1
IN VITRO EVALUATION OF HUMAN CYTOCHROME P450 INHIBITION BY
ORGANOSULFUR COMPOUNDS FROM GARLIC
Hélène Martin, Sophie Bergerot, Néjia Dahech, Anne Desplan, Lucien Guenot, Marie-France
Vernevaut and Marie-Hélène Siess *
Unité Mixte de Recherche de Toxicologie Alimentaire, Institut National de la Recherche
Agronomique, 17 rue Sully, 21065 Dijon Cedex, France.
Keywords : organosulfur compounds ; garlic ; human CYP ; CYP 1A2 ; CYP 2E1 ; CYP 2A6
* Corresponding author : Marie-Hélène Siess
e-mail : siess@avignon.inra.fr tel + 33432722016
Abbreviations : AM, allyl mercaptan ; AMS, allyl methyl sulfide ; AMSO, allyl methyl
sulfoxide ; AMSO2 allyl methyl sulfone ; COH, coumarin hydroxylase ; CZX, chlorzoxazone
hydroxylase ; DAS, diallyl sulphide ; DADS, diallyl disulfide ; EROD, ethoxyresorufin Odeethylase ; KI, Apparent dissociation constant for inhibitor ; kinact, constant of inactivation for
inhibitor ; SAC, S-allyl cysteine
2
Abstract
The aim of this study was to assess the inhibiting effect of several garlic sulfur compounds on
human cytochromes P450 (CYP) involved in the activation of carcinogen : CYP 1A2, CYP
2E1 and CYP 2A6. We have determined the effects of sulfur compounds derived from garlic
such as diallyl sulfide diallyl disulfide and allicin as well as several compounds which were
demonstrated to be metabolites after garlic consumption such as S-allyl cysteine, allyl
mercaptan , allylmethyl sulfide , allylmethyl sulfoxide and allylmethyl sulfone. Most of these
components had weak or no effects on these CYPs, for concentrations up to 500 µM. Allicin
was the only compound which had a significant effect. It caused a loss of the coumarin
hydroxylase and chlorzoxazone hydroxylase activitiesy which was time and concentration
dependent. This suggests that allicin would be an irreversible inhibitor of CYP 2E1 and CYP
2A6 dependent activities. Overall the present data do not support the hypothesis that garlic
exerts a beneficial effect on carcinogenesis in man via the modulation of enzymes involved in
the activation of carcinogens.
3
1. Introduction
Epidemiologic studies showed that the consumption of garlic was associated with a reduced
risk of cancer incidence in humans (Fleischauer and Arab, 2001). The organosulfur
compounds specific of garlic, seem to be the principal agents responsible for this
chemoprotective action. Many investigators have shown that these compounds inhibited
chemically-induced tumor development in various tissues, in rat and mouse (Bianchini and
Vaino, 2001 ; Le Bon and Siess, 2000). A mechanism that may be responsible for this
protective effects is the interaction of these compounds with the metabolism and activation of
carcinogens either by inhibiting certain enzymes or by enzyme induction. Several studies with
animals or cells have shown an inhibitory effect of garlic compounds on some the activities of
cytochrome P450 enzymes (CYP). In vivo administration of allylsulfides to rats decreased the
activity of CYP 2E1 and can therefore block the activation of nitrosamine or other compounds
activated by this CYP (Brady et al., 1991 ; Guyonnet et al., 2000). Allyl sulfides such as
diallyl disulfide (DADS) and diallyl trisulfide were shown to inhibit CYP1-mediated
bioactivation of benzo(a)pyrene in HepG2 cells (Chun et al., 2001).
So far the effects of garlic compounds on human CYP have been the subject of little research.
A recent in vitro study with the use of human recombinant microsomes demonstrated that
allicin a garlic constituent was a potent inhibitor of CYP2C9 and CYP 2C19 but not 1A2,
2D6 , or 3A4 (Zhou et al., 2002). An other in vitro study suggested that whole garlic
preparations may inhibit CYP 2C9, 2C19, 3A4 and 3A5 (Foster et al., 2001). DADS and
dipyridyl sulfides were shown to inhibit human CYP 2A6 (Fujita and Kamataki, 2001) as
well as CYP2E1 and CYP2A3 (Morris et al., 2004). Knowledge of the inhibitory properties of
garlic sulfur compounds may elucidate whether inhibition of CYP-mediated bioactivation
reactions contributes to their anticarcinogenic activity in man.
