1. Participant N° : P 12
Unité Mixte de Recherche de Toxicologie Alimentaire INRA-ENSBANA, 17 rue Sully, BP 86510,
21065 Dijon Cedex, France ; Tel : 33 3 80693221 ; Fax : 33 3 80693225 ; E-mail : siess@dijon.inra.fr
Scientific team
Research leader
Dr. Marie-Hélène Siess
(12 person months)
Duties: Co-ordination of this task and carginogenic-metabolising enzymes.
Scientists and engineers
Dr. Anne-Marie Le Bon
(16 person months)
Duties: genotoxicity
Dr. Raymond Bergès
(6 person months)
Duties: Hepatocarcinogenesis
Dr. Caroline Teyssier
(16 person months), has left the lab March 2002
Duties: Metabolism and bioavailibility
of organosulfur compounds
Post-doctoral fellows : Dr E. Germain (Nov/2001Oct/2002)
(12 person months)
Duties: metabolism of sulfur compounds
Research technicians
Marie-France Pinnert
(6 person months)
Duties: Immunohistological methods
for preneoplasic foci
Patrick Tassin and Michel Ponchelet
(6 person months)
Duties: Animal care
Marie-France Vernevaut
(12 person months)
Duties: Enzymes assays and
Immunoblots
Christine Belloir
(16 person months)
Duties: genotoxicity tests
Joëlle Chevalier
(16 person months)
Duties: Metabolism of organosulphur compounds
Contractual links to other participants : yes (P4, P5)
The laboratory has studied for 5 years the chemo-preventive effects of Allium species and is involved
in projects on Allium (onion) and Cancer granted by French ministries (AQS 98.P.0470).
Objectives
The research Unit of Food Toxicology belongs to INRA (Institut National de la Recherche
Agronomique). The laboratory is a part of the Department : „ Nutrition and Food Safety “. The
objectives of this laboratory are to improve knowledge concerning the mode of action of
microconstituents that protect against toxic effects especially those involved in chemical
carcinogenesis. Various approaches are used : (i) regulation of drug metabolising enzymes, (ii)
influence of these microconstituents on DNA alterations mutagenesis and carcinogenesis caused by
chemicals agents and (iii) metabolism and bio-availability of microconstituents. The food
microconstituents belong to different chemical classes : polyphenols (flavonoids, phenolic terpenes,
pro-anthocyanidins), organosulfur compounds from Allium, carotenoids. The proposed project is highly
relevant to ongoing studies on the anticarcinogenic effects of microconstituents of vegetables.
Workplan
WP6
 : milestones completed
Year 1
 In vitro metabolism of sulphur compounds by human and rat subcellular fractions from liver
 In vitro effects of garlic compounds on human CYP P450 isoenzymes
 In vitro evaluation of the antigenotoxic properties of garlic compounds in Hep G2 cells
Year 2
 Determination of the bioavailability of diallyl disulfide in rat by measuring the concentrations of the
metabolites in blood and in main organs.
 Evaluation of the antigenotoxic properties of garlic extracts in tissues of rat
 In vivo effects of garlic extracts on several carcinogen metabolizing enzymes (Phase I and phase
II).
Year 3
Determination of the bioavailability of allicin and garlic powder in rat by measuring the concentrations
of the metabolites in blood and in main organs : In progress
 Evaluation of the effects of subcellular fractions from rats treated with garlic extracts on the
mutagenicity of carcinogens using the Ames test
 In vitro effects of metabolites of garlic compounds on human CYP isoenzymes (additionnal
milestone)
Year4
Investigation of the antiinitiating effects of garlic extracts using the medium term hepatocarcinogenesis
model in the rat.
WP7
Year4
Identification and quantification of sulphur compounds in man by measuring their concentrations in
blood and urine after ingestion of a garlic preparation.
