The molecular mechanisms of diallyl disulfide and diallyl sulfide induced hepatocyte cytotoxicity

Chemico-Biological Interactions 180 (2009) 79–88

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The molecular mechanisms of diallyl disulfide and diallyl sulfide induced hepatocyte cytotoxicity D. Truong, W. Hindmarsh, P.J. O’Brien ∗ Graduate Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, 144 College St, Toronto, Ontario, Canada M5S 3M2

a r t i c l e

i n f o

Article history: Received 20 October 2008 Received in revised form 29 January 2009 Accepted 16 February 2009 Available online 23 February 2009 Keywords: Diallyl disulfide Diallyl sulfide Hydrogen sulfide Acrolein Rat hepatocytes Cytotoxic mechanisms

a b s t r a c t Diallyl disulfide (DADS) and diallyl sulfide (DAS) are the major metabolites found in garlic oil and have been reported to lower cholesterol and prevent cancer. The molecular cytotoxic mechanisms of DADS and DAS have not been determined. The cytotoxic effectiveness of hydrogen versus allyl sulfides towards hepatocytes was found to be as follows: NaHS > DADS > DAS. Hepatocyte mitochondrial membrane potential was decreased and reactive oxygen species (ROS) and TBARS formation was increased by all three allyl sulfides. (1) DADS induced cytotoxicity was prevented by the H2 S scavenger hydroxocobalamin, which also prevented cytochrome oxidase dependent mitochondrial respiration suggesting that H2 S inhibition of cytochrome oxidase contributed to DADS hepatocyte cytotoxicity. (2) DAS cytotoxicity on the other hand was prevented by hydralazine, an acrolein trap. Hydralazine also prevented DAS induced GSH depletion, decreased mitochondrial membrane potential and increased ROS and TBARS formation. Chloral hydrate, the aldehyde dehydrogenase 2 inhibitor, however had the opposite effects, which could suggest that acrolein contributed to DAS hepatocyte cytotoxicity. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Numerous organosulfur compounds have been reported to be responsible for the pharmacologically active role of garlic. Organosulfur compounds taken together constitute less than 3.5% of fresh garlic (w/w). Diallyl disulfide (DADS), diallyl sulfide (DAS), and allyl mercaptan (AM) were reported to lower cholesterol levels and prevent cancer [1–6]. Tumor cells were particularly susceptible to DADS [6]. The chemoprotective action of DADS and DAS was suggested to result from their ability to inhibit carcinogen bioactivating enzymes, e.g., CYP 2E1. They also induced detoxification enzymes, e.g., glutathione-S-transferase (GST), epoxide hydrolase, quinone reductase and UDP-glucuronosyl transferase [7,8]. The primary health benefit of garlic extract herbal products was suggested to result from the metabolite allicin (diallyl thiosulfox-

Abbreviations: DADS, diallyl disulfide; DAS, diallyl sulfide; NaHS, sodium hydrosulfide; H2 S, hydrogen sulfide; PM, propyl mercaptan; AM, allyl mercaptan; AMS, allyl methyl sulfide; AMSO, allyl methyl sulfoxide; AMSO2 , allyl methyl sulfone; DASO, diallyl sulfone; DASO2 , diallyl sulfoxide; ROS, reactive oxygen species; TBARS, thiobarbituric acid reactive species; ALDH2 , aldehyde dehydrogenase 2; DTT, dithiothreitol; GST, glutathione-s-transferase. ∗ Corresponding author. Tel.: +1 416 978 2716; fax: +1 416 978 8511. E-mail address: [email protected] (P.J. O’Brien). 0009-2797/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2009.02.008

ide) produced when alliin was hydrolyzed by alliinase. Allicin was also reduced by the liver hepatocytes to form DADS, DAS, AM, propyl mercaptan (PM), and hydrogen sulfide (H2 S) [9–13]. DADS was also reduced by primary rat hepatocytes to form AM, allyl methyl sulfide (AMS), allyl methyl sulfoxide (AMSO) and sulfone (AMSO2 ) [14,15]. Human or rat liver microsomal P450 dependent monooxygenase and NADPH catalyzed DADS oxidation to DADS sulfoxide (allicin). Rat liver cytosol GST and GSH catalyzed DADS reduction to AM that was then methylated to AMS by unknown mechanisms. Liver microsomes and NADPH then catalyzed the oxidation of AMS to AMSO and AMSO2 [16]. Jin and Baillie [17] identified 10 GSH conjugates in the bile of rats dosed with DAS (200 mg/kg). CYP 2E1 also catalyzed the oxidation at the sulfur atom to form reactive diallyl sulfoxide (DASO), diallyl sulfone (DASO2 ) and DASO2 monoepoxide, which reacted with GSH [17]. Black et al. [18] detected significant levels of CYP 2E1 N-alkylprotophyrin IX adducts in the livers of mice given DASO2 (100 mg/kg) or recombinant rat and human CYP2E1 incubated with DASO2 (1–4.0 mM). Three GSH acrolein conjugates of DAS, DASO, and DASO2 were identified in the bile of rats dosed with DAS [18]. Garlic fed to rats, cats, dogs, and sheep caused hemolytic anemia, liver and lung toxicity but their molecular toxic mechanisms have not been investigated [19–22]. Hepatocyte cytotoxicity induced by DADS and DAS has been compared and possible molecular cytotoxic mechanisms were proposed for each agent.

