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Laboratories of Organic Chemistry in Life Science [Y. N., H. O.] and Biosignals and Response [S. M.], Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502; Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology, Kinki University, Wakayama 649-6493 [A. M.]; Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, 565-0871 [M. I.]; and Laboratory of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Nagoya 464-8601 [Y. M., Y. K., T. O., K. U.], Japan
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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GST isozyme (GSTP1). Our studies demonstrated for the first
time that the addition of BITC to the cells resulted in an immediate
increase in the reactive oxygen intermediates (ROIs) detected by a
fluorescence probe, 2',7'-dichlorofluorescin diacetate. The level of
the ROIs in the cells treated with BITC (10 µM) was
50-fold higher than those in the control cells. Furthermore,
glutathione depletion by diethyl maleate significantly enhanced
BITC-induced ROI production and accelerated the BITC-induced elevation
of the GST activity, whereas pretreatment of the cells with glutathione
inhibited both the ROI production and GST induction. The
structure-activity relationship of the isothiocyanates also indicated
that the ROI-producing activities closely correlated with their
GST-inducing potencies. Moreover, the GSTP1 enhancer I-containing
region was found to be essential for induction of the
GSTP1 gene by intracellular ROI inducers such as
BITC and diethyl maleate. These data suggest the involvement of the
redox regulation on the induction of GSTP1 by BITC. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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,ß-unsaturated aldehydes (2
, 3)
. A
number of studies support the fact that certain food phytochemicals
protect against cancer. An important group of compounds that have this
property are organosulfur compounds, such as ITCs. ITCs are compounds
that occur as glucosinolates in a variety of cruciferous vegetables,
such as Brassica species. Many ITCs are effective
chemoprotective agents against chemical carcinogenesis in experimental
animals. ITCs inhibit rat lung, esophagus, mammary gland, liver, small
intestine, colon, and bladder tumorigenesis (4, 5, 6, 7, 8)
.
Previous studies have also reported that ITCs inhibit phase I enzymes
(cytochrome P-450) that are required for the bioactivation of
carcinogens and increase the carcinogen excretion or detoxification by
the phase II detoxification enzymes (9
, 10)
. ROIs are involved in many biological processes. Recent findings have revealed that both the GST gene expression and induction of the transcription factor, such as activated protein-1, might be related with intracellular oxidative stress (11 , 12) . In addition, it has been shown that JNK, which activates the transcriptional factor such as c-Jun, was induced by ITCs mediated by oxidative stress (13) . The protective effect exerted by overexpressed Bcl-2 and antioxidants provide substantial evidence for the involvement of ROIs in the ITC-mediated JNK activation and apoptosis and point to oxidative stress as the probable mediator for signal transduction. However, the exact nature and the role of ITC-induced ROI production on GST induction are still not established and are the topics of this report.
In a recent study, we have screened a number of fruits for sources of GST inducers and described the isolation and identification of BITC as a major GST inducer from papaya.4 In addition, we have also observed that, among a total of 20 isothiocyanates and their derivatives, BITC was found to be the most potent inducer of GST activity in the cells. Here we report the molecular mechanism underlying GST induction in the cells treated with BITC. Our studies demonstrated for the first time that the addition of BITC to cultured rat liver epithelial cells resulted in an immediate increase in ROIs detected by a H2DCF-DA fluorescence probe. Furthermore, the agent, such as DEM enhancing the BITC-induced ROI production, accelerated the GST induction by BITC, whereas the antioxidant GSH significantly inhibited them. These results suggested that ROIs are involved in the GST induction by BITC.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Cell Cultures.
RL34 cells were obtained from the Health Science Research Resources
Bank (Osaka, Japan; Ref. 14
). The cells were grown as
monolayer cultures in DMEM supplemented with 5% heat-inactivated fetal
bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.3
mg/ml L-glutamine, 0.11 mg/ml pyruvic acid, and 0.37%
NaHCO3 at 37°C in a atmosphere of 95% air and
5% CO2. Postconfluency cells were exposed to the
test compounds in the medium containing 5% fetal bovine serum.