Determination of the levels of phase II enzymes (glutathion S-transferase) in human lymphocytes
Evaluation of the DNA alteration in human lymphocytes and the antimutagenic effects of urine
Deliverables
DH 8
DH 9
DH10
DH 13
DH 14
DH 15
DH 20
DH 21
DH 22
DH 27
In vitro metabolism of diallyl disulfide and allicin (paper send to an editor)
In vitro effects of sulphur compounds on human P450 iso-enzymes (publication in
preparation by Feb 2003)
In vitro evaluation of the anti-geno-toxic properties of garlic compounds in Hep G2 cells
(presentation of a poster + publication in preparation)
In vivo metabolism of diallyl disulfide (paper accepted for publication in Xenobiotica +
presentation of a poster)
Effects of garlic extracts on carcinogen-metabolising enzymes in the rat (publication in
preparation by Feb 2003)
Publication of the in vivo anti-genotoxic properties of garlic in rat (publication in
preparation)
Modulation of the mutagenicity in the Ames test
Performing intervention study with report and analysis of results
Short report on the results concerning biomarkers of cancer in human intervention study
In vivo metabolism of diallyl disulfide and garlic powder
Research activities during the third reporting period
1 - Determination of the bioavailability of garlic powder in rat by measuring the concentrations
of the metabolites in blood and in main organs (in progress)
The aim of the study is to identify and analyse the metabolites available in vivo after a single
oral administration of garlic powder to rats. The organic sulphur metabolites present in the stomach,
liver, intestine, plasma and urine are measured by GC-MS, over a period of 15 days.
Male Wistar rats 8-weeks old are fasted for 18 hours before and 6 hours after garlic
administration. Garlic powder is mixed with water and is administered by gavage at a dose of 5 g / kg
body weight. After oral administration, the animals are maintained separately in metabolic cages.
Urine (kept under cold conditions) is collected every 24 hours. Animals are sacrificed by
exsanguination at , 20, 40, minutes, 1, 2, 4, 6 and 24 hours after oral dosing and then every two days
during 15 days. Livers and stomachs and intestine (40 cm) are removed and weighed. Blood and urine
are immediately centrifuged. All tissues are stored at -20°C prior to analysis.
Tissues are homogenised in distilled water. A protein precipitation is followed by an extraction of the
supernatant three times with dichloromethane. The extracts are then concentrated by evaporation
under a nitrogen stream to a final volume of 500 µl. p-cymene is added nonane as an internal
standard. 2 µl aliquot is injected into the GC MS chromatograph (Perkin Elmer turbomass with a splitsplitless injector). Total ion chromatograms and mass spectra are recorded. For the first acquisition ,
GC-MS is used in scan mode from m/z 28-300 amu in order to ascertain the identification of
compounds by comparison with standards. For the 2 nd acquisition , GC-MS is used in selected ion
monitoring mode.
The first analyses show the formation of allyl methyl sulphide (AMS), allyl methyl sulphoxide
(AMSO) and allyl methyl sulphone (AMSO2) in most tissues. AMSO2 is the most abundant and the
most persistent compound. The next step of this milestones will be the quantification of these
metabolites in different tissues and the determination of pharmacokinetic parameters.
Difficulties : Caroline Teyssier has left our laboratory on March 2002. Her duties were :
metabolism and bioavailability of garlic sulfur compouds. No replacement was possible. Marie-Hélène
Siess has taken in charge these duties and moreover her own duties (coordination of the WP6 and
study of the carcinogen metabolizing enzymes). Therefore the milestone on the metabolism of garlic
compounds is delayed.
2 - Evaluation of the effects of subcellular fractions from rats treated with garlic extracts on the
mutagenicity of carcinogens using the Ames test
The objective is to determine the effects of consumption of garlic powders by rats on the mutagenicity
of chemical carcinogens in the Ames test. The underlying hypothesis is that modulation of the
activities of carcinogen-metabolizing enzymes which has been demonstrated in the second year (DH
14), results in a decrease or in an increase of the mutagenicity of genotoxic compounds.
Therefore, in one hand, we evaluated the effects of hepatic microsomes from garlic-treated
rats, which contain cytochromes P450, on the mutagenicity of indirect-acting mutagens such as a food
heterocyclic amine (PhIP), cyclophosphamide (CP), aflatoxin B1 (AFB1). On the other hand, we
studied the effects of the cytosolic fractions, which contain the major phase II enzymes, on the
mutagenicity of ultimate metabolites (styrene oxide, 4-nitroquinoline-oxide) which are detoxified by
phase II enzymes.
