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Initiation and post-initiation chemopreventive effects of diallyl sulfide in esophageal carcinogenesis
Cancer Letters, 64 (1992) 39 - 42 Ekevier Scientific Publishers lreland
39 Ltd.
Initiation and post-initiation chemopreventive sulfide in esophageal carcinogenesis Michael J. Wargovicha,
Osamu
effects of dialIy1
Imadaa and L.C. Stephensb
‘Section of Gastrointestinal Oncology and Digestive Diseases, Department of Medical Oncology and bDepartment of Vete~nary medicine and Surgery, The university of Texas, M.D. Anderson Cancer Center, Houston, TX 77030 (USA) (Received (Accepted
7 March 1992) 10 March 1992)
Summary
~ia~~y~sulfide @AS), one of a number of organosulfur compounds accounting for the flauor and smell associated with garlic, has been shown to inh~bjt a number of chemically induced forms of cancer. In this study, DAS was examined for its ~hemopreuentjue effects in both the initiation and post-initiation phases of nitrosomethylbenzylamine-induced esophageal carcinogenesis in the Sprague Dawfey rat. Although highly inhibjtory during initiation, DAS is ineffective when given after the carcinogen. DIG, though not effectjue as a preventive in post-initiation, was not found to promote esophageal carcinogenesis. Keywords:
chemoprevention; altliums; esophageal cancer:. diallyl sulfide
Introduction
Organosulfur compounds from a&urn vegetables have proven to be interesting and potentially important chemopreventive agents. Studies in our laboratory have focussed on the Correspondence Gastrointestinal M.D. Anderson
to: Michael J. Wargovich, Section of Oncology, and Digestive Diseases, Box 78, Cancer Center, Houston, Texas 77030, USA.
Printed and Published
in Ireland
compound diallyl sulfide (DAS), a lipophilic thioether derived from oxidized allicin, which is produced when a garlic clove is crushed. It is representative of many such agents in garlic [4]. Earlier studies showed that DAS had pronounced chemopreventive activity when tested in the dimethylhydrazine model for colon cancer and in the nitrosomethylbenzylamine (NMBA)-induced esophageal carcinogenesis model in the rat [ 11,131. DAS and other closely related fiavor chemicals from garlic have also been shown to inhibit experimentally induced lung tumors and forestomach tumors in mice [9,14]. The most likely mechanism of action of DAS and related analogues is believed to involve specific inhibition of hepatic cytochrome P-450 IIEl, an isozyme linked to the bioactivation of low molecular weight aliphatic carcinogens such as dimethyIhydrazine and certain nitrosamines. Brady et al. [2,3] have clearly detected a marked inhibitory effect on this isozyme; others have reported interference with carcinogen metabolism when DAS is administered [5,6]. Thus, DAS seems to be predominantly active in suppressing the initiation phase of gastrointestinal carcinogenesis. Testing of new chemopreventive agents only rarely encompasses the full measure of efficacy by examining for effects in all stages of carcinogenesis. For the most part, new agents
40
are tested by inco~oration into the animal diet, and the test diets are administered for the duration of the experimental protocol. Certain chemopreventives, however, have been shown to have untoward effects when administered in the promotional phases of carcinogenesis, in some cases, even stimulating tumor promotion [1,7,8,12]. The goal of this study was to determine the effect of DAS in the NMBA rat model for esophageal cancer when the compound was given either during initiation or during the promotion phase of carcinogenesis .
Detroit, MI), once weekly for 5 weeks. We chose the post-initiation dose based upon our previous report in which we found complete suppression of NMBA tumorigenesis when 200 mg/kg was administered during the initiation phase of carcinogenesis [13]. Two control groups were included, i.e., rats receiving DAS by gavage at 10 or 100 mg/kg for 5 weeks but treated with saline in lieu of NMBA. The experiment was terminated at 15 weeks and all rats were necropsied. A veterinary pathologist assisted in the histotyping of tumors encountered in the NMBA-seated rats.