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2. Materials and methods

2.4. Mitochondrial oxygen uptake

2.1. Materials

Hydrogen and allyl sulfides were preincubated with 200 ␮l of isolated rat mitochondria (8 mg protein/ml) for 20 min. The entire 200 ␮l of preincubated medium was added to a 2 ml Clark type oxygen electrode reaction chamber containing 1600 ␮l of 0.1 M phosphate buffer pH 7.4, 37 ◦ C, 20 ␮l of 0.4 mM (100×) tetramethylphenylenediamine (TMPD) and 20 ␮l of 1 mM (100×) ascorbic acid.

1-Bromoheptane, 2,4-dinitrofluorobenzene (DNFB), 2,7-dichlorofluorescin diacetate (DCFH-DA), rhodamine 123, DADS, DAS, aminobenzotriazole and phenylimidazole were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Collagenase (from Clostridium histoliticum) was purchased from Worthington Biochemicals Corporation (Freehold, NJ). HEPES and bovine serum albumin were obtained from Boehringer–Mannheim (Montreal, Canada). All other chemicals used were of the highest purity that was commercially available. 2.2. Isolation and incubation of hepatocytes Hepatocytes were isolated from adult male Sprague–Dawley rats (220–250 g), by collagenase perfusion of the liver as described by Moldeus et al. [23]. Cell viability was measured by the trypan blue exclusion method using a light microscope, and the hepatocytes used in this study were at least 85–90% viable. Hepatocytes were preincubated in Krebs–Henseleit bicarbonate buffer (pH 7.4) supplemented with 12.5 mM HEPES for 30 min under a constant flow of 95% O2 /5% CO2 at 37 ◦ C in continuously rotating 50 ml round bottom flasks before addition of chemicals. Stock solutions of chemicals including DADS and DAS were made either in incubation buffer or in DMSO. The final DMSO concentration did not exceed 0.5% (v/v), and was non-toxic to cells. The flasks containing hepatocytes and DADS or DAS were sealed for the first 20 min in order for equilibrium to be established to ensure reproducible toxicity. GSH depleted hepatocytes were prepared by incubating hepatocytes with 150 ␮M bromoheptane for 30 min which did not affect P450 activities or hepatocyte viability [24]. P450 inhibited hepatocytes were prepared by incubating hepatocytes with 500 ␮M aminobenzotriazole for 30 min [25]. CYP 2E1 inhibited hepatocytes were prepared by incubating hepatocytes with 300 ␮M phenylimidazole [26] or 200 ␮M DAS for 30 min and did not affect hepatocyte viability. Mitochondrial aldehyde dehydrogenase 2 (ALDH2) inhibited hepatocytes were prepared by incubating hepatocytes with 1 mM of chloral hydrate for 30 min and did not affect hepatocyte viability [27]. 2.3. Determination of mitochondrial membrane potential ( m ) decrease The uptake and retention of the cationic fluorescent dye, rhodamine 123, was used to measure mitochondrial membrane potential. This assay is based on the selective accumulation of rhodamine 123 in the mitochondria by facilitated diffusion. When the mitochondrial potential is decreased, the amount of rhodamine 123 that enters the mitochondria is decreased, as there is no facilitated diffusion. Thus, the amount of rhodamine 123 in the supernatant is increased and the amount in the pellet is decreased. Samples (500 ␮l) were taken from the cell suspension incubated at 37 ◦ C, and centrifuged at 1000 rpm for 1 min. The cell pellet was then resuspended in 2 ml of fresh incubation medium containing 1.5 ␮M rhodamine 123 and incubated at 37 ◦ C in a thermostatic bath for 10 min with gentle shaking. Hepatocytes were separated by centrifugation and the amount of rhodamine 123 appearing in the incubation medium was measured fluorimetrically using a Shimadzu RF5000U fluorescence spectrophotometer set at 490 nm excitation and 520 nm emission wavelengths. The capacity of mitochondria to take up the rhodamine 123 was calculated as the difference in fluorescence intensity between control and treated cells [28].

2.5. UV determination of catalase activity Hydrogen and allyl sulfides were preincubated in a 1 ml 0.1 M TRIS buffer solution (pH 7.4) containing 33 ␮g of catalase for 10 min. At 10 min, 80 ␮l of incubation medium was transferred to a 1.5 ml quartz cuvette containing 910 ␮l of buffer and 10 ␮l of H2 O2 from a 9.79 M stock solution was added and mixed to initiate the reaction. The absorbance of H2 O2 at  240 nm was followed for 10 min. Reaction mixtures were blanked each time before H2 O2 addition. 2.6. Determination of reactive oxygen species (ROS) To determine the rate of hepatocyte ROS generation, 2,7dichlorofluorescin diacetate (DCFH-DA) was added to the hepatocyte incubate. DCFH-DA penetrates hepatocytes and became hydrolyzed to non-fluorescent dichlorofluorescin. Dichlorofluorescin then reacted with ‘ROS’ to form the highly fluorescent dichlorofluorescin that effluxed the cell. One milliliter samples were withdrawn at 30, 60 and 120 min. These samples were then centrifuged for 1 min at 50 × g. The cells were resuspended in buffer and 1.6 ␮M DCFH-DA was added. The cells were then incubated at 37 ◦ C for 10 min. The fluorescence intensity of ROS product was measured using a Shimadzu RF5000U fluorescence spectrophotometer. Excitation and emission wavelengths were 500 and 520 nm, respectively [29]. 2.7. Lipid peroxidation (TBARS) Hepatocyte lipid peroxidation was determined by measuring the amount of thiobarbituric acid reactive substances (TBARS) formed during the decomposition of lipid hydroperoxides by following the absorbance at 532 nm using a Pharmacia Biotech Ultrospec 1000 [30]. Briefly, 1 ml aliquots of hepatocyte suspension (106 cells/ml) were treated with trichloroacetic acid (70% w/v) and thiobarbituric acid (0.8% w/v). The suspension was then boiled for 20 min and read at 532 nm. Malondialdehyde (MDA) formation was expressed as UV absorbance at 532 nm. 2.8. Quantitation of cellular GSH GSH and GSSG were measured by HPLC analysis of deproteinized samples (25% meta-phosphoric acid) after derivatization with iodoacetic acid and DNFB as described by Reed et al. [31]. A Waters HPLC system (Model 150 pumps, WISP 710B auto injector and model 410 UV/vis detector) equipped with a Waters ␮Bondapak® NH2 (10 ␮m) 3.9 mm × 300 mm column was used. 2.9. Mass spectrometry analysis GC–MS analysis of allyl mercaptan and diallyl disulfide was carried out as described by Germain et al. using a DB-5 capillary GC column (30 m × 0.25 mm × 0.25 ␮m film thickness) [14]. Identification of allyl mercaptan and diallyl disulfide was performed using library matching with 80% or greater probability match considered a positive match. Allyl mercaptan and diallyl disulfide in dichloromethane was identified by retention time matching to