Enzyme Assay.
GST activity was measured using 1-chloro-2,4-dinitrobenzene as a
substrate according to the method of Habig et al.
(15)
.
Western Blot Analysis.
The ITC-treated and untreated cells were rinsed twice with PBS (pH 7.0)
and lysed by incubation at 37°C for 10 min with a solution containing
0.8% digitonin and 2 mM EDTA (pH 7.8). Each whole-cell
lysate was then treated with the Laemmli sample buffer for 3 min at
100°C (16)
. The samples (20 µg) were run on a 12.5%
SDS-PAGE slab gel. One gel was used for staining with Coomassie
brilliant blue, and the other was transblotted on a nitrocellulose
membrane with a semidry blotting cell (Trans-Blot SD;
Bio-Rad), incubated with Block Ace (40 mg/ml) for blocking, washed, and
treated with the antibody.
RNA Preparation and Northern Blot Analysis.
The rat GSTP1 cDNA probe was obtained from the Japanese Cancer Research
Resource Bank (17)
. The probe was labeled with
[-32P]dCTP by random oligonucleotide priming
(Amersham). The preparation of the total RNA from RL34 cells and
Northern blot analysis were carried out as reported previously
(9
, 18)
.
GSH Assay.
Measurement of GSH in the cells was spectrophotometrically performed
using the commercial kit GSH-400 (Bioxytech). Confluent monolayer cells
were exposed to test compounds for the indicated times, and at the end
of the incubation period, cell monolayers were washed twice with PBS
(pH 7.0) and extracted with the 5% metaphosphoric acid solution
containing 5 mM EDTA. After centrifugation
(10,000 x g for 20 min), 50 µl of 12
mM chromogenic reagent in 0.2
M HCl were added to the resulting supernatant
(300 µl) and mixed thoroughly. After 50 µl of 7.5
M NaOH were added and mixed, the mixture was
incubated at 25°C for 10 min, and then the absorbance was determined
spectrophotometrically at 400 nm.
Intracellular Oxidative Products Determination.
Intracellular oxidative products were detected by
H2DCF-DA as an intracellular fluorescence probe
(19
, 20)
. Briefly, the cells under confluency were treated
with H2DCF-DA (50 µM) for 30 min at
37°C. After washing twice with PBS, the test compound was added to
the complete medium and incubated for another 30 min. A flow cytometer
(CytoACE 150; JASCO, Tokyo, Japan) was used to detect DCF formed by the
reaction of H2DCF with the intracellular
oxidative products. Experiments were repeated two times with similar
results. The data are expressed as one representative histogram. Images
of the cellular fluorescence were acquired using a confocal laser
scanning microscope (Fluoro-View; Olympus Optical Co., Ltd., Tokyo,
Japan) with a x40 objective (488-nm excitation and 518-nm emission).
CAT Assay.
A 3.0-kb fragment between -2.9 kb (EcoRI) and 59 bp
(AccI) of the GSTP1 gene was inserted into the
HindIII site of pSV0CAT and designated as ECAT
(21)
. 1CAT was constructed from the ECAT using the
appropriate restriction enzymes (21)
. RL34 cells were
transfected with 10 µg of plasmid construct by the calcium phosphate
coprecipitation procedure described by Chen and Okayama
(22)
. Cells were harvested 48 h after transfection.
Cell lysates were obtained after five freeze-thaw cycles in 0.25
M Tris-HCl (pH 7.5). The protein was equalized
and used for the CAT assay. The degree of acetylation was determined by
reading the intensity of the spots using the Fuji-BAS 2000 system (Fuji
Photo, Tokyo, Japan).
