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In vivo antigenotoxic effects of dietary allyl sulfides in the rat
CANCER LETTERS
ELSEVIER
Cancer Letters 114 (1997) 131-134
In vivo antigenotoxic effects of dietary ally1 sulfides in the rat Anne-Marie Le Bon*, Corinne Roy, Christine DuPont, Marc Suschetet Unite’ de Toxicologic Nutritionnelle,
INRA, 17 rue Sully, 21034 Dijon cedex, France
Abstract The effects of dietary administration of diallyl sulfide (DAS), diallyl disulfide (DADS) and ally1 mercaptan(AM) on the genotoxicity of different chemicals were studied in two experimental systems:(i) measurementof hepatic DNA single-strand breaks induced in rats by aflatoxin Bl (AFBl), N-nitrosodimethylamine (NDMA) or methylnitrosourea (MNU); (ii) mutagenicity of AFBl or NDMA on Sulmonellu typ4imurium TAlOO using hepatic S9 from rats fed ally1 sulfides as the activation system. All compounds strongly reduced hepatic DNA breaks induced by AFBl and NDMA but did not modify the genotoxicity of MNU. In the Ames test, the mutagenicity of NDMA was strongly inhibited by hepatic S9 from rats fed either compound.The mutagenicity of AFBl was al80reducedbut to a lesserextent. Such effects are likely related to the modulation of drug-metabolizing enzymes which play a key role in metabolic activation as well as detoxication of NDMA and AFBl. 0 1997 Elsevier Science Ireland Ltd. Keywords: Ally1 sulfides; Aflatoxin Bl; N-Nitrosodimethylamine; DNA single-strand breaks; Ames test
1. Introduction
Garlic is one plant to which anticancer properties have been attributed on the basis of epidemiologic observations and laboratory studies [l]. The possible anticarcinogenic propertiesof garlic may be attributed to various organosulfur compounds, including ally1 sulfides. Experimental studies on animal models have shown an inhibition of chemically-induced carcinogenesisin different organs by ally1 sulfide administered during the initiation step [2]. Ally1 sulfides are known for inducing several phaseI and phase II enzymes [3,4]. In a recent three-step medium hepatocarcinogenesis assay [5], we observed that DAS and DADS diets fed to rats in the initiation phase strongly * Corresponding author.
reduced the number and the size of preneoplasic foci initiated by AFBl. When initiation was induced by N-nitrosodiethylamine, DAS reduced the size of preneoplastic foci, but not their number. Inasmuch as the number of foci is closely related to a first mutational event (initiation index) and the size of foci reflects the clonal expansion rate of initiated cells (promotion index), it is not clear whether the most likely mechanism for ally1 sulfides chemoprevention is a specific inhibition of genotoxicity or if it is related to their ability to diminish mechanismsof proliferation/promotion. In order to establish whether in vivo antigenotoxic activity could explain the anti-initiating action of ally1 sulfides, two approacheswere employed: 1. The effects of DAS, DADS and ally1 mercaptan (AM, a DADS metabolite) exposureon liver DNA single-strand breaks (SSBs) induced in rats by a
0304-3835/97/$17.00 0 1997 Elsevier Science Ireland Ltd. All rights reserved PII SO304-3835(97)04642-9
NDMA or APB1 (mciirect-acting MNU (a direcr-acting carcinopen) were investigated. 3. The mutagenicity of NDMA. whose ac?r\atioa is catalyzed predominantly by C‘YP 7Ei and ot APB 1. whose activation IS modulated hj various CYP and whose detoxification depends predom,nantly on (XT, was investigated using the Ames mutagenicity assay as end-point. single
dose
carcinogens)
of
or
Post mitochondrial supernatant (S9) from rats fed DAS, DADS or AM were used as activation systems.
i:xpresscd as elutmn constant computed on the third fraction: K (ml ’ 1=I j--in i % of DNA retained on the filter)l/eiuted volume.
mutagenicity studies were performed accord ing ro Maron and Ames 161 employing S&zonelltr ,i,~~7hi~lrl~riUrN TA 100. The mutagenic response <)I ,ZFBI and NDMA was checked using the pre-incuhation procedure with S9 mix containing 10% and 30% lrepatic S9 preparations and adjusted to pH 7.1 and The
9.3, rcyeclivcl~.