Materials
Biostatistical
and Methods
and diets Male Sprague - Dawley rats were obtained from Harlan-Sprague Dawley, Inc. (Indianapolis, IN) and housed in sawdust-bedded cages in a room with controlled humidity and a X/12 h (light/dark) cycle. Water was available ad libitum, All rats weighed 110 - 125 g at the start of the experiment. After 1 week of acclimatization, all rats were placed on an AIN-76A pelleted diet (Dyets, Inc., Bethlehem, PA) and they remained on this diet until the end of the experiment; animals were weighed weekly. All research procedures were approved by the Institutional Animal Care and Use Committee, protocol 1188807842. Animals
Esophageal
tumorigenesis
The aim of this study was to test two doses of DAS during the initiation phase of NMBA carcinogenesis and one dose in the postinitiation phase. Therefore, animals were randomized into groups of 20. For the initiation experiment, rats were gavaged with either 10 mg/kg or 100 mg/kg DAS (98% pure, Aldrich Co., Milwaukee, WI) in corn oil once weekly for 5 weeks, 3 h prior to carcinogen injection. The post-initiation group received DAS at 200 mg/kg weekly by gavage only after carcinogen treatments were completed. All rats treated with the carcinogen were injected with 3 mg/kg NMBA (Ash Stevens Co.,
analysis
Tumor incidence was evaluated for significance by chi-square 2 x 2 contingency analysis. The differences in means of total tumors, papillomas and squamous cell carcinomas were determined by Student’s t-test; P < 0.05 was determined to be significant. Results and Discussion In this study animal body weight gain did not differ significantly among all treatment groups throughout the experiment. As shown in Table I, DAS, although a very effective inhibitor of neoplasia when administered during initiation of NMBA carcinogenesis, was ineffective when administered after the carcinogen. When given during initiation, DAS caused a dose-related reduction in tumor incidence. However, when DAS was administered at 200 mg/kg weekly for 5 weeks after carcinogen treatment had ceased we observed no inhibition in either total tumor incidence or in number of papillomas or invasive squamous cell carcinomas per rat. This is all the more interesting in light of our previous finding that the 200 mg/kg dose of DAS when given concomitantly with NMBA initiation completely inhibited esophageal tumorigenesis [ 131. The data suggest that DAS specifically modifies either the metabolic activation of NMBA or augments detoxification of the carcinogen. Depletion of cytochrome P-450 and inactivation of specific isoforms implicated in the metabolism of nitrosamine car-
41
Table I. cancer.
Effect of diallyl sulfide
Group (n-20)
DAS DAS NMBA NMBA NMBA NMBA”’ l
SCC, squamous
in initiation/post-initiation.
Dose of DAS
Tumor
fmgikg)
(%I
10 100 0 10 100 200
0 0 96 90 63” 96
incidence
Phases
of nitrosomethylbenzylamine-induced
Tumors per rat
Papillomas per rat
SCC’ per rat
0 0 3.48 3.34 1.63’ 3.42
0 0 2.65 3.03 1.53’ 2.60
0 0 0.82 0.62 0.10” 0.69
l
l
esophageal
cell carcinomas.
l*P c 0.05; significantly different from NMBA only. l
l
*Post-initiation
experiment.
