cancer Letters. 57 (1991) 121-129 Elsevier Scientific Publishers Ireland
Ltd.
A.K. Maurya and S.V. Singh Diuision of Experimental
Therapeutics. Deportment of Oncology, Uniuersity of Miami School of Medicine,
Miami,
Florida
(U.S.A.)
(Received 12 December 1990) (Revision received 28 January 1991) (Accepted 30 January 1991)
Summary
red after treating mice with 50 pmol DAS. GSH peroxidase activity towards t-butylhydroperoxide increased in a dose-dependent fashion in the mice stomach treated with DAS. Even though this activity towards hydrogen peroxide was similar in mice treated with 50 or 75 pmol DAS, fhese values were significantly higher than that of the control. GSH reducrase was also elevated in the stomach of mice treated with 75 pmol DAS. These results suggest that DAS may exert anti-neoplastic effect by modulating GSH dependent detoxification enzymes.
Diallyl sulfide (DAS), an organosulfur compound identified as the flavor component in garlic, has been shown to inhibit chemically induced neoplasia of forestomach and lung in mice. Even though the exact mechanism(s) of anti-neoplastic activity of DAS is not known, several independent studies suggest that this effect may, at least in part, be due to the elevation of glutathione-S-transferase (GST) activity. To gain further insight into the mechanism(s) of anti-carcinogenic activity of DAS, we have determined effect of orally administered DAS (25, 50 and 75 pmol) on levels of (Y, p and ?r class GSTs and glutathione (GSH) peroxidase and GSH reductase activities of female A/J mice stomach. Western blotting revealed presence of (Y, p and n class GSTs in mice stomach. A significant increase in all the three classes of GSTs was observed in the stomach of mice treated with DAS. Maximum increase in GST CY and a was evident by treating the animals with 75 pmol DAS whereas maximum induction of GST p occur-
Keywords: glutathione; glutathione transferase; diallyl sulfide; glutathione peroxidase; glutathione reductase Introduction Garlic and onion oils have been shown to have anti-carcinogenic activity 141. Several independent studies suggest that anti-neoplastic effect of these plant oils may primarily be due to the presence of an ally1 group containing organosulfur compounds [27-291. In a recent study, eight organosulfides were tested for their inhibitory effects on benzo[a]pyrene
Correspondence to: Dr. Shivendra V. Singh. University of Miami School of Medicine, P.O. Box 016960 (R-71), Miami, FL
33101,
U.S.A.
0304-3835/91/$03.50 Published and Printed
0 1991 Elsevier Scientific in Ireland
Publishers
Ireland
Ltd
122
(B[a]P) in . d uce d neoplasia of forestomach and lung in female A/J mice [27]. Among these compounds, diallyl sulfide (DAS), ally1 methyl disulfide, ally1 methyl trisulfide, and diallyl trisulfide inhibited B[a]P induced forestomach tumors whereas pulmonary adenoma was inhibited only by DAS and ally1 methyl disulfide [27]. Saturated analogs of these compounds, in which propyl group was substituted for the ally] group, did not inhibit either of these carcinomas [27]. DAS has also been shown to inhibit 1,2-dimethylhydrazine-induced colonic tumors in mice [31] and N-nitrosomethylbenzylamine-induced esophageal cancer in rats [32]. In addition, tumor promotion in dimethylbenzanthracene-induced skin cancer is inhibited by topical application of garlic and onion oil [4]. Although the mechanism(s) of protective effect of organosulfur compounds against chemicafly induced carcinogenesis is not completely understood, some of the studies suggest that this may, at least in part, be due to the elevation of glutathione-Stransferase (GST) activity [27-291. GST is a family of multifunctional enzyme proteins which detoxifies a wide variety of electrophilic xenobiotics including many carcinogens, by catalyzing their conjugation to glutathione (GSH) [8,12]. Other functions of GSTs include chemical removal of some xenobiotics through non-catalytic binding [14,25] and protection from oxidants through expression of selenium independent GSH peroxidase II activity [3,19]. Mammalian GST isoenzymes have been grouped into three classes, namely (Y, p and x [15]. Despite reports on elevated GST activity in DAS treated mice tissues [27,29], effect of this compound on different classes of GST isoenzymes has not been determined. In order to determine the role of GSTs in anti-neoplastic activity of organosulfides, their effect on different classes of GST isoenzymes/subunits must-be determined. In this study, we have investigated the effect of DAS on (Y, p and ?r class GST isoenzymes of female A/J mice stomach. We have also compared GSH peroxidase and
GSH reductase activities in the stomach control and DAS-treated mice. Materials
