Antimutagenic activity of organosulfur compounds from Allium is associated with phase II enzyme induction

Mutation Research 495 (2001) 135–145

Antimutagenic activity of organosulfur compounds from Allium is associated with phase II enzyme induction Denis Guyonnet, Christine Belloir, Marc Suschetet, Marie-Hélène Siess, Anne-Marie Le Bon∗ Institut National de la Recherche Agronomique, Unité Mixte de Recherche de Toxicologie Alimentaire, BP 86510, 17 rue Sully, 21065 Dijon Cedex, France Received 19 December 2000; received in revised form 15 May 2001; accepted 16 May 2001

Abstract In a previous study, we showed that naturally occurring organosulfur compounds (OSCs) from garlic and onion modulated the activation of carcinogen via the alteration of cytochromes P450. The present study was undertaken to determine the incidence of the in vivo induction of phase II enzymes by individual OSCs on the genotoxicity of several carcinogens. Diallyl sulfide (DAS), diallyl disulfide (DADS), dipropyl sulfide (DPS) and dipropyl disulfide (DPDS), were administered by gavage (1 mmol/kg) to male SPF Wistar rats for 4 consecutive days. The effects of treatments on phase II enzymes and on the genotoxicity of carcinogens were evaluated with hepatic cytosols and microsomes from OSCs-treated rats. DADS strongly increased all the phase II enzymes activities examined, i.e. total glutathione S-transferase (GST) activity, mu GST activity, quinone reductase (QR) activity and epoxide hydrolase (EH) activity. In addition, DADS strongly increased the protein level of rGSTP1. QR activity, total and mu GST activities were also increased by DAS and DPDS whereas DPS increased only mu GST activity and QR activity. To assess the repercussions of these inductions on the genotoxicity of carcinogens, the effects of cytosols or microsomes from OSCs-treated rats on the mutagenicity of (+)-anti-7␤,8␣-dihydroxy-9␣,10␣-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), styrene oxide (SO) and 4-nitroquinoline 1-oxide (4-NQO) were measured in the Ames test. DADS showed a very effective antimutagenic activity against BPDE, SO and 4-NQO. DAS reduced the mutagenicity of BPDE and SO. In contrast, DPS and DPDS showed little efficient antimutagenic activity since they only reduced the mutagenicity of BPDE and 4-NQO, respectively. Interestingly, DADS appeared to be as effective as ethoxyquin, a model inducer of phase II enzymes, in both inducing phase II enzymes and inhibiting the mutagenicity of carcinogens. This study demonstrated that the antimutagenic activities of OSCs against several ultimate carcinogens were closely related to their ability to induce phase II enzymes. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Organosulfur compounds; Antimutagenicity; Phase II enzymes; Ethoxyquin; Ames test; Liver subcellular fractions

Abbreviations: BaP, benzo[a]pyrene; DAS, diallyl sulfide; DADS, diallyl disulfide; DPS, dipropyl sulfide; DPDS, dipropyl disulfide; CYP, cytochrome P450; GST, glutathione S-transferase; EH, epoxide hydrolase; QR, quinone reductase; UGT, UDP-glucuronosyltransferase; OSCs, organosulfur compounds; GSH, glutathione BPDE, (+)-anti-7␤,8␣-dihydroxy-9␣,10␣-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene; SO, styrene oxide; 4-NQO, 4-nitroquinoline 1-oxide; DCNB, 1,2-dichloro-4-nitrobenzene; CDNB, 1-chloro-2,4-dinitrobenzene ∗ Corresponding author. Tel.: +33-3-80-69-32-15; fax: +33-3-80-69-32-25. E-mail address: [email protected] (A.-M. Le Bon). 1383-5718/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 ( 0 1 ) 0 0 2 0 5 - 4