2. Materials and methods 3. Results
For 2 weeks, male weanling Wistar rats had free access to a purified diet. Thereafter. during 2 weeks, three groups received the same diet containing 0.2% of either DAS or DADS or AM dissolved in corn oit of the diet. Because ally1 sulfides provoked a temporary dietary restriction, three pair-fed control groups were given an amount of cantrol diet equivalent to the amount of experimental diet consumed by the treated groups. Two separate experiments were carried out using this same protocol: the first one in order to evaluate DNA damage after genotoxic treatment (four rats/ group). the second one to prepare liver S9 fractions according to the method of Maron and Ames f61 (six rats/group). 2.2. Alkaline elution of hqmtic DNA At the end of experimental period, rats were injected i.p. with a single dose of AFT31 (2 m&kg) or NDMA (IO mg/kgj or MNU (50 mg/kgj and killed 3 h later (AFB 1 and NDMA) or 2 h later (MNlij. Hepatic DNA damage was evaluated as single-strand breaks by the alkaline elution technique according to thenprocedure described by Kohn et al. [ 7 J. DNA alkaline elution was performed for 15 h with 0.02 M !Sa?EDTA adjusted to pH 12.1 with NaOH. The amount of DNA eluted and remaining on the tilter was determined by the microfluorimetric procedure described by Cesarone et al. (131.The results were
Hepam DNA Gngle-strand breaks induced by APB 1 were strongly reduced by ally1 sulfide pretreatment (Table 1). DADS had the most pronounced inhibitory effect. DNA damage being reduced by 82%. 411 three ally1 s&ides significantly reduced hepatic DNA single-strand breaks induced by NDMA. DAS and DADS exhibited a similar inhibition rate (50%) whereas the inhibitory effect of AM was less pronounccd iW/;i. Liver genotoxicity induced by %ING was not moditied by any treatment. Liver S9 from ally1 sulfide-treated rats markedly decreased the mutagenicity of NDMA as compared to liver SC)from control rats (Fig. I ). The mutagenicity ;,f AFBl is only slightly reduced when the activation
A.-M. L,e Bon et al. /Cancer Letters 114 (1997) 131-134
DAS
2000
/ 1000
9 $
0 2000
DADS
. 3 s c
1000
it z! + .u, =
0 2000
AM
1000
0 0
12
NDMA
3
(mg/ plate)
0
25
AFBl
50
100
(ng/ plate)
Fig. 1. Mutagenicity of NDMA and AFBl in the Ames test using liver S9 fractions from control (0) and ally1 sulfides-treated (0) rats.
systemwas derived from animals fed DAS or AM. No difference has been discerned between S9 from animals fed DADS and S9 from control animals.