cinogenesis have been reported [2,3]. In the colon and liver, following acute exposure to DAS, levels of Phase II detoxification enzymes such as glutathione-S-transferase are known to be increased [5,6. lo]. Neither Phase I or Phase II enzymes were measured in the current study because NMBA treatment rapidly produced malignant transformation in the entire esophagus of lthe Sprague - Dawley rat, resulting in little histologically ‘normal’ tissue. DAS, though ineffective in the post-initiation phase in terms of chemoprevention, also did not promote esophageal cancer in this study. Initial, encouraging data from experiments with potential chemopreventives such as indole-3-carbinol and retinoids have been tempered by the fact that each of these agents when tested in the promotional phase of carcinogenesis were shown to promote tumorigenesis [ 1,7,&E]. These undesirable findings inject a note of caution and discourage the precipitous incorporation of such chemopreventive agents into clinical chemoprevention efficacy trials in man. At the very least it would seem prudent to test the candidate efficacious dose for most chemopreventive a!gents found during initiation in a post-initiation setting. DAS, already shown to be an effective chemopreventive in a number of animal models for cancer, appears
to have activity relegated only to the earliest phases of carcinogenesis and it would therefore seem to be a very proficient inhibitor of carcinogenic nitrosamines. Acknowledgements The authors wish to recognize the expert technical assistance of Cindy Palmer, Marco Velasco and Teresa Joseph. The study was supported by grant 89B25 from the American Institute for Cancer Research, NC1 Cancer Center Support Grant CA-16 772 and from the Wakunaga Pharmaceutical Co, Ltd., Osaka, Japan. References Bailey, G.S., Dashwood, R.H., Fong,
A.T., Williams, D.E., Scanlan, R.A. and Hendricks, J.D. (1991) Modulation of mycotoxin and nitrosamine carcinogenesis by indole-3-carbinol; quantitative analysis of inhibition versus promotion. IARC Sci. Publ., 105, 275- 280. Brady, J.F., Li, D.C., fshizaki, H. and Yang C.S. (1988) Effect of diallyl sulfide on rat liver microsomal nitrosamine metabolism and other monooxygenase activities. Cancer Res., 48, 5937 - 5940. Brady, J.F., Wang, N.H., Hong, J.Y., Xiao, F., Li, Y., Yoo, J.S., Ning, S.R., Lee, N.J., Fukuto, J.M. and Gapac, J.M. (1991) Modulation of rat hepatic microsomal monooxygenase enzymes and cytotoxicity by diallyl sulfide. Toxicol. Appl. Pharamacol., 108, 342-354.
4
Fenwick, G.F. and Hanley, A.B. The genus Allium, part 1. (1985) Crit. Rev. Food Sci. Nutr., 22, 199- 217.
5
Gudi, V.A. and Singh, S.V. (1991) Effect of diallyl a naturally occurring anticarcinogen on glutathione dent detoxification enzymes in female CD-1 mouse Biochem. Pharmacof., 42, 1261- 1265. Maurya, A.K. and Singh, S.V. (1991) Differential tion of glutathione transferase isoenzymes of stomach by diallyl sulfide, a naturally occurring cinogen. Cancer Lett., 57, 121- 129.
6
7
sulfide, depentissues.
10
inducmouse anticar-
11
McCormick, D.L., Bagg, B.J. and H&in, T.A. (1987) Comparative activity of dietary or topical exposure to three retinoids in the promotion of skin tumor induction in mice. Cancer Res., 47, 5989 - 5993.
8
Pence,
B.C., Buddingh, F. and Yang, dietary factors in the ple dimethylhydrazine carcinogenesis: indole-3carbinol. J Natl. Cancer Inst.,
S.P. (1986) Multienhancement of main effect of 77, 269-276.
9
Sparnins, V.L., Barany, G. and Wattenberg, L.W. (1988) Effects of organosulfur compounds from garlic and onions on benzo[a]pyrene-induced neoplasia and glutathione-S-
12
13
transferase activity in the mouse. Carcinogenesis, 9, 131-134. Sumiyoshi, H. and Wargovich R.J. (1990) Chemoprevention of 1,2-dimethylhydrazine-induced colon cancer by naturally occurring organosulfur compounds. Cancer Res., 50, 5084 - 5087. Wargovich, M. J. (1987) Diallyl sulfide, a flavor component of garlic [AIlium sotiuum], inhibits dimethylhydrazineinduced colon cancer. Carcinogenesis, 8, 487 - 489. Wargovich, M.J. and Hong W.K. (1990) Promotion of nitrosomethylbenzylamine-induced esophageal cancer by retinyl acetate. Proc. Am. Assoc. Cancer Res., 31, 163. Wargovich, M.J., Woods, C., Eng, V.W.S., Stephens, L.C. and Gray, K.N. (1988) Chemoprevention of nitrosomethylbenzylamine-induced esophageal cancer in rats by the thioether, diallyl sulfide. Cancer Res., 48, 6872 - 6875.
14
Wattenberg, L.W., Sparnins, V.L. and Barany, G. (1989) Inhibition of n-nitrosodiethylamine carcinogenesis in mice by naturally occurring organosulfur compounds and monoterpenes. Cancer Res., 49, 2689 - 2692.
