Critical role of allyl groups and disulfide chain in induction of Pi class glutathione transferase in mouse tissues in vivo by diallyl disulfide, a naturally occurring chemopreventive agent in garlic
Chhanda Bose1,*,
Jianxia Guo2,*,
Ludwika Zimniak1,
Sanjay K. Srivastava2,
Sharda P. Singh1,
Piotr Zimniak1,3,4 and
Shivendra V. Singh2,5
1 Department of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205,
2 Department of Pharmacology and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261,
3 Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 and
4 Central Arkansas Veterans Healthcare System, Medical Research, Little Rock, AR 72205, USA
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Abstract
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We have shown previously that the chemoprotective activity of diallyl disulfide (DADS), a naturally occurring anticancer agent in garlic, against benzo[a]pyrene (BP)-induced forestomach carcinogenesis in mice correlates strongly with its inductive effects on the expression of Pi class glutathione (GSH) transferase mGSTP1-1. The present structureactivity relationship studies were designed to define the role of allyl groups and the disulfide chain in mGSTP1-inducing activity of DADS. Hepatic mGSTP1 mRNA levels rose rapidly upon treatment of mice with DADS, reached a maximum between 12 and 24 h (
5.7-fold induction) and fell to control levels by 48 h after DADS treatment. Induction of mGSTP1 mRNA in the forestomach was maximal between 6 and 12 h after DADS treatment (
4.7-fold induction). The mGSTP1 mRNA expression was either unaltered (liver) or moderately increased (forestomach) upon treatment of mice with dipropyl disulfide (DPDS), which is a naturally occurring saturated analog of DADS. These results indicated that the allyl groups are critical for the mGSTP1-inducing activity of DADS. A statistically significant increase in the expression of mGSTP1 mRNA was also observed in the liver and forestomach of mice treated with diallyl monosulfide (DAMS), albeit to a much lesser extent compared with DADS. These results indicated that the oligosulfide chain length in garlic organosulfides (OSCs) is equally important for their mGSTP1-inducing activity. The role of the disulfide chain in DADS-mediated induction of mGSTP1 was further investigated by testing a pair of alkadienes (1,7-octadiene and 1,8-nonadiene) having structural similarity to DADS. Both DADS and the alkadienes carry allyl groups at both ends of a linear molecule and the distance between the allylic carbon atoms is similar in both compounds, but the central disulfide chain of DADS is replaced with an alkyl chain in the alkadienes. The alkadienes were either ineffective or moderately active in increasing mGSTP1 expression. In conclusion, the results of the present study clearly indicate that the presence of terminal allyl groups as well as the central disulfide chain is required for maximum induction of mGSTP1 in vivo by garlic-derived OSCs.
Abbreviations: BP, benzo[a]pyrene; (+)-anti-BPDE, (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; DADS, diallyl disulfide; DAMS, diallyl monosulfide; DAU, diallyl urea; DPDS, dipropyl disulfide; GSH, glutathione; mGSTP1-1, Pi class murine glutathione transferase isoform; 1,7-OD, 1,7-octadiene; 1,8-ND, 1,8-nonadiene; OSCs, organosulfur compounds.
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Introduction
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Medicinal properties of the vegetables of genus Allium such as garlic are well documented, and include anti-thrombotic effects, lipid-lowering and cardiovascular properties, anti-bacterial effects and anticancer properties (reviewed in refs 15). The evidence for the antineoplastic activity of Allium vegetables derives both from epidemiological data and from laboratory studies. Epidemiological studies have concluded that increased consumption of garlic and other Allium vegetables is associated with reduced incidence of gastric cancers (6,7). Laboratory studies indicate that the anticarcinogenic activity of Allium vegetables, including garlic, is due to organosulfides (OSCs) that are metabolically generated upon cutting or chewing of these vegetables (4,5,8).