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1. Introduction Epidemiologic studies have reported that regular consumption of garlic and onion is inversely related to the risk of cancer, particularly with regard to gastric cancer [1–4]. These anticarcinogenic effects, as other biological properties of garlic and onion, are attributed to specific organosulfur compounds (OSCs) present in very high levels in these vegetables. The protective effects of these naturally occurring substances against cancer have been supported by many experimental studies with animal models [5–7]. OSCs may exert their protective effects at all main stages of the carcinogenic process [5,6,8,9] and are especially efficient inhibitors of the initiation phase of chemically-induced carcinogenesis in different organs in rats and mice [5,7,10–12]. The underlying mechanisms of the anticarcinogenic effects of OSCs on the initiation stage are not precisely known. One mechanism may be the modification of carcinogen metabolism via the modulation of drug-metabolizing enzymes. OSCs were shown to alter cytochromes P450 (CYP) and phase II detoxication enzymes in different tissues in rats and mice [5,13–18]. CYP play a key role in the metabolism of chemical carcinogens because they catalyze either detoxication reactions or chemical activation resulting in the formation of toxic metabolites. The outcome of this metabolism depends on CYP isozymes and on the carcinogen. OSCs are slight inducers of the CYP1A family, strong inducers of CYP2B and efficient inhibitors of CYP2E1 in the rat liver [14–16,19]. In a previous study, we showed that these CYP modifications can lead to an enhancement or a decrease in the mutagenicity of carcinogens in the Ames test [20]. In contrast to CYP, phase II enzymes are generally involved in detoxication pathways by catalyzing the elimination of reactive intermediates of carcinogens. Therefore, induction of phase II enzymes is considered as a major mechanism of protection against carcinogenesis and mutagenesis [21,22], and the ability of some compounds to induce phase II enzymes has been used to predict their chemopreventive potency [23,24]. Among the phase II enzymes, glutathione S-transferases (GST) play a pivotal role in cellular protection against carcinogens by conjugating their electrophile metabolites with glutathione (GSH) [25,26]. Experimental studies have shown that OSCs

strongly induce GST activity in different organs in rats and mice [5,6,13,16,17]. They also induce other phase II enzymes such as epoxide hydrolase (EH), quinone reductase (QR) and UDP-glucuronosyltransferases (UGT) [13,15–17,27]. To date, the level of phase II enzyme induction required to protect the organism against genotoxic effects of carcinogen remains to be determined. Complementary studies are required to characterize the implication of the induction of phase II enzymes in the anticarcinogenic effects of OSCs. In this study, the incidence of the in vivo induction of phase II enzymes by individual OSCs on the genotoxicity of mutagens was evaluated in the Ames test. We studied the effects of four OSCs, namely diallyl sulfide (DAS), diallyl disulfide (DADS), dipropyl sulfide (DPS) and dipropyl disulfide (DPDS). These OSCs have different capacities to induce phase II enzymes in rat liver [13,16]. We focused our study on the liver because hepatic metabolism is a major factor for determining the concentration of mutagen or carcinogen in the body. The effects of OSCs were compared with those of ethoxyquin (EQ), a prototype phase II enzyme inducer in rat [28,29]. We evaluated the effects of liver subcellular fractions (microsomes and cytosols) from OSC-treated rats on the mutagenicity of the ultimate metabolite of various carcinogens. Specific substrates of EH, DT-diaphorase and GST subunits M1, M2 and P1 were used to assess the incidence of the induction of these phase II enzymes by OSCs. The ultimate carcinogenic metabolite of benzo[a]pyrene (BaP), (+)-anti-7␤,8␣-dihydroxy-9␣,10␣-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) [30] and the genotoxic metabolite of styrene, styrene oxide (SO), were used to examine the incidence of the modification of rGSTP1 and rGSTM1/M2, respectively. SO was also used to determine the effect of microsomal EH modulation since this enzyme catalyzes the inactivation reaction of SO into glycol styrene [31]. Finally, the incidence of QR modification was evaluated using 4-nitroquinoline 1-oxide (4-NQO), a tongue carcinogen in rat [32], as substrate. This study demonstrated that hepatic subcellular fractions from OSC-treated rats inhibited the genotoxicity of these ultimate carcinogens. These effects are closely related to the induction of phase II enzymes which are involved in the detoxication pathways of these carcinogens.

