Inhibition of N-nitrosodimethylamine demethylase in rat and human liver microsomes by isothiocyanates and their glutathione, L-cysteine, and N- acetyl-L-cysteine conjugates

Article abstract of DOI:10.1021/tx9502094

Natural and synthetic isothiocyanates and their conjugates were examined for their inhibitory effects toward rat and human liver microsomal N-dimethylnitrosoamine demethylase (NDMAd) activity using a radiometric NDMAd assay. Substrate concentrations of 30 and 60 μM were used to probe the activity of cytochrome P4502E1 isozyme through the α-hydroxylation of NDMA. It was found that alkyl isothiocyanates such as sulforaphane and allyl isothiocyanate displayed very weak inhibition, whereas the arylalkyl isothiocyanates such as benzyl and phenethyl isothiocyanate showed significant inhibition toward rat liver NDMAd activity with IC₅₀ values of 9.0 and 8.3 μM, respectively. More interestingly, glutathione conjugates of benzyl, phenethyl, and 6-phenylhexyl isothiocyanates all inhibited NDMAd at the comparable concentrations. In the phenethyl isothiocyanate conjugates series, there exist marked differences in their inhibitory activity; i.e., its conjugates with L-cysteine (IC₅₀ = 4.3 μM) and with glutathione (IC₅₀ = 4.0 μM) are more potent than its conjugate of N-acetylcysteine (IC₅₀ = 24.0 μM). The same trend was also observed for the human liver microsomal NDMAd activity. The half-lives of these conjugates were determined in the presence of other free thiols from L-cysteine or glutathione using an HPLC system. It was shown that isothiocyanates are released from their conjugates and react with the free thiols present in the solution. The longer half-life of N-acetylcysteine conjugate of phenethyl isothiocyanate as compared to the other conjugates is consistent with its lower inhibitory activity. The inhibition of NDMAd, and therefore cytochrome P4502E1, by isothiocyanate conjugates is most likely due to the action of the free isothiocyanates released from the conjugates. Since cytochrome P4502E1 and other isozymes play important roles in the activation of the tobacco-specific nitrosoamines, these results provide a basis for investigating the potential of isothiocyanate conjugates as chemopreventive agents.

Full text of DOI:10.1021/tx9502094

9
32
Chem. Res. Toxicol. 1996, 9, 932-938
In h ibition of N-Nitr osod im eth yla m in e Dem eth yla se in
Ra t a n d Hu m a n Liver Micr osom es by Isoth iocya n a tes
a n d Th eir Glu ta th ion e, L-Cystein e, a n d
1
N-Acetyl-L-cystein e Con ju ga tes
†
†
‡
‡
Ding J iao, C. Clifford Conaway, Mong-Heng Wang, Chung S. Yang,
Werner Koehl,† and Fung-Lung Chung*
,§
,†
Division of Carcinogenesis and Molecular Epidemiology, Naylor Dana Institute for Disease
Prevention, American Health Foundation, Valhalla, New York 10595, and Laboratory for Cancer
Research, College of Pharmacy, Rutgers University, Piscataway, New J ersey 08854
Received December 14, 1995X
Natural and synthetic isothiocyanates and their conjugates were examined for their inhibitory
effects toward rat and human liver microsomal N-dimethylnitrosoamine demethylase (NDMAd)
activity using a radiometric NDMAd assay. Substrate concentrations of 30 and 60 µM were
used to probe the activity of cytochrome P4502E1 isozyme through the R-hydroxylation of
NDMA. It was found that alkyl isothiocyanates such as sulforaphane and allyl isothiocyanate
displayed very weak inhibition, whereas the arylalkyl isothiocyanates such as benzyl and
phenethyl isothiocyanate showed significant inhibition toward rat liver NDMAd activity with
IC50 values of 9.0 and 8.3 µM, respectively. More interestingly, glutathione conjugates of benzyl,
phenethyl, and 6-phenylhexyl isothiocyanates all inhibited NDMAd at the comparable
concentrations. In the phenethyl isothiocyanate conjugates series, there exist marked
differences in their inhibitory activity; i.e., its conjugates with L-cysteine (IC50 ) 4.3 µM) and
with glutathione (IC50 ) 4.0 µM) are more potent than its conjugate of N-acetylcysteine (IC50
)
24.0 µM). The same trend was also observed for the human liver microsomal NDMAd
activity. The half-lives of these conjugates were determined in the presence of other free thiols
from L-cysteine or glutathione using an HPLC system. It was shown that isothiocyanates are
released from their conjugates and react with the free thiols present in the solution. The longer
half-life of N-acetylcysteine conjugate of phenethyl isothiocyanate as compared to the other
conjugates is consistent with its lower inhibitory activity. The inhibition of NDMAd, and
therefore cytochrome P4502E1, by isothiocyanate conjugates is most likely due to the action
of the free isothiocyanates released from the conjugates. Since cytochrome P4502E1 and other
isozymes play important roles in the activation of the tobacco-specific nitrosoamines, these
results provide a basis for investigating the potential of isothiocyanate conjugates as
chemopreventive agents.