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Table 1 Garlic sulfur metabolites induce hepatocyte cytotoxicity. Treatment

Cytotoxicity (% of trypan blue uptake)

Incubation time (min)

30

Hepatocytes only +NaHS (0.5 mM) +DADS (1.0 mM) +Hydroxocobalamin (100 ␮M) +DAS (2.0 mM) +Hydralazine (0.2 mM) +AM (5.0 mM) +AM (2.0 mM) +Hydralazine (0.2 mM) +DTT (2.0 mM) +chloral hydrate (1.0 mM) (ALDH2 inhibitor) +Cyclosporin A (5 ␮M) +TEMPOL (200 ␮M) +Quercertin (50 ␮M)

22 32 34 24 29 23 24 21 23 26 24 22 24 23

60

Dithiothreitol (DTT) added 10 min after metabolites +DADS (1.0 mM) + DTT (1.0 mM) +DAS (2.0 mM) + DTT (2.0 mM)

23 ± 2** 26 ± 3

27 ± 3** 34 ± 2

25 ± 3** 56 ± 3

29 ± 2** 73 ± 3

30 min preincubation with 5 ␮M cyclosporin A +Cyclosporin A + DADS (1.0 mM) +Cyclosporin A + DAS (2.0 mM)

22 ± 3 24 ± 3

33 ± 3** 25 ± 3**

43 ± 3** 48 ± 2**

49 ± 3** 56 ± 2**

ROS scavengers added after garlic sulfur metabolites +DADS (1.0 mM) + TEMPOL (200 ␮M) +DADS (1.0 mM) + quercetin (50 ␮M) +DAS (2.0 mM) + TEMPOL (200 ␮M) +DAS (2.0 mM) + quercetin (50 ␮M)

22 24 23 22

± ± ± ±

3 2 3 3

36 38 31 28

± ± ± ±

3** 3** 3 3

41 47 43 50

± ± ± ±

3** 3** 2** 3**

57 62 62 59

± ± ± ±

3** 2** 3** 3**

GSH-depleted hepatocytes +1-Bromoheptane + NaHS (0.2 mM) + DADS (0.5 mM) + DAS (0.5 mM)

22 34 35 25

± ± ± ±

2 2* 3* 2

21 59 43 34

± ± ± ±

3 3* 2* 2*

25 72 66 60

± ± ± ±

2 2* 3* 3*

28 94 78 71

± ± ± ±

2 2* 3* 2*

± ± ± ± ± ± ± ± ± ± ± ± ± ±

2 2* 2* 2 3 3 3 3 3 3 3 2 3 3

21 47 53 27 37 27 39 23 23 24 22 24 23 25

120 ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3 2* 3* 3 2* 3** 3 3 3 3 3 3 2 2

25 64 70 39 63 33 54 29 25 29 25 28 29 27

± ± ± ± ± ± ± ± ± ± ± ± ± ±

180 2 2* 3* 3 2* 3** 3 3 2 2 2 3 2 3

27 88 88 46 78 37 62 26 30 32 28 25 25 26

± ± ± ± ± ± ± ± ± ± ± ± ± ±

2 3* 3* 3 3* 3** 3 3 3 3 3 3 2 3

Hepatocytes were incubated in Krebs–Henseleit solution pH 7.4 at 37 ◦ C under the atmosphere of 95%O2 /5%CO2 . All of the chemicals were present in the suspensions during the experiment. All modulating chemicals used were not hepatotoxic. The samples were taken at mentioned time intervals and cell death was assessed by trypan blue exclusion. The results from three independent experiments are presented as mean ± S.E. ↑Indicates treatment induced significantly more cytotoxicity compared to hepatocytes treated with garlic sulfur metabolites only at p < 0.05. * Indicates treatment induced significantly higher cytotoxicity compared to hepatocytes only control at p < 0.05. ** Indicates treatment induced significantly less cytotoxicity compared to hepatocytes treated with garlic sulfur metabolites only at p < 0.05.

that of a neat sample of allyl mercaptan and diallyl disulfide. 1.0 mM of dithiothreitol was added to 1.0 mM of diallyl disulfide in dichloromethane and a 2 ␮l sample was injected into the GC–MS within 30 min.

