Workpackage number :
Phase:
WP 6 ; Cancer
24-36 months
Start date :
Completion date :
February 1 2000
January 31 2004
Current status Ongoing
N°s of partners involved : P12 , P5, P13
Person-month per partner : P5: 10, P12: 54, P13: 24; total : 88
Already devoted person months per partner: P5 : 10 , P12 : 50 , P13 : 5, total: 65
Objectives
This task will assess the anti-carcinogenic potential of garlic in relation with the bio-availability and the metabolism of garlic sulphur compounds. Several complementary approaches will be employed to elucidate their mechanisms of action on different physiological and biological events involved in carcinogenesis:
- inhibition of bio-activation of carcinogens
- stimulation of detoxification of carcinogens
- prevention of geno-toxicity
- inhibition of the initiation of liver carcinogenesis
- influence on apoptosis in tumour and normal cells
In vitro studies will allow to determine the effects of pure sulfur compounds on different targets
(Carcinogen-metabolising enzymes, alterations of DNA, apoptosis) and to understand their mechanisms of action. In vivo studies take into account the dietary situation : garlic extracts will be administered to rats through the diet, and the determination of their effects on targets will be assessed after different steps such as absorption, distribution to target tissues and metabolism, which can not always be taken into account in studies in vitro .
Description of work
Bio-availability and metabolic fate of organo-sulfur compounds from garlic will be studied by characterisation of the different pathways of bio-transformation of these compounds (diallyl disulfide, allicin, garlic powder) using rat and human sub-cellular fractions and by identifying and quantifying the components present in the blood and several organs after administration of these test substances to rats, by means of HPLC, GC-MS, LC-MS (P12).
Effects of garlic on carcinogen-metabolising enzymes : In vitro the effects of isolated compounds
(allicin, diallylsulfide) will be investigated on several CYP P450 iso-enzymes (CYP1A1, 2E1, 2A6, 3A4) involved in carcinogen activation in man. For compounds having an inhibitory effect, characteristics of the inhibition will be studied: reversible inhibition of mechanism-based inactivation. In vivo rats will be treated with garlic extracts incorporated in their diet and several phase I enzymes (involved in activation: CYP 1A1,2, 2B1,2, 2E1, 3A) and phase II enzymes (involved in detoxification: glutahione transferase, UDP-glucuronyl transferase and quinone reductase) which are important in carcinogen metabolism will be measured in the liver. In addition immunoblots will be performed to assess the microsomal levels of P450 iso-enzymes and the cytosolic levels of glutathione transferase sub-units.
These determinations will assess the potential inducing or repressing effects of garlic on carcinogen metabolising enzymes in conditions which are related to dietary conditions ( P12).
Protection of garlic against carcinogen geno-toxicity : In vitro the antigeno-toxic effects of isolated sulphur garlic compounds will be evaluated in HepG2 cells using the comet assay. In vivo rats will be fed with garlic extracts/powder incorporated in their diet, geno-toxic carcinogens will be given and the
DNA damage will be determined in different tissues, as well as the effects of sub-cellular fractions on the mutagenicity of carcinogens using the Ames test ( P12).
Prevention of hepato-carcinogenesis initiation in the rat : The capacities of garlic extracts to act as cancer anti-initiators will be assessed using a two step medium term hepato-carcinogenesis assay (i.e. resistant hepatocyte model adapted from Farber’s group model), which allows the detection of

preneoplastic foci expressing the placental form of glutathione transferase (GST-P) as end-point
(immuno-histochemically and morphometrically) after administering extracts to rats for two weeks and submission to hepatectomy and injection of carcinogens and submission to a selection procedure (
P12).
Investigations on cyto-toxicity, apoptosis and signal transduction : First cyto-toxicity of garlic compounds and garlic extracts will be tested by the MTT test and the sulfo-rhodamine assay. Second the apoptotic activity will be monitored by morphological characteristics and DNA degradation analysis
(measurement of diploid DNA content by flow cytometry). Molecular mechanism of apoptosis will be evaluated by identifying second messenger, transcriptional factors (AP-1, NF-kB) and transcription products (c-myc, TNF, p53, bcl-2 gene family) (P13).
