1. Marie-Hélène SIESS et al.
WP 6 CANCER
Marie-Hélène SIESS et al.
Participants :
P12 : UMR de Toxicologie Alimentaire, INRA, Dijon
P5 : IRBI, University F.Rabelais, Tours
P13 : University of Muenchen, Department of Pharmacy,
Center of Drug Research, Muenchen

2. RATIONALE
 Cancer is one of the first causes of death and morbidity within EU
 Cancer is preventable by plant food (eating fruits and vegetables
 The project is focused on the mechanisms of action of garlic on critical cellular and molecular events in the process of carcinogenesis
The probability to have a cancer in our lives is evaluated to 1 out of 2 for men and 1 out of 3 for women.
One european citizen out of 4 dies from a cancer.
Several means to combat cancer and several progresses have been made in therapy.
Among the others prevention is a valuable (effective approach to combat .
There is a conse,nsus about the role of consuming fruits & vegetables and the reduction of the risk of cancer.
In this context our WP was focused on

3. ORIGINAL OBJECTIVES
 to investigate the role of garlic and its sulfur compounds on the process of carcinogenesis through cellular, animal and clinical studies
 in vivo studies (rat) with garlic powders
 in vitro studies (cells) with isolated sulfur compounds
 to clarify the metabolism of sulfur compounds of garlic which could be effective on carcinogenesis

4. Bioavailability and metabolism of sulfur compounds from garlic

5. APPROACHES
In vivo : administration of pure DADS or garlic in rats and measurements of the concentrations of metabolites in several tissues
Ex vivo : perfusion of a rat liver by pure DADS and identification of metabolites specifically formed in the liver
In vitro : metabolism of DADS by rat and human subcellular fractions to validate the hypothetical pathways

6. Proposed scheme for the metabolism of DADS in the rat
Allylmercaptan
Allylglutathione sulfide S
glutathione SH
Allylmethylsulfide
Allylmethylsulfoxide
Allylmethylsulfone
Metabolites detected in vitro
Metabolites detected in vivo

7. CONCLUSION
 The metabolism of DADS is partly elucidated in rat and man
DADS is rapidly absorbed and extensively metabolized.
AMSO and AMSO2 are probably excretion compounds
Similar metabolites profiles are observed after garlic administration

8. Role of garlic on the process of carcinogenesis : In vivo studies with rat

9. OBJECTIVES
 To assess the anticarcinogenic effect of garlic consumption on the appearence of liver preneoplasic foci initiated by chemical carcinogens
 To elucidate the mechanisms of the anticarcinogenic action
To determine if the anticarcinogenic effect is correlated with the level of sulfur compounds in garlic

10. STUDIED MATERIALS Variety and location Variety Printanor FR
Variety Printanor SP
Fertilisation SO4
kg /ha
100
200
400
Alliin content
nmoles/mg 58 75 90
330
Experimental
groups SO
S100
S200
S400

11. Activating and protecting mechanisms of carcinogenesis
Activating mechanisms
Protecting mechanims
Activation of chemical pro-carcinogens
Activation of pro-oncogenes
Promoting effects
Angiogenesis
Detoxication of carcinogens
Protection of DNA
Tumor supressing genes
Antipromoting effects
Stimulation of immunity
Carcinogenesis is made of a series of events which transform a normal cell to a tumoral cell.
Overall the formation of maligant cells results from a balance between mechanisms which promote or activate the process and mechanisms which slow down or inhibit the process
Initiation is made of a series of events whereby a carcinogen induces alterations in the genetic make-up of the cell. This results in a lesion that can be inherited and which confers to the cell the potential for neoplastic growth.

12. PROTOCOL OF HEPATOCARCINOGENESIS
Sacrifice
weeks
Garlic diet
Promotion
Initiation
Detection of
preneoplastic foci
Carcinogen administration
Aflatoxin B1 (AFB1) :
10 x 0.025mg/kg
Diethylnitrosamine (DEN) :
10 X 2.5 mg/kg

13. RESULTS
Effects of garlic on the appearance of preneoplastic foci induced by AFB1 in the liver
AFB1
AFB1 + Garlic powder S400

14. RESULTS
Effects of garlic on the number of preneoplastic foci induced by AFB1 in the liver a b
Correlation of the nb foci with S content of garlic
r =
P = 2.93 E-04
Means and SEM (n=10).
Means having different letters are significantly different (P 0.05, Newman Keuls’ test).

15. RESULTS
Effects of garlic on the area of preneoplastic foci induced by AFB1 in the liver b a
Correlation of area foci with S content of garlic
r =
P = 0.006
Means and SEM (n=10).
Means having different letters are significantly different (P 0.05 Newman Keuls’ test).