39 Ltd.
Initiation and post-initiation chemopreventive sulfide in esophageal carcinogenesis Michael J. Wargovicha,
Osamu
effects of dialIy1
Imadaa and L.C. Stephensb
‘Section of Gastrointestinal Oncology and Digestive Diseases, Department of Medical Oncology and bDepartment of Vete~nary medicine and Surgery, The university of Texas, M.D. Anderson Cancer Center, Houston, TX 77030 (USA) (Received (Accepted
7 March 1992) 10 March 1992)
Summary
~ia~~y~sulfide @AS), one of a number of organosulfur compounds accounting for the flauor and smell associated with garlic, has been shown to inh~bjt a number of chemically induced forms of cancer. In this study, DAS was examined for its ~hemopreuentjue effects in both the initiation and post-initiation phases of nitrosomethylbenzylamine-induced esophageal carcinogenesis in the Sprague Dawfey rat. Although highly inhibjtory during initiation, DAS is ineffective when given after the carcinogen. DIG, though not effectjue as a preventive in post-initiation, was not found to promote esophageal carcinogenesis. Keywords:
chemoprevention; altliums; esophageal cancer:. diallyl sulfide
Introduction
Organosulfur compounds from a&urn vegetables have proven to be interesting and potentially important chemopreventive agents. Studies in our laboratory have focussed on the Correspondence Gastrointestinal M.D. Anderson
to: Michael J. Wargovich, Section of Oncology, and Digestive Diseases, Box 78, Cancer Center, Houston, Texas 77030, USA.
Printed and Published
in Ireland
compound diallyl sulfide (DAS), a lipophilic thioether derived from oxidized allicin, which is produced when a garlic clove is crushed. It is representative of many such agents in garlic [4]. Earlier studies showed that DAS had pronounced chemopreventive activity when tested in the dimethylhydrazine model for colon cancer and in the nitrosomethylbenzylamine (NMBA)-induced esophageal carcinogenesis model in the rat [ 11,131. DAS and other closely related fiavor chemicals from garlic have also been shown to inhibit experimentally induced lung tumors and forestomach tumors in mice [9,14]. The most likely mechanism of action of DAS and related analogues is believed to involve specific inhibition of hepatic cytochrome P-450 IIEl, an isozyme linked to the bioactivation of low molecular weight aliphatic carcinogens such as dimethyIhydrazine and certain nitrosamines. Brady et al. [2,3] have clearly detected a marked inhibitory effect on this isozyme; others have reported interference with carcinogen metabolism when DAS is administered [5,6]. Thus, DAS seems to be predominantly active in suppressing the initiation phase of gastrointestinal carcinogenesis. Testing of new chemopreventive agents only rarely encompasses the full measure of efficacy by examining for effects in all stages of carcinogenesis. For the most part, new agents
40
are tested by inco~oration into the animal diet, and the test diets are administered for the duration of the experimental protocol. Certain chemopreventives, however, have been shown to have untoward effects when administered in the promotional phases of carcinogenesis, in some cases, even stimulating tumor promotion [1,7,8,12]. The goal of this study was to determine the effect of DAS in the NMBA rat model for esophageal cancer when the compound was given either during initiation or during the promotion phase of carcinogenesis .
Detroit, MI), once weekly for 5 weeks. We chose the post-initiation dose based upon our previous report in which we found complete suppression of NMBA tumorigenesis when 200 mg/kg was administered during the initiation phase of carcinogenesis [13]. Two control groups were included, i.e., rats receiving DAS by gavage at 10 or 100 mg/kg for 5 weeks but treated with saline in lieu of NMBA. The experiment was terminated at 15 weeks and all rats were necropsied. A veterinary pathologist assisted in the histotyping of tumors encountered in the NMBA-seated rats.