4. Discussion
Numerous studies indicate that DNA damage,and SPmediated mutagenesisalso, can be either increased or decreasedas a result of drug-metabolizing alterations. However, the direction and magnitude of modulation varies depending on the carcinogen, the inducer and the experimental conditions [9,10]. Our study clearly demonstratesthat feeding rats with ally1 sulfides preventsDNA-SSBs in hepatocytes after a single injection of either AFB 1 or NDMA. This result implies that less reactive(s) metabolites(s)have been formed from AFBl and NDMA as a consequence of specific modulation of drug-metabolizing enzymes. The no protective effect of ally1 sulfide against DNA damage provoked by the direct-acting carcinogenMNU strengthensthis hypothesis.As CYP 2El is predominantly involved in the activation of
133
NDMA, prevention of NDMA genotoxicity is consistent with the suppressionof CYP 2El expression by ally1 sulfides. Ally1 sulfide administration to rats induces several CYPs including CYP 2B1/2 which would have a tendency to increase the production of the reactive AFBl-epoxide. An increase in DNA damagewould then be expected. However, simultaneous induction of detoxification pathways (EH, UGT, GST) would tend to the detoxification of the carcinogen. These results are consistent with those of hepatocarcinogenesis. Inasmuch as ally1 sulfides caused significant changes in drug-metabolizing enzymes activities, it was conceivable that hepatic preparations from ally1 sulfide-treated rats may modulate metabolic activation of chemical carcinogens in the S9-mediated mutagenesisassay.In fact, the inhibition of the mutagenic effect of the promutagensby liver S9 from rats fed ally1 sulfides was dramatically marked in the case of NDMA. This is compatible with the inhibition of CYP 2El previously reported. The mutagenicity of AFBl was influenced very little by ally1 sulfide treatment. Since ally1 sulfides induce numerous enzymes associatedwith the metabolism of AFB 1, it was surprising to observeno effect on the bioactivation of this carcinogen. Several phase I, but also phase II, enzymesare involved in AFB 1 metabolism.However, the S9 mix would lack cofactors for conjugating enzymes. Therefore we probably failed to observe the effects of induction of transferasesby ally1 sulfides. Moreover, despite of induction of CYP 2B by ally1 sulfides, no enhancementof the mutagenicity of AFBl was observed. Taken together,the results of the presentstudy raise the possibility that ally1 sulfides have antigenotoxic potency through modulation of drug-metabolizing enzymes.However, other mechanismscannot be discarded.
References [l] Steinmetz, K.A., Kushi, L.H., Bostick, R.M., Folson, A.R. and Potter, J.D. (1994) Vegetables, fruit, and colon cancer in the Iowa women’s health study. Am. J. Epidemiol., 139, 1-15. [2] Wargovich, M.J. (1992) Inhibition of gastrointestinal cancer by organosulfur compounds in garlic. In: Cancer Chemopre-
vention. pp 195-203. Editors: L.W. Wattenbcrg, M, Lipkin, C.W. Boone and G.J. Kelloff. CRC Press.Boca Raton. FL, [3] Brady, J.F., Li, D., Ishizaki, H. and Yang, C.S. (1988) Effect of diallyl sulfide in rat liver microsomal nitrosaminemetabolism and other monooxygenaseactivities. Cancer Res.. 48. 5937-5940. 141Haber, D., Siess, M.H.. De Waziers. 1.. Beaune, P. and Suachetet, M. (1994) Modification of hepatic drug-metabolizing enzymes in rat fed naturally occuring allyi sulphides.Xenobiotica, 24, 169- 182. [51 Haber-Mignard, D., Suschetet,M.. Berg&, R.. Astorg, P. and Siess,M.H. (1996) Inhibition of aflatoxin 81 and N-nitrosodiethylamine-inducedliver preneoplasticfoci in rats fed naturally occuring ally1 sulfides. Nutr. Cancer. 25, 61 - 70. [6] Maron, D.M. and Ames, B.N. (1983)Revised methodsfor the Salmonella mutagenicity test. Mutat. Res.. 113. I73 2! 5. 171Kohn, K.W., Erickson, L.C.. Ewig. R.A.G. and Friedman.
CA. (1976) Fracnonatronof DNA from mammalian cells by alkaline elution. Biochemistry. IS. 4629-4637. [Xl Cesarone,C.F., Bolognesi. C and Santi. I,. (1979) Improvctr microfluorimetric DNA determination in biological material using 33258 Hoechst.Anal. B&hem.. IfkI, 188-197. I’,] Lin. D.X., .Malaveille. C . Park, S.S., Gelboin. H.V. am! Bartscb. H. I 1090) Contribution of DNA methylation and benzylation to i$nitroso-N-benzyl-methylamine-induced mutagenesisin bacteria: effects of rat liver cytochromeP4SO isozymes and ghnathione transferascs.Carcinogenesis, Il. If551--lh58. i if)] Hamilton. J.W. and Bloom, SE. (1986) Correiation between induction of xenobiotic metabolism and DNA damage from chemical carcinogensin the chick embryo in viva. Carcinorenesis. 7. I101 -1106.