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2. Material and methods 2.1. Chemicals DAS (purity 97%, CAS no. 592-88-1), DADS (purity 80%, remainder other allyl sulfides, CAS no. 2179-57-9), DPS (purity 97%, CAS no. 111-47-7) and DPDS (purity 98%, CAS no. 629-19-6) were obtained from Aldrich (Strasbourg, France). They were used without further purification. 4-NQO (CAS no. 56-57-5), SO (CAS no. 96-09-3), CDNB (CAS no. 97-00-7), DCNB (CAS no. 99-54-7) and EQ (CAS no. 91-53-2) were purchased from Sigma-Aldrich Chimie (Saint-Quentin Fallavier, France). BPDE (CAS no. 58917-67-2) was obtained from Midwest Research Institute (Kansas City, MO). Antibodies raised against rGSTP1 were obtained from Biotrin International (Dublin, Ireland). Salmonella typhimurium strain TA100 was provided by Dr. B. Ames (Department of Biochemistry, University of California, Berkeley, USA). 2.2. Animals and treatments Male SPF Wistar rats, 5 weeks old, from Iffa Credo (L’Arbresle, Lyon, France) were housed in individual stainless steel cages and maintained at 21◦ C, with constant humidity and a 12 h light–dark cycle. During the experiment, they were allowed free access to a semi-liquid purified diet as previously described [19]. The treatment of rats with each OSC (1 mmol/kg for 4 consecutive days) was done as previously described [20]. EQ (0.5% (w/w)) was administered for 6 consecutive days in the diet. 2.3. Preparation of hepatic subcellular fractions Twenty-four hours after the last treatment, the animals were killed by cervical dislocation following 16 h of fasting. Livers were removed and pooled. Liver microsomes and cytosols were prepared as previously described [19,20]. Microsomes and cytosols were stored in aliquots of 1 ml at −80◦ C. 2.4. Enzyme assays The protein level of microsomes and cytosols was measured by the method of Bradford [33], adapted for

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automatic measurement using a Cobas Fara II centrifugal analyzer (Roche Instruments, Basel, Switzerland). Total GST and mu GST activities were, respectively, measured with 1,2-dichloro-4-nitrobenzene (DCNB) and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates, according to the method of Habig et al. [34]. The other enzyme assays were performed as previously described [19]. 2.5. Western blotting analysis Western blotting analysis of rGSTP1 was performed as previously described [13]. Antibodies raised against rGSTP1 were used at a dilution of 1:400. 2.6. Mutagenicity assays The Ames test was performed with S. typhimurium TA100 according to Maron and Ames [35] with slight modifications. A bacterial suspension obtained after an overnight culture of 12 h was used. The effects of treatments on the mutagenicity of 4-NQO, BPDE, BaP and SO were determined by a liquid preincubation method using microsomes or cytosols as the detoxication system. The choice of hepatic subcellular fractions was done according to the cellular localization of the enzymes studied. Cofactors (GSH or NADH) were added when needed (Table 1). Experimental conditions, i.e. preincubation time, percentage of liver subcellular fraction, order of addition of the different constituents in the medium were specifically determined for each test (Table 1). These adjustments were necessary owing to the great instability of BPDE in aqueous solution or because of the toxicity of the mutagen (SO, 4-NQO) for the bacterial strain. The percentage of liver subcellular fraction for each test was determined in order to obtain efficient metabolism of the mutagen. This was checked by comparing the mutagenicity induced by each mutagen alone with the mutagenicity induced by the same mutagen in presence of detoxication system with or without cofactors (GSH, NADH). Then, the time of preincubation retained for each test was chosen to give an optimal metabolism of each mutagen by liver subcellular fraction. After the preincubation period, the mixtures were diluted with soft agar, plated onto minimal glucose

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Table 1 Experimental conditions used in the mutagenicity assays Enzyme studied

Mutagens

Subcellular fraction

Cofactor

Preincubation time

Observations

rGSTP1

BPDE

Cytosols (1%)

GSH (5 mM)

5 min

Mutagen added at the last moment

QR EH RGSM1/M2

4-NQO SO SO

Cytosols (0.2%) Microsomes (20%) Cytosols (5%)

NADH (8 mM)

20 min Without preincubation 10 min

GSH (5 mM)

agar plates and further incubated for 48 h at 37◦ C to allow the development of histine revertant colonies (His+). The number of His+ revertants was counted on two repetitions of triplicate plates for each dose of mutagen. 2.7. Statistical analysis Data were compared by analysis of variance followed by a Dunnett’s test to compare the treated groups to the control group (control rats). P ≤ 0.05 was chosen as indicating significance. Calculations were made with the SAS system (Cary, NC).