The effects of two garlic powders, obtained from the French trial 2001, were studied. Powders
were prepared from garlic (Printanor) grown on soil with no addition of sulphate (G0) or from garlic
grown on soil fertilized with 200 kg/ha (G200). The G200 powder contains a higher amount of alliin.
2-1 Methodology and study materials
Animals and dietary treatments. Male Wistar rats, 4 weeks old, were housed in individual stainless
cages and maintained at 21°C with a 12-h light-dark cycle. First, they were fed ad libitum a purified
diet then they received the same diet (control group) or a diet containing 5% garlic powder (G0 group:
no fertilisation; G200 group : 200 kg/ha SO4) for 2 weeks. Garlic powders were incorporated into the
diet at the expense of sucrose and casein.
Preparation of hepatic subcellular fractions. At the end of the experimental period, animals were killed
by exsanguination. Livers were removed and pooled. Hepatic subcellular fractions (microsomes and
cytosol) were prepared by differential centrifugation and stored in small aliquots at –80°C. The protein
levels of the microsomal and cytosolic fractions were measured by the method of Bradford (1976)
adapted for automatic measurement using a Cobas Fara II centrifugal analyser (Roche Instruments).
Mutagenicity assays. The effects of treatments on the activation and the detoxification of mutagens
were determined by using hepatic subcellular fractions as the metabolization system in the Ames test.
The Ames test was performed with Salmonella typhimurium strains TA98 and TA100 according to
Maron and Ames (1983) with slight modifications (Guyonnet et al, 2000 and 2001). Briefly, the test
was carried out by incubating the mutagen with the subcellular fraction, the bacteria and cofactors at
37°C for a short period. Then the mixtures were diluted with soft agar, plated into minimal glucose
agar plates and further incubated for 48h at 37°C to allow the development of histidine revertant
colonies (His+). The number of His+ revertants, which is related to the genotoxic potency of mutagens,
was counted on two repetitions of triplicate plates for each dose of mutagen.
2-2 Results and discussion
The effects of hepatic subcellular fractions from rats fed garlic are summarized in table 1.
Results show that only activation of phase 1 substrates is modified by garlic feeding.
Hepatic microsomal fractions from G200-treated rats were somewhat more effective than microsomes
from control rats in converting CP to mutagens (figure 1). Microsomes from rats fed garlic powders
increased the mutagenicity of AFB1 (figure 2). No difference could be discerned between G0 group
and control group. Microsomes from rats fed G0 diet reduced the mutagenicity of the heterocyclic
amine, PhIP (table 1). In contrast, an increased of PhIP mutagenicity is observed by microsomes from
rats fed a diet containing the garlic powder high in alliin (G200). However these effects are not very
important since most of the measures do not significantly differ from the control group.
No effect of garlic diets on detoxification of ultimate mutagens was observed : 4-nitroquinoline-oxide
and styrene oxide mutagenicity was not modified by cytosols from garlic-treated rats (table 1).
In conclusion, hepatic subcellular fractions from rats treated with garlic powders slightly
modified the genotoxicity of mutagens when compared with control. Moderate effects on activation of
mutagens and no effect on detoxification of ultimate mutagens have been observed. The modulation
of phase 2 enzymes induced by garlic diet is probably not enough important to inhibit mutagenicity of
ultimate genotoxins. However, the results from the in vivo study, using the Comete test (DH 15),
showed that consumption of garlic caused a reduction of AFB1 genotoxicity. This discrepancy could
be explained by the fact that phase 2 enzymes which are involved in AFB1 detoxication are not taken
into account in this protocol (Ames test).
3 - In vitro effects of metabolites of garlic compounds on human CYP P450 isoenzymes
This is an additionnal milestone. In the course of your work on the metabolism of sulfur
compouds we have detected new metabolites which were formed after ingestion of a garlic
compounds (diallyl disulfide) : allylmercaptan (AM), allyl methylsulfide (AMS), allyl methyl sulphoxide
(AMSO), allyl methyl sulphone (AMSO2) and allylgltathione sulfide (AGS) (see DH13). Therefore, it
was of interest to assess their effects on human CYPs which are involved in carcinogen activation.