Milestones and Deliverables
Milestones Deliverables
2000
 In vitro metabolism of sulphur compounds by human and rat subcellular fractions from liver
(P12): done
 In vitro effects of garlic sulfur compounds on human CYP isoenzymes (P12): done
 In vitro evaluation of the antigenotoxic properties of garlic compounds in Hep G2 cells (P12): done
2001
 Determination of the bioavailability of diallyl disulfide in rat by measuring the concentrations of the metabolites in blood and in main organs
(P12): done
 In vivo effects of garlic extracts on several carcinogen metabolizing enzymes (Phase I and phase II) (P12): done
 Evaluation of the antigenotoxic properties of garlic extracts in tissues of rat (P12): done
DH 8 In vitro metabolism of diallyl disulfide : paper send to Drug metabolism and Disposition : done
DH 9 Publication on the i n vitro effects of sulphur compounds on human P450 iso-enzymes : in preparation by Feb 2003
DH 10 In vitro evaluation of the anti-geno-toxic properties of garlic compounds in Hep G2 cells : presentation of a poster + publication in preparation
2002
 Determination of the bioavailability of garlic powder in rat by measuring the concentrations of the metabolites in blood and in main organs
(P12) : in progress.
 In vitro effects of metabolites of garlic compounds on human CYP isoenzymes
(additionnal milestone) (P12): done
 Evaluation of the effects of subcellular fractions from rats treated with garlic extracts on the mutagenicity of carcinogens using the Ames test
(P12) : done
 Determination if garlic compounds/preparations are able to induce apoptosis in tumour cells and not in normal cells and identification of potential signaling molecules in apoptosis (P13) : done
DH 13 In vivo metabolism of diallyl disulfide : paper accepted for publication in Xenobiotica + presentation of a poster : done
DH 14 Publication on the effects of garlic extracts on carcinogen-metabolising enzymes in the rat :in preparation by Feb 2003
DH 15 Publication of the in vivo anti-genotoxic properties of garlic in rat : in preparation
2003
 Investigation of the anti-initiating effects of garlic extracts using the medium term hepatocarcinogenesis model in the rat (P12).
DH 20 Publication on the modulation of the mutagenicity by garlic in the Ames test
DH 27 Publication on in vivo metabolism of garlic

powder
DH 25 Publication on the effects of garlic compounds and extracts on apoptosis
Progress 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 (P12)
During the previous reporting periods (2000 and 2001), we have investigated the bioavailability and the metabolism of an isolated garlic sulfur compound, diallyl disulfide, in the rat and in human tissues. Two manuscripts have been written and sent to editors (DH 8 and DH 13). One of this manuscript is now in press in Xenobiotica ( In vivo metabolism of diallyl disulphide in the rat:
Identification of two new metabolites, by Germain E. et al.).
However information on the bioavailability of sulfur compounds when garlic (and not isolated compounds) is ingested is essential to explain its beneficial effects on health. Therefore the aim of the current 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.
The first analyses show the formation of allyl methyl sulphide (AMS), allyl methyl sulphoxide
(AMSO) and allyl methyl sulphone (AMSO
2
) in most tissues. AMSO
2
is the most abundant and the most persistent compound. The next step of this current study will be the quantification of these metabolites in different tissues and the determination of their pharmacokinetic parameters.
Caroline Teyssier left the lab in March 2002. Her duties were : the metabolism of sulfur compounds from garlic. MarieHélène 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 milestone on the metabolism of garlic powder. Next year a postdoc will be recruited and will take in charge this part of the program to fullfill this metabolic study.
2 - In vitro effects of metabolites of garlic compounds on human CYP isoenzymes (additional milestone P12)
In the course of our study on the metabolism of sulfur compouds we have detected new metabolites which were formed after ingestion of a garlic compounds : allylmercaptan (AM), allyl methylsulfide (AMS), allyl methyl sulphoxide (AMSO), allyl methyl sulphone (AMSO
2
) and allylgltathione sulfide (AGS) (see DH13). Therefore, it was of interest to assess their effects on human
CYPs which are involved in carcinogen activation such as :
- CYP 1A2 which is active towards hydrocarbons
- CYP 2A6 which is involved in the activation of nitrosamines
- CYP 2E1 which is able to metabolize nitrosamines and numerous low molecular weight chemicals
The metabolites (DADS, DADSO, AM, AMS, AMSO, AMSO2 and AGS) were studied at two doses :
50 and 500 µM.