3. Results The effects of the treatments on total and mu GST activities, QR activity and EH activity are shown in Table 2. DADS and EQ significantly induced all the

Bacterial strain added at the end of the preincubation

phase II enzyme activities measured. The increase in phase II enzyme activities produced by DADS was comparable to that produced by the model inducer EQ. EQ produced the largest increase in total GST activity (3.4-fold), mu GST activity (2.9-fold) and EH activity (1.5-fold) whereas the strongest increase in QR activity was observed with DADS (6.1-fold). DAS and DPDS treatments significantly enhanced total GST activity by 1.6- and 1.4-fold, respectively, whereas the induction produced by DPS was non-significant (1.3-fold). DAS, DPS and DPDS significantly increased mu GST activity (∼1.4-fold) as well as QR activity (∼1.5-fold). At the same time, DAS, DPS and DPDS were ineffective in increasing EH activity. Western blotting analysis showed that DADS and EQ strongly increased the level of rGSTP1 protein whereas this protein was not detectable in control rats and in DAS-, DPS- and DPDS-treated rats (Fig. 1). In all mutagenicity assays, we compared the effects of hepatic subcellular fractions of OSC-treated rats

Table 2 Effects of DAS, DADS, DPS, DPDS and EQ on phase II enzyme activities in cytosols or microsomes in rat livera Treatments

Total GSTb

Control DAS DADS DPS DPDS EQ

1169 1875 3350 1476 1596 3994

± ± ± ± ± ±

58 32∗ 49∗ 59 19∗ 188∗

mu GST (nmol/min/mg protein)c 25.7 36.7 57.0 30.3 33.3 74.0

± ± ± ± ± ±

0.3 0.3∗ 0.6∗ 0.3∗ 0.7∗ 0.6∗

QRd 389 547 2382 607 658 2134

EHe (nmol/min/mg protein) ± ± ± ± ± ±

4 7∗ 19∗ 7∗ 23∗ 40∗

Values are means ± S.E.M. of three repetitions. GST activity was expressed in nmols of CDNB conjugate per min per mg of cytosolic proteins. c GST activity was expressed in nmols of DCNB conjugate per min per mg of cytosolic proteins. d QR activity was expressed in nmols of NADH oxidized per min per mg of cytosolic proteins. e EH activity was expressed in nmols of [14 C] glycol styrene per min per mg of microsomal proteins. ∗ Significantly different from the corresponding control value (Dunnett’s test, P ≤ 0.05). a

b

28.6 30.7 37.4 30.4 27.8 41.9

± ± ± ± ± ±

0.8 0.1 0.3∗ 3.3 2 1.5∗

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Fig. 1. Western blotting analysis for the expression of rGSTP1 in the liver of control, ethoxyquin and OSCs-treated rats, 20 ␮g of cytosolic proteins was loaded onto the gel.

with those obtained with hepatic subcellular fractions of EQ-treated rats. It is noteworthy that hepatic subcellular fractions of EQ-treated rats demonstrated a strong antimutagenic action against all the genotoxic compounds used in this study when compared with control rats (Figs. 2–5). Hepatic cytosols from control, DAS- and DPS-treated rats reduced the mutagenicity induced by 4-NQO in the same way (Fig. 2). Cytosols from DADS-treated rats significantly inhibited the mutagenicity induced by 4-NQO when compared with control rats.

Nevertheless, this antimutagenic effect was less marked than the one produced by cytosols from rats fed EQ-containing diet. Cytosols from DPDS-treated rats also decreased the mutagenicity of 4-NQO when compared with cytosols from control rats, although to a lesser extent than DADS-treated rat cytosols. Cytosols from control rats notably reduced the mutagenicity of BPDE (Fig. 3). Cytosols from DAS-, DADS- and DPS-treated rats were more effective than control cytosols in inhibiting BPDE mutagenicity. DADS demonstrated the greater antimutagenic effect

Fig. 2. Effect of hepatic cytosols from control rats, ethoxyquin- and OSC-treated rats on 4-NQO-mediated mutagenesis. The mutagenicity assay was carried out using S. typhimurium strain TA100 and 0.2% (v/v) of cytosolic preparations. The system was supplemented with NADH (8 mM). The spontaneous reversion rate was 127 ± 5. Results are presented as a mean ± S.E.M. of two series of triplicates. (∗) Significantly different from the control group (Dunnett’s test, P ≤ 0.05).

