WP 6 UMR de Toxicologie Alimentaire INRA Dijon WP 6 Cancer : General objectives inhibition of...

WP 6 UMR de Toxicologie Alimentaire INRA Dijon

WP 6 Cancer : General objectives

inhibition of bio-activation of carcinogens

stimulation of detoxification of carcinogens

prevention of genotoxicity

inhibition of the initiation of liver carcinogenesis

influence on apoptosis in tumour and normal cells

To elucidate the mechanisms of action of garlic and sulphur

compounds on different biological events involved in carcinogenesis:

To know the transformation and the fate of garlic sulphur compounds

ingested up to their targets


WP 6 Cancer : Milestones 2002

P12 : Determination of the bioavailability of garlic powder in rat by measuring the concentrations of the metabolites in blood and in main organs in progress

P12 : Evaluation of the effects of subcellular fractions from rats treated with garlic extracts on the mutagenicity of carcinogens using the Ames test in progress

P13 Determination of the specificity of garlic compouds/preparation toward apoptosis in specific types of human tumor cell lines

P12 : In vitro effects of garlic compounds on human CYP isoenzymes


In vivo metabolism of garlic (in progress)

to investigate the in vivo fate of garlic compounds and their metabolites after a single oral administration

OBJECTIVES


Proposed scheme for the metabolism of DADS in rat

SS

O

S

S

SH

Allylmercaptan AM

Allylmethylsulfide AMS

Allylmethylsulfoxide AMSO

Allylmethylsulfone AMSO2

S

glutathione

Allylglutathione sulfide AGS


Garlic powder i.g. 1.5 g / rat

Plasma

Liver

Urine

homogenization

protein precipitation (TCA, 30%)

Extraction with CH2CL2 (3 times)

Sample analysis (in progress)

Detection and Identification:

GC-MS, LC-MS

EXPERIMENTAL (in progress)


Evaluation of the effects of hepatic fractions from rats fed garlic extracts

on the mutagenicity of carcinogens in the Ames test

Objectives : to assess if the modulation of drug metabolizing enzyme

activities induced by garlic ingestion modifies activation/detoxication

of mutagens.

Anne-Marie Le Bon et al.


Glucuronosyl transferases (UGT)

Glutathione transferases (GST)

Phase 1 Phase 2

ADN Mutagenesis

Quinone reductase (QR)

Cytochromes P450 1A (EROD)

2B (PROD)

2E1 (PNPH)

(CYP)

RHROH

OR RO+

ROR’

3A (NO)


Dietcontaining

garlic powder

(5%, 15 days)

EXPERIMENTAL DESIGN

Cytosol

Microsomes

livermale Wistar rat

homogenization

centrifugation

Ames test

Enzyme activities

G 0 (0 kg/ha SO4) G 200 (200 kg/ha SO4)


Effects of garlic ingestion on liver enzyme activities

EROD PROD PNPH NO UGT GST QR

% o

f co

ntr

ol

0

100

200

300

400

*

*

* *

*

*

***

**

C

G 0

G 200

Phase 1 enzymes(CYP)

Phase 2 enzymes(transferases)

(1A) (2B) (2E1) (3A)


AMES TEST

plating on minimal glucose

agar plate

incubation 48 heuresat 37 °C

His+ revertantcoloniescounting

. .. . ...

hepatic fraction

bacteriaPro-mutagen

ormutagen

Salmonellatyphimurium

Salmonella typhimurium His - His+

Mutagen


Effects of hepatic microsomes on PhIP mutagenicity

Rev

erta

nts

His

+ /

pla

te

PhIP (ng / plate )

0 500 1000 2000 3000

0

50

100

150

200

250

300

Control

G 0

G 200

*


Effects of hepatic microsomes on aflatoxin B1 (AFB1) mutagenicity

Rev

erta

nts

His

+/ p

late

AFB1 (ng / plate )

0 6 13 25 50 100

0

200

400

600

800

1000

*

**

*

ControlG 0

G 200


G 0

G 200

Mutagens(Enzyme involved in metabolism)

PhIP(CYP1A1)

CP(2B)

AFB1(2B, 3A)

SO(GST)

4-NQO(QR)

: increased effect

: no effectcompared to control

: slight effect

: decreased effect

n.d. : not determined

NDMA(2E1)

n.d.

n.d.

Phase 1 substrates Phase 2 substrates

PhIP : heterocyclic amine

CP : cyclophosphamide

AFB1 : aflatoxin B1

SO : styrene oxide

4-NQO : 4-nitrosoquinoline oxide

NDMA : N-nitrosodimethylamine


CONCLUSIONS

Garlic diet : weak effect on enzyme activities

no consequence on the reduction of mutagenesis in the Ames test

Different results between this ex-vivo study and the in vivo study (protection)

Induction of GST by garlic = mechanism of protection?