CYP 1A2 is strongly inbited (87 %) by DADSO and lightly inhibited (27 %) by AM. The other metabolites have no effect. CYP 2 A6 is inhibited by DADSO, AGS (40 %) and less by AMSO2 (10 %).
CYP 2E1 is strongly inhibited (60 %) by DADSO and also by DADS (47 %). AM, AMS and SAC produce moderate inhibitions.
Therefore, even if the concentrations of metabolites (500 µM) inhibiting human CYP are rather high, these data suggest that some metabolites of garlic sulfur compounds may reduce the genotoxicity of carcinogens which are metabolized by these CYP.
These data and those obtained the first year (Milestone : In vitro effects of garlic compounds on human CYP P450 isoenzymes) will be combined in a unique paper on the effects of garlic sulfur compounds and their metabolites on human CYP P450 isoenzymes (DH 9)

3 - Evaluation of the effects of subcellular fractions from rats treated with garlic extracts on the mutagenicity of carcinogens using the Ames test (P12)
The objective of this current study 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), 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.
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. Microsomes from rats fed garlic powders increased the mutagenicity of AFB1. 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.
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).
Two papers on the antigenotoxic effects of garlic compounds or garlic powders are in preparation or will be written next year (DH 10 and 15). However Anne-Marie Le Bon who is in charge of these papers has a maternity leave until March 2003. The writing will be postponed until she will come back in the laboratory.
4 - Determination if garlic compounds/preparations are able to induce apoptosis in tumour cells and not in normal cells and identification of potential signaling molecules in apoptosis (P13)
We were interested if garlic sulphur compounds can induce apoptosis. For that reason we incubated a leukaemic T-cell line (Jurkat) with AMSO, AMSO
2
, DADS and various garlic extracts for
24h. Cell viability was determined by the MTT-assay. For all garlic constituents only DADS reduced, at concentrations as high as 100 µM, cell-viability to ~ 55 %. Treatment with up to 100 µM AMSO and
AMSO
2
reduced cell viability to 75 %. Overall the effective concentrations are very high and therefore unlikely to mediate the chemo-preventive effect of garlic.
Additionally we used DMSO and aqueous garlic extract of Spanish and French garlic powders.
In concentrations up to 500
µg
/ ml
DMSO garlic extracts, France 2000, Spain 2000 & 2001, decreased viability about 20 % in comparison to untreated cells.
The garlic extract from France 2001 showed heavy bacterial and fungal contaminations, therefore we were not able to perform this assay. Further we experienced strong bacterial and fungal contaminations with all aqueous extracts and even with ethanolic extracts, so we are not able to make any predictions on possible cytotoxic effects.
So far just DADS showed a markedly cytotoxic effect on Jurkat cells, however this effect occurs only in very high concentrations. As we observed before DMSO garlic extracts are not mediating cytotoxic effects.

5 - Conclusion
Carcinogenesis is a complex and multistep process. At this stage of the project the experimental data accumulated have demonstrated a variety of biological effects for garlic or sulfur compouds which may be beneficial in the prevention of cancer :
- In vitro we demonstrated that DADS and DADSO are inactivators of human CYPs which are involved in carcinogen activation . However this effect occurs only at very high concentrations
- sulfur compounds from garlic or metabolites of these compounds can prevent the genotoxicity of carcinogens in a human cell-line. They may act by scavenging ultimate metabolites and/or by modulating the activity of carcinogen metabolizing enzymes.
- In vivo in the rat fed with garlic powder , we observed a modulation of hepatic enzymes involved in carcinogen activation or detoxication . This would have a consequence on the prevention of the genotoxicity of chemicals which are metabolized by these enzymes. This hypothesis was confirmed by checking the antigenotoxic effect of feeding garlic powders using the Cometes test.
- DADS but not garlic powders showed a markedly cytotoxic effect on leukaemic T-cells.
However this effect occurs only at very high concentrations.
However other experimental data were not convincing in showing that garlic can prevent cancer.
Actually hepatic subcellular fractions from rats fed with garlic powders has no effect in preventing the genotoxicity of ultimate mutagens. The modulation of detoxication enzymes induced by garlic diet is probably not enough important to inhibit mutagenicity of some ultimate genotoxins.
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.
6 th 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).