Materials
Biostatistical
and Methods
and diets Male Sprague - Dawley rats were obtained from Harlan-Sprague Dawley, Inc. (Indianapolis, IN) and housed in sawdust-bedded cages in a room with controlled humidity and a X/12 h (light/dark) cycle. Water was available ad libitum, All rats weighed 110 - 125 g at the start of the experiment. After 1 week of acclimatization, all rats were placed on an AIN-76A pelleted diet (Dyets, Inc., Bethlehem, PA) and they remained on this diet until the end of the experiment; animals were weighed weekly. All research procedures were approved by the Institutional Animal Care and Use Committee, protocol 1188807842. Animals
Esophageal
tumorigenesis
The aim of this study was to test two doses of DAS during the initiation phase of NMBA carcinogenesis and one dose in the postinitiation phase. Therefore, animals were randomized into groups of 20. For the initiation experiment, rats were gavaged with either 10 mg/kg or 100 mg/kg DAS (98% pure, Aldrich Co., Milwaukee, WI) in corn oil once weekly for 5 weeks, 3 h prior to carcinogen injection. The post-initiation group received DAS at 200 mg/kg weekly by gavage only after carcinogen treatments were completed. All rats treated with the carcinogen were injected with 3 mg/kg NMBA (Ash Stevens Co.,
analysis
Tumor incidence was evaluated for significance by chi-square 2 x 2 contingency analysis. The differences in means of total tumors, papillomas and squamous cell carcinomas were determined by Student’s t-test; P < 0.05 was determined to be significant. Results and Discussion In this study animal body weight gain did not differ significantly among all treatment groups throughout the experiment. As shown in Table I, DAS, although a very effective inhibitor of neoplasia when administered during initiation of NMBA carcinogenesis, was ineffective when administered after the carcinogen. When given during initiation, DAS caused a dose-related reduction in tumor incidence. However, when DAS was administered at 200 mg/kg weekly for 5 weeks after carcinogen treatment had ceased we observed no inhibition in either total tumor incidence or in number of papillomas or invasive squamous cell carcinomas per rat. This is all the more interesting in light of our previous finding that the 200 mg/kg dose of DAS when given concomitantly with NMBA initiation completely inhibited esophageal tumorigenesis [ 131. The data suggest that DAS specifically modifies either the metabolic activation of NMBA or augments detoxification of the carcinogen. Depletion of cytochrome P-450 and inactivation of specific isoforms implicated in the metabolism of nitrosamine car-
41
Table I. cancer.
Effect of diallyl sulfide
Group (n-20)
DAS DAS NMBA NMBA NMBA NMBA”’ l
SCC, squamous
in initiation/post-initiation.
Dose of DAS
Tumor
fmgikg)
(%I
10 100 0 10 100 200
0 0 96 90 63” 96
incidence
Phases
of nitrosomethylbenzylamine-induced
Tumors per rat
Papillomas per rat
SCC’ per rat
0 0 3.48 3.34 1.63’ 3.42
0 0 2.65 3.03 1.53’ 2.60
0 0 0.82 0.62 0.10” 0.69
l
l
esophageal
cell carcinomas.
l*P c 0.05; significantly different from NMBA only. l
l
*Post-initiation
experiment.
