Carcinogenesis vol.25 no.5 pp.787--791, 2004
DOI: 10.1093/carcin/bgg161
Diallyl sulfide inhibits the oxidation and reduction reactions of stilbene estrogens
catalyzed by microsomes, mitochondria and nuclei isolated from breast tissue
of female ACI rats
Ronald D.Thomas1,3, Mario R.Green1, Chantell Wilson1
and Sakeenah Sadrud-Din2
1
Environmental Toxicology Program, College of Pharmacy and
Pharmaceutical Sciences and 2Biology Department, College of Arts and
Sciences, Florida A&M University, Tallahassee, FL 32307, USA
3
To whom correspondence should be addressed
Email: [email protected]
Previously, it has been demonstrated that microsomal,
mitochondrial and nuclear enzymes isolated from the
liver of male Sprague--Dawley rats catalyzed the oxidation
of diethylstilbestrol (DES) to DES quinone. In the present
study we have shown that diallyl sulfide (DAS) inhibits the
oxidation of DES to DES quinone in all three subcellular
fractions (microsomes, mitochondria and nuclei) isolated
from breast tissue of female ACI rats. UV analysis of
mitochondrial and microsomal fractions revealed that
DAS decreased the rate of DES oxidation to DES quinone
and DAS also decreased the rate in which DES quinone was
reduced to DES. Lineweaver--Burk plots of the rate of DES
quinone formation at various DES and DAS concentrations
demonstrated that DAS inhibited the oxidation of DES and
the reduction of DES quinone in a non-competitive fashion.
In both microsomal and mitochondrial oxidation reactions
the Km remained constant whereas the Vmax decreased with
increasing DAS (0, 186 and 373 mM) concentrations (microsomes Km ˆ 80 mM; Vmax ˆ 5.56, 4.16 and 3.33 nmol/mg
protein/min; mitochondria Km ˆ 35.7 mM; Vmax ˆ 3.45,
2.44 and 1.82 nmol/mg protein/min). Results were similar
for reduction reactions. HPLC analysis revealed that a
concentration of 186 mM DAS inhibited the mitochondrial,
microsomal and nuclear oxidation by 27, 35 and 40%,
respectively. A concentration of 373 mM DAS inhibited
the mitochondrial, microsomal and nuclear oxidation by
50, 52 and 60% respectively. The data provide direct evidence that the breast tissue contain the metabolic machinery required to oxidize DES to reactive intermediates that
may lead to genetic instability and cancer. This inhibition
may play a role in the chemoprevention of stilbene estrogen-induced breast cancer.
Introduction
It has been shown that estrogens including diethylstilbestrol
(stilbene estrogen) are metabolized to reactive intermediates,
such as semiquinone and quinone (1,2). Microsomal, mitochondrial and nuclear enzymes are all involved in the oxidation and reduction of DES (3,4). The reactive metabolites of
diethylstilbestrol have been shown to covalently bind to DNA (5)
Abbreviations: DAS, diallyl sulfide; DES, diethylstilbestrol; PMSF,
phenylmethylsulfonyl fluoride.
Carcinogenesis vol.25 no.5 # Oxford University Press 2004; all rights reserved.
and nuclear proteins (6). The metabolism of estrogen is
believed to play a major role in estrogen-induced cancer (7).
Several sulfur containing compounds with chemopreventive
properties have been isolated from garlic, the most effective
one being diallyl sulfide (DAS) (8). Diallyl sulfide has been
found to inhibit dimethylhydrazine-induced colon carcinoma
in female C57NL/6 J mice (9), N-nitrosoamine induced
esophageal carcinoma in male Sprague--Dawley rats (10), Nnitrosodiethylamine induced pulmonary adenoma in female
A/J mice (11), benzo[a]pyrene induced forestomach tumor and
pulmonary adenoma in female A/J mice (12) and hepatocarcinogenesis induced by 1,2-dimethylhydrazine in Fisher 344 rats
(13). It has been shown that DAS inhibits the production of
DES-induced DNA adducts presumably via metabolic modulation (14). Whether or not DAS will inhibit estrogen induced
cancer or estrogen metabolism is yet to be determined.
In the present study the inhibition of microsomal, mitochondrial and nuclear oxidation of DES to DES quinone by DAS
has been investigated. This study provides direct evidence for
the inhibition of the oxidation and reduction of DES by DAS in
various organelles. DES reactive intermediates generated during oxidation are able to covalently bind to both mitochondrial
and nuclear DNA (15). Analogous in vivo oxidation of DES to
reactive metabolites and covalent modifications in DNA by
reactive products may be factors in DES-induced cancer.