ELSEVIER
Cancer Letters 114 (1997) 131-134
In vivo antigenotoxic effects of dietary ally1 sulfides in the rat Anne-Marie Le Bon*, Corinne Roy, Christine DuPont, Marc Suschetet Unite’ de Toxicologic Nutritionnelle,
INRA, 17 rue Sully, 21034 Dijon cedex, France
Abstract The effects of dietary administration of diallyl sulfide (DAS), diallyl disulfide (DADS) and ally1 mercaptan(AM) on the genotoxicity of different chemicals were studied in two experimental systems:(i) measurementof hepatic DNA single-strand breaks induced in rats by aflatoxin Bl (AFBl), N-nitrosodimethylamine (NDMA) or methylnitrosourea (MNU); (ii) mutagenicity of AFBl or NDMA on Sulmonellu typ4imurium TAlOO using hepatic S9 from rats fed ally1 sulfides as the activation system. All compounds strongly reduced hepatic DNA breaks induced by AFBl and NDMA but did not modify the genotoxicity of MNU. In the Ames test, the mutagenicity of NDMA was strongly inhibited by hepatic S9 from rats fed either compound.The mutagenicity of AFBl was al80reducedbut to a lesserextent. Such effects are likely related to the modulation of drug-metabolizing enzymes which play a key role in metabolic activation as well as detoxication of NDMA and AFBl. 0 1997 Elsevier Science Ireland Ltd. Keywords: Ally1 sulfides; Aflatoxin Bl; N-Nitrosodimethylamine; DNA single-strand breaks; Ames test
1. Introduction
Garlic is one plant to which anticancer properties have been attributed on the basis of epidemiologic observations and laboratory studies [l]. The possible anticarcinogenic propertiesof garlic may be attributed to various organosulfur compounds, including ally1 sulfides. Experimental studies on animal models have shown an inhibition of chemically-induced carcinogenesisin different organs by ally1 sulfide administered during the initiation step [2]. Ally1 sulfides are known for inducing several phaseI and phase II enzymes [3,4]. In a recent three-step medium hepatocarcinogenesis assay [5], we observed that DAS and DADS diets fed to rats in the initiation phase strongly * Corresponding author.
reduced the number and the size of preneoplasic foci initiated by AFBl. When initiation was induced by N-nitrosodiethylamine, DAS reduced the size of preneoplastic foci, but not their number. Inasmuch as the number of foci is closely related to a first mutational event (initiation index) and the size of foci reflects the clonal expansion rate of initiated cells (promotion index), it is not clear whether the most likely mechanism for ally1 sulfides chemoprevention is a specific inhibition of genotoxicity or if it is related to their ability to diminish mechanismsof proliferation/promotion. In order to establish whether in vivo antigenotoxic activity could explain the anti-initiating action of ally1 sulfides, two approacheswere employed: 1. The effects of DAS, DADS and ally1 mercaptan (AM, a DADS metabolite) exposureon liver DNA single-strand breaks (SSBs) induced in rats by a
0304-3835/97/$17.00 0 1997 Elsevier Science Ireland Ltd. All rights reserved PII SO304-3835(97)04642-9
NDMA or APB1 (mciirect-acting MNU (a direcr-acting carcinopen) were investigated. 3. The mutagenicity of NDMA. whose ac?r\atioa is catalyzed predominantly by C‘YP 7Ei and ot APB 1. whose activation IS modulated hj various CYP and whose detoxification depends predom,nantly on (XT, was investigated using the Ames mutagenicity assay as end-point. single
dose
carcinogens)
of
or
Post mitochondrial supernatant (S9) from rats fed DAS, DADS or AM were used as activation systems.
i:xpresscd as elutmn constant computed on the third fraction: K (ml ’ 1=I j--in i % of DNA retained on the filter)l/eiuted volume.
mutagenicity studies were performed accord ing ro Maron and Ames 161 employing S&zonelltr ,i,~~7hi~lrl~riUrN TA 100. The mutagenic response <)I ,ZFBI and NDMA was checked using the pre-incuhation procedure with S9 mix containing 10% and 30% lrepatic S9 preparations and adjusted to pH 7.1 and The
9.3, rcyeclivcl~.