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Fig. 3. Effect of hepatic cytosols from control rats, ethoxyquin- and OSC-treated rats on BPDE-mediated mutagenesis. The mutagenicity assay was carried out using S. typhimurium strain TA100 and 1% (v/v) of cytosolic preparations. The system was supplemented with GSH (5 mM). The spontaneous reversion rate was 110 ± 4. Results are presented as a mean ± S.E.M. of two series of triplicates. (∗) Significantly different from the control group (Dunnett’s test, P ≤ 0.05).

Fig. 4. Effect of hepatic cytosols from control rats, ethoxyquin- and OSC-treated rats on SO-mediated mutagenesis. The mutagenicity assay was carried out using S. typhimurium strain TA100 and 5% (v/v) of cytosolic preparations. The system was supplemented with GSH (5 mM). The spontaneous reversion rate was 99 ± 8. Results are presented as a mean ± S.E.M. of two series of triplicates. (∗) Significantly different from the control group (Dunnett’s test, P ≤ 0.05).

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Fig. 5. Effect of hepatic microsomes from control rats, ethoxyquin- and OSC-treated rats on SO-mediated mutagenesis. The mutagenicity assay was carried out using S. typhimurium strain TA100 and 20% (v/v) of cytosolic preparations. The spontaneous reversion rate was 115 ± 6. Results are presented as a mean ± S.E.M. of two series of triplicates. (∗) Significantly different from the control group (Dunnett’s test, P ≤ 0.05).

and this protective effect was close to the inhibitory effect produced by cytosols from EQ-treated rats. The effects of DPDS treatment depended on BPDE concentration: a reduction of BPDE mutagenicity was observed at the lowest dose whereas an increase of BPDE mutagenicity or no effect occurred at higher doses. SO mutagenicity was reduced in the same way by cytosols from control, DPS- and DPDS-treated rats (Fig. 4). Cytosols from DAS- and DADS-treated rats were significantly more efficient than cytosols from control rats in inhibiting SO mutagenicity. The antimutagenic effect of DADS was slightly higher than DAS but was less marked that the inhibitory effect produced by EQ. When microsomes were used as the detoxication system, control, DPS and DPDS treatments reduced the mutagenicity of SO in the same way, except that DPS treatment significantly decreased SO mutagenicity at a dose of 300 ␮g of SO per plate (Fig. 5). Microsomes from DAS-treated rats also reduced SO mutagenicity but this inhibitory effect was restricted to the lowest doses of SO. Microsomes from DADS-treated rats strongly inhibited SO mutagenicity

when compared with microsomes from control rats. Again, the strongest reduction in SO mutagenicity was observed with microsomes from EQ-treated rats.

4. Discussion OSCs were shown to mediate significant chemopreventive activities against the initiation stage of carcinogenesis induced by various chemical carcinogens [5,7,10–12]. Concomitant with this, they were demonstrated to modify phase I and II enzymes when administered to rats or mice [5,13–18]. However, the exact involvement of these modifications in the anticarcinogenic properties of OSCs needs to be assessed. There is evidence that induction of phase II enzymes is a relevant mechanism for the anticarcinogenic action of several chemopreventive agents [21,22]. On the basis of the ability of OSCs to induce phase II enzymes in the rat liver, the antimutagenic effects of hepatic subcellular fractions from OSC-treated rats against various chemical carcinogens were investigated. This study showed that the inhibition of the