Furthermore, the inhibition of the production of these reactive
metabolites may prevent DES-induced cancer.
Materials and methods
Chemicals
NADH, diethylstilbestrol, phenylmethylsulfonyl fluoride (PMSF), cumene
hydroperoxide, b-napthoflavone, digitonin and diallyl sulfide were purchased
from Sigma Chemical Co., St Louis, MO. Female ACI rats 7--8 weeks old were
purchased from Harlan, Indianapolis, IN.
Animal treatment
Ten female ACI rats were treated for 4 days with a daily dose of b-napthoflavone (50 mg/kg i.p.) to induce the IA family of cytochrome p-450 (2). The
rats were killed by carbon dioxide exposure. The breast tissue was removed,
weighed and homogenized in 1:10 wt/vol (0.25 M sucrose, 1.0 mM EDTA,
2.5 mM PMSF). Microsomes, mitochondria and nuclei from the breast tissue
were isolated by differential centrifugation. The organelles were used to
catalyze the oxidation and reduction reactions of DES. The oxidation and
reduction products generated by the organelles were analyzed by UV absorption and HPLC analysis.
Preparation of nuclei
Nuclei were isolated according to the procedure of Rickwood et al. (16). In
brief, tissue was homogenized in 2 vol of 0.25 M sucrose solution containing
1.0 mM PMSF. The homogenate was filtered through cheesecloth and centrifuged for 10 min at 1000 g. The pellet obtained was suspended in 0.25 M
sucrose and 4 vol of 2.3 M sucrose was under-laid. The nuclei were collected
by centrifugation at 100 000 g for 60 min. The nuclei were then washed with
0.25 M sucrose containing 0.2 mM PMSF. The purity of nuclei was checked
after staining with hematoxylin by phase contrast microscopy. Glucose 6phosphatase and cytochrome C oxidase activity was assayed to determine
microsomal and/or mitochondrial contamination (17,18). It was determined
787
R.D.Thomas et al.
that there was 51% contamination of both microsomes and mitochondria
based on enzymatic activity.
Microsomal preparation
Breast tissue homogenate was centrifuged at 12 000 g for 30 min to eliminate
nuclei and mitochondria. The supernatant was centrifuged at 120 000 g to
collect microsomes. The purity of the microsomes was assessed by both
morphological and biochemical analyses. Microsomes were stained with
eosin and hematoxylin. Phase contrast microscopy did not reveal any nuclear
contamination. The determination of cytochrome C oxidase (17) demonstrated
51% mitochondrial contamination.
Mitochondrial preparation
Breast tissue was centrifuged at 3000 g for 30 min to remove nuclei and
cellular debris. The supernatant was centrifuged at 11 000 g to collect mitochondria. The mitochondria was treated with 1.6% digitonin to remove the
outer membrane and cellular contamination. The mitoplasts (mitochondria
without outer membrane) was collected by centrifugation for 30 min at
11 000 g for 30 min (19). The purity of mitoplasts was assessed by both
morphological and biochemical analyses. Mitoplasts were stained with eosin
and hematoxylin. Phase contrast microscopy did not reveal any cellular contamination. The determination of cytochrome C oxidase activity (17), an enzymatic marker of mitochondria, showed 100--110 mmol/mg protein/min specific
activity. Microsomal contamination was assessed by measuring the activity of
glucose 6-phosphatase, an enzymatic marker of endoplasmic reticulum (18).
The activity of glucose 6-phosphatase was 601 pmol/mg protein/min in microsomes. However, the activity of glucose 6-phosphatase was 21 pmol/mg
protein/min in our mitochondrial preparations. These values demonstrate that
our mitoplasts were free of nuclei and microsomes. This is in agreement with
the report of Niranjan et al. (20,21).
Oxidation reaction system
The reaction condition for the conversion of DES to DES quinone as described
by Roy and Liehr (2) was used. The reaction condition consists of 120 mM
cumene hydroperoxide, 420 mg/ml mitoplasts or 346 mg/ml of microsomes, in
a final volume of 1.0 ml of 10 mM phosphate buffer, pH 7.5. Various
concentrations (0--120 mM) of DES were used to determine the kinetic constants of the reactions. Various concentrations of DAS (186 and 373 mM) were
added to determine the type of inhibition that DAS has on the oxidation of
DES. No cumene hydroperoxide (oxidation cofactor) was used in control
reactions. The conversion of DES to DES quinone was monitored as a gradual
increase in UV absorption in the range of 280--400 nm. The oxidation product
formation was also analyzed by HPLC (2). DES metabolites were extracted
from the oxidation reactions with ethyl ether. The extraction recovery of
synthetic DES quinone was 95 5%. The identity of DES quinone was
confirmed by GC/MS analyses.