Processing of garlic generates a number of structurally different water-soluble as well as oil-soluble OSCs (1). Diallyl disulfide (DADS) is the predominant oil-soluble OSC in essential garlic oil (9). Cellular studies have shown that DADS inhibits proliferation of human colon, lung, skin, prostate, hepatoma and breast cancer cells (1013). The antiproliferative activity of DADS is believed to be due to its ability to induce apoptotic cell death and to arrest cells in G2/M phase (1317). DADS-induced apoptosis in the human breast cancer cell line MDA-MB-231 correlated with up-regulation of the pro-apoptotic protein Bax, down-regulation of the anti-apoptotic protein Bcl-XL and activation of caspase-3 (13). The G2/M arresting activity of DADS was shown to involve inhibition of the kinase activity of p34cdc2 (15). We have shown previously that the growth of H-ras oncogene transformed tumor xenografts in vivo is significantly inhibited by oral administration of DADS (18). In vivo anticancer activity of DADS against human breast and colon cancer xenografts has also been documented (13,19).
In addition to the antiproliferative activity in cell culture and tumor xenograft models, DADS is highly effective in prevention of chemically induced cancers in animal models. Wattenberg et al. (20) were the first to demonstrate that N-nitrosodiethylamine-induced neoplasia of the forestomach in female A/J mice is inhibited by >90% upon p.o. DADS administration prior to the carcinogen challenge (20). Intragastric intubation of DADS prevented colon and renal neoplasia in multi-organ carcinogenesis model in male F344 rats (21). The 7,12-dimethylbenz[a]anthracene-induced and 12-O-tetradecanoylphorbol-13-acetate-promoted skin papilloma formation in SENCAR mice is significantly inhibited by topical application of DADS (22). In addition, dietary administration of DADS to rats effectively prevented azoxymethane-induced invasive adenocarcinoma of the colon and N-methyl-N-nitrosourea and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced mammary carcinogenesis (2325).
We have shown previously that benzo[a]pyrene (BP)-induced forestomach tumorigenesis in mice is inhibited significantly upon oral administration of DADS (26). Our studies also revealed that the chemopreventive activity of DADS against BP-induced cancer in mice correlates strongly with its inductive effects on the hepatic and target organ expression of Pi class glutathione (GSH) transferase isoform mGSTP1-1 (26,27), which is highly efficient in detoxification of the ultimate carcinogenic metabolite of BP, (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE] (27). In contrast to the significant advances in our understanding of the biological mechanism of BP-induced cancer prevention by DADS (2629), the contribution of the structural elements of the DADS molecule (allyl groups and disulfide chain) to the mGSTP1-1-inducing activity of DADS remains elusive. The present structureactivity relationship studies were designed to fill this gap in our knowledge. The results clearly indicate that both the allyl groups and the disulfide chain are essential for DADS-mediated induction of mGSTP1 mRNA in vivo
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Materials and methods
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Materials
Female A/J mice (68 weeks old) were purchased from the National Cancer Institute (Frederick, MD). DADS and its structural analogs, including dipropyl disulfide (DPDS), diallyl monosulfide (DAMS), 1,7-octadiene (1,7-OD), 1,8-nonadiene (1,8-ND) and diallyl urea (DAU) were obtained from Sigma (St Louis, MO).
Treatment of animals and tissue collection
Female A/J mice were fed AIN-76 diet (ICN Biomedicals, Aurora, OH) for 1 week before treatment. Animals were divided in eight groups of 12 mice/group. Group 1 (control) received two oral injections, 48 h apart, of 0.1 ml cottonseed oil (vehicle for DADS, DPDS and DAMS). Group 2 received two oral treatments (48 h apart) of 0.1 ml phosphate-buffered saline (vehicle for 1,7-OD, 1,8-ND and DAU). Groups 38 were treated orally (twice, 48 h apart) with 25 µmol of DADS, DPDS, DAMS, 1,7-OD, 1,8-ND and DAU, respectively (in 0.1 ml of cottonseed oil or phosphate-buffered saline). The dose and schedule of DADS administration was similar to that used in tumorigenesis bioassays (26). Three mice from each group were killed at 6, 12, 24 and 48 h after second vehicle or test compound administration. Livers were removed, washed with ice-cold sterile PBS, and immediately frozen in liquid nitrogen. Forestomach tissues were removed, cut longitudinally to remove food contents, washed with sterile PBS and immediately frozen in liquid nitrogen. The liver and forestomach tissues were stored at 80°C until used.