In the current study, we have investigated the inhibitory capacity of garlic sulfur compounds
towards several CYPs involved in the metabolism of carcinogens. The following CYPS were
examined : CYP 1A2 involved in the activation of various chemical carcinogens such as
heterocyclic and aromatic amines, aflatoxin B1 and certain nitroaromatic compounds (Eaton
et al., 1995), CYP 2A6 involved in the activation of nitrosamines or other chemicals
(Yamasaki et al., 1992) and CYP 2E1 involved in activation of nitrosamines and other low
molecular weight carcinogens (Guengerich et al., 1991). CYP 1A2, 2A6 and 2E1 expressed in
human B-lymphoblastoid cells were used as an enzyme source. The deethylation of
ethoxyresorufin, the hydroxylation of coumarin and the hydroxylation of chlorzoxazone were
4
used to assess inhibition of CYP 1A2, CYP 2A6 and CYP 2E1 activities, respectively. We
have determined the effects of sulfur compounds derived from garlic such as allicin, DADS
and diallyl sulfide (DAS) (Figure 1). Indeed after cutting the bulb, allin which is present as
such in the bulb, is cleaved by alliinase to sulfenic acids, whose condensation products lead to
allicin. Decomposition of allicin gives sulfides as major breakdown products Lawson, 1998).
We also examined the effects of some compounds which were demonstrated to be metabolites
of garlic compounds such as S-allyl cystein (SAC), allyl mercaptan (AM), allylmethyl
sulphide (AMS), allylmethyl sulfoxide (AMSO) and allylmethyl sulfone (AMSO2), after
garlic consumption(Egenschwindt et al., 1992 ; , Germain et al., 2002 ; Taucher et al., 1996)
(Figure 1). Most of these components had weak or no effects on these CYPs, for
concentrations up to 500 µM. Allicin was the only compound which had a significant effect.
In order to get a better understanding of its mechanisms of action, the underlying inhibitory
mechanisms were investigated in more details.
SH
Allyl mercaptan (AM)
S
Allyl methyl sulfide (AMS)
O
S
Allyl methyl sulfoxide (AMSO)
O
S
Allyl methyl sulfone (AMSO2)
O
S
Diallyl sulphide (DAS)
S
S
Diallyl disulfide (DADS)
5
O
Allicin
S
S
NH2
S-allylcysteine (SAC)
S
CO2H
Figure 1 : Structures of garlic sulfur compounds used in this study
2. Materials and methods
2.1. Chemicals
AM, AMS, DAS and DADS were purchased from Sigma Aldrich Chemical (Strasbourg,
France). DADS (purity of 80 %) was further purified under reduced pressure whereas AM (80
% purity) and AMS were used without further purification. Allicin was synthesized from
DADS according to the method of Block and O’Connor (1974). The oxidising agent was
metachloroperbenzoic acid. The purity was 78 % as judged by HPLC. The contaminants were
DADS and DAS. AMSO and AMSO2 were obtained by oxidation with sodium metaperiodate
in a methanolic solution thawed on ice overnight (Furniss et al., 1989). 7hydroxychlorzoxazone was obtained from Ultrafine Chemicals (Salford, UK). All other
reagents were analytical or HPLC grade and were obtained from from Sigma Aldrich
Chemical (Strasbourg, France).
2.2. Enzymes
Microsomes from human B-lymphoblastoid cells expressing cDNA encoding human CYP
P450 1A2, 2E1 and 2A6 were purchased from Gentest Corp., MA USA. These cells contain a
vector with a human CYP cDNA and human CYP reductase cDNA
2.3. Monooxygenases activities
Catalytic activities known to be specific to CYP1A2 (ethoxyresorufin O-deethylation, EROD)
CYP 2A6 (coumarin 7-hydroxylation, COH) and CYP 2E1 (chlorzoxazone 6-hydroxylation,
CZX) were measured according to procedures previously described.
6
EROD activity was determined by the method described by Burke et al. (1985) The reaction
mixture contained : 0.1 M Tris-HCl buffer pH 7.7, 25 mM MgCl2, 2µM 7-ethoxyresorufin,
and microsomes (70 pmoles CYP 1A2 /ml) in a final volume of 0.4 ml. Reaction was started
with 0.25 mM NADPH. Resorufin was determined fluorometrically using a Cobas Fara II
centrifugal analyser with excitation and emission wavelengths fixed at 522 nm at 584 nm,
respectively. Quantification of resorufin formed was made by addition to the reaction medium
of 50 pmoles of resorufin at the end of each reaction.