cinogenesis have been reported [2,3]. In the colon and liver, following acute exposure to DAS, levels of Phase II detoxification enzymes such as glutathione-S-transferase are known to be increased [5,6. lo]. Neither Phase I or Phase II enzymes were measured in the current study because NMBA treatment rapidly produced malignant transformation in the entire esophagus of lthe Sprague - Dawley rat, resulting in little histologically ‘normal’ tissue. DAS, though ineffective in the post-initiation phase in terms of chemoprevention, also did not promote esophageal cancer in this study. Initial, encouraging data from experiments with potential chemopreventives such as indole-3-carbinol and retinoids have been tempered by the fact that each of these agents when tested in the promotional phase of carcinogenesis were shown to promote tumorigenesis [ 1,7,&E]. These undesirable findings inject a note of caution and discourage the precipitous incorporation of such chemopreventive agents into clinical chemoprevention efficacy trials in man. At the very least it would seem prudent to test the candidate efficacious dose for most chemopreventive a!gents found during initiation in a post-initiation setting. DAS, already shown to be an effective chemopreventive in a number of animal models for cancer, appears
to have activity relegated only to the earliest phases of carcinogenesis and it would therefore seem to be a very proficient inhibitor of carcinogenic nitrosamines. Acknowledgements The authors wish to recognize the expert technical assistance of Cindy Palmer, Marco Velasco and Teresa Joseph. The study was supported by grant 89B25 from the American Institute for Cancer Research, NC1 Cancer Center Support Grant CA-16 772 and from the Wakunaga Pharmaceutical Co, Ltd., Osaka, Japan. References Bailey, G.S., Dashwood, R.H., Fong,
A.T., Williams, D.E., Scanlan, R.A. and Hendricks, J.D. (1991) Modulation of mycotoxin and nitrosamine carcinogenesis by indole-3-carbinol; quantitative analysis of inhibition versus promotion. IARC Sci. Publ., 105, 275- 280. Brady, J.F., Li, D.C., fshizaki, H. and Yang C.S. (1988) Effect of diallyl sulfide on rat liver microsomal nitrosamine metabolism and other monooxygenase activities. Cancer Res., 48, 5937 - 5940. Brady, J.F., Wang, N.H., Hong, J.Y., Xiao, F., Li, Y., Yoo, J.S., Ning, S.R., Lee, N.J., Fukuto, J.M. and Gapac, J.M. (1991) Modulation of rat hepatic microsomal monooxygenase enzymes and cytotoxicity by diallyl sulfide. Toxicol. Appl. Pharamacol., 108, 342-354.
4
Fenwick, G.F. and Hanley, A.B. The genus Allium, part 1. (1985) Crit. Rev. Food Sci. Nutr., 22, 199- 217.
5
Gudi, V.A. and Singh, S.V. (1991) Effect of diallyl a naturally occurring anticarcinogen on glutathione dent detoxification enzymes in female CD-1 mouse Biochem. Pharmacof., 42, 1261- 1265. Maurya, A.K. and Singh, S.V. (1991) Differential tion of glutathione transferase isoenzymes of stomach by diallyl sulfide, a naturally occurring cinogen. Cancer Lett., 57, 121- 129.
6
7
sulfide, depentissues.
10
inducmouse anticar-
11
McCormick, D.L., Bagg, B.J. and H&in, T.A. (1987) Comparative activity of dietary or topical exposure to three retinoids in the promotion of skin tumor induction in mice. Cancer Res., 47, 5989 - 5993.
8
Pence,
B.C., Buddingh, F. and Yang, dietary factors in the ple dimethylhydrazine carcinogenesis: indole-3carbinol. J Natl. Cancer Inst.,
S.P. (1986) Multienhancement of main effect of 77, 269-276.
9
Sparnins, V.L., Barany, G. and Wattenberg, L.W. (1988) Effects of organosulfur compounds from garlic and onions on benzo[a]pyrene-induced neoplasia and glutathione-S-
12
13
transferase activity in the mouse. Carcinogenesis, 9, 131-134. Sumiyoshi, H. and Wargovich R.J. (1990) Chemoprevention of 1,2-dimethylhydrazine-induced colon cancer by naturally occurring organosulfur compounds. Cancer Res., 50, 5084 - 5087. Wargovich, M. J. (1987) Diallyl sulfide, a flavor component of garlic [AIlium sotiuum], inhibits dimethylhydrazineinduced colon cancer. Carcinogenesis, 8, 487 - 489. Wargovich, M.J. and Hong W.K. (1990) Promotion of nitrosomethylbenzylamine-induced esophageal cancer by retinyl acetate. Proc. Am. Assoc. Cancer Res., 31, 163. Wargovich, M.J., Woods, C., Eng, V.W.S., Stephens, L.C. and Gray, K.N. (1988) Chemoprevention of nitrosomethylbenzylamine-induced esophageal cancer in rats by the thioether, diallyl sulfide. Cancer Res., 48, 6872 - 6875.
14
Wattenberg, L.W., Sparnins, V.L. and Barany, G. (1989) Inhibition of n-nitrosodiethylamine carcinogenesis in mice by naturally occurring organosulfur compounds and monoterpenes. Cancer Res., 49, 2689 - 2692.