Northern blot analysis for mGSTP1 mRNA expression
Total RNA was prepared from the liver or forestomach tissues using TRI-Reagent (30). Fifteen micrograms of RNA were separated on a 1.2% agaroseformaldehyde gel, blotted to Zeta-Probe membrane, and hybridized to a 32P-labeled 148 bp probe specific for mGSTP1 that spans exon 1 and part of the genomic 5' flanking sequence [positions 54175564 of the mGSTP gene, GenBank accession number U15654 (31)]. The blot was washed at increasing stringency (final wash in 0.1x SSC, 0.1% SDS, 60°C), and band intensities were determined using a PhosphorImager. The values were corrected for equal RNA loading by stripping the blot and re-probing it with an 800 bp PstIXbaI fragment from human glyceraldehyde 3-phosphate dehydrogenase cDNA. Statistical significance of difference in mGSTP1 mRNA expression between control and treated group was determined by two-tailed t-test corrected for multiple comparisons.
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Results and discussion
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We have shown previously that the chemopreventive activity of DADS against BP-induced forestomach tumorigenesis in mice correlates strongly with its inductive effects on mGSTP1-1 expression in the liver as well as in the target organ (26,27). However, no data are yet available to indicate whether allyl groups and/or the disulfide chain are required for DADS-mediated induction of mGSTP1. In the present study, we have systematically investigated the role of allyl groups and the disulfide chain in mGSTP1-inducing activity of DADS using its structural analogs shown in Figure 1
.

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Fig. 1. Chemical structures and molecular models of test compounds. A, DADS; B, DPDS; C, DAMS; D, 1,7-OD; E, 1,8-ND; F, DAU. The molecular models were built and their geometry was optimized using the software LabViewer (Accelrys, San Diego, CA ). Distances between allylic carbon atoms are shown in angstroms. The atomic color scheme is as follows: carbon in gray, hydrogen in white, sulfur in yellow, oxygen in red and nitrogen in blue.
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Figure 2A
shows the effect of DADS administration on hepatic and forestomach mGSTP1 mRNA levels. The hepatic mGSTP1 mRNA levels rose rapidly upon oral administration of DADS, reached a maximum between 12 and 24 h, and fell to control levels by 48 h after DADS treatment. The DADS-mediated induction of hepatic mGSTP1 mRNA was
5.7-fold compared with vehicle-treated controls. As can be seen in Figure 2A
, the DADS-mediated induction of mGSTP1 mRNA was relatively more rapid in the forestomach than in the liver reaching a maximum between 6 and 12 h. The DADS treatment resulted in
4.7-fold increase in forestomach mGSTP1 mRNA levels compared with controls. Similar to liver, however, the levels of mGSTP1 mRNA in the forestomach fell to control levels by 48 h after DADS administration.

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Fig. 2. Levels of mGSTP1 mRNA in the liver (closed circles) and forestomach (open circles) of animals treated with DADS (A), DPDS (B) and DAMS (C) relative to vehicle-treated controls. Fifteen micrograms of RNA each were separated on a 1.2% agaroseformaldehyde gel, blotted to Zeta-Probe membrane, and probed with a 32P-labeled 148 bp sequence specific for mGSTP1. Band intensities were determined using a PhosphorImager. The values were corrected for equal RNA loading by stripping the blot and re-probing it with an 800 bp PstIXbaI fragment from human glyceraldehyde 3-phosphate dehydrogenase cDNA. Asterisks denote a significant difference (P < 0.05 by two-tailed t-test corrected for multiple comparisons) between the reported value and the vehicle-treated control at the corresponding time point.
