metabolism of diallyl disulfide by human liver microsomal ...

840 TEYSSIER ET AL.
TABLE 6
Correlation between DADS oxidation and CYP isoform specific activities in a
panel of human liver microsomes
Enzyme Activity CYP
Correlation
Coefficient (r)
EROD 1A2 0.07
COH 2A6 0.06
TDH 2C9 0.52 a
MpH 2C19 0.05
DOD 2D6 0.05
PNPH 2E1 0.63 b
(�-1)LAH 2E1 0.40 c
NfO 3A4 0.19
(�)LAH 4A 0.07
a p � .001, n � 33
b p � .001, n � 25
c p � .05, n � 26
DADSO 2) that should interact with the enzyme and should not be
released from the active site of the enzyme.
Egen-Schwind et al. (1992) have studied the metabolism of
DADSO in a perfused rat liver. They observed that while passing
through the liver, DADSO was metabolized to DADS. The discrepancy
could be explained by the models that were used in both studies.
They did not add NADPH during the liver perfusion. Due to the
sensitivity of DADSO to temperature, a dismutation of DADSO in
DADS could be possible. We observed the formation of DADS when
DADSO was incubated with microsomes at 37°C without NADPH. In
addition, Jin and Baillie (1997) studied the metabolism of DAS in rat.
They proposed that the reduction of allyl sulfoxide to DAS was
impossible with respect to the glutathione conjugates observed with
rat fed with DAS or allyl sulfoxide.
Flavin or CYPs are the only enzymes present in microsomes that
can catalyze NADPH- and oxygen-dependent oxidation of xenobiotics.
The inhibition of CYP by 1-aminobenzotriazole, a suicide inhibitor,
as well as the irreversible inactivation of FMO by heating
induced a decrease of the rate of DADS oxidation. Moreover, the
DADS oxidation was observed with microsomes prepared from cells
expressing human FMO3. These results suggest a contribution of both
CYP and FMO-containing monooxygenases. Some results allow the
evaluation of the FMO implication in this oxidation: its K m is much
higher than the one obtained for FMOs associated with CYP in human
microsomes (10.15 versus 0.61 mM). The similar comparison made
with cDNA-expressed isoenzymes gave 10.15 mM for FMO3 and
0.03 mM for CYP2E1. When the incubation of microsomes and
DADS was made in the presence of inhibited CYP, the DADS oxidase
activity is very low, whereas when the FMOs were inactivated this
activity was slightly decreased. Each of these results suggests that
FMOs are less active than CYP in the metabolism of DADS. Nevertheless,
to our knowledge, this is the first study that describes the
implication of FMOs in the oxidation of sulfur compounds issued
from garlic.
Three approaches have been developed to identify the CYP isoenzymes
involved in DADS oxidation. They were based on: 1) the use
of cDNA-expressed CYP isoenzymes, 2) the use of selective chemical
inhibitors, and 3) the study of the correlation of DADS oxidation
activity with marker activities of CYP isoenzymes. Several pieces of
evidence indicate that CYP2E1 is the major cytochrome P-450 responsible
for the metabolic process: 1) the rate of formation of
DADSO was substantially inhibited by the CYP2E1 inhibitors diethyldithiocarbamate
and chlorzoxazone, 2) PNPH and (�-1)LAH activities
(two marker activities of CYP2E1) in 25 individual human liver
microsomes exhibited the best correlation with the formation of
DADSO, and 3) with a K m of 0.03 mM and a V max/K m ratio of 2010
calculated with cDNA-expressed CYP2E1, this isoenzyme exhibited
the highest affinity and the highest intrinsic clearance among the
isoforms tested. Even if the quantity of CYP2E1 in human microsomes
was evaluated to 7% of whole CYP versus 18% for CYP2C and
29% for CYP3A (Shimada et al., 1994), the relative involvement of
CYP2E1 is predominant.
Nevertheless, our results suggest the involvement of other CYPs.
Many isoenzymes, such as CYP2A6, CYP2C9, CYP2C19, CYP2D6,
and CYP2E1 were able to oxidize DADS to DADSO. Their K m values
showed that in a competitive context like in human microsomes, only
CYP2E1 would be involved. The apparent velocity of CYP2E1 was
not the highest one. We demonstrated that DADSO was a mechanismbased
inhibitor of CYP2E1 (Martin, Teyssier and Siess, publication in
preparation). This means that the more DADSO is produced, more
CYP2E1 is inhibited. This observation may serve as an explanation
for the low V max of CYP2E1.
In the inhibitory experiments, tranylcypromine, an inhibitor described
as being specific of CYP2C19 (Postlind et al., 1998), strongly
inhibited DADS oxidase activity, suggesting the participation of this
isoenzyme. However, the specificity of this inhibitor should be reconsidered
because Draper et al. (1997) recently demonstrated that this
molecule is among the strongest inhibitors of the coumarin hydroxylase
activity (CYP2A6 activity marker), and we showed the PNPH
activity inhibition by tranylcypromine. Therefore, CYP2C19 does not
seem to be involved in DADS oxidation.
DADS has a chemical structure similar to DAS; they differ only by
one sulfur atom. These two molecules are metabolized by CYP2E1
and produce a mechanism-based inhibitor of CYP2E1. The metabolism
of DADS and its pathway is one more similarity between these
two molecules issued of garlic.
In conclusion, the oxidation of DADS to DADSO in human liver
microsomes is mainly mediated by CYPs but also by FMOs. Among
the CYP isoenzymes, CYP2E1 seems to be the most involved isoenzyme
even if a few other isoenzymes are also able to catalyze this
reaction.
Acknowledgments. We thank Jacques Auger for providing
DADSO and Marie-France Vernevaut for her technical assistance.
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