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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, after exposure of the RL34 cells to 10
µM BITC for 24 h, the GST activity was
increased 2.3-fold, compared with that of the BITC analogue, PITC. To
examine the GST isozymes responsible for the increase in the GST
activity of the BITC-treated RL34 cells, an immunoblot analysis was
carried out. In RL34 cells, no detection of the class GST isozyme,
GSTA1, which is the most abundant GST isozyme in normal rat liver, and
the constitutive expression of the class GSTP1 have been reported
previously (9)
. As shown in Fig. 1B
, the
protein level of GSTP1 significantly increased in the cells treated
with BITC for >16 h. Another class GST isozyme, GSTA3, was
slightly induced (data not shown). These results indicated that the
induction of GST activity by BITC mainly resulted from the enhanced
expression of GSTP1. In addition, the Northern blot analysis of the
total mRNA from RL34 cells treated with 10 µM
BITC was performed using an oligonucleotide probe complementary to the
GSTP1 mRNA sequence. As shown in Fig. 1C
, the treatment of
RL34 with BITC resulted in a 5.2-fold increase in GSTP1 mRNA after
24 h. Therefore, the increment in GSTP1 mRNA coincided with a
substantial rise in the GSTP1 protein level and GST activity, whereas
the effect of PITC on the GSTP1 protein and gene expression was almost
negligible (Fig. 1, B and C
).
|
). To assess the role of the oxidative stress in the
induction of GST, we examined the effects of the pretreatment of the
cells with GSH or DEM prior to the BITC treatment. Because the maximal
induction of the stress signaling pathways, such as JNK activation, was
observed within 12 h after ITC treatment (13)
, the
cells were exposed to 10 µM BITC for 1 h,
and after washing out the BITC followed by another 16 h of
incubation, the GST activity was measured. As shown in Fig. 2B
, both GST activities were significantly elevated in the
cells treated with BITC, even for 1 h. The GSTP1 protein was also
significantly induced by BITC treatment (data not shown). Pretreatment
of GSH for 1 h partially blocked the effect of BITC on the GST
induction (Fig. 2B
), whereas pretreatment before BITC
stimulation as well as treatment alone of a potent thiol blocker, DEM,
significantly and additionally enhanced the GST activity. These results
suggested the involvement of redox regulation in the induction of GST
by BITC.
|
, the BITC-induced increase in intracellular fluorescence,
even at 10 min, was observed in the fluorescence images acquired by a
confocal laser scanning microscope, whereas the control cells treated
only with H2DCF-DA showed a weak accumulation of
DCF attributable to the ROIs produced by the basal oxidative
metabolism. The level of ROIs induced by different concentrations of
BITC was increased in a dose-dependent manner. The level at 10
µM BITC was 50-fold higher than that of the
control (Fig. 3B
, entries 1 and 3).
These data indicated that BITC is a potential inducer of intracellular
ROIs.
|
, weakly active AITC showed a slight production of ROIs, and inactive
PITC did not produce ROIs. To determine whether the intracellular ROI
accumulation was negatively regulated by the GSH level, we examined the
effects of intracellular GSH modifiers, such as BSO, DEM, and
antioxidants. BSO significantly reduced the GSH level (data not shown)
but only slightly enhanced the ROI production, although reducing the
GSH level almost comparable with BITC (data not shown), whereas the
strong intracellular thiol blocker, DEM, significantly enhanced the ROI
production. Moreover, the peroxide production by BITC was partially but
significantly suppressed by GSH, quercetin, and the NADPH oxidase
inhibitor, DPI, whereas the XOD inhibitor, allopurinol, was ineffective
(Fig. 4
). The intracellular production of ROIs was confirmed by the
lack of inhibition by the membrane-impermeable, ROI-scavenging enzymes,
catalase and superoxide dismutase.
|
). We first tested the effect of BITC on both
ECAT gene and 1CAT gene expressions. As shown in
Fig. 5B
, BITC (5 µM) could enhance
the activity of the ECAT gene 3.3-fold of the control in
these cells, whereas BITC could not activate the GPEI deletion mutant
1CAT. Next, we examined the effects of the pretreatment of the cells
with GSH or DEM prior to the BITC treatment. Pretreatment of GSH (10
mM) completely blocked the BITC-induced
enhancement of the ECAT activity. However, DEM, which induced the
accumulation of DCF attributable to the ROIs (Fig. 4
), significantly
enhanced not only the basal activity (3.8-fold) but also the
BITC-stimulated activity of ECAT (1.4-fold; Fig. 5B
). These
results suggest that GPEI is the ROI response element and that BITC may
act on this element via production of the intracellular ROIs.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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The present results indicated that BITC elevated the GST activity
in normal rat liver epithelial cells (RL34), even at the concentration
of 10 µM (Fig. 1A
). To determine which
isozymes participate in the induction of the GST activity by BITC, an
immunoblot analysis was done using the GST class-specific antibodies.