2. Materials and methods 3. Results
For 2 weeks, male weanling Wistar rats had free access to a purified diet. Thereafter. during 2 weeks, three groups received the same diet containing 0.2% of either DAS or DADS or AM dissolved in corn oit of the diet. Because ally1 sulfides provoked a temporary dietary restriction, three pair-fed control groups were given an amount of cantrol diet equivalent to the amount of experimental diet consumed by the treated groups. Two separate experiments were carried out using this same protocol: the first one in order to evaluate DNA damage after genotoxic treatment (four rats/ group). the second one to prepare liver S9 fractions according to the method of Maron and Ames f61 (six rats/group). 2.2. Alkaline elution of hqmtic DNA At the end of experimental period, rats were injected i.p. with a single dose of AFT31 (2 m&kg) or NDMA (IO mg/kgj or MNU (50 mg/kgj and killed 3 h later (AFB 1 and NDMA) or 2 h later (MNlij. Hepatic DNA damage was evaluated as single-strand breaks by the alkaline elution technique according to thenprocedure described by Kohn et al. [ 7 J. DNA alkaline elution was performed for 15 h with 0.02 M !Sa?EDTA adjusted to pH 12.1 with NaOH. The amount of DNA eluted and remaining on the tilter was determined by the microfluorimetric procedure described by Cesarone et al. (131.The results were
Hepam DNA Gngle-strand breaks induced by APB 1 were strongly reduced by ally1 sulfide pretreatment (Table 1). DADS had the most pronounced inhibitory effect. DNA damage being reduced by 82%. 411 three ally1 s&ides significantly reduced hepatic DNA single-strand breaks induced by NDMA. DAS and DADS exhibited a similar inhibition rate (50%) whereas the inhibitory effect of AM was less pronounccd iW/;i. Liver genotoxicity induced by %ING was not moditied by any treatment. Liver S9 from ally1 sulfide-treated rats markedly decreased the mutagenicity of NDMA as compared to liver SC)from control rats (Fig. I ). The mutagenicity ;,f AFBl is only slightly reduced when the activation
A.-M. L,e Bon et al. /Cancer Letters 114 (1997) 131-134
DAS
2000
/ 1000
9 $
0 2000
DADS
. 3 s c
1000
it z! + .u, =
0 2000
AM
1000
0 0
12
NDMA
3
(mg/ plate)
0
25
AFBl
50
100
(ng/ plate)
Fig. 1. Mutagenicity of NDMA and AFBl in the Ames test using liver S9 fractions from control (0) and ally1 sulfides-treated (0) rats.
systemwas derived from animals fed DAS or AM. No difference has been discerned between S9 from animals fed DADS and S9 from control animals.