In tr od u ction
Organic isothiocyanates from natural sources have
been shown to inhibit tumorigenesis induced by environ-
mental carcinogens such as polycyclic aromatic hydro-
carbons and nitrosamines in rodents (1-6). Mechanistic
studies of the inhibitory effects of arylalkyl isothiocyan-
ates against lung tumorigenesis induced by the tobacco-
responsible for metabolic activation of NNK to methy-
lating and pyridyloxobutylating species (7-10). Inhibi-
tion of metabolic activation of NNK reduces the level of
DNA methylation and pyridyloxobutylation and, conse-
quently, decreases the number of neoplasms in the lungs
of the NNK-treated A/J mice and F344 rat (3, 7).
Previous studies from this laboratory showed that benzyl
isothiocyanate (BITC) and phenethyl isothiocyanate
specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-
(PEITC) decreased metabolic activation of N-nitrosodi-
2
1
-butanone (NNK), showed that arylalkyl isothiocyanates
methylamine (NDMA) and the NDMA-induced methy-
lation of hepatic DNA in rats (11). NDMA, a widely-
occurring environmental carcinogen, induces liver and
lung cancer in rat and A/J mice, respectively (12, 13).
The R-hydroxylation of NDMA at a low concentration to
generate the methanediazonium ion is primarily cata-
lyzed by microsomal P4502E1 (14). PEITC is a competi-
tive inhibitor and also a suicidal inhibitor of this reaction
are potent competitive inhibitors of mouse and rat P450s
*
To whom correspondence should be addressed.
American Health Foundation.
Rutgers University.
†
‡
§
Present address: Werner Koehl, Department of Food Chemistry
and Environmental Toxicology, University of Kaiserslautern, 67663
Kaiserslautern, Germany.
X
Abstract published in Advance ACS Abstracts, J uly 15, 1996.
A part of this work was presented at the 86th Annual Meeting of
(15).
1
the American Association for Cancer Research, March 18-22, 1995,
In this study, we examined several natural isothiocy-
Toronto, Canada.
anates for their activities in the inhibition of NDMA
metabolism (structures in Chart 1). Allyl isothiocyanate
(AITC), sulforaphane (SFO), BITC, and PEITC are of
particular interest because they are found in various
cruciferous vegetables consumed by humans (4). Fur-
thermore, glutathione (GSH) conjugation is a major
2
Abbreviations: NDMA, N,N-dimethylnitrosamine; NDMAd, N,N-
dimethylnitrosamine demethylase; P450, cytochrome P450; NNK,
-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; SFO, (-)-1-isothiocy-
anato-4(R)-(methylsulfinyl)butane (sulforaphane); AITC, allyl isothio-
cyanate; BITC, benzyl isothiocyanate; PEITC, phenethyl isothiocyan-
ate; PHITC, 6-phenylhexyl isothiocyanate; NAC, N-acetyl-L-cysteine;
GSH, glutathione, SAR, structure-activity relationship.