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mutagenicity of various carcinogens by hepatic subcellular fractions from OSC-treated rats was related to their capacity to induce phase II enzymes. OSCs are known to be potent inducers of GST [11,15–19]. In this study, we confirm that DADS is a better effective inducer of total GST and mu GST activities than DAS, DPS and DPDS. These differences between OSCs are due to the specific modifications of GST subunit levels produced by each OSC. DADS increases the protein level of all the major hepatic GST subunits and especially GST of the alpha class, rGSTM1 and rGSTP1, whereas the induction of GST subunits produced by DAS, DPS and DPDS was less marked and restricted to GST subunits of the alpha and mu classes [13,18]. The induction of mu GST activity is of great interest in terms of chemoprevention. The mu GST subunits represent the major GST subunits in rat liver [26] and are involved in the detoxication of mutagenic metabolites of carcinogens such as BaP-4,5-oxide and SO [25,36]. The antimutagenic effects of DADS and DAS against SO-mediated mutagenesis demonstrated in this study were consistent with their ability to increase mu GST activity. The effective increase of mu GST activity produced by DADS is in accordance with its ability to induce the protein level of both rGSTM1 and M2 in the rat liver [13]. The intensity of the increase of mu GST activity produced by DPS and DPDS seems to be insufficient to mediate an inhibitory effect against SO mutagenicity in our experimental conditions. Taking together, these results suggest that induction of the different classes of GSTs by OSCs can afford protection against a large array of carcinogens. However, in some cases, the toxicity of a compound is increased after conjugation with GSH [37]. For instance, dihaloalkanes are activated by the class theta GST T1-1 [38]. Some chemopreventive agents such as indole-3-carbinol have been shown to induce this GST isoenzyme and in relation with that, the bioactivation of dihaloalkanes was induced [39]. It would therefore be interesting to examine whether OSCs are able to activate these chemicals. OSCs are also able to induce epoxide hydrolase which is involved, for instance, in SO detoxication [31]. In this study, only DADS increased EH activity but previous studies have shown that DAS, DPS and DPDS were able to induce EH activity in the liver [13,15]. In agreement with the ability of DADS to increase EH activity, microsomes from DADS-treated

rats strongly decreased the mutagenicity of SO. Despite the lack of increase of EH activity, microsomes from DAS-treated rats decreased the mutagenicity of SO, but to a lower extent than DADS. Several studies have shown the antimutagenic action of OSCs against BaP. DADS decreased the formation of micronuclei or DNA breaks induced by BaP in HepG2 cells [40] and a mixture of DAS, DADS and diallyl trisulfide inhibited micronuclei formation induced by BaP in mouse bone marrow cells [41]. Moreover, DADS was an effective inhibitor of BaP-induced forestomach cancer in mice [11]. DAS also inhibited BaP-induced cancer of forestomach and lung in mice [5,11] whereas DPS and DPDS showed some anticarcinogenic properties against BaP-induced lung cancer in mice [5,11,42]. We have previously shown that DAS, DPS and DPDS increased the activation of BaP into mutagenic metabolites whereas DADS did not affect BaP activation when compared to control treatment (Table 3) [20]. Therefore, the induction of phase II enzymes is likely the main mechanism by which these OSCs exert their chemoprotective effects against BaP-induced carcinogenesis. Induction of phase II enzymes would counterbalance the induction of CYP1A1 produced by certain OSCs. Numerous phase II enzymes (QR, GSTM2, GSTP1, UGT) are involved in detoxication pathways of BaP [25,43–45]. Among these, GSTs play a critical role owing to their ability to detoxify BPDE, the most mutagenic metabolite of BaP [30]. In this study, we demonstrated that cytosols from DAS-, DADS- and DPS-treated rats decreased the mutagenicity of BPDE. The induction of distinct GST subunits by DAS, DADS and DPS is likely to be responsible for their differential inhibitory effects on BPDE mutagenicity. In rats, GSTP1 has the highest activity toward anti-BPDE [43]. rGSTM2 also catalyzes this reaction but with a lower catalytic efficiency than rGSTP1 whereas rGSTM1 has little activity towards BPDE [25,43]. As demonstrated in a previous study [13], only DADS was an effective inducer of rGSTP1. A positive correlation was also found between the ability of OSCs to prevent BaP-induced forestomach cancer in mice and their effectiveness in inducing mGSTP1 [11]. Therefore, the strong antimutagenic activity of DADS against BPDE can be attributed to an increase rate of GSH-conjugation of BPDE through the induction of rGSTP1. The antimutagenic effects of DAS and