2.10. Statistical analysis Statistical comparisons for hepatocyte studies were carried out using the one-way ANOVA with post hoc Dunnett’s analysis to

Table 2 DAS induced cytotoxicity is decreased by cytochrome P450 inhibitors and increased by an ALDH2 inhibitor. Treatment

Cytotoxicity (% of trypan blue uptake)

Incubation time (min)

30

Hepatocytes only +DADS (1.0 mM) +DAS (2.0 mM) +Aminobenzotriazole (500 ␮M) +Phenylimidazole (300 ␮M)

24 36 31 22 21

± ± ± ± ±

2 2* 3 3 3

23 57 40 25 23

± ± ± ± ±

2 3* 2* 3 3

28 72 63 23 27

± ± ± ± ±

3 3* 3* 3 2

26 82 74 28 25

± ± ± ± ±

3 3* 3* 2 3

30 min preincubation with P450 inhibitors +Aminobenzotriazole (P450 inhib.) (500 ␮M) + DAS (2.0 mM) +Phenylimidazole (CYP3E1 inhib.) (300 ␮M) + DAS (2.0 mM) (CYP 2E1 inhibitor) +DAS (200 ␮M) + DAS (2.0 mM) +Aminobenzotriazole (500 ␮M) + DADS (1.0 mM)

20 23 26 29

± ± ± ±

3 3 3 3

25 33 28 50

± ± ± ±

2** 2* 3** 3*

33 47 45 67

± ± ± ±

3** 2* , ** 3* , ** 2*

38 55 50 78

± ± ± ±

2** 3* , ** 2* , ** 3*

30 min preincubation with ALDH2 inhibitor +Chloral hydrate (1 mM) + DAS (2.0 mM) +Chloral hydrate (1 mM) + DADS (1.0 mM)

49 ± 3* , *** 31 ± 3*

60

120

77 ± 2* , *** 46 ± 3*

180

92 ± 2* , *** 67 ± 3*

100* , *** 81 ± 2*

Hepatocytes were incubated in Krebs–Henseleit solution pH 7.4 at 37 ◦ C under the atmosphere of 95%O2 /5%CO2 . All the chemicals were present in the suspensions during the experiment. All modulating chemicals used were not hepatotoxic. The samples were taken at mentioned time intervals and cell death was assessed by trypan blue exclusion. The results from three independent experiments are presented as mean ± S.E. * Indicates treatment induced significantly higher cytotoxicity compared to hepatocytes only control at p < 0.05. ** Indicates treatment induced significantly less cytotoxicity compared to hepatocytes treated with garlic sulfur metabolites only at p < 0.05. *** Indicates treatment induced significantly more cytotoxicity compared to hepatocytes treated with garlic sulfur metabolites only at p < 0.05.

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Table 3 Hepatocyte intracellular GSH depletion by cytotoxic concentrations of garlic sulfur metabolites. Treatment

Cytotoxicity at 60 min (% trypan blue uptake)

Hepatocytes only +NaHS (0.5 mM) +DTT (0.5 mM) +DADS (1.0 mM) +DTT (1.0 mM) +Aminobenzotriazole (500 ␮M) 30 min preinc. +Chloral hydrate (1 mM) 30 min preinc. +Hydralazine (0.2 mM) +DAS (2.0 mM) +Hydralazine (0.2 mM) +Chloral hydrate (1 mM) 30 min preinc. +Aminobenzotriazole (500 ␮M) 30 min preinc. +Phenylimidazole (300 ␮M) 30 min preinc. +DAS (200 ␮M) 30 min preinc. +DTT (2.0 mM) +Hydralazine (0.2 mM) +Chloral hydrate (1 mM) 30 min preinc. +Aminobenzotriazole (500 ␮M) 30 min preinc. +Phenylimidazole (300 ␮M) 30 min preinc. +DAS (200 ␮M) 30 min preinc. +DTT (2.0 mM)

23 41 37 57 29 53 59 48 43 26 64 29 37 39 46 25 27 26 22 24 24

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2 3* 3* 3* 3** 3* 3* 3* 3* 3** 3* 3** 3* 3* 3* 2 3 3 2 3 3

Cytotoxicity at 120 min (% trypan blue uptake) 27 67 49 68 31 72 64 74 57 37 84 43 51 49 54 28 24 30 26 25 27

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3 3* 3* , ** 2* 3** 2* 2* 3* 3* 3* , ** 3* 3* , ** 3* 3* 3* 3 3 2 2 3 3

Hepatocyte [GSH] at 30 min (nM/106 cells) 56 42 52 36 52 34 33 29 48 52 28 49 47 44 52 51 50 59 54 49 53

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Hepatoctye [GSH] at 90 min (nM/106 cells) 51 ± 31 ± 43 ± 11 ± 49 ± 13 ± 12 ± 14 ± 20 ± 46 ± BDL 37 ± 32 ± 34 ± 24 ± 53 ± 46 ± 49 ± 52 ± 54 ± 52 ±

2 2, *** 3 2*** 2 2*** 2*** 3*** 2 2 2*** 2 2*** 2*** 2 3 2 2 2 2 3

2 2*** 2 2*** 2 2*** 2*** 3*** 2*** 2 2*** 2*** 3*** 3*** 3 3 2 3 2 2

Method for measuring GSH used was as described by Reed et al. [31]. Data from three independent experiments are presented as mean ± S.E. * Indicates treatment induced significantly higher cytotoxicity compared to hepatocytes only control at p < 0.05. ** Indicates treatment induced significantly less cytotoxicity compared to hepatocytes treated with garlic sulfur metabolites only at p < 0.05. *** Indicates treated hepatocytes had significantly lower GSH levels compared to control hepatocytes at p < 0.05. BDL = below detection limit.