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Figure 2B
shows the effects of oral administration of 25 µmol DPDS, which is a naturally occurring saturated analog of DADS, on hepatic and forestomach mGSTP1 mRNA expression. We hypothesized that if the disulfide chain alone is sufficient for DADS-mediated induction of mGSTP1, an increase in the levels of mGSTP1 transcript should be observed in response to DPDS administration. DPDS treatment did not affect the expression of mGSTP1 mRNA in the liver. However, a moderate but statistically significant increase in mGSTP1 mRNA expression was apparent in the forestomach of DPDS-treated mice when compared with controls. The level of forestomach mGSTP1 mRNA was increased between 1.5 and 1.7-fold upon DPDS administration (Figure 2B
). These results indicated that the presence of a disulfide chain affects mGSTP1-inducing activity of DADS at least in the target organ.
The role of the disulfide chain in DADS-mediated induction of mGSTP1 was further examined by determining the mGSTP1-inducing activity of DAMS (Figure 1C
), which is a naturally occurring monosulfide analog of DADS. As shown in Figure 2C
, a statistically significant increase in mGSTP1 mRNA expression was observed in both the liver and forestomach of DAMS-treated mice when compared with the controls. However, DAMS was a far less potent inducer of mGSTP1 expression than DADS. Interestingly, the kinetics of mGSTP1 induction appeared to be different between DAMS and DADS-treated groups. For example, unlike DADS, the hepatic levels of mGSTP1 mRNA rose more rapidly in response to DAMS administration. These results indicated that the mGSTP1-inducing activity of garlic OSCs is dependent upon the number of sulfur atoms, which also directly affects the in vivo kinetics of the mGSTP1 induction in a tissue-specific manner. Data on DPDS and DAMS also indicated that the allyl groups together with the disulfide chain are required for maximal induction of mGSTP1 by garlic OSCs.
The positive correlation of mGSTP1-inducing potency of OSCs with the number of sulfur atoms suggested that the disulfide chain could reflect a requirement for an appropriate spacing of the allyl groups for optimal activity. This question was approached by testing a pair of unsaturated alkadienes shown in Figure 1D and E
. These compounds are similar to DADS in that their terminal bonds are unsaturated, but have the central disulfide chain replaced with an alkyl chain. In DADS, the distance between the allylic carbon atoms is
4.7 Å (Figure 1A
). The two alkadienes (1,7-OD and 1,8-ND) were chosen to cover the same range of distance between their allylic carbons (4.05.0 Å). Thus, alkadienes should induce mGSTP1 expression if the disulfide chain serves solely to provide an appropriate spacing of the allyl groups in DADS. On the other hand, alkadienes should be ineffective if the disulfide chain has direct functions beyond an appropriate positioning of the allyl groups. As shown in Figure 3A and B
, the expression of forestomach mGSTP1 mRNA was moderately increased upon treatment of mice with 1,7-OD (between 1.3- and 1.8-fold over control) and 1,8-ND (between 1.3- and 2.2-fold over the control). While hepatic mGSTP1 mRNA expression was not altered upon 1,7-OD treatment (Figure 3A
), mGSTP1 mRNA levels were reduced significantly in the livers of 1,8-ND-treated mice (Figure 3B
). Collectively, these results indicated that the disulfide chain might have a direct role in the mGSTP1-inducing activity of DADS.

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Fig. 3. Levels of mGSTP1 mRNA in the liver (closed circles) and forestomach (open circles) of mice treated with 1,7-OD (A), 1,8-ND (B) and DAU (C) relative to vehicle-treated controls. Asterisks denote a significant difference (P < 0.05 by two-tailed t-test corrected for multiple comparisons) between the reported value and the vehicle-treated control at the corresponding time point.