and
of
Methods
Chemicals Diallyl sulfide was purchased from Aldrich Chemical Co., Milwaukee, WI. Sources of all other chemicals used in this study were same as described previously [23]. Treatment of animals Female A/J mice (7- ll-week-old) were purchased from Jackson Laboratories, Bar Harbor, ME. Mice were divided into groups of three. Control group of animals were given 0.1 ml cottonseed oil by oral intubation and experimental groups of mice received 25, 50 or 75 pmol of DAS per animal dissolved in 0.1 ml cottonseed oil. Animals were sacrificed 48 h after the treatment, tissues were dissected out, washed with ice-cold phosphate buffered saline (PBS) and stored at - 20°C until used. Preparation of supernatant fraction A 10% (w/v) homogenate of whole tissue was prepared in 10 mM potassium phosphate buffer (pH 7) containing 1.4 mM 2-mercaptoethanol using a polytron. Homogenates were centrituged at 14 000 x g for 1 h to obtain supematant fractions. Enzyme
assays
GST activity towards l-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid was determined according to the method of Habig et al. [lo]. GSH peroxidase and GSH reductase activities were determined by the methods described by Awasthi et al. [2] and Beutler [5], respectively. Protein content was determined according to the method of Bradford [6]. Statistical analyses were performed by Student’s t-test. Western
blotting
Antibodies raised against human a, ~1and x class GST isoenzymes were same as described
123
previously [ 11. Western blotting was performed by the method of Towbin et al. [30]. Results
and
Discussion
To determine the effect of DAS on GST activity of mice stomach, animals were administered single or double doses of the desired concentrations of DAS followed by sacrifice 48 h post-treatment. The time point of DAS treatment, i.e. 48 h was chosen based on a previous study [27], where DAS was administered 48 h prior to the carcinogen treatment. Data presented in Table I shows the effect of 25, 50 and 75 pmol DAS treatment on GST activity of mice stomach. Treatment of animals with single dose of 25,50, and 75 pmol DAS increased GST activity by 1.46, 1.86 and 2.97 fold, respectively, when compared with control. Similarly, two doses of
Table 1. Effect of oral administration of diallyl sulfide on GST activity of female A/J mice stomach.
Treatment dose of diallyl sulfide Gmol)
None (cottonseed oil) 25 50 75
GST activity towards (unjtsa/mg protein) Single treatmentb
25,50 and 75 pmol DAS treatment also elevated GST activity by 1.44, 2.00 and 2.25, respectively, when compared with the control. Since increased GST activity in mice stomach by single and double dose DAS was comparable, in subsequent experiments, the animals were treated with single dose of DAS. Mammalian GST isoenzymes have been placed into three major classes, IX, p and x, based on their substrate specificities, structural homologies and immunological cross-reactivities [15]. Even though, GST isoenzymes belonging to CY,p and K classes have overiapping substrate specificities [ 151, certain class of isoenzymes exhibit preferential substrate specificities. For example, a class GST isoenzymes indicate preference towards ethacrynic acid [ 15,241 whereas selenium independent GSH peroxidase II activity is maximally expressed by a class GST isoenzymes [15,22]. To determine the effect of DAS on R class isoenzyme, GST activity towards ethacrynic acid was also determined (Table II). This activity in the stomach of control and 25 pmol DAS treated mice were comparable. On the other hand, ethacrynic acid conjugating activity was higher by 1.18 and 1.97-fold in the stomach of
CDNB
Two treatmentsC
463.3
ZII 33.0d
577.6
677.3 862.6 1380.0
f 97.9’ * 30.6’ f 26.4’
834.0 1156.6 1300.0
Table
Il.
conjugating
Effect of diallyl sulfide on ethacrynic activity of female A/J mice stomach.
f 17.9
Treatment dose of diallyl sulfide
+ 40.5’ =t 15.2’ f 69.2’
bmol)
“One unit of enzyme catalyzed conjugation of 1 nmol GSH to CDNB/min at room temperature. bAnimals were given test compound in 0.1 ml cottonseed oil and sacrificed 48 h after the treatment. ‘Mice were treated twice with DAS, 48 h apart and sacrificed 48 h after the second administration dValues represent mean l SD, ” = 3. ‘P zz 0.05, treated versus control. 'P 5 0.01, treated versus control.
None (cottonseed 25 50 75
acid
GST activity towards ethacrynic acid (unitsa/mg protein) oil)
46.0 50.0 54.3 91.0
f f + f
2.6b 2.0 2.5’ 1.7d
aOne unit of GST catalyzed conjugation of 1 nmol ethacrynic acid to GSH/min at 25°C. + S.D. of three deterbValues represent mean minations. ‘P zz 0.05, treated versus control. dP 5 0.01, treated versus control.