Reduction reaction system
The reduction reaction conditions for the conversion of DES quinone to DES
as described by Roy and Liehr (2) was used. The reaction condition consists of
50 mM NADH, 420 mg/ml mitoplasts or 346 mg/ml of microsomes, and
in a final volume of 1.0 ml of 10 mM phosphate buffer, pH 7.5. Various
concentrations (0--60 mM) of DES quinone were used to determine the kinetic
constants of the reactions. Various concentrations of DAS (186 and 373 mM)
were added to determine the type of inhibition that DAS has on the reduction of
DES quinone. No NADH (reduction cofactor) was used in control reactions.
The conversion of DES quinone to DES was monitored as a gradual decrease
in UV absorption in the range of 280--400 nm. The formation of reduction
products was analyzed by HPLC (2). DES quinone and its metabolites were
extracted with ethyl ether.
HPLC analysis
The oxidation and reduction products were extracted with water saturated ethyl
ether. The reaction mixture was dried under nitrogen and metabolites were
reconstituted in methanol. An appropriate amount (10--50 ml) of sample was
injected into the HPLC. A methanol/water gradient consisting of 36--83%
methanol was run using a C14 reverse phase column from 0 to 30 min at a
flow rate of 1 ml/min. The UV detection was performed at a wavelength of
254 nm. The statistical significance was determined using a one-way ANOVA
on SAS statistical software.
Results
Inhibition of DES oxidation by DAS
Mitoplasts were incubated in the presence of DES and cumene
hydroperoxide using the oxidation reaction system described
in the methods. DES quinone was detected by UV spectroscopy. The UV spectral analysis of the mitochondrial mixture
containing DES and cumene hydroperoxide revealed a gradual
increase in the absorbance at 312 nm. The spectral pattern was
identical to that of synthetic DES quinone (Figure 1). In the
control reactions, no DES quinone was produced. In oxidation
reactions that contained DAS (373 mM) the production of DES
quinone was reduced by 50%. The rate of DES quinone formation in the presence of mitoplasts and cumene hydroperoxide was dependent on the concentration of DES (Figure 2). A
Lineweaver--Burk plot of rate of formation of DES quinone at
various concentrations of DES yielded a Km of 35.7 mM and
Vmax of 3.45 nmol/mg protein/min. The kinetic constants of the
reactions were determined by using various concentrations
of DES (0--120 mM) and two concentrations of DAS (186
and 373 mM). With increasing concentrations of DAS the
Km remained constant whereas the Vmax decreased (3.45,
2.44 and 1.82 nmol/mg protein/min) (Figure 2). The data
indicate that the mitochondria are capable of metabolizing
Fig. 1. Oxidation of DES to DES quinone catalyzed by mitoplasts. (A) The control reaction, i.e. contains no cumene hydroperoxide. (B) The complete reaction.
(C) The complete reaction with 373 mM DAS. The oxidation was monitored by UV spectroscopy. The lowest absorbencies were recorded at time 0. An increase in
absorbency was recorded every 30 s.
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Diallyl sulfide inhibits redox cycling of DES
Fig. 2. Influence of various substrate concentrations of DES on the rate of oxidation of DES to DES quinone by mitoplasts: The reaction mixture consisted of
mitoplasts (0.42 mg equivalent protein), 120 mM cumene hydroperoxide, and various concentrations of DES (0--100 mM) in a final volume of 1 ml 10 mM
phosphate buffer, pH 7.5. A Lineweaver--Burk plot of rate of formation of DES quinone and its inhibition by DAS revealed a constant Km with decreasing
Vmax . Values represent the means of four experiments.
Table I. Kinetic constants of oxidation and reduction reactions
Oxidation
Km
Reduction
Microsomes
Mitochondria
Microsomes
Mitochondria
80 mM
35.7 mM
100 mM
50 mM
5.56
3.45
22.0
2.0
4.16
VmaxI
186 mM DAS
2.44
14.0
1.5
3.33
VmaxII
373 mM DAS
1.82
12.0
1.25
Vmax0
0 mM DAS
The unit for Vmax is nmol/mg protein/min.