16. Metabolic pathways of Aflatoxin B1 (AFB1)
AFM1
AFQ1
CYP 1A
CYP 2B
Phase II enzymes
AFB1-glutathione
AFB1-epoxide
CYP 2B
CYP 3A
AFB1
DNA
Initiation of carcinogenesis

17. Effects of garlic on carcinogen metabolizing enzymes
RESULTS
Effects of garlic on carcinogen metabolizing enzymes
Treatments S0
S200
S400
CYP 1A - 
CYP 2B
CYP 3A
GST
 
UGT
CYP : cytochrome P450
GST : glutathione transferase
UGT : UDP-glucuronosyltransferase

18. RESULTS Effects of garlic on DNA damage induced by AFB1 in rat liver
Means (n=5) having different letters are significantly different (P 0.05 Newman Keuls ’s test)
Undamaged DNA
Broken DNA = comet
genotoxic
The comet assay is a very simple , rapid and sensitive technique to analyse DNA damge at the individual cellular level. Under highly alcaline conditions, single-, double strand-breaks and alkali-labile lesions are detected

19. Initiation of carcinogenesis
Non toxic
metabolites
phase 2
(transferases)
phase 1
(CYP)
Procarcinogen
Ultimate metabolite
DNA
Initiation of carcinogenesis

20. RESULTS
Effects of garlic on the apparition of preneoplastic foci induced by DEN in the liver
Means and SEM (n=10).

21. Initiation of carcinogenesis
Metabolic pathways of nitrosamine
CYP 2E1
spontaneous
DEN
OH-DEN
CH3+
DNA
DEN undergoes an oxidative N demethylation by CYP 2E1 resulting in the production of a methyl ion, a potent alkylator of DNA
Initiation of carcinogenesis

22. Effects of garlic on CYP 2E1 activity
RESULTS
Effects of garlic on CYP 2E1 activity a b b b
Means and SEM (n=5).
Means having different letters are significantly different (P 0.05 Newman Keuls ’s test)

23. RESULTS
Effects of garlic on DNA damage induced by nitrosamine in rat liver b c a
Means (n=5) having different letters are significantly different (P 0.05 Newman Keuls ’s test)

24. CONCLUSION
 Garlic powder ingestion reduced preneoplasic foci induced by AFB1.
Mechanisms of action :
 induction of CYP 1A and/or GST and UGT
 no effect on CYP 2B and 3A
 A significant correlation was found between allin content and the anticarcinogenic efficacy
 No protection against the carcinogenicity induced by DEN
even if there is an inhibition of the genotoxicity of nitrosamine
 Mechanisms of action unknown

25. Antigenotoxic effects of sulfur compounds in human cells

26. OBJECTIVES
To assess if garlic compounds prevent DNA alterations induced by mutagenic compounds in a human cell line, HepG2 cells.
The studied molecules were :
Range : 5 to 100 µM
DADS (diallyl disulfide)
DADSO (allicin)
SAC (S-allyl cysteine)
AM (allyl mercaptan)
DNA alterations were measured using the comet test.
Undamaged DNA
Broken DNA = comet
Genotoxic compound

27. PROTOCOLS
Garlic compound
Pro-mutagen
 Pre-treatment studies : cells are treated first with the garlic compound
and then with the pro-mutagen
Hypothesis : inhibition or induction of xenobiotic metabolizing enzymes
Mutagens : benzopyrene (BaP)
aflatoxin B1 (AFB1)
nitrosodimethylamine (NDMA)
CYP 1A
CYP 1A, 3A
CYP 2E1
 Co-treatment studies : cells are treated with both the garlic compound
and the direct-acting mutagen at the same time
Hypothesis : scavenging of mutagenic compounds
Garlic compound + mutagen
Mutagens : hydrogen peroxide (H2O2)
methyl methane sulfonate (MMS)
4-nitrosoquinoline oxide (4-NQO)

28. Pre-treatment studies
RESULTS
Pre-treatment studies
DADS
DADSO AM
SAC
BaP  
AFB1  
() 
()
NDMA  
OSC display anti G activity when administered before the genotoxic agent, suggesting an effect of these compounds on the metabolic pathway of different carcinogens.
 : inhibition of the genotoxicity
() : slight inhibition
- : no effect

29. RESULTS Co-treatment studies  () DADS DADSO SAC AM H2O2 MMS 4-NQO
 : inhibition of the genotoxicity
() : slight inhibition
- : no effect

30. CONCLUSIONS
 Sulfur compounds from garlic prevent the genotoxicity of carcinogens in a human cell line
They act through different mechanisms of action :
modulation of enzymes involved in activation or detoxication
scavenging of ultimate species

31. WP6 : GENERAL CONCLUSION
 The metabolism of sulfur compounds such diallyl disulfide is partly elucidated in rat and man.
New metabolites and pathways are identified in the rat.
 Garlic consumption prevents the initiation step of chemical hepatocarcinogenesis in the rat. Some mechanism of action are elucidated.
The efficacy is correlated with the S-content of garlic
In human cells, garlic sulfur compounds inhibit the genotoxicity induced by different chemicals.

32. Expected impacts of the WP 6 outputs
 To contribute to improve the knowledge for the prevention of cancer (scientific community)
 To contribute to policy design on the prevention of cancer (health public agencies)
 To contribute to fully optimize the health effects of garlic (breeders, farmers, agrofood industries)

33. Deliverables WP6 Po Po Po
Po = poster

34. Participants of my lab
Scientists : Anne-Marie, Caroline,
Engineer : Raymond
Technicians : Christine, Joëlle, Lucien, Marie-France,
Marie-France
Post-docs : Emmanuelle and Varsha
Students : Caroline, Néjia, Sophie, Marlène, Anne,
Céline

37. initiation of carcinogenesis
APPROACHES
non toxic
metabolites
phase 2
(transferases)
phase 1
(CYP)
pro-carcinogen
ultimate metabolite
Bio-
activation
phase 1
(CYP)
Activity/expresion of XME
Alteration of DNA (Comet test
Ames test)
Preneoplastic foci
(Immunohistology)
DNA
initiation of carcinogenesis