determine the differences between treatments and control, and post hoc Tukey’s analysis to determine the differences between treatments with a probability of P < 0.05 being considered significant, otherwise, as indicated in table legends. Results represent the mean ± S.E. of three independent samples.

atocytes from DAS but not from DADS induced GSH depletion and cytotoxicity (Table 3). However, hepatocytes preincubated with the CYP 2E1 inhibitor phenylimidazole (54%) [26] only partially protected hepatocytes from DAS. DAS inhibited its own metabolism and protected hepatocytes by inhibiting CYP 2E1. Equimolar dithiothreitol (DTT) (a disulfide reductant) added 10 min after DADS significantly decreased hepatocyte cytotoxicity; however, adding an equimolar concentration of DTT did not protect hepatocytes from DAS (Table 1). Glutathione (GSH) depleted hepatocytes were more sensitive to all of the garlic sulfur metabolites (Table 1). Table 3 shows that all of the garlic sulfur metabolites significantly depleted hepatocyte GSH at 30 min, with more GSH depleted at 90 min with an order of effectiveness of DADS > DAS. Adding an equimolar concentration of DTT 10 min after DADS

3. Results Table 1 shows that the relative hepatocyte cytotoxic potency of garlic sulfur metabolites was H2 S (0.5 mM) > DADS (1.0 mM) > DAS (2.0 mM). Allyl mercaptan (AM, a DADS metabolite) was not cytotoxic at 2.0 mM. Hepatocytes preincubated with aminobenzotriazole to inhibit cytochrome P450s (50% inhibition) [25] completely protected hep-

Table 4 Garlic sulfur metabolites decreased hepatocyte mitochondrial membrane potential. Treatment

Mitochondrial membrane potential, 

Incubation time (min)

30

60

120

120

Hepatocytes +NaHS (0.5 mM) +DTT (0.5 mM) +DADS (1.0 mM) +DTT (1.0 mM) +Cyclosporin A (5 ␮M) 30 min preinc. +DAS (2.0 mM) +DTT (2.0 mM) +DAS (200 ␮M) 30 min preinc. +Chloral hydrate (1 mM) 30 min preinc. +Hydralazine (0.2 mM) +Cyclosporin A (5 ␮M) 30 min preinc. +AM (2.0 mM) +DTT (2.0 mM) +Cyclosporin A (5 ␮M) +DAS (200 ␮M) +Chloral hydrate (1 mM) +Hydralazine (0.2 mM)

480 ± 7 523 ± 8* 493 ± 7 554 ± 9* 489 ± 10 477 ± 6 517 ± 5* 504 ± 6* 492 ± 8 534 ± 6* , *** 474 ± 8 465 ± 10 470 ± 8 467 ± 9 482 ± 11 498 ± 10 459 ± 10 471 ± 9

496 ± 7 563 ± 8* 532 ± 9* 632 ± 7* 492 ± 9 487 ± 7 564 ± 9* 541 ± 7* 541 ± 7* 642 ± 7* , *** 487 ± 9 482 ± 11 499 ± 7 516 ± 9 481 ± 10 479 ± 12 466 ± 11 519 ± 11

493 ± 5 678 ± 8* 579 ± 11* 681 ± 9* 499 ± 11 503 ± 7 591 ± 5* 574 ± 5* 543 ± 8* 694 ± 12* , *** 511 ± 8 516 ± 10 489 ± 5 513 ± 12 494 ± 8 492 ± 10 510 ± 9 515 ± 9

23 ± 3 59 ± 3** 46 ± 3** 62 ± 3** 29 ± 3 33 ± 2 58 ± 2** 56 ± 2** 44 ± 3** 87 ± 3** 37 ± 3** 43 ± 2** 25 ± 3 27 ± 3 24 ± 3 24 ± 2 28 ± 2 25 ± 3

m

(rhodamine 123 uptake)

Cytotoxicity (% trypan blue uptake)

Treatment conditions are the same as described in Table 4.1. The results from three independent experiments are presented as mean ± S.E. * Indicates treatment induced significantly higher mitochondrial membrane depolarization compared to hepatocyte only control at p < 0.05. ** Indicates treatment induced significantly higher cytotoxicity compared to hepatocytes only control at p < 0.05. *** Indicates treatment induced significantly higher mitochondrial membrane depolarization compared to hepatocytes treated with DAS (2.0 mM) only at p < 0.05.

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Table 5 Hepatocyte ROS formation induced by garlic sulfur metabolites. Treatment

ROS production (fluorescent intensity units)

Incubation time (min)

30

Hepatocytes only +NaHS (0.5 mM) +TEMPOL (200 ␮M) +quercetin (50 ␮M) +DADS (1.0 mM) +DTT (1.0 mM) +Cyclosporin A (5 ␮M) 30 min preinc. +TEMPOL (200 ␮M) +Quercetin (50 ␮M) +DAS (2.0 mM) +DTT (2.0 mM) +DAS (200 ␮M) 30 min preinc. +Chloral hydrate (1 mM) 30 min preinc. +Hydralazine (0.2 mM) +Cyclosporin A (5 ␮M) 30 min preinc. +TEMPOL (200 ␮M) +Quercetin (50 ␮M) +AM (2.0 mM) +TEMPOL (200 ␮M) +Quercetin (50 ␮M) +DTT (1.0 mM) +Cyclosporin A (5 ␮M) +DAS (200 ␮M) +Chloral hydrate (1 mM) +Hydralazine (0.2 mM)

240 287 231 237 325 233 398 251 247 312 294 231 331 251 223 238 254 243 221 238 248 253 243 247 255