3-1 Methodology and study materials
Human liver samples were provided by the Service de Chirurgie Digestive Thoracique et
Cancérologie of the General Hospital of Dijon, France. The samples were frozen in liquid nitrogen and
stored at – 80°C until use for microsome preparation. The microsomes were prepared by differential
centrifugation. Microsomal proteins were quantified by the method of Bradford.
Microsomes from human B-lymphoblastoid cells expressing cDNA encoding human CYP 1A2,
2A6 and 2E1 were purchased from Gentest Corp. (MA USA). These cells contain a vector with a
human CYP cDNA and human CYP reductase cDNA.
The following CYP activities were measured :
- ethoxyresorufin O-deethylase (EROD) as a marker of CYP 1A2 which is active towards
hydrocarbons
- coumarin hydroxylase (COH), as a marker of CYP 2A6 which is involved in the activation
of nitrosamines
- chlorzoxazone hydroxylase (CZX) as a marker of CYP 2E1 able to metabolize
nitrosamines and numerous low molecular weight chemicals
The metabolites are the following : DADS, allicin (DADSO), AM, AMS, AMSO, AMSO2 and AGS. They
were studied at two doses : 50 and 500 µM.
3–2 Results and discussion
CYP 1A2 is strongly inbited (87 %) by DADSO and lightly inhibited (27 %) by AM. The other
metabolites have no effect (figure 3).
CYP 2 A6 is inhibited by DADSO, AGS (40 %) and less by AMSO2 (10 %) (figure 3)
CYP 2E1 is strongly inhibited (60 %) by DADSO and also by DADS (47 %). AM, AMS and SAC
produce moderate inhibitions (figure 3).
In conclusion : even if the concentrations of metabolites (500 µM) inhibiting human CYP are rather
high, these data suggest that metabolites of garlic sulfur compounds could reduce the genotoxicity of
carcinogens which are metabolized by these CYP.
Significant difficulties or delays experienced during the third period reporting
Caroline Teyssier left the lab in March 2002. Her duties were : the metabolism of sulfur
compounds from garlic. Marie-Hélène Siess have taken in charge these duties moreover her own
duties (study of drug metabolizing enzymes and management of the WP6). This has delayed the
milestones about the metabolism of garlic powder. Next year a postdoc which will be recruited and will
take in charge this part of the program.
Anne-Marie Le Bon have a maternity leave from November 2002 to March 2003. Her duties are on the
genotoxicity. No replacement is possible.Therefore the writing of the paper (DH 10) is postponed
Sub-contracted work during the third reporting period
None
EXPLOITATION AND DISSEMINATION ACTIVITIES
Papers accepted for publication or sent to an editor :
Publications
In vivo metabolism of diallyl disulphide in the rat: Identification of two new metabolites. By Germain E.,
Auger J., Ginies C., Siess M-H. and Teyssier C. 2002 Accepted for publication in Xenobiotica, paper in
press.
In vitro and ex vivo metabolism of diallyl disulfide rat and human liver enzymes.
Germain E., Chevalier J. Siess M-H. and Teyssier C. send to Drug Metabolism and Disposition
Posters
In vitro and ex vivo metabolism of diallyl disulfide, a characteristic sulfur compoud of garlic , in rat
Germain E., Chevalier J. Steib M., Siess M-H. and Teyssier C.
6th International ISSX meeting Munich , GER october 2001
Evaluation of the antigenotoxic potential of garlic sulfur compounds in HepG2 cells
C. Belloir, M.H. Siess, C. Daurat and AM. Le Bon
3th annual meeting of the french network NACRe (Nutrition and Cancer network), Dijon FR Nov 2002
Metabolism of diallyl disulfide, a naturally occurring component of garlic, in the rat.
Germain E., Chevalier J. Steib M., Siess M-H. and Teyssier C.
3th annual meeting of the french network NACRe (Nutrition and Cancer network), Dijon FR Nov 2002
ETHICAL ASPECTS AND SAFETY PROVISIONS
Ethical aspects were considered for the animal studies All necessary facilities and permits to work with
animals (rats and mice) are available. The animal house at INRA in Dijon is licensed by the Ministry of
Agriculture for nutritional and toxicological experiments (agreement numbers 3273 and A21200). The
regulations are included in decree 87-848 of October 19, 1987 (OJ 10/20/87) and by-law of April 19,
1988 (OJ 04/27/88).