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The mGSTP1-inducing OSCs (DAMS and DADS) are poorly soluble in aqueous media. It is possible that hydrophobicity, by influencing the intracellular distribution of the compounds, affects their mGSTP1-inducing ability, either negatively or positively. We approached this question by determining the effect of oral administration of DAU on hepatic and forestomach mGSTP1 expression (Figure 3C
). DAU is a structural analog of DADS in which the central disulfide chain is replaced by a hydrophilic moiety that preserves a distance between allylic centers found in DADS (Figure 1F
). This compound is soluble in aqueous media. As shown in Figure 3C
, the expression of mGSTP1 mRNA was increased by
1.62.2-fold in the liver and 1.42.0-fold in the forestomach of DAU-treated mice. However, DAU was a far less potent inducer of mGSTP1 than DADS. These results confirm that the mGSTP1-inducing activity of garlic OSCs is in part dependent on the presence of the oligosulfide moiety, and further demonstrate that this requirement is not abolished by increasing the water solubility of the test compound.
Previous studies, including those from our laboratory, have demonstrated that the garlic-derived OSCs, particularly DADS, are highly potent inhibitors of BP-induced tumorigenesis in mice when administered before the carcinogen challenge (26,32). Tumorigenic activity of BP, which is a ubiquitous environmental and dietary pollutant, is attributed to its activated diol epoxide metabolite (+)-anti-BPDE (33,34). While metabolic conversion of BP to (+)-anti-BPDE is catalyzed by cytochrome P450-dependent monooxygenases and epoxide hydrolase (35,36), GSTs play an important role in its detoxification (3739). Kinetic studies of purified GST isoenzymes of different classes have revealed that the Pi class isoform GSTP1-1 is highly effective in catalyzing the GSH conjugation and hence detoxification of (+)-anti-BPDE (3739). Consistent with in vitro kinetic data, GSTP1-transfected cells are significantly more resistant to (+)-anti-BPDE-induced DNA damage than vector-transfected control cells (40,41). In vivo evidence for the protective effect of GSTP1-1 against chemically induced cancers derives from studies in GSTP knockout mice (42). Deletion of the mGSTP gene cluster has been shown to increase the sensitivity of mice to chemically induced cancers (42). Together, these studies provide convincing evidence for the importance of GSTP1-1 in the protection against carcinogenesis induced by BP and similar acting chemical carcinogens.
Previous studies from our laboratory indicated that increased detoxification of (+)-anti-BPDE through induction of GSTs is an important mechanism for chemopreventive activity of garlic-derived OSCs against BP-induced cancer in mice (2629). This conclusion is based on our findings of a strong positive correlation between chemopreventive efficacy of OSCs and their effects on the induction of mGSTP1-1 not only in liver, which is the major site for xenobiotic biotransformation, but also in the target organ (26). The present structureactivity relationship studies were undertaken to gain insights into the role of allyl groups and the oligosulfide chain in mGSTP1-1-inducing activity of OSCs. Our results indicate that maximal induction of mGSTP1-1 by garlic-derived OSCs requires the presence of allyl groups as well as the disulfide chain. The mGSTP1-1-inducing activity of OSC is substantially reduced upon substitution of the terminal allyl groups with saturated propyl groups. Likewise, the allylic OSC with a disulfide chain (DADS) is a far more potent inducer of hepatic as well as forestomach mGSTP1 expression than the corresponding monosulfide indicating that a disulfide bridge is equally important for maximal induction of this isoenzyme by garlic OSCs. Data on alkadienes indicate that the disulfide chain of DADS may have a direct role in induction of mGSTP1, rather than only to provide an appropriate spacing of the allyl groups. Finally, a comparison of the mGSTP1-inducing activity of DADS and DAU indicates that the hydrophobicity of allylic OSCs may be an additional determinant of their mGSTP1-inducing activity.
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Notes
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* These authors contributed equally 
5 To whom correspondence should be addressed Email: singhs{at}msx.upmc.edu 
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Acknowledgments
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This investigation was supported by USPHS grant CA55589, awarded by the National Cancer Institute. The authors thank Karen Lew for preparation of figures.
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Received April 5, 2002;
revised June 13, 2002;
accepted June 24, 2002.