60 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

10 7* 9 11 13* 8 10* 9 11 7* 11* 11 19* , *** 10 10 10 12 11 10 10 11 13 11 12 10

260 398 261 272 437 271 374 259 273 373 370 273 447 289 264 253 257 249 245 244 251 278 278 272 263

Cytotoxicity (% trypan blue uptake)

120 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

8 9* 10 7 9* 10 9* 8 9 12* 10* 9 10* , *** 10 11 11 11 11 10 11 9 10 10 10 11

386 463 364 381 561 392 499 374 392 485 471 433 555 419 374 371 386 380 362 376 363 397 394 382 396

120 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

9 10* 9 10 12* 9 10* 8 9 12* 10* 10* 10* , *** 9 10 9 10 10 11 13 11 10 12 11 10

21 53 30 34 65 27 39 39 44 61 52 37 91 33 45 38 41 32 27 24 26 23 30 23 25

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3 2** 2 3** 3** 3 3** 2** 3** 3** 2** 3** 3** 2 3** 2** 3** 3 3 2 3 2 3 3 3

Treatment conditions are the same as described in Table 4.1. The results from three independent experiments are presented as mean ± S.E. * Indicates treatment induced significantly higher ROS formation compare to hepatocyte control at p < 0.05. ** Indicates treatment induced significantly higher cytotoxicity compared to hepatocytes only control at p < 0.05. *** Indicates treatment induced significantly higher ROS formation compared to hepatocytes treated with DAS (2.0 mM) only at p < 0.05.

prevented further intracellular hepatocyte GSH depletion. This correlated with the decrease in DADS induced hepatocytes cytotoxicity by DTT. Hydralazine a specific acrolein scavenger also prevented DAS induced intracellular hepatocyte GSH depletion. This also correlated with the protection of hepatocytes by hydralazine against DAS (Table 3).

Adding the ROS scavengers TEMPOL, or quercetin immediately after DADS significantly protected hepatocytes at all time points tested, whereas, adding TEMPOL, or quercetin immediately after DAS only protected at 120 and 180 min (Table 1). DADS and DAS caused a significant hepatocyte mitochondrial membrane potential decrease as early as 30 min after exposure

Table 6 Hepatocyte lipid peroxidation induced by garlic sulfur metabolites. Treatment

TBARS formation (nmol/106 cells)

Incubation time (min)

30

Hepatocytes only +NaHS (0.5 mM) +TEMPOL (200 ␮M) +Quercetin (50 ␮M) +DADS (1.0 mM) +DTT (1.0 mM) +Cyclosporin A (5 ␮M) 30 min preinc. +TEMPOL (200 ␮M) +Quercetin (50 ␮M) +DAS (2.0 mM) +DTT (2.0 mM) +DAS (200 ␮M) 30 min preinc. +Chloral hydrate (1 mM) 30 min preinc. +Hydralazine (0.2 mM) +Cyclosporin A (5 ␮M) 30 min preinc. +TEMPOL (200 ␮M) +Quercetin (50 ␮M) +AM (2.0 mM) +TEMPOL (200 ␮M) +Quercetin (50 ␮M) +DTT (1.0 mM) +Cyclosporin A (5 ␮M) +DAS (200 ␮M) +Chloral hydrate (1 mM) +Hydralazine (0.2 mM)

218 410 223 231 429 243 332 225 221 339 346 224 422 234 232 245 238 214 201 238 257 223 237 209 241

60 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

11 10* 10 10 14* 11 12* 11 10 9* 10* 11 12* , *** 11 12 10 12 12 11 12 12 11 11 10 12

225 397 227 230 449 215 357 232 235 369 354 339 453 240 238 258 241 222 237 232 248 238 245 213 236

Cytotoxicity (% trypan blue uptake) 120

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

12 13* 9 11 13* 10 11* 9 12 14* 12* 9* 10* , *** 9 12 11 11 10 10 10 12 12 11 9 12

225 494 223 237 577 255 393 235 241 478 482 478 559 249 297 263 249 245 253 234 253 244 252 248 253

120 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

9 10* 9 11 11* 10 11* 12 11 10* 11* 10* 12* , *** 12 11 110 9 12 11 10 11 10 9 11 10

23 59 29 30 62 31 43 42 40 67 58 41 88 34 46 36 34 28 24 29 25 27 24 26 22

Treatment conditions are the same as described in Table 4.1. The results from three independent experiments are presented as mean ± S.E. * Indicates treatment induced significantly higher TBARS formation compared to control at p < 0.05. ** Indicates treatment induced significantly higher cytotoxicity compared to hepatocytes only control at p < 0.05. *** Indicates treatment induced significantly higher TBARS formation compared to hepatocytes treated with DAS (2.0 mM) only at p < 0.05.

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2 3** 3 3 4** 3 2** 2** 3** 3** 3** 3** 3** 3 2** 2** 2** 3 3 2 2 3 2 3 3

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Table 7 Inhibition of mitochondrial respiration by NaHS and DADS. Treatments

nmol O2 consumed in reaction/ml/min

Mitochondria only +NaHS (0.5 mM) 20 min preincubation +Hydroxocobalamin (0.1 mM) added at 10 min +DADS (1.0 mM) 20 min preincubation +GSH (2.0 mM) +Hydroxocobalamin (0.1 mM) added at 10 min +DTT (1.0 mM) +DAS (2.0 mM) 20 min preincubation

32.5 5.0 16.7 21.2 7.44 30.6 22.7 31.2

GSH-depleted mitochondria only +DADS (2.0 mM) 20 min preincubation

31.0 ± 2 32.6 ± 2

± ± ± ± ± ± ± ±

2 1* 2* 2* 1** 2 2* 3

At 37 ◦ C [O2 ] ␮M/ml H2 O = 0.217; Phosphate buffer 0.1 M, pH 7.4 37 ◦ C. The results from three independent experiments are presented as mean ± S.E. Mitochondrial GSH was depleted by preincubating with 100 ␮M 1-bromoheptane for 30 min. The concentration of mitochondria used was 870 ␮g/ml. * Indicates treated mitochondria had significantly lower oxygen consumed in reaction compare to mitochondria only at p < 0.05. ** Indicates GSH given to DADS treated mitochondria had significantly lower oxygen consumed in reaction compared to mitochondria treated with DADS only at p < 0.05.