4. Discussion
Numerous studies indicate that DNA damage,and SPmediated mutagenesisalso, can be either increased or decreasedas a result of drug-metabolizing alterations. However, the direction and magnitude of modulation varies depending on the carcinogen, the inducer and the experimental conditions [9,10]. Our study clearly demonstratesthat feeding rats with ally1 sulfides preventsDNA-SSBs in hepatocytes after a single injection of either AFB 1 or NDMA. This result implies that less reactive(s) metabolites(s)have been formed from AFBl and NDMA as a consequence of specific modulation of drug-metabolizing enzymes. The no protective effect of ally1 sulfide against DNA damage provoked by the direct-acting carcinogenMNU strengthensthis hypothesis.As CYP 2El is predominantly involved in the activation of
133
NDMA, prevention of NDMA genotoxicity is consistent with the suppressionof CYP 2El expression by ally1 sulfides. Ally1 sulfide administration to rats induces several CYPs including CYP 2B1/2 which would have a tendency to increase the production of the reactive AFBl-epoxide. An increase in DNA damagewould then be expected. However, simultaneous induction of detoxification pathways (EH, UGT, GST) would tend to the detoxification of the carcinogen. These results are consistent with those of hepatocarcinogenesis. Inasmuch as ally1 sulfides caused significant changes in drug-metabolizing enzymes activities, it was conceivable that hepatic preparations from ally1 sulfide-treated rats may modulate metabolic activation of chemical carcinogens in the S9-mediated mutagenesisassay.In fact, the inhibition of the mutagenic effect of the promutagensby liver S9 from rats fed ally1 sulfides was dramatically marked in the case of NDMA. This is compatible with the inhibition of CYP 2El previously reported. The mutagenicity of AFBl was influenced very little by ally1 sulfide treatment. Since ally1 sulfides induce numerous enzymes associatedwith the metabolism of AFB 1, it was surprising to observeno effect on the bioactivation of this carcinogen. Several phase I, but also phase II, enzymesare involved in AFB 1 metabolism.However, the S9 mix would lack cofactors for conjugating enzymes. Therefore we probably failed to observe the effects of induction of transferasesby ally1 sulfides. Moreover, despite of induction of CYP 2B by ally1 sulfides, no enhancementof the mutagenicity of AFBl was observed. Taken together,the results of the presentstudy raise the possibility that ally1 sulfides have antigenotoxic potency through modulation of drug-metabolizing enzymes.However, other mechanismscannot be discarded.
References [l] Steinmetz, K.A., Kushi, L.H., Bostick, R.M., Folson, A.R. and Potter, J.D. (1994) Vegetables, fruit, and colon cancer in the Iowa women’s health study. Am. J. Epidemiol., 139, 1-15. [2] Wargovich, M.J. (1992) Inhibition of gastrointestinal cancer by organosulfur compounds in garlic. In: Cancer Chemopre-
vention. pp 195-203. Editors: L.W. Wattenbcrg, M, Lipkin, C.W. Boone and G.J. Kelloff. CRC Press.Boca Raton. FL, [3] Brady, J.F., Li, D., Ishizaki, H. and Yang, C.S. (1988) Effect of diallyl sulfide in rat liver microsomal nitrosaminemetabolism and other monooxygenaseactivities. Cancer Res.. 48. 5937-5940. 141Haber, D., Siess, M.H.. De Waziers. 1.. Beaune, P. and Suachetet, M. (1994) Modification of hepatic drug-metabolizing enzymes in rat fed naturally occuring allyi sulphides.Xenobiotica, 24, 169- 182. [51 Haber-Mignard, D., Suschetet,M.. Berg&, R.. Astorg, P. and Siess,M.H. (1996) Inhibition of aflatoxin 81 and N-nitrosodiethylamine-inducedliver preneoplasticfoci in rats fed naturally occuring ally1 sulfides. Nutr. Cancer. 25, 61 - 70. [6] Maron, D.M. and Ames, B.N. (1983)Revised methodsfor the Salmonella mutagenicity test. Mutat. Res.. 113. I73 2! 5. 171Kohn, K.W., Erickson, L.C.. Ewig. R.A.G. and Friedman.
CA. (1976) Fracnonatronof DNA from mammalian cells by alkaline elution. Biochemistry. IS. 4629-4637. [Xl Cesarone,C.F., Bolognesi. C and Santi. I,. (1979) Improvctr microfluorimetric DNA determination in biological material using 33258 Hoechst.Anal. B&hem.. IfkI, 188-197. I’,] Lin. D.X., .Malaveille. C . Park, S.S., Gelboin. H.V. am! Bartscb. H. I 1090) Contribution of DNA methylation and benzylation to i$nitroso-N-benzyl-methylamine-induced mutagenesisin bacteria: effects of rat liver cytochromeP4SO isozymes and ghnathione transferascs.Carcinogenesis, Il. If551--lh58. i if)] Hamilton. J.W. and Bloom, SE. (1986) Correiation between induction of xenobiotic metabolism and DNA damage from chemical carcinogensin the chick embryo in viva. Carcinorenesis. 7. I101 -1106.