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Table 3 Compilation of the effects induced by liver subcellular fractions from rats fed OSCs on the mutagenicity of genotoxic compounds in the Ames test (this study and [20])a Treatment

DAS DADS DPS DPDS

Phase I substrates (P450 involved in activation)

Phase II substrates (enzyme involved in detoxication)

BaP (1A1)

PhIP (1A2)

CP (2B, 2C11)

NPiP (2B)

DMN (2E1)

BPDE (GSTP1, GSTM2)

SO (GSTM1, GSTM2)

4-NQO (QR)

SO (EH)

 0  

   

 0  

  



0

0 0

0 0

0 0

a (0): Effect equivalent to that induced by the control treatment; (): enhancement of activation when compared with control; ( ): decrease of activation when compared with control. The number of symbols is indicative of the intensity of the effect. The protective effects are underlined.

DPS against BPDE are likely due to the induction of mu GST subunits responsible for the increase of GST activity measured with the model substrate DCNB. In contrast, DPDS was unable to inhibit BPDE-mutagenicity despite an increase of mu GST activity. It was demonstrated that DPDS was most effective in inducing rGSTM1 than rGSTM2 [13,18]. This specific pattern of induction of mu class GST would explain why DPDS is devoid of antimutagenic activity against BPDE. In addition to GST, the induction of QR may account for the chemoprotective effects of OSCs against BaP since QR inactivates quinones generated during the oxidative metabolism of BaP [30,46] and therefore prevents mutations generated by BaP-3,6-quinone [45,47]. Using 4-NQO as a prototype quinone, we observed that the potent induction of liver QR produced by DADS is related to a strong inhibition of 4-NQO mutagenicity. The other OSCs tested were slight inducers of QR activity and, as a result of this, they showed few or no antimutagenic activity against 4-NQO. In this study, we have compared the effects of OSCs to those of EQ, one of the most extensively chemopreventive compounds studied. EQ is a potent inducer of different phase II enzymes such as QR and GST in rat [28]. EQ notably induces rGSTP1 and rGSTA5 at protein and mRNA levels [28,29]. This study demonstrates that EQ exerted strong antimutagenic action which is in agreement with its ability to induce phase II enzymes. Interestingly, DADS appears to have the same profile as EQ in both inducing phase II enzymes and inhibiting the mutagenicity of carcinogens. Especially DADS and EQ were found to be very effective at inducing QR

and rGSTP1. Both DADS and EQ have been found to afford protection against the initiation of chemical carcinogenesis in different experimental models. Administration of DADS or EQ inhibits aflatoxin B1-initiated preneoplastic foci in the rat liver [7,48], reduces dimethylbenz(a)anthracene-initiated mammary tumors in rats [10,49] and prevents BaP-induced forestomach cancer in mice [11,50]. The present report provides evidence that DADS possesses significant antimutagenic properties and shows similarities with EQ, a known chemopreventive agent. These protective effects are closely related to its ability to markedly increase phase II enzymes but a previous study has also shown that DADS reduced the mutagenicity of numerous carcinogens via modulation of phase I enzymes (Table 3) [20]. Therefore, it seems that DADS can exert a broad range of antimutagenic effects since it has the capacity to act at different stages of carcinogen metabolism and against an array of structurally diverse compounds. On the basis of its antimutagenic and anticarcinogenic actions, DADS can be regarded as a promising natural cancer chemopreventive agent. We are aware that the dose of DADS we employed was rather high compared to the possible intake of this substance through normal human intake of garlic. However, activities of phase II enzymes can be induced by low amounts of DADS [17]. Therefore, DADS could afford protection against genotoxic compounds at dose levels similar to those that could be attained by human intake. In contrast, the antimutagenic properties of DAS, DPS and DPDS were less marked (Table 3). These compounds are less efficient than DADS in preventing mutagenicity of ultimate metabolites. Furthermore, in the first part

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of this work [20], we showed that these compounds can either enhance or decrease the bioactivation of chemicals. The antimutagenic effects of these compounds will therefore depend on the mutagen and on the balance between activation and detoxication.

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