Among the GST isozymes, GSTP1 significantly increased 16 h after
the BITC treatment (Fig. 1B
), suggesting that the increase
in GST activity was largely attributable to the elevated synthesis of
this protein. The increase in the GSTP1 protein level coincided with
the substantial rise in the GSTP1 mRNA level (Fig. 1C
). A
recent study using transgenic mice lacking the class GST
demonstrated that this class of GST was involved in the metabolism of
carcinogens, such as 7,12-dimethylbenz[a]anthracene, in
mouse skin and had a profound effect on tumorigenicity
(25)
. These results suggest that the class GST isozyme
could be one of the important determinants in cancer susceptibility,
particularly in diseases where exposure to polycyclic aromatic
hydrocarbons is involved.
It has been shown that the gene expression of GSTA1 is related to the
intracellular oxidative stress presumably mediated by hydroxyl radicals
(·OH) or the pro-oxidative potential of GSTA1 inducers (11
, 12)
. In addition to GSTA1, oxidative stress has been reported to
enhance the expression of genes encoding other antioxidant enzymes,
including the 
-glutamyl cysteine synthase (26)
, heme
oxygenase (27)
, and heat shock protein 90
(28)
. Thus, it is increasingly recognized that an adequate
amount of oxidative stress stimulates a variety of signal
transduction pathways under circumstances that do not result in cell
death. In a recent study, the treatment of RL34 cells with the major
end product of the oxidized fatty acid metabolism results in GSTP1
induction (9)
and shows a quick cellular GSH depletion,
the generation of intracellular ROIs, and the activation of stress
signaling pathways (10)
. The finding that at least 1 h of exposure to BITC was sufficient to evoke the elevation of GST
activity (Fig. 2B
) is consistent with the previous
observation (13)
that BITC induced the oxidative
stress-dependent JNK activation within 1 h. These data suggest
that ITCs may induce GST and/or other phase II detoxification enzymes
through the stress signaling pathway involving oxidative stress and JNK
or p38 cascade, similar to other stimuli, including hydrogen peroxide
(10)
, UV light (29)
, osmotic stress
(30)
, the DNA-damaging agent (31)
,
inflammatory cytokine (32)
, and lipid peroxidation
products (10)
.
In the present study, we showed that the BITC-induced enhancement
of GST activity was blocked by the antioxidant GSH and enhanced by the
thiol blocker DEM (Fig. 2B
). These results suggest that the
initial signal for GST induction is likely to be transduced to a
plausible cytosolic sensor(s) or receptor(s). Talalay and Zhang
(33)
have reported recently that the total intracellular
concentrations of the ITCs [ITC and the corresponding dithiocarbamate;
R-NH-C(
S)-SG] correlated closely and was linear with their
potencies as inducers of phase II detoxification enzymes. For example,
BITC was quickly and significantly accumulated in Hepa 1c1c7 cells, but
very little inactive PITC was detected within the cells
(33)
. They have also suggested the possibility that
intracellular GSH as a target of direct alkylation with ITCs plays a
negative regulating role in the phase II detoxification enzyme
induction, on the basis of the result that depletion of GSH by BSO
increased the inducer potencies of several ITCs (33)
. We
indeed observed that the treatment of BITC for 15 min resulted in a
significant depletion of the intracellular GSH level (Fig. 2A
). However, based on the observation that BSO, which
reduces the intracellular GSH level, showed no GST-inducing potency
(data not shown), it is unlikely that down-regulation of intracellular
GSH is simply an initial signal for GST induction.