(Table 4), which correlated with increased hepatocyte ROS and TBARS formation (Tables 5 and 6). Hepatocytes treated with equimolar concentration of DTT following DADS addition prevented the mitochondrial membrane potential decrease, ROS formation and TBARS formation. Hepatocytes preincubated with the mitochondrial transition pore inhibitor cyclosporin A [32] also prevented the DADS induced decrease in mitochondrial membrane potential, but did not affect ROS and TBARS formation (Tables 4–6). Preincubating hepatocytes with cyclosporin A also prevented the DAS induced mitochondrial membrane potential decrease as well as ROS and TBARS formation. Furthermore, hepatocytes preincubated with cyclosporin A were also significantly protected from DADS and DAS (Table 1). Adding DADS to a buffer solution containing GSH and mitochondria significantly decreased mitochondrial respiration by 77% within 20 min; however, DADS did not inhibit mitochondrial respiration by GSH depleted mitochondria. DADS alone inhibited mitochondrial respiration uptake by 34%. This was reversed when hydroxocobalamin (a compound that complexes and autoxidizes hydrogen sulfide) [33] was added to the incubating solution of DADS and mitochondria at 10 min (Table 7). However DAS had no effect on mitochondrial respiration. Jin and Baillie identified the glutathione adducts: S-(3hydroxypropyl)-glutathione and S-(2-carboxyethyl) glutathione in the bile of rats dosed with DAS, which was attributed to acrolein formation from DAS [17]. Preincubating hepatocytes with chloral hydrate to inhibit mitochondrial aldehyde dehydrogenase (ALDH2) (74% inhibition) [27] significantly increased DAS cytotoxicity as early as 30 min, but did not affect DADS induced cytotoxicity (Table 2). However, adding hydralazine to scavenge acrolein prevented DAS induced cytotoxicity [34] (Table 3). Adding hydralazine or chloral hydrate to control hepatocytes did not affect hepatocyte viability (Table 1). Hydralazine treated hepatocytes prevented DAS from inducing a decrease in mitochondrial membrane potential, ROS or TBARS formation (Tables 4–6). Chloral hydrate an ALDH2 inhibitor [27] markedly increased cytotoxicity or hepatocyte GSH depletion induced by 2 mM DAS. Aminobenzotriazole, phenylimidazole, or 200 ␮M DAS significantly reduced hepatocyte GSH depletion (induced by DAS) (Table 3). The acrolein trap hydralazine added immediately after DAS prevented cytotoxicity or intracellular GSH depletion at 90 min. Inhibition of hepatocyte ALDH2 by chloral hydrate did not affect DADS hepatot-

Fig. 1. The results from three independent experiments are presented as mean ± S.E. *Indicates significantly lower catalase activity compared to control at p < 0.05.

cyte GSH depletion (Table 3). ALDH2 inhibited hepatocytes were also more susceptible to DAS induced mitochondrial membrane potential decrease, and ROS and TBARS formation (Tables 4–6). The ROS scavengers TEMPOL or quercetin added to hepatocytes immediately after DADS, or DAS prevented ROS and TBARS formation (Tables 5 and 6). NaHS treated hepatocytes showed a significant mitochondrial membrane potential decrease and ROS or TBARS formation beginning at 30 min (Tables 4–6). Adding TEMPOL and quercetin prevented ROS and TBARS formation at all time points tested (Tables 5 and 6). Furthermore, NaHS reduced mitochondrial respiration by 85% of control within 20 min of preincubation (Table 7). Adding garlic sulfur metabolites to a solution containing catalase inhibited the rate of H2 O2 decomposition catalyzed by catalase. The order of catalase inhibition potency by the garlic metabolites was NaHS > > DADS > DAS (Fig. 1). GSH depleted hepatocytes were more susceptible to NaHS (Table 1), and NaHS treated hepatocytes showed significant GSH depletion at 30 min with more GSH depleted at 90 min. DTT added 10 min after NaHS was added to hepatocytes prevented intracellular GSH depletion (Table 3). 4. Discussion DADS and DAS are bioactive sulfur metabolites in garlic and onion that have been shown to inhibit cholesterol biosynthesis and tumor cell proliferation [6,5,35]. However, the molecular toxic mechanisms of these compounds towards freshly isolated rat hepatocytes have not been investigated. A novel hypothesis on their possible cytotoxic mechanisms is presented. Cytochrome P450 inhibited hepatocytes were significantly more resistant to DAS compared to CYP2E1 inhibited hepatocytes and suggested that DAS was metabotically activated by other P450s as well as CYP 2E1. Brady et al. [36] first demonstrated that DAS was metabolized to diallyl sulfoxide and diallyl sulfone (DASO2 ) in vivo. DAS inhibited P-450 2E1 catalyzed metabolism by a competitive inhibitor mechanism whereas DASO2 demonstrated suicide inactivation of P-450 2E1 [36]. Hernandez and Forkert showed that mice pretreated with DASO2 (1.25–200 mg/kg) significantly decreased the mutant frequency in the small intestine induced by vinyl carbamate (a precarcinogenic CYP 2E1 substrate) [37]. Using ion spray LC–MS/MS, Jin and Baillie [17] identified 10 metabolites of DAS that formed GSH conjugates and demonstrated that extensive oxidation of DAS in vivo occurred at the sulfur atom, the allylic carbon, and the terminal double bonds. CYP 2E1 preferentially catalyzed the oxidation of the sulfur atom to form sulfoxide (DASO) and sulfone (DASO2 ). The suicide inhibition of CYP 2E1 occurred when the enzyme itself mediated activation of the DASO2 olefinic ␲-bond