124
crease by 50 pmol DAS treatment, such an increase was not apparent in Western blot analysis (data not shown). These results suggest that Western blotting may not be sensitive enough to detect such a difference. Effect of DAS treatment on cy and p class GSTs was also determined by Western blotting (Fig. 2A and B). Using anti GST (Yand equal amount of cytosolic protein (60 pg), immunoreactive bands were observed in the lanes containing supernatants from 50 and 75 pmol DAS treated mice stomach (Fig. ZA, lanes 4 and 5, respectively). These results indicate that o class GST isoenzyme concentration was very low in female A/J mice stomach and treatment of animals with 25 pmol DAS had no effect on this GST isoenzyme. GST a!
mice treated with 50 and 75 pmol DAS, respectively, as compared to that of control (Table II) indicating a significant increase in GST r level in the stomach of mice treated with 75 pmol DAS. To confirm this finding, Western blotting was performed using antibodies raised against GST a of human placenta and equal amount (75 pg) of cytosolic protein from control and 75 pmol DAS treated mice stomach (Fig. 1). Western blot data was in good agreement with the enzyme activity determinations and revealed a markedly higher GST x content in the stomach of 75 pmol DAS treated mice (Fig. 1, lane 3) when compared to the control (Fig. 1, lane 2). Even though, ethacrynic acid conjugating activity showed 18% (P I 0.05) in-
Fig-l. natant
Western containing
GST T of human respectively.
blot
analysis
75 pg protein placenta.
Lanes
using
antibodies
were
subjected
2 and
raised
against
to polyacrylamide
3 contained
supernatants
GST
K of human
gel electrophoresis. from
control
placenta. Lane
and 75 pmol
Aliquots 1 contained
DAS-treated
of the
super-
homogenous mice stomach.
125
GST p content was relatively higher in the supernatants from 50 and 75 pmol DAS (Fig. 2B, lanes 4 and 5, respectively) treated mice stomach when compared to that of untreated mice (Fig. 2B, lane 2). Interestingly, GST p content in the stomach of 75 pmol DAS
content, however, increased in the stomach of mice treated with 50 and 75 pmol DAS; 75 pmol dose exhibiting greater effect (Fig. 2A, lane 5). On the other hand, GST p was detected in the supernatants from control as well as DAS treated mice stomach (Fig. 2B).
1
2
3
4
5
Fig. 2. Western blot analysis using antibodies raised against (A) 01class GST and (B) b class GST of human liver. Equal amount of cytosolic protein (60 pg) from control and DAS treated mice stomach supernatant fractions were subjected to electrophoresis. Lane 1 in gels (A) and (B) contained affinity purified GST from rat liver as positive control. Lanes 2 to 5 in both gels contained supernatants from control, 25 pmol, 50 lmol and 75 pmol DAS-treated mice stomach, respectively.
126
treated mice was lower (Fig. 2B, lane 5) when compared to that treated with 50 pmol DAS (Fig. 2B, lane 4). Effect of DAS treatment on GSH peroxidase activity of mice stomach was also determined (Table III). Two types of GSH peroxidases, selenium dependent and independent, have been characterized in mammalian tissues [ 18,191. Selenium containing GSH-peroxidase is a tetramer and uses both lipid hydroperoxides such as t-butylhydroperoxide (tBHP) and hydrogen peroxide as substrates whereas selenium independent GSH peroxidase II (an activity expressed by dimeric GST isoenzymes) reduces only lipid hydroperoxides (t-BHP) [3,18,19]. Thus, the effect of DAS on selenium dependent and independent GSH peroxidases was evaluated by determining the enzyme activity towards t-BHP and hydrogen peroxide (Table III) GSH peroxidase activity in control mice stomach was slightly higher towards t-BHP as compared to that with hydrogen peroxide indicating that both types of GSH peroxidases were expressed in this tissue. GSH peroxidase activity with t-BHP was higher by 1.59 and 2.34-fold in the stomach of mice treated with 50 and 75 pmol DAS, respectively as compared to that of control (Table III). On the other hand, this activity towards hydrogen peroxide was higher by 1.56 and 1.46 in the stomach of mice treated with 50 and 75 pmol DAS, respectively. These results indicated that whereas 50 pmol DAS
Table Ill. Effect of diallyl sulfide treatment Treatment dose of diallyl sulfide
Control 50 75
(cottonseed
on GSH peroxidase
GSH peroxidase
oil)