It is noteworthy that the treatment of BITC quickly and significantly
enhanced the intracellular ROI production in RL34 cells detected by the
fluorescence probe, H2DCF-DA (Fig. 3
).
H2DCF-DA reacts peroxidase dependently or
spontaneously with some types of ROIs, including hydrogen peroxide,
lipid hydroperoxide, hypochlorous acid, and peroxinitrite (34
, 35)
. The experiments using the membrane-impermeable catalase and
superoxide dismutase also suggested that the BITC-induced ROI
generation might occur within the cells. In the structure-activity
relationship study of ITCs, the ROI-producing activities correlated
closely with their GST-inducing potencies (Fig. 4
). These results
provide clear evidence for the intracellular ROI production induced by
BITC and suggest that intracellular ROIs may be involved in the
BITC-stimulated GST induction.
The oxidative stress-inducible effect of BITC was blocked by the
pretreatment of an antioxidant, such as GSH or quercetin (Fig. 4
). In
fact, RL34 cells exhibited superoxide dismutase- and XOD
inhibitor-inhibitable superoxide anion radical generation when ferric
nitrilotriacetate was treated for 24 h (36)
. It is
unlikely that XOD is responsible for the BITC-induced ROI production
because activation of XOD is required for the conversion time lag of
xanthine dehydrogenase, a major form of XOD protein in healthy tissue
(37)
and because of the lack of interference by the enzyme
inhibitor, allopurinol (Fig. 4
). The treatment of BSO, only in part,
mimicked the elevation of cellular oxidative stress (Fig. 4
). It has
been reported that BSO mainly depleted GSH by blocking GSH synthesis
and GSH reductase but did not appreciably affect the mitochondrial GSH
pools (38)
. On the other hand, the pretreatment of DEM,
which blocks the intracellular thiol groups, including mitochondrial
GSH (39)
, only produced ROIs and enhanced the BITC-induced
oxidative stress (Fig. 4
). Moreover, pretreatment of DPI, acting
not only as a NAD(P)H oxidase inhibitor (40)
but also an
inhibitor of mitochondrial reactive oxygen species production
(41)
, resulted in a significant decrease in the
BITC-induced ROI accumulation (Fig. 4
). These results strongly
suggested that ROIs detected in the cells exposed to BITC may originate
from the mitochondria, one of the major ROI-producing organella (Fig. 6
). We have indeed observed that BITC enhances ROI production in isolated
mitochondria.5
|
). As mentioned above, the
ITC-induced oxidative stress is reported to be involved in the
ITC-induced activation of JNK (13)
, which phosphorylates
transcriptional factors, such as c-Jun, ATF-2, and Elk-1, and strongly
augments their transcriptional activity (42, 43, 44, 45)
.
Identification of the factor(s) that binds GPEI and activates the
GSTP1 gene expression is required for further understanding
of the regulation of GSTP1 induction by BITC. In addition, it is
noteworthy that the class GST isozyme is closely related to the
regulation of JNK signaling (46)
. It is within the range
of possibility that the primary target of BITC or BITC-induced ROIs is
GSTP1 itself, the modification of which may result in direct
stimulation of the JNK signaling pathway. The observation that GST
activity was inhibited by the incubation with BITC for 30
min6
also supports this hypothesis. In conclusion, intracellular ROIs are likely to mediate the BITC-induced GSTP1 gene expression, based on the observations that: (a) a short exposure time (1 h) to BITC is sufficient to evoke the elevation of GST activity; (b) ROI-producing activities closely correlated with their GST inducing potencies; and (c) DEM enhanced the BITC-induced ROI production and accelerated both the basal and BITC-induced GST activities and the GSTP1 gene expression, whereas the antioxidant GSH inhibited them. Although many researchers have confirmed that ITCs are promising and effective anticarcinogen candidates, some of the ITCs showed an enhancement of carcinogenicity or lack of chemopreventive effects in rat liver and kidney, especially dosed at the postinitiation phase (47 , 48) . Lee (49) has postulated that ITCs are oxidatively converted to the corresponding isocyanates, causing chromosome aberration, mutation, and cancer (50) . These events may correlate with the intracellular ROI-producing potentials of the ITCs. In fact, cytotoxicity of ITCs at high dose can be inhibited by the antioxidant, N-acetylcysteine (51) . As mentioned above, the profound relationship between GSTP1 and the stress signaling pathways (46) indicated that GSTP1 is one of the most important components that could influence key cellular functions, including growth, apoptosis, and transformation. Because the cancer preventive or promoting potential, threshold, and target organ of ITCs have to be distinguished in detail, further mechanistic studies on intracellular oxidative stress and the subsequent events induced by the ITCs are essential to provide supporting information.