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85

Scheme 1. DADS and DAS mechanism of toxicity.

to a reactive epoxide. The autocatalytic destruction of CYP2E1 by DASO2 reactive metabolites was hypothesized to be responsible for the chemopreventative effects of DAS in vivo against CPY2E1 bioactivated carcinogens [17]. Our data also showed that DAS inhibited its own metabolism, which prevented DASO2 reactive metabolite formation (proposed by Jin and Baillie). We therefore speculate that acrolein the DAS reactive metabolite (identified by Jin and Baillie) contributed to DAS induced hepatocyte cytotoxicity. Aldehyde dehydrogenase (ALDH2) inhibited hepatocytes were more susceptible to DAS than control hepatocytes. Furthermore, intracellular GSH levels were completely depleted by DAS. The mitochondrial membrane potential was also decreased, whereas ROS formation and lipid peroxidation were all markedly increased when DAS was added to aldehyde dehydrogenase inhibited hepatocytes. The results suggest that DAS is a mitochondrial toxin and is consistent with the acrolein metabolite contributing to cytotoxicity [38]. Preincubating DAS with mitochondria alone did not affect

mitochondrial respiration, which suggests reactive metabolite(s) catalyzed by microsomal P450s were responsible for mitochondrial toxicity. Furthermore, the acrolein trap hydralazine protected hepatocytes against DAS cytotoxicity and prevented DAS induced intracellular hepatocyte GSH depletion, mitochondrial membrane potential decrease, and ROS or TBARS formation. Burcham and Pyke [34] previously showed that hydralazine trapped acrolein and prevented protein–protein cross-linking. The proposed mechanism for DAS induced hepatocyte cytotoxicity is summarized in Scheme 1. DADS cytotoxicity was not prevented by P450 inhibitors but GSH depleted hepatocytes were approximately two times more susceptible towards DADS compared to control hepatocytes. Adding DADS to hepatocytes also depleted intracellular GSH in 30 min, which did not recover at 90 min. GSH has been shown to conjugate DADS as the DADS–GSH conjugate was identified after adding radiolabelled GSH to DADS and human liver cytosol. AM was also formed and

Fig. 2. GC–MS results for a dichloromethane solution containing DADS GC–MS analysis was carried out according to methods in [14] with a JB5 column. DADS was identified from its retention time of 5.18 min.

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Fig. 3. GC–MS results for a dichloromethane solution containing DADS and DTT GC–MS analysis was carried out according to methods in Ref. [14] with a JB5 column. DADS was identified from its retention time of 5.18 min. AM was identified from its retention time of 3.31 min.

was methylated to AMS, which was then oxidized to form AMSO and AMSO2 [14]. DADS (LD50 = 1.0 mM) was five times more cytotoxic to hepatocytes than AM (LD50 = 5.0 mM) (Table 1) and dithiothreitol, a disulfide reductant, added at 10 min prevented DADS cytotoxicity. When DTT was added to a solution containing DADS minutes before treating hepatocytes with the same DADS solution, hepatocyte cytotoxicity was prevented suggesting that DTT reduced DADS to AM (Figs. 2 and 3). DADS may have mediated hepatocyte cytotoxicity by directly inhibiting mitochondrial function as the mitochondrial membrane potential was decreased at 30–60 min when hepatocytes were treated with DADS. ROS production was also elevated at 30 min and lasted until 120 min. Both DTT and the mitochondrial permeability transition pore inhibitor cyclosporin A prevented the decrease in the hepatocyte mitochondrial membrane potential, ROS formation, and lipid peroxidation induced by DADS. Adding the ROS scavengers TEMPOL and quercetin also inhibited ROS formation, lipid peroxidation and partially prevented DADS cell death. A cell-free mitochondrial respiration assay showed that DADS inhibited mitochondrial oxygen uptake within 20 min of exposure, which suggests that DADS induced hepatocyte ROS production could be due to DADS inhibition of the electron transport chain enzyme cytochrome c oxidase. Elevated ROS levels in DADS treated hepatocytes could also be partly attributed to the inhibition of catalase by DADS,

and/or the stimulation of ferrous iron release by hydrogen sulfide formed from GSH reacting with DADS [33]. Previous research from our laboratory demonstrated that disulfide metabolites of thiono-sulfur drugs induced hepatocyte cytotoxicity by inhibiting mitochondrial membrane potential, which led to the release of Ca2+ into the cytosol. The mitochondrial membrane potential decrease was prevented for these disulfides when dithiothreitol (DTT) was added to maintain cellular thiol status [39]. Sundaram and Milner showed that adding DADS to human tumor cells in culture also resulted in a dose-dependent increase in intracellular free calcium concentration leading to apoptotic cell death [35]. Taken together, we speculate that DADS may induce hepatocyte cytotoxicity by a ROS mediated mechanism induