenberg, L. W. Chemoprevention of carcinogenesis by minor
cytosolic proteins. Antibodies raised against GST M1 and GST M2 diluted 1:400.
nonnutrient constituents of the diet. In Food, Nutrition ande Western blot analysis performed with 20 µg of cytosolic proteins; antibodies raised
Chemical Toxicity; Parke, D. V., Ioannides, C., Walker, R., Eds.;
against GST P diluted 1:500; nd, not detected; +, visible band.
Smith-Gordon: London, U.K., 1993; pp 287-300.
(5) Smith, T. J.; Yang, C. S. Effect of food phytochemicals on
xenobiotic metabolism and tumorigenesis. In Food Phytochemi-
present in the powder such as alliin or γ-glutamyl-S-allyl-L-
cals for Cancer PreVention I: Fruits and Vegetables; Ho, C.-T.,
cysteine, which are similar to DADS, could be the active
Osawa, T., Huang, M.-T., Rosen, R. T., Eds.; ACS Symposium
compounds. All of these compounds possess an allyl group,
Series 547; American Chemical Society: Washington, DC, 1994;
which appeared to be critical for this effect (13, 37). (ii) When
the garlic powder is ingested, some sulfur compounds could be
(6) Bianchini, F.; Vainio, H. Allium vegetables and organosulfur
metabolized to DADS. DADS and other volatile sulfur com-
compounds: do they help prevent cancer ? EnViron. Health
pounds were detected in urine following garlic oil ingestion by
Perspect. 2001, 109, 893-902.
(7) Le Bon, A. M.; Siess, M. H. Organosulfur compounds from
In this study, we have shown that consumption of garlic
Allium and the chemoprevention of cancer. Drug Metab. Drug Interact. 2000, 17, 51-79.
powder modulated the activities of carcinogen metabolizing
(8) Guyonnet, D.; Belloir, C.; Suschetet, M.; Siess, M. H.; LeBon,
enzymes. In the case of UGT, the concentration of alliin in garlic
A. M. Liver subcellular fractions from rats treated by organo-
determines its capacity to increase this enzyme activity. Increas-
sulfur compounds from Allium modulate mutagen activation.
ing the sulfur compounds content in garlic bulbs to maximize
Mutat. Res. 2000, 466, 17-26.
its beneficial effects against cancer is a worthwhile consider-
(9) Kwak, M. K.; Kim, S. G.; Kwak, J. Y.; Novak, R. F.; Kim, N.
ation. However, it is important to note that the modulating
D. Inhibition of cytochrome P4502E1 expression by organosulfur
effects of garlic were observed with doses administered at much
compounds allylsulfide, allylmercaptan and allylmethyl sulfide
higher levels than the possible intake through a normal human.
in rats. Biochem. Pharmacol. 1994, 47, 531-539.
Therefore, it would be of great interest to explore whether lesser
(10) Haber, D.; Siess, M. H.; Canivenc-Lavier, M. C.; Le Bon, A.
doses might produce comparable effects. Moreover, it would
M.; Suschetet, M. Differential effects of dietary diallyl sulfideand diallyl disulfide on rat intestinal and hepatic drug-metaboliz-
be interesting to appreciate the effect of garlic consumption on
ing enzymes. J. Toxicol. EnViron. Health 1995, 44, 423-434.
drug metabolizing enzymes in man. This would be useful to
(11) Pan, J.; Hong, J. Y.; Ma, B. L.; Ning, S. M.; Paranawithana, S.
explain the preventive effect of garlic against cancer, reported
R.; Yang, C. S. Transcriptional activation of cytochrome P450
2B1/2 genes in rat liver by diallyl sulfide, a compound derived from garlic. Arch. Biochem. Biophys. 1993, 302, 337-342. ABBREVIATIONS USED
(12) Wu, C. C.; Sheen, L. Y.; Chen, H. W.; Kuo, W. W.; Tsai, S. J.;
Lii, C. K. Differential effects of garlic oil and its three major
CYP, cytochrome P450; GST, glutathione S-transferase; QR,
organosulfur components on the hepatic detoxification system
quinone reductase; UGT, UDP glucuronosyltransferase; DADS,
in rats. J. Agric. Food Chem. 2002, 50, 378-383.
Garlic and Hepatic Drug Metabolizing Enzymes in Rat
J. Agric. Food Chem., Vol. 51, No. 26, 2003
(13) Hu, X.; Benson, P. J.; Srivastava, S. K.; Mack, L. M.; Xia, H.;
(30) Guengerich, F. P.; Martin, M. V.; Beaune, P. H.; Kremers, P.;
Gupta, V.; Zaren, H. A.; Singh, S. V. Glutathione S-transferases
Wolff, T.; Waxman, D. J. Characterization of rat and human
of female A/J mouse liver and forestomach and their differential
liver microsomal cytochrome P-450 forms involved in nifedipine
induction by anti-carcinogenic organosulfides from garlic. Arch.
oxidation, a prototype for genetic polymorphism in oxidative
Biochem. Biophys. 1996, 336, 199-214.
drug metabolism. J. Biol. Chem. 1986, 261, 5051-5060.
(14) Cho, J. Y.; Kim, S. G. Differential induction of hepatic
(31) Habig, W. H.; Pabst, M. J.; Jakoby, W. B. Glutathione S-
microsomal epoxide hydrolase by alkyl sulphides and alkyl ethers
transferases. The first enzymatic step in mercapturic acid
in rat. Xenobiotica 1997, 27, 759-767.
formation. J. Biol. Chem. 1974, 249, 7130-7139.
(15) Hatono, S.; Jimenez, A.; Wargovich, M. J. Chemopreventive
(32) Mulder, G. J.; van-Doorn, A. B. A rapid NAD+-linked assay
effect of S-allylcysteine and its relationship to the detoxification
for microsomal uridine diphosphate glucuronyltransferase of rat
enzyme glutathione S-transferase. Carcinogenesis 1996, 17,
liver and some observations on substrate specificity of the
enzyme. Biochem. J. 1975, 151, 131-140.
(16) Singh, S. V.; Pan, S. S.; Srivastava, S. K.; Xia, H.; Hu, X.; Zaren,
(33) Burke, M. D.; Thompson, S.; Weaver, R. J.; Wolf, C. R.; Mayer,
H. A.; Orchard, J. L. Differential induction of NAD(P)H:
R. T. Cytochrome P450 specificities of alkoxyresorufin O-
quinone oxidoreductase by anti-carcinogenic organosulfides from
dealkylation in human and rat liver. Biochem. Pharmacol. 1994,
garlic. Biochem. Biophys. Res. Commun. 1998, 244, 917-920.
(17) Guyonnet, D.; Siess, M. H.; Le Bon, A. M.; Suschetet, M.
(34) Haber-Mignard, D.; Suschetet, M.; Berges, R.; Astorg, P.; Siess,
Modulation of phase II enzymes by organosulfur compounds
M. H. Inhibition of aflatoxin B1- and N-nitrosodiethylamine-
from allium vegetables in rat tissues. Toxicol. Appl. Pharmacol.
induced liver preneoplastic foci in rats fed naturally occurring
1999, 154, 50-58.
allyl sulfides. Nutr. Cancer 1996, 25, 61-70.
(18) Guyonnet, D.; Belloir, C.; Suschetet, M.; Siess, M. H.; Le Bon,
(35) Wargovich, M. J. Diallyl sulfide, a flavor component of garlic
A. M. Antimutagenic activity of organosulfur compounds fromAllium is associated with phase II enzyme induction. Mutat. Res.
(Allium sativum), inhibits dimethylhydrazine-induced colon
2001, 495, 135-145.
cancer. Carcinogenesis 1987, 8, 487-489.
(19) Hu, X.; Benson, P. J.; Srivastava, S. K.; Xia, H.; Bleicher, R.
(36) Surh, Y. J.; Lee, R. C.; Park, K. K.; Mayne, S. T.; Liem, A.;
J.; Zaren, H. A.; Awasthi, S.; Awasthi, Y. C.; Singh, S. V.
Miller, J. A. Chemoprotective effects of capsaicin and diallyl
Induction of glutathione S-transferase pi as a bioassay for the
sulfide against mutagenesis or tumorigenesis by vinyl carbamate
evaluation of potency of inhibitors of benzo[a]pyrene-induced
and N-nitrosodimethylamine. Carcinogenesis 1995, 16, 2467-
cancer in a murine model. Int. J. Cancer 1997, 73, 897-902.
(20) Park, K. A.; Kweon, S.; Choi, H. Anticarcinogenic effect and
(37) Bose, C.; Guo, J.; Zimniak, L.; Srivastava, S. K.; Singh, S. P.;
modification of cytochrome P450 2E1 by dietary garlic powder
Zimniak, P.; Singh, S. V. Critical role of allyl groups and
in diethylnitrosamine-initiated rat hepatocarcinogenesis. J. Bio-
disulfide chain in induction of Pi class glutathione transferase
chem. Mol. Biol. 2002, 35, 615-622.
in mouse tissues in vivo by diallyl disulfide, a naturally occurring
(21) Ip, C.; Lisk, D. J. Modulation of phase I and phase II xenobiotic-
chemopreventive agent in garlic. Carcinogenesis 2002, 23,
metabolizing enzymes by selenium-enriched garlic in rats. Nutr.Cancer 1997, 28, 184-188.
(38) Munday, R.; Munday, J. S.; Munday, C. M. Comparative effects
(22) Freeman, G. G.; Mossadeghi, N. Influence of sulfate nutrition
of mono-, di-, tri-, and tetrasulfides derived from plants of the
on the flavour components of garlic (Allium satiVum) and wild
Allium family: redox cycling in vitro and hemolytic activity
onion (A.Vineale). J. Sci. Food Agric. 1971, 22, 330-334.
and Phase 2 enzyme induction in vivo. Free Radical Biol. Med.
(23) Lawson, L. D. Garlic: a review of its medicinal effects and
2003, 34, 1200-1211.
indicated active compounds. In Phytomedicines of Europe:
(39) Schaffer, E. M.; Liu, J. Z.; Milner, J. A. Garlic powder and allyl
Chemistry and Biological ActiVity; Lawson, L. D., Bauer, R.,
sulfur compounds enhance the ability of dietary selenite to inhibit
Eds.; ACS Symposium Series 691; American Chemical Soci-
7,12-dimethylbenz[a]anthracene-induced mammary DNA ad-
ety: Washington, DC, 1998; pp 176-209.
ducts. Nutr. Cancer 1997, 27, 162-168.
(24) Arnault, I.; Christides, J. P.; Mandon, N.; Haffner, T.; Kahane,
(40) Polasa, K.; Krishnaswamy, K. Reduction of urinary mutagen
R.; Auger, J. High-performance ion-pair chromatography method
excretion in rats fed garlic. Cancer Lett. 1997, 114, 185-186.
for simultaneous analysis of alliin, deoxyalliin, allicin and
(41) Hayes, J. D.; Pulford, D. J. The glutathione S-transferase
dipeptide precursors in garlic products using multiple mass
supergene family: regulation of GST and the contribution of
spectrometry and UV detection. J. Chromatogr. 2003, 991, 69-
the isoenzymes to cancer chemoprotection and drug resistance. Crit. ReV. Biochem. Mol. Biol. 1995, 30, 445-600.
(25) Haber, D.; Siess, M. H.; de Waziers, I.; Beaune, P.; Suschetet,
(42) Ip, C.; Lisk, D. J.; Stoewsand, G. S. Mammary cancer prevention
M. Modification of hepatic drug-metabolizing enzymes in rat
by regular garlic and selenium-enriched garlic. Nutr. Cancer
fed naturally occurring allyl sulphides. Xenobiotica 1994, 24, 1992, 17, 279-286.
(43) Liu, J.; Lin, R. I.; Milner, J. A. Inhibition of 7,12-dimethylbenz-
(26) Bradford, M. M. A rapid and sensitive method for the quantitation
[a]anthracene-induced mammary tumors and DNA adducts by
of microgram quantities of protein utilizing the principle of
garlic powder. Carcinogenesis 1992, 13, 1847-1851.
protein-dye binding. Anal. Biochem. 1976, 72, 248-254.
(44) Bock, K. W. Roles of UDP-glucuronosyltransferases in chemical
(27) Omura, T.; Sato, R. The carbon monoxide-binding pigment of
liver microsomes. I. Evidence for its hemoprotein nature. J. Biol.
carcinogenesis. Crit. ReV. Biochem. Mol. Biol. 1991, 26, 129- Chem. 1964, 269, 2370-2378.
(28) Burke, M. D.; Thompson, S.; Elcombe, C. R.; Halpert, J.;
(45) Bartzatt, R.; Blum, D.; Nagel, D. Isolation of garlic derived sulfur
Haaparanta, T.; Mayer, R. T. Ethoxy-, pentoxy- and benzyl-
compounds from urine. Anal. Lett. 1992, 25, 1217-1224.
oxyphenoxazones and homologues: a series of substrates todistinguish between different induced cytochromes P-450. Bio-Received for review June 25, 2003. Revised manuscript received chem. Pharmacol. 1985, 34, 3337-3345. October 6, 2003. Accepted October 8, 2003. This work was financially
(29) Tassaneeyakul, W.; Veronese, M. E.; Birkett, D. J.; Miners, J. supported by the grant QLK1-CT-1999-498 from the European Union
O. High-performance liquid chromatographic assay for 4-nitro-
within the Quality of Life program FP5.
phenol hydroxylation, a putative cytochrome P-4502E1 activity, in human liver microsomes. J. Chromatogr. 1993, 616, 73-78.