4
S0893-228x(95)00209-8 CCC: $12.00 © 1996 American Chemical Society

Inhibition of P450 2E1 by Isothiocyanate
Chem. Res. Toxicol., Vol. 9, No. 6, 1996 933
Ch a r t 1. Str u ctu r es of th e Isoth iocya n a tes
Stu d ied
Ta ble 1. Molecu la r F or m u la , Ma ss, a n d HP LC Ca p a city
F a ctor s (k′) of Isoth iocya n a te Con ju ga tes
mass
formula
calcd
obsda
HPLC k′ b
AITC-GSH
BITC-GSH
PEITC-GSH
PHITC-GSH
PEITC-Cys
PEITC-NAC
C14H22N4O6S2
C18H24N4O6S2
C19H26N4O6S2
C23H34N4O6S2
C12H16N2O2S2
3 2
C14H18N O S
406.48
456.54
470.57
526.68
284.40
326.40
407.0
457.2
471.3
527.3
285.1
327.0
1.09
3.33
4.15
7.76
4.32
6.24
metabolic pathway of isothiocyanates in rodents and
humans since more than half of isothiocyanates admin-
istered were excreted as the N-acetylcysteine (NAC)
conjugates in the urine (16-19). It is plausible that the
GSH and L-Cys conjugates of isothiocyanates may be
involved in the inhibition of P450s in vivo. To examine
this possibility, the GSH, Cys, and NAC conjugates of
several isothiocyanates were synthesized and structurally
characterized. They were assayed for their inhibitory
activities toward hepatic microsomal N-nitrosodimeth-
ylamine demethylase (NDMAd) in vitro. We also studied
the exchange of isothiocyanate conjugate among thiol
groups as a plausible mechanism for enzyme inhibition
by the conjugates.
2
a
The molecular mass was determined by using an electrospray
ionization MS spectrometry. b The capacity factor was calculated
as k′ ) (tR - t )/t , where t is the retention time of the unretained
0
0
0
peak (formaldehyde was used), and tR is the retention time of the
conjugate. The compounds were eluted with a gradient of mobile
phase (20% to 50% of acetonitrile over 30 min) at 1 mL/min.
procedures were exercised to avoid personnel contamination by
infectious agents (23).
Micr osom es. Male F344 rats were purchased from Charles
River Breeding Laboratories, Inc. (Kingston, NY). Rats were
fed NIH-07 diet and maintained under the following standard
conditions: 20 ( 2 °C, 50 ( 10% relative humidity, and a 12-h
light, 12-h dark cycle. Microsomes were prepared as previously
described from livers of 2 rats at 3 months of age and stored at
-
80 °C until use (24). The human liver microsomes were
Ma ter ia ls a n d Meth od s
prepared using a published procedure (25) from a sample kindly
provided by Dr. F. Peter Guengerich through the Tennessee
Donor Service (Nashville, TN). The protein concentrations were
determined by Bradford method (26) using the Coomassie Plus
Protein Assay Reagent (Pierce, Rockford, IL).
Ra d iom etr ic NDMA Dem eth yla se Assa y. A previous
published procedure was used for this assay (27). The final
incubation mixture (100 µL) in a 2-mL Eppendorf tube contained
In str u m en ts. A Waters HPLC system (Waters Corp., Mil-
ford, MA) equipped with an automated gradient controller, two
Model 501 pumps, and a Waters 990 photodiode array detector
was used for the analysis. The 1D and 2D NMR spectra were
recorded on a Bruker AM 360 MHz NMR spectrometer (Bruker
Instruments Inc., Billerica, MA). A Finnigan MAT TSQ700
mass spectrometer (Finnigan Corp., San J ose, CA) was used to
record electrospray mass spectra of all isothiocyanate conju-
gates.
1
4
6
0 µM of radioactive NDMA (a 1:2 mixture of [ C]NDMA and
NDMA, 0.1 µCi), rat liver microsomal proteins (129 µg), an
NADPH generating system (0.4 mM NADP , 10 mM glucose
+
Ch em ica ls. AITC, BITC, and PEITC of the highest purity
were purchased from Aldrich Chemical Co. (Milwaukee, WI).
SFO was custom synthesized by LKT Laboratories, Inc. (St.
6
-phosphate, 0.2 unit of glucose-6-phosphate dehydrogenase),
buffer at pH 7.4 (50 mM Tris, 10 mM MgCl , 150 mM KCl),
2
and an inhibitor (1-100 µM). AITC, BITC, and conjugates were
dissolved in water or buffer at the assay concentrations. PEITC
has much lower solubility in water than the other two isothio-
cyanates. Most organic solvents cannot be used since they are
inhibitors of NDMA demethylase (28). Acetonitrile was used
as a vehicle to deliver PEITC into the Eppendorf tubes, and then
+
Paul, MN). NDMA, NADP , glucose 6-phosphate, glucose-6-
phosphate dehydrogenase, Cys, NAC, and GSH were from
1
4
Sigma Chemical Co. (St. Louis, MO). [methyl- C]-N-Nitrosodi-
methylamine (49 mCi/mmol) was custom synthesized by SRI
International Co. (Menlo Park, CA). The labeled NDMA was
purified before use by passing through a chromatography
column filled with anion exchange resin Dowex 1 (200-400
mesh, Sigma Chemical Co., St. Louis, MO). All other chemicals
from commercial sources were reagent grade.
2
was removed with a stream of N before adding the other
ingredients. Aqueous PEITC solution (up to 1 mM) could also
be prepared by adding methyl-â-cyclodextrin (Sigma Chemical
Co.) at a concentration that did not inhibit NDMA demethylase.
These two methods of introducing PEITC into the aqueous
incubation system gave comparable results. The result for
PEITC was the average value. The former procedure was
applied in the assay of all other isothiocyanates. However,
neither of these methods was successfully applied to the more
lipophilic PHITC. All incubations were carried out for 20 min
and done in duplicate and the average values were used. In
experiments using human liver microsomes, 97 µg of microsomal
The GSH conjugates of AITC, BITC, PEITC, and PHITC and
the Cys and NAC conjugates of PEITC were synthesized using
published procedures (16, 20). The GSH conjugate of SFO was
not prepared due to a limited amount of SFO available. The
purity of products was established by thin layer liquid chroma-
tography (TLC) and reversed-phase HPLC. A single spot was
observed under UV and after ninhydrin spray of TLC plates
(
Silica gel GHLF, 250 µm, 10 × 20 cm, purchased from Analtech,
Inc., Newark, DE). Molecular weights and structures of the
conjugates were confirmed by electrospray ionization MS and
14
protein and 30 µM [ C]NMDA with the same composition as
the previously described were used. All operations were con-
ducted in a fume hood, and safety procedures for handling
radioactive chemicals and biohazards were followed as described
in the “Hazardous Materials” section. The incubations were
carried out in triplicate with each inhibitor concentration. The
NDMAd activity measured by the amount of HCHO formed was
plotted against concentration of the inhibitor. The IC50 values,
the concentrations that inhibit HCHO formation by 50%, were
determined from these results. The standard deviation of IC50
was calculated from three separate experiments.
Exch a n ge Rea ction of Isoth iocya n a te Con ju ga tes w ith
L-Cystein e or GSH u n d er P h ysiologica l Con d ition s. The
exchange reaction of isothiocyanate conjugates with thiol was
conducted in a 2.0-mL Eppendorf tube at 37 °C in a water bath
shaker. The isothiocyanate-GSH and L-cysteine were dissolved
1
D and 2D NMR spectroscopy. For NMR measurements, the
samples were dissolved in DMSO-d
6
, and 2D COSY spectra were
recorded with 1K data size and a total of 256 experiments each
with 32 scans (21, 22). Unambiguous NMR spectral assign-
ments were made using 1D and 2D spectra. Molecular formula,
weights, and capacity factors (k′) of all conjugates are sum-
1
marized in Table 1. Their H-NMR spectral assignments are
listed in Table 2.
Ha za r d ou s Ma ter ia ls: NDMA is a volatile carcinogen.
Precautions were taken when using radioactive compounds and
carcinogens, e.g., wearing double disposable gloves, performing
all experiments under a fume hood, and disposing waste ac-
cording to appropriate radiation safety guidelines. In the
handling of human tissues, proper precautions and safety

9
34 Chem. Res. Toxicol., Vol. 9, No. 6, 1996
J iao et al.
Ta ble 2. 1H-NMR Assign m en ts of Isoth iocya n a te Con ju ga tes in DMSO-d 6 Solu tion a
AITC-GSH
3.31
1.92, 1.83
2.30
BITC-GSH PEITC-GSH
PHITC-GSH
PEITC-Cys
PEITC-NAC
γ-Glu
RH(1H)
âH(2H)
γH(2H)
J Rb
3.33
3.30
3.31
1.90, 1.83
2.30
1.91, 1.83
2.30
1.92, 1.83
2.30
6.8
6.8
6.8
6.8
Cys
Gly
NH(1H) 8.52
8.59
4.52
3.28, 3.79
8.6
4.6
8.50
4.48
3.23, 3.77
8.5
4.6
9.9
8.53
4.49
3.23, 3.75
8.8
4.7
7.55
RH(1H)
âH(2H)
J NR
4.50
3.30, 3.87
8.6
3.53
3.61, 3.27
4.10
3.38, 3.19
5.9
4.8
3.8
J Râ
4.6
4.6
5.8
14.4
J Râ′
10.0
10.0
13.8
10.0
13.5
J bb′
13.4
13.3
14.4
NH(1H) 8.56
8.61
3.70
8.59
3.75
5.7
8.59
3.69
5.7
RH(2H)
J NR
3.70
5.7
NHC(dS)S NH(1H) 10.21
10.58
10.18
10.03
8.50 (br pk)
11.81
3
2
1
2
1
6
5
4
3
2
1
2
1
2
1
alkyl
CH2dCHCH2- PhCH2-
PhCH2CH2- PhCH2CH2CH2CH2CH2CH2- PhCH2CH2- PhCH2CH2-
1,(2H) 3.58
1
2
,(2H) 4.21
4.38
1,(2H) 3.51
2,4,(4H) 1.55
3,5,(4H) 1.30
6,(2H) 2.55
1,(2H) 3.75
2,(2H) 2.91
1,(2H) 3.70
2,(2H) 2.91
2
,(1H)5.83
J HH ) 14.2 2,(2H) 2.87
3
3
3
3
,(2H) 5.17,5.10
J 1,2 ) 7.6
J 1,2 ) 7.7
J 1,2 ) 7.7
3
J 2,3cis ) 17.2
J 2,3trans ) 10.3
J 3,3 ) 5.6
CH3CO(3H) 1.60
3
2
3
J 5,6 ) 7.2
ArH(5H)
7.30
7.25
7.22
7.25
7.25
a
Chemical shifts (δ) are in ppm relative to (TMS) and coupling constants (J ) are in Hz. The NMR spectra were recorded on a 360 MHz
spectrometer.
Ta ble 3. In h ibition (IC50) of NDMA Dem eth yla se Activity
in Ra t Liver Micr osom es a n d th e Ha lf-Lives (t1/2) of
Isoth iocya n a te Con ju ga tes in th e Aqu eou s Solu tion
Con ta in in g Th iol a t P h ysiologica l Con d ition
in a phosphate buffer (20 mM, pH 7.4, degassed) to make a 1.0
and 3.0 mM solution, respectively. The reaction was then
initiated by mixing 0.75 mL of each solution. A 5 µL aliquot of
the reaction mixture was taken every 20 min for HPLC analysis.
The HPLC system consisted of a Vydac guard column (4.6 × 20
mm), a Vydac 218TP54 reverse-phase C18 analytical column (4.6
inhibitors
SFO
IC50 ( SD (µM)a
t1/2 (min)b
>100
×
250 mm) and a Partsil 5 ODS-3 reverse-phase C18 analytical
AITC
BITC
PEITC
77.0 ( 4.4
9.0 ( 0.3
8.3 ( 1.9
column (4.6 × 250 mm), (Whatman, Clifton, NJ ) in sequence.
Samples were eluted isocratically with a mobile phase of 40%
of acetonitrile in water (0.1% trifluroacetic acid (TFA)), or a 1:1
ratio of acetonitrile and water (0.1% TFA) for PHITC-GSH and
PEITC-NAC at a flow rate of 1 mL/min. The sample peaks were
monitored by UV detection at 254 nm wavelength. These
conditions offered the best separation of the GSH and L-Cys
conjugates. In the case of reaction of PEITC-GSH with L-Cys,
a synthetic standard of the cysteine conjugate of PEITC was
used as a reference. The newly formed peak of this reaction
was found to coelute with the standard conjugate, and they had
identical UV spectra. The formation of PEITC-GSH when
PEITC-Cys was incubated with GSH solution was also estab-
lished using the synthetic standard of PEITC-GSH. Other
isothiocyanate-Cys conjugates were identified by their char-
acteristic UV spectra and HPLC retention times. For the
reaction between BITC-GSH and PEITC-Cys, their solutions
AITC-GSH
BITC-GSH
PEITC-GSH
PHITC-GSH
PEITC-CYS
PEITC-NAC
24.7 ( 2.2
2.2 ( 0.2
4.0 ( 1.3
8.8 ( 0.5
4.3 ( 1.2
24.0 ( 1.7
27
25
18
48
14
>50
a
Determined using the radiometric NDMA demethylase assay
described in Materials and Methods. Each value was obtained
from three determinations. b Determined by the reaction of con-
jugates (0.5 mM) and free thiol (1.5 mM) in a phosphate buffer
(20 mM, pH 7.4) and at 37 °C.
creased upon conjugation with GSH. The IC50 values of
each isothiocyanate and their conjugates were deter-
mined and are summarized in Table 3. SFO, a major
isothiocyanate in broccoli and a potent phase II enzyme
inducer in hepatoma cell culture (29), was not inhibitory
toward P4502E1 activity. Similarly, AITC, abundant in
brown mustard and also a phase II enzyme inducer (30,
(1.0 mM in phosphate buffer) were mixed at 1:1 ratio. The
reaction course was followed by the HPLC method as the above.
Resu lts
3
1), showed very weak inhibitory activity. In contrast,
In h ibition of Rod en t Hep a tic NDMA Dem eth yl-
a se Activity by Isoth iocya n a tes a n d Th eir Con ju -
ga tes. The inhibition of NDMAd activity in rat liver
microsomes was determined by a radiometric NDMAd
assay (27). It has been reported that most organic
solvents used for dissolving substrates or inhibitors in
the enzyme assays are inhibitors of P4502E1 (28).
Isothiocyanates such as SFO, AITC, and BITC were
sufficiently soluble in aqueous solution at the concentra-
tions examined. PEITC and more lipophilic isothiocy-
anates such as PHITC, however, were poorly soluble in
water. PEITC was assayed using methods described in
the Materials and Methods to circumvent the problem
of poor solubility. The solubility also significantly in-
both BITC and PEITC significantly inhibited P4502E1
activity in rat liver microsomes. GSH conjugates of
AITC, BITC, and PEITC were more potent than their
parent isothiocyanates. Although our previous studies
showed that PHITC was a much stronger inhibitor of
NNK-induced lung tumorigenesis in A/J mouse and of
NNK metabolism in A/J mouse lung microsome than
PEITC (7, 32), its GSH conjugate was not as potent as
PEITC-GSH. PEITC-Cys and PEITC-GSH were both
potent inhibitors of NDMAd activity, whereas PEITC-
NAC was much less inhibitory. Similar trends were also
observed in the mouse liver microsomes.3 In summary,
GSH conjugates of BITC, PEITC, and PHITC and PEITC-

Inhibition of P450 2E1 by Isothiocyanate
Chem. Res. Toxicol., Vol. 9, No. 6, 1996 935
F igu r e 1. Dose-dependent inhibition of NDMA demethylation
activity in human liver microsomes by PEITC and its conju-
gates. Each data point represents the average of three incuba-
tions. Incubation mixture (0.1 mL) contains 97 µg of human liver
microsomal protein suspended in a pH 7.4 Tris buffer, a NADPH
generating system, 30 µM radioactive NDMA, and an inhibitor
(1-100 µM). Blanks were done without adding NADPH gener-
ating system, and controls were done without adding inhibitors.
Incubation was carried out at 37 °C for 20 min. While Cys, NAC,
and GSH had little effect on the NDMA demethylase activity,
PEITC and its conjugates inhibited the enzyme activity with
IC50 values: 6 µM (PEITC), 9 µM (PEITC-Cys), 8 µM (PEITC-
GSH), and 85 µM (PEITC-NAC).
F igu r e 2. HPLC chromatograms showing time-dependent
formation of PEITC-Cys upon reaction of PEITC-GSH with
L-Cys. The reaction was initiated by mixing 0.5 mM of PEITC-
GSH and 1.5 mM of L-Cys in a phosphate buffer (1.5 mL, pH
7
.4) at 37 °C. Aliquots of sample (5 µL) were analyzed by HPLC
Cys all significantly inhibited NDMAd activity, but AITC-
GSH and PEITC-NAC were considerably less active.
In h ibition of NDMA Dem eth yla se Activity in
Hu m a n Liver Micr osom es by P EITC a n d Its Con -
ju ga tes. It has been shown that activation of NDMA in
human liver microsomes was primarily catalyzed by
P4502E1 (33, 34). To examine the ability of isothiocy-
anates and their conjugates to inhibit human NDMAd
activity, i.e., P4502E1 activity, we assayed PEITC and
its conjugates in human liver microsomes. The inhibition
curves for the selected compounds were plotted in Figure
at an interval of 20 min.
Since the latter two compounds have to be formed
through the reaction between free isothiocyanates and
free sulfhydryl groups of Cys or GSH, this result firmly
established that the exchange of isothiocyanate conju-
gates is via the release of free isothiocyanates as inter-
mediates.
The kinetics of the exchange reaction of different
conjugates varies. For example, the three conjugates of
PEITC exhibited marked differences for the exchange
process, i.e., the rate to reach equilibrium in descending
order: PEITC-Cys, PEITC-GSH, and PEITC-NAC (Fig-
ure 3). To compare the exchange processes among
different conjugates, the half-life for each conjugate to
reach equilibrium in the presence of free thiol groups (of
Cys in most cases) was determined (Table 3). Their half-
lives vary depending upon the particular isothiocyanate
conjugate. In the GSH conjugate series, the PHITC
conjugate showed a much longer half-life than the
conjugates of other isothiocyanates.
1
, together with those of Cys, GSH, and NAC as controls.
While Cys, GSH, and NAC had little effect on human
P4502E1 activity within the concentration range tested,
PEITC and its conjugates significantly inhibited the
P4502E1 activity in a dose-dependent manner. Consis-
tent with results obtained from rat liver microsomes,
PEITC, PEITC-Cys, and PEITC-GSH had similar IC50
values (6-9 µM), whereas PEITC-NAC was much less
potent (IC50 ) 85 µM).
GSH Con ju ga t es of Isot h iocya n a t e E xch a n ge
w ith F r ee Th iol Gr ou p s in Solu tion . The observation
that GSH conjugates of isothiocyanate inhibited NDMAd
activity prompted us to examine the reaction of isothio-
cyanate-GSH conjugates with free thiols of proteins, one
of the reactions believed to be involved in the inactivation
of enzymes (35). Upon incubation of PEITC-GSH con-
jugate with L-cysteine in pH 7.4 buffer at 37 °C, we
observed a time-dependent formation of the L-Cys con-
jugates of isothiocyanates by using a RP-HPLC system
Discu ssion
Our data showed that NDMAd in rat liver microsomes
was significantly inhibited by BITC and PEITC at
micromolar concentrations, but weakly inhibited by SFO
and AITC. Thus, arylalkyl isothiocyanates appeared to
have stronger inhibitory effects than the alkyl isothio-
cyanates. In agreement with these observations, the
GSH conjugate of AITC showed considerably weaker
inhibition of NDMAd than the GSH conjugates of BITC
and PEITC. In the inhibition of NNK metabolism in
mouse lung microsomes, PHITC is a much more potent
inhibitor than PEITC (10). However, such a trend was
not observed in the conjugated series for NDMA demeth-
ylation, probably due to the retarded dissociation of
PHITC-GSH. Another possibility is that a different
(Figure 2). The newly formed peak coeluted with the
synthetic standard of PEITC-Cys and showed a UV
spectrum identical to that of PEITC-Cys. Conversely,
PEITC-GSH was formed when PEITC-Cys was incubated
with GSH under the same conditions. Similarly, when
a mixture of BITC-GSH and PEITC-Cys was incubated,
both BITC-Cys and PEITC-GSH peaks were observed.
3
D. J iao, C. Conaway, and F.-L. Chung, unpublished data.

9
36 Chem. Res. Toxicol., Vol. 9, No. 6, 1996
J iao et al.
is likely that some of these functional groups are modified
by isothiocyanates. Moreover, the NADPH-P450 reduc-
tase is also susceptible to the modification of sulfhydryl
groups and may also be a target for isothiocyanates (39).
The GSH conjugates of isothiocyanates have been shown
to release isothiocyanates with or without the catalysis
of glutathione S-transferases (20, 40, 41). The inhibitory
effect of isothiocyanate conjugates in the NDMAd assay
could be attributed to the ability of conjugates to generate
free isothiocyanate. This notion is supported by the
present observation that the isothiocyanates released by
their GSH or other thiol conjugates are subsequently
conjugated with free thiol groups via this pathway. The
lower IC50s of the conjugates as compared to those of the
corresponding isothiocyanates suggest, however, the pos-
sibility that conjugates can directly inhibit NMDAd. The
differences in IC50 between the conjugates and the parent
isothiocyanates could be due partially to the poor solubil-
ity of the isothiocyanates (see Materials and Methods).
Our observations that the conjugates were considerably
less active than the isothiocyanates in pentoxyresorufin
dealkylase (2B1) and ethoxyresorufin dealkylase (1A)
assays in which dimethyl sulfoxide was used to solubilize
3
the substrates seem to support this possibility. Ad-
ditional evidence against the conjugates being inhibitors
comes from studies using an S-hexyl-GSH analog with a
thioether linkage which precludes dissociation reaction.
This compound showed no inhibitory activity against 2E1
in mouse liver microsome incubations.3 Although the
direct inhibition by the conjugates cannot be completely
ruled out, the results so far obtained are consistent with
the mechanism of dissociation in the inhibition by the
conjugates.
Since the exchange of conjugation among different thiol
groups requires release of the free isothiocyanate from
one conjugate and subsequent formation of a new con-
jugate, the difference in their half-lives to reach equilib-
rium could have resulted from either the different
dissociation rate of isothiocyanate conjugates or the
different reactivity of isothiocyanates toward free thiols
or a combination of both. Although this system does not
allow us to examine the detailed kinetic features of either
equilibrium involved, it may reflect a certain complexity
of the interactions between isothiocyanate conjugates and
protein thiols in microsomes. When examined carefully,
however, the data may reveal some qualitative informa-
tion on the dissociation of the conjugates. For instance,
in the series of PEITC conjugates, it may be assumed
that the reactivity of PEITC toward the sulfhydryl group
of Cys and GSH is similar or identical; the different half-
lives could be attributed solely to different dissociation
rates of PEITC-Cys, PEITC-GSH, and PEITC-NAC in
descending order. It is noted that PEITC-NAC was much
less potent than PEITC-Cys and PEITC-GSH in the
inhibition of NDMAd in both rodent and human liver
microsomes. Since most of incubations in the assay were
completed within an hour, PEITC-Cys and PEITC-GSH
dissociated readily and had sufficient time to interact
with microsomal enzymes. Thus, they were more potent
inhibitors than PEITC-NAC, which had a much longer
half-life (>50 min) and probably would be much less facile
to interact with enzymes during the incubation period.
However, such an interpretation based on the different
dissociation rates of conjugates needs to be confirmed
with an accurate measurement of dissociation constants
for those conjugates.
F igu r e 3. Time-dependent changes of the composition of
PEITC conjugates in reaction mixture. (a) PEITC-Cys (0.5 mM,
pH 7.4) + GSH (1.5 mM, pH 7.4); (b) PEITC-GSH (0.5 mM, pH
7
7
.4) + L-Cys (1.5 mM, pH 7.4); (c) PEITC-NAC (0.5 mM, pH
.4) + L-Cys (1.5 mM, pH 7.4).
structure-activity relationship may be involved in in-
hibiting the activation of different carcinogens. Current
evidence indicates that the metabolism of NDMA is
primarily catalyzed by P4502E1 in liver microsomes,
whereas the metabolism of NNK in mouse lung is
primarily catalyzed by P4502A1 and 2B1 (8, 36). More
investigations regarding the selectivity of isothiocyanates
toward the inhibition of specific P450 enzyme are needed
for a better assessment of their chemopreventive poten-
tials.
In this study we have demonstrated that isothiocyan-
ate conjugates are inhibitory toward NDMAd in rat liver
microsomes. Similar trends were observed for PEITC
and its conjugates in human liver microsomes. The
inhibitory activities of both isothiocyanates and their
conjugates in the microsomal systems appear to operate
through a common mechanism. It was postulated that
isothiocyanates interact with the substrate binding site
of P450 and react with thiol, amino, and hydroxyl groups
of the protein (37). Since there are more than a half
dozen Cys and many amino and hydroxyl group contain-
ing residues in rat and human P4502E1 proteins (38), it

Inhibition of P450 2E1 by Isothiocyanate
Chem. Res. Toxicol., Vol. 9, No. 6, 1996 937
It has become increasingly evident that many chemi-
cally reactive metabolites may be transported as GSH
conjugates in vivo (42). It is plausible that the previously
observed inhibitory activity of isothiocyanates against
NNK-induced tumorigenesis in mouse lung may be, in
part, mediated by transportation and subsequent dis-
sociation of their respective GSH conjugates. Therefore,
the discovery of the inhibitory activity of NDMAd by
isothiocyanate conjugates provides an important insight
into the molecular mechanism by which isothiocyanates
inhibit nitrosamine-induced carcinogenesis. The PEITC-
Cys conjugate is less toxic than PEITC itself and has been
shown to be absorbed upon oral administration to mice
resulting in the stimulation of glutathione S-transferase
production (43). It is of considerable interest to study
whether isothiocyanate conjugates act as inhibitors in
animal tumor bioassays. Future studies will focus on the
in vivo metabolism of isothiocyanates and the potential
of isothiocyanate conjugates as chemopreventive agents.
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Ack n ow led gm en t. This work was supported by
Grant CA 46535 from the National Cancer Institute. This
is Paper No. 23 in the series “Dietary Inhibitors of
Chemical Carcinogenesis”. We would like to thank
Zhonglin Zhao and Paul Yonkers for their technical
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