(demonstrated for DADS, see Guyonnet et al, Carcinogenesis,

2002)


Working hypothesis:

Anticarcinogenic properties of garlic compouds could be explained by the inhibition of several CYPs which are involved in the activation of carcinogens

Objectives :

To assess the inhibitory effects of garlic compounds and compounds issued from the metabolism of DADS, on human CYP activities

In vitro effects of garlic compounds on human CYP enzyme activities

Marie-Hélène Siess et al.



Compounds selected:

Garlic compounds : S-allylcysteine, allicine, diallyldisulfide

Metabolites of DADS : Allylmercaptan, allylmethyl sulfide,

allylmethylsulfone allylmethylsulfoxide, allylglutathione sulfide

Doses :

50 µM and 500 µM


In vitro effects of garlic compounds on human CYPs enzyme activities

CYP 1A2

CYP 2A6

CYP 2E1

CYP 3A4

Activation of carcinogens

Heterocyclic amines

Tobacco nitrosamines

Nitrosamines, solvents ...

Aflatoxine B1, polycyclic aromatic hydrocarbons

Enzyme activities

Ethoxyresorufine deethylase

Coumarine hydroxylase

Chlorzoxazone hydroxylase

Nifedipine oxidase

Enzyme activities measured with microsomes isolated from human livers


Control AM AMS AMSOAMSO2DADSDADSO DAS SAC AGS

% o

f con

tro

l act

ivity

0

20

40

60

80

100

120

140

Effects of garlic compounds on human CYP 1A2

N = 4 different human samples


Control AM AMS AMSOAMSO2DADSDADSO DAS SAC AGS

% o

f con

tro

l act

ivity

0

20

40

60

80

100

120

Effects of garlic compounds on human CYP 2A6


Control AM AMS DADS DADSO DAS SAC

% o

f con

tro

l act

ivity

0

20

40

60

80

100

120

Effects of garlic compounds on human CYP 2E1


Control AMSO AMSO2 DADSO AGS

% o

f con

tro

l act

ivity

0

20

40

60

80

100

120

140

160

Effects of metabolites of DADS on human CYP 2E1



CONCLUSIONS

Most garlic compounds fail to inhibit CYP enzyme activities, at 50 µM

Allicine is the most active inhibitor of CYP 1A2

CYP 2E1 is inhibited (40-60 %), by allicine, DADS, allyl glutathione

sulfide and allyl mercaptan (500 µM)


WP 6 Cancer : General conclusions

Are animal and human studies succesful in showing that garlic can prevent cancer ?

YES NO

Increase of detoxication enzymes (rat)

Decrease of CYPs (animal, human samples)

In vivo decrease of the mutagenicity of some carcinogens (animal)

Increase of CYP enzymes (rat)

Decrease of CYPs for high concentrations (human samples)

Ex vivo no decrease of the mutagenicity of some carcinogens (animal)


Bottelnecks:

There will be some delay in the experiments because :

Caroline left the lab on february 2002. A postdoc will be involved in the program in 2003.

Anne-Marie will be absent from the lab (November 2002 -March 2003) for a maternity leave. No replacement is possible.


Next steps :2002 and 2003

In vivo metabolism of garlic in rat (in progress)

Hepatocarcinogenesis protocol will be performed to confirm the antigenotoxic effects of garlic powders

Participation to the human intervention study


Stocks of garlic powders available at September 2002 in my freezer

Varieties : Printanor (Morasol, Messidrome are not in my freezer)

Field experiments : 2000 and 2001

Countries ; France and Spain

Analysis:

2000 trials : analyses from Jacques and Thomas are different

2001 trials : analyses were done by Jacques on individual or pooled levels of fertilisation


Overview of the garlic powders

Printanor FR 2000 ~ 8000 g

Printanor SP 2000 ~ 17273 g

Fertilization Rep 1 Rep 2 Rep 3 Rep 40 2305 3260 3815 3190100 2820 3095 2945 3160200 2175 2465 2740 3520400 2910 3260 3340 2650

Fertilization Rep 1 Rep 2 Rep 3 Rep 40 530 180 570 355100 680 800 1390 570200 660 800 450 670400 640 590 925 6902000 400 330 370 540

Fertilization0 163850 2642100 1973200 1740

Printanor FR 2001 ~ 20750 g

Printanor SP 2001 ~ 13140 g

Levels of fertilisation mixed before making the powders

In red color : samples burnt and not available


Pre-treatment studies

DADS DADSO SACAM

NDMA

AFB1 ()

BaP

In vitro evaluation of antigenotoxic properties of garlic compounds

in HepG2 cells (comet test)

Garlic compound Pro-mutagen

: inhibition of the genotoxicity() : slight inhibition : no effect

WP 6 UMR de Toxicologie Alimentaire INRA Dijon WP 6 Cancer : General objectives inhibition of...

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