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Acknowledgments |
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FOOTNOTES |
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1 Supported by Grants-in-Aid for JSPS
Research Fellow grant (to Y. N.) from the Ministry of Education,
Science, Sports and Culture of Japan and supported in part by Program
for Promotion of Basic Research Activities for Innovative
Biosciences. 
2 To whom requests for reprints should be
addressed, at Laboratory of Organic Chemistry in Life Science, Division
of Applied Sciences, Graduate School of Agriculture, Kyoto University,
Kyoto 606-8502, Japan (Y. N.) or Laboratory of Food and Biodynamics,
Nagoya University Graduate School of Bioagricultural Sciences, Nagoya
464-8601, Japan. Phone: 81-52-789-4127; Fax: 81-52-789-5741; E-mail: uchidak{at}agr.nagoya-u.ac.jp (K. U.).
3 The abbreviations used are: GST, glutathione
S-transferase; ITC, isothiocyanate; CAT, chloramphenicol
acetyltransferase; GSH, glutathione; ROI, reactive oxygen intermediate;
JNK, c-Jun NH2-terminal kinase; BITC, benzyl
isothiocyanate; DCF, dichlorofluorescein; H2DCF-DA,
2',7'-dichlorofluorescin diacetate; DEM, diethyl maleate; PITC, phenyl
isothiocyanate; BSO, D,L-buthionine
(S,R)-sulfoximine; AITC, allyl
isothiocyanate; DPI, diphenylene iodonium; XOD, xanthine oxidase; GPEI,
GSTP1 enhancer I.
4 Y. Nakamura, Y. Morimitsu, T. Uzu, H. Ohigashi,
A. Murakami, Y. Naito, Y. Nakagawa, T. Osawa, and K. Uchida. A
glutamine S-transferase inducer from papaya: Rapid screening
identification and structure-activated relationship of isothiocyanates,
submitted for publication.
5 Y. Nakamura, A. Yoshihiro, H. Ohigashi, and K.
Uchida, manuscript in preparation.
6 Y. Nakamura and K. Uchida, unpublished
observation.
Received 9/13/99. Accepted 11/30/99.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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B, and glutathione S-transferase gene expression. J. Biol. Chem., 271: 13422-13429, 1996.-glutamylcysteine synthetase activity in rat lung epithelial L2 cells. J. Biol. Chem., 269: 26512-26517, 1994.This article has been cited by other articles:
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(BITC) and 
(PITC), mean values of
three independent experiments; bars, SD.
B, immunoblot analysis of GSTP1. Each whole-cell lysate
was treated with a Laemmli sample and was then run on a 12.5% SDS-PAGE
slab gel. The gel was transblotted on a nitrocellulose membrane and
treated with the antibody. C, Northern blot analysis of
GSTP1 mRNA. Total cellular RNA (20 µg) was fractionated on 1%
agarose/formaldehyde gel and transferred onto a nitrocellulose filter,
and the RNA blot was then hybridized with a 32P-labeled
cDNA probe for GSTP1 (top) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH;
bottom). The autoradiographs were scanned to quantify
GSTP1 mRNA induction relative to control of the ratio
(GSTP1/GAPDH).
) or 1CAT (