DES to DES quinone and DAS is capable of inhibiting this
metabolism. Based on the kinetic constants the nature of this
inhibition seems to be non-competitive. Results from the oxidation in microsomal reactions were similar to those in mitochondria. The microsomes were capable of oxidizing DES
to DES quinone and DAS inhibited this oxidation (data not
shown).
The kinetic constants for the mitochondrial and microsomal
oxidation reactions are summarized in Table I. The kinetic
constants were not determined for nuclear oxidation reactions
due to the small amount of nuclei that could be isolated from
the breast tissue.
Inhibition of the reduction of DES quinone by DAS
Mitoplasts were incubated in the presence of DES quinone and
NADH using the reduction reactions as described in the methods. The disappearance of DES quinone was detected by UV
spectroscopy. The UV spectral analysis of the mitochondrial
mixture containing DES quinone and NADH revealed a gradual decrease in the absorbance at 312 nm (Figure 3). In the
control reactions little DES quinone was reduced. The amount
of DES quinone reduced was decreased by 50% in the reduction reactions that contained DAS (373 mM). The rate of DES
quinone disappearance in the presence of mitoplasts and
NADH was dependent on the concentration of DES quinone
(Figure 4). A Lineweaver--Burk plot of rate of disappearance
of DES quinone at various concentrations of DES yielded a Km
of 50.0 mM and Vmax of 2.0 nmol/mg protein/min. The kinetic
constants of the reactions were determined by using various
concentrations of DES quinone (0--60 mM) and two concentrations of DAS (186 and 373 mM). With increasing concentrations of DAS the Km remained constant whereas the Vmax
decreased (2.0, 1.5 and 1.25 nmol/mg protein/min, respectively) (Figure 4). The data indicate that mitochondria are
capable of reducing DES quinone to DES and DAS is capable
of inhibiting this reduction. Based on the kinetic constants, the
nature of this inhibition seems to be non-competitive. Results
from the reduction in microsomal reactions were similar to
those in the mitochondria. The microsomes were capable of
reducing DES quinone to DES and DAS inhibited this reduction (data not shown).
The kinetic constants for the mitochondrial and microsomal
reduction reactions are summarized in Table I. The kinetic
constants were not determined for nuclear reduction reactions
due to the small amount of nuclei that could be isolated from
the breast tissue.
HPLC results
HPLC analysis of the production of oxidation and reduction
products of DES in microsomes, mitochondria and nuclei were
similar to that demonstrated by UV analysis. DAS inhibited
oxidation and reduction reactions of DES in a dose-dependent
fashion. A concentration of 186 mM DAS inhibited the mitochondrial, microsomal and nuclear oxidation by 27, 35 and
40%, respectively. A concentration of 373 mM DAS inhibited
the mitochondrial, microsomal and nuclear oxidation by 50, 52
and 60% respectively (Table II). Similar results were seen in
reduction reactions (Table III).
Discussion
It is commonly recognized that the liver is the major organ of
xenobiotic biotransformation. We have demonstrated for the
first time that organelles (mitochondria, microsomes and
nuclei) isolated from the breast of female ACI rats catalyze
the oxidation and reduction reactions of DES. This is significant in that it provides evidence that breast tissue is capable of
metabolizing estrogens into reactive products that can cause
DNA damage. Redox-cycling of DES has been demonstrated
to produce reactive oxygen species such as superoxide radicals
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R.D.Thomas et al.
Fig. 3. Reduction of DES quinone to DES catalyzed by mitoplasts. (A) The control reaction, i.e. contains no NADH. (B) The complete reaction. (C) The complete
reaction with 373 mM DAS. The reduction was monitored by UV spectroscopy. The highest absorbencies were recorded at time 0. A decrease in
absorbency was recorded every 30 s.
Fig. 4. Influence of various concentrations on the reduction of DES quinone catalyzed by mitoplasts in the presence NADH: The reaction mixture consists of
mitoplasts (0.42 mg equivalent protein), 50 mM NADH, and various concentrations of DES quinone (0--44 mM) in a final volume of 1 ml 10 mM of
phosphate buffer, pH 7.5. A Lineweaver--Burk plot of rate of formation of DES and its inhibition by DAS revealed a constant Km with decreasing Vmax . Values
represent the means of four experiments.