treatment caused increase in the activity of both GSH peroxidases, 75 pmol DAS treatment further increased only GSH peroxidase II activity and the selenoenzyme remained unaffected. Increased GSH peroxidase II activity in the stomach of 50 and 75 pmol DAS treated mice (Table III) was in good agreement with the elevated GST a (Fig. 2A) because this activity has been shown to be maximally expressed by CYclass GST isoenzymes [15,22]. GSH reductase activity was also compared in 14,000 g supernatant fractions from control and DAS treated mice stomach (Fig. 3). This activity was comparable in the stomach of control and those treated with 25 or 50 pmol DAS. On the other hand, GSH reductase activity was significantly higher (1.64 fold) in 75 pmol DAS treated mice stomach than that of control (Fig. 3). Organosulfur compounds, abundant in garlic and onion oils, have been shown to inhibit polycyclic aromatic hydrocarbon such as B[a]P-induced neoplasia of lung and forestomach in mice. B[a]P is known to be mutagenic and carcinogenic after only metabolic activation by monooxygenases to reactive intermediates which bind to DNA [9]. Since some of the reactive metabolites of B[a]P are substrates for GST [ll, 171, it is logical to suspect that cellular levels of GST, along with monooxygenases, will affect carcinogenic-potential of B[a]P. Even though, the effect of DAS on monooxygenases re-
activity of female A/J
activity (unitsa/mg
protein)
t-Butylhydroperoxide
Hydrogen
374.6 597.6 878.0
353.6 552.0 517.0
“One unit of enzyme catalyzed conversion bValues represent mean + S.D., n = 3. 'P 5 0.01,treated versus control.
f f f
20.1b 19.3’ 15.7’
of 1 pmol GSH to GSSG/min
mice stomach.
peroxide
f 40.9 f 45.5’ f 45.7’
at 37°C.
127
0
0
25 Diallyl sulfide
50
75
f.mOl)
Fig. 3. Effect of DAS treatment on GSH reductase activity of mice stomach. Values represent mean f S. D. (n = 3). a; P I 0.01, treated versus control.
mains to be determined, this study indicates that bulk GST activity is significantly increased in the stomach of DAS treated mice. Our results are consistent with those published by other investigators [27,29] and suggest that GST, an important component of cellular defense system, may contribute to the anticarcinogenic activity of DAS. Results of this study also demonstrate that aI, cc.and n class GSTs of mice stomach are differentially induced by DAS treatment. For example, a significant induction of GST (Yand P occurred by treating the animals with 75 pmol DAS. On the other hand, maximum induction of p class GST was observed by treating mice with 50 pmol DAS and the levels of this GST isoenzyme decreased at both, higher and lower doses of DAS. The ultimate carcinogenic metabolite of B[a]P is suggested to be 70,8a-dihydroxy-9a, lOa-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) [7,26]. GST mediated conjugation of BPDE with GSH by rat and human isoenhave been documented [20,21]. zymes Among human GST isoenzymes (GST (Y and
p of human liver and GST x of human placenta), maximum activity towards BPDE is exhibited by class n GST followed by GST p and this represents a poor substrate for CYclass isoenzymes [20]. Similarly, conjugation of BPDE to GSH is most efficiently catalyzed by a a class GST (GST 7-7) of rat lung [21]. Even though substrate specificity of mice GST isoenzymes towards BPDE has not been determined, it is likely that mice GSTs may exhibit catalytic patterns similar to those of rat and human GST isoenzymes. These arguments suggest that anti-neoplastic effect of DAS may, at least in part, be attributed to the induction of CY,Jo and a class GST isoenzymes in stomach of treated mice. GSH is the major soluble cellular thiol and has many important functions within the cell. In addition to its contributions in regulating protein and DNA synthesis [16], GSH serves as an essential co-factor for many enzymes [13]. GSH also plays a pivotal role in detoxification processes through GST and GSH peroxidase activities. Adequate concentration of GSH is maintained within cells through enzymes of GSH redox cycle including GSH reductase [13,16]. Thus, GSH peroxidase and GSH reductase activities play important roles in cellular defense as well as maintenance of cellular functions. Even though we did not quantitate GSH levels in this study, GSH peroxidase and GSH reductase activities were elevated in the mice stomach as a result of DAS treatment. Thus, it is likely that both these enzymes may also contribute to antineoplastic activity of DAS. Acknowledgments This investigation was supported by an American Cancer Society-Florida Division (Nora I. Rodd, F89UM-6) grant. The technical assistance of Ms. Anjali Mascaranhas is acknowledged. We also thank Dr. Yogesh C. Awasthi of the University of Texas Medical Branch at Galveston for his generous gift of antibodies against human GST isoenzymes.
128
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sheets:
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