COH activity was determined according to Maurice et al. (1991). The reaction mixture
contained : 0.05 M phosphate buffer pH 7.4, 0.1 mM coumarin, 1 mM NADPH, and
microsomes (70 pmoles CYP 2A6 /ml), the final volume being 0.4 ml. 7-hydroxycoumarin
formed was determined fluorometrically using the Cobas Fara II analyser with excitation and
emission wavelengths fixed at 380 nm at 450 nm, respectively. Quantification of 7hydroxycoumarin formed was made by addition to the reaction medium of 2 nmoles of 7hydroxycoumarin at the end of each reaction.
CZX activity was determined by the following method adapted from Leclercq et al. (1998).
The reaction mixture contained 0.1 mM Tris HCl bufferpH 7.4, 0.5 mM chlorzoxazone, 2mM
MgCl2, 1mM NADPH and microsomes (70 pmoles CYP 2E1/ml) in a final volume of 0.5 ml.
Incubations were initiated by addition of NADPH and were carried out at 37 °C for 10min.
The reactions were stopped by addition of 1N HCl. After removal the precipitated proteins by
centrifugation, 5µl of the supernatant was analysed by HPLC. Samples were
chromatographed on an ODS column, 5µm particle size. A solvent gradient of 0.1 %
diethylamine buffer pH 3 and acetonitrile was used in the proportions of 20 % acetonitrile for
3.5 min, increasing to 75 % over 2 min, then maintaining this concentration for 1 min and
then decreasing to 20 % and maintaining this last concentration for 2 min. The flow rate was
1.8 ml/min. Calibration curves were constructed by injecting quantities of 6hydroxychlorzoxazone ranging from 0.25 to 5 nmoles, added to the reaction medium. UV
detection was performed at 287 nm.
2.4. Inactivation reactions
Two different procedures were used for studying the inactivation of CYP 2A6 and CYP 2E1.
In the first one incubation mixture contained buffer, various concentrations of allicin added as
an aqueous solution and microsomal proteins (5 mg/ml final concentration) in a final volume
of 0.5 ml. The reaction was initiated by the addition of 1 mM NADPH or water in the case of
reaction without NADPH. At t=0 and regularly thereafter (from 0 to 16 min) aliquots were
7
removed from the incubation medium and diluted 10-fold into a second stage incubation
medium. The residual activity was determined in the conditions described above.
The other procedure is the following : various concentrations of allicin were incubated with
buffer and NADPH. At various times (from 0-16 min) COH assay or CZX assay were
initiated by addition of coumarin or chlorzoxazone respectively. The resulting rate of activity
was analysed as described above.
2.5. Estimation of kinetic constants
All constants were determined from a treatment analogous to the Michaelis-Menten analysis
using the SAS program (Cary, NC, USA).
The constants for enzymatic inactivation (KI), i. e. the maximal rate constant of inactivation at
saturating levels of inactivator (kinact) and the apparent dissociation constant for the inhibitor
KI were calculated by fitting the following expression :
k=[kinact*[I] / KI + [I]]
were k is the slope of the line, obtained for each inhibitor concentration in a semilogarithmic
plots of remaining activity versus preincubation time, and [I] is the inhibitor concentration.
3. Results and discussion
Inhibition of catalytic activities of CYPS by 8 organosulfur compounds was examined. The
chemical structures of the chemicals is shown in figure 1. The inhibition of CYP1A2, CYP
2A6 and CYP 2E1 activities by these chemicals is summarized in Table 1. EROD activity was
inhibited only by AM and allicin. AM inhibition was weak whereas allicin caused a 87 %
inhibition at 500 µM. For COH activity, on the eight compounds examined only allicin caused
a moderate inhibition at 500 µM. For CZX activity AM, AMS, DADS and allicin caused an
inhibition . The % of inhibition was the most important for allicin
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Table 1 Effects of garlic compounds (500 µM) on ethoxyresorufin deethylase (EROD),
coumarin hydroxylase (COH) and chlorzoxazone hydrolase (CZX) activities in human CYPs
Residual activity
Chemical
EROD (CYP 1A2)
COH (CYP 2A6)
CZX (CYP 2E1)
None
100
100
100
AM
73
98
77
AMS
91
98
86
AMSO
87
96
not measured
AMSO2
92
89
not measured
DADS
91
100
53
Allicin
13
69
41
DAS
91
97
91
SAC
91
94
92
Activities are expressed from control and are the means of two determinations. Control values
are 8.58 pmoles/min/mg proteins for EROD, 0.55 nmoles /min/mg protein for COH and 1.05
nmoles/min/mg protein for CZX activities.
As allicin was the only compound which had a significant effect, its inhibitory mechanisms
was investigated in more detail. Allicin was preincubated with recombinant microsomes and
NADPH for different times ranging from 0-16 min and the residual COH or CZX activities
were measured. The loss of the activity was shown to be time- and concentration-dependent
and there was no lag time for this inactivation (Figure 2 and 3). In the absence of allicin ,
there was no signficant loss of activity. Some inhibition of these activities was already found
at time O. After 16-min incubation of 400 µM of allicin with CYP 2A6, about 70 % of the
COH activity was inhibited whereas for CZX only 28 % was inhibited. This suggest that
allicin is a slower , time dependent inhibitor for CYP 2E1 than for CYP 2A6.
9
0
.
6
0 µM
0
.
4
50 µM
Activy(nmoles/inmg)
100 µM
200 µM
0
.
2
400 µM
0
.
0
0 2 4 6 8 1
0 1
2 1
4 1
6 1
8
P
r
e
i
n
c
u
b
a
t
i
o
n
t
i
m
e
(
m
i
n
)
Figure 2 Effect of preincubation time and allicin concentration on coumarin hydroxylase
activity in human CYP 2A6 microsomes. Allicin was added to the incubation mixture and
preincubated for indicated periods before the reaction was started by the addition of the
substate coumarin
10
0
.
5
0
0
.
4
5
0 µM
0
.
4
0
0
.
3
5
0
.
3
0
50 µM
Activy(nmoles/ig)
0
.
2
5
100 µM
0
.
2
0
200 µM
0
.
1
5
400 µM
0
.
1
0
02468
1
0
1
2
1
4
1
6
1
8
P
r
e
i
n
c
u
b
a
t
i
o
n
t
i
m
e
(
m
i
n
)
Figure 3 Effect of preincubation time on the inhibition of chlorzoxazone hydroxylase by
allicin. Allicin was added to the incubation mixture and preincubated for indicated periods
before the reaction was started by the addition of the substate chlorzoxazone.
Kinetic constants were determined by linear regression analysis as the slopes of inactivation
for each allicin concentration. According to the equations of Kitz and Wilson (1962), we
demonstrated that the inactivation obeyed saturation kinetics, from which kinact and K I were
calculated. Table 2 summaries inactivation constants values after two determinations of
activities.
Table 2 Kinetic parameters of inactivation of human CYP 2A6 and CYP 2E1 by allicin
Kinetic constants
KI (µM)
k inact (min –1)
CYP 2A6
CYP 2E1
362 [75-649]
178 [0-653]
0.054 [0.029-0.079]
0.116 [0-0.222]
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The inhibition of COH activity by allicin could be partially prevented by preincubation with
glutathione. This was also observed for CZX activity. The inhibitory effect of allicin could be
attributed to its rapid reaction with SH groups of CYP. This has been yet demonstrated for
other enzymes such as SH-protease papain, and alcohol dehydrogenase. (Rabinkov et al .
1998).
In conclusion, these in vitro data demonstrate that organosulfur compounds from garlic
produce only minor changes on CYP 1A2, CYP 2E1 and CYP2A6 mediated metabolism.
Most of these components had weak or no effects on these CYPs, for concentrations up to 500
µM. Allicin was the only compound which had a significant effect. There are some
indications that this compounds would be an irreversible inhibitor of CYP 2E1 and CYP 2A6.
Overall these data do not support the hypothesis that suggests that metabolites of garlic sulfur
compounds could prevent carcinogenesis in man by inhibiting the activation of carcinogens.
Acknowledgements
We thank Pr. Jacques Auger (Université F. Rabelais, Tours, France) for the synthesis of
allicin and S-allyl cysteine. This research was carried out in the framework of the Garlic &
Health project which has been partly financed by the European Quality of Life and
Management of Living Resources Programme (QLK1-CT-1999-00498).
12
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