Table II. HPLC analysis of oxidative reactions
Table III. HPLC analysis of reduction reactions
Conditions
Conditions
Control (-ChP)
Complete system
‡DAS (186 mM)
(% Inhibition)
‡DAS (373 mM)
(% Inhibition)
DES quinone (pmol/min/mg protein)
Microsomes
Mitoplasts
Nuclei
ND
3.12 0.20
2.04 0.15a
35%
1.49 0.12b
52%
ND
2.45 0.16
1.66 0.14a
27%
1.23 0.12b
50%
ND
0.196 0.038
0.117 0.014a
40%
0.078 0.020b
60%
Complete system: 346 mg/ml microsomes, 420 mg/ml mitoplast and
450 mg/ml nuclei, 120 mM cumene hydroperoxide (ChP), 100 mM DES,
186 mM and 373 mM DAS in 1 ml 10 mM potassium phosphate buffer
pH 7.5. Each value in the table represents the mean and standard error of
the mean of five individual reactions.
a
Values are statistically different from the control at P 5 0.05.
b
Values are statistically different from the control and the 186 mM
DAS reactions at P 5 0.05.
and DES quinone (1). These reactive molecules can cause
DNA damage and ultimately mutations that cause cancer.
The demonstration of the redox-cycling of DES by mitoplasts
and nuclei are of significance in that these are not traditional
790
Control (-NADH)
Complete system
‡DAS (186 mM)
(% Inhibition)
‡DAS (373mM)
% Inhibition)
DES (pmol/min/mg protein)
Microsomes
Mitoplasts
Nuclei
ND
0.92 0.12
0.90 0.14
3.1%
0.54 0.12a
41%
ND
1.27 0.19
0.99 0.14a
22%
0.89 0.12a
30%
ND
0.16 0.033
0.10 0.014a
38%
0.075 0.020a
53%
Complete system: 346 mg/ml microsomes, 420 mg/ml mitoplasts and
450 mg/ml nuclei, 50 mM NADH, 44.8 mM DES, 186 mM and 373 mM
DAS and 1 ml 10 mM potassium phosphate buffer pH 7.5. Each value in
the table represents the mean and standard error of the mean of five individual
reactions.
a
Indicates that values are statistically different from the control at P 5 0.05.
organelles of metabolic study. However, they contain the most
critical macromolecule (DNA) in regards to carcinogenesis.
The role that the mitochondria play in carcinogenesis is not
clear. However there are many correlations with alterations in
the mitochondria and carcinogenesis. It has been shown that
mitochondrial DNA is the primary site of attack by reactive
Diallyl sulfide inhibits redox cycling of DES
metabolites of benzo[a]pyrene, aflatoxin B1, 7,12-dimethylbenz[a]anthracine and 3-methylcholanthrene (22--24). In the
case of benzo[a]pyrene the adduct level in the mitochondria
was 40 times higher than that found in genomic DNA and these
adducts persist longer in mitochondrial DNA (21). The
mitochondria of tumor cells are frequently structurally and
functionally different from those isolated from normal cells
(22--24). This evidence supports the idea that mitochondrial
metabolism may play a crucial role in carcinogenesis. In
addition to demonstrating that these organelles can metabolize
DES to reactive intermediates, we have demonstrated that DAS
inhibits this metabolism in a non-competitive fashion in mitochondria and microsomes. This inhibition may help explain the
mechanism of the chemopreventive actions of DAS.
Furthermore, we have shown that DAS when given with
DES, inhibits the formation of DES-induced DNA adducts
in vitro and in vivo (14). This demonstrates that direct inhibition of DES metabolism plays a role in the inhibition of DESinduced DNA adduct formation. We have also shown that
DAS administered several days prior to DES, inhibits the
formation of DES-induced DNA adducts (14). This implies
that some process in addition to the direct inhibition of metabolism by DAS is involved in the inhibition of DES-induced
DNA adducts. It has been shown that DAS induces glutathione
S-transferase, epoxide hydrolase and UDP-glucuronosyltransferase in the liver (25). We propose that the expression of these
genes may be augmented by DAS to afford protection from the
production of DES induced breast cancer. The results of this
study will help elucidate the mechanism of estrogen-induced
breast cancer. The data will provide a foundation for the
further investigation of the chemopreventive properties of
DAS and structurally similar compounds.
Acknowledgement
This study was supported by the United States Department of Defense Breast
Cancer Research Project. Grant # DAMD17-98-1-8081.
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Received May 1, 2003; revised August 6, 2003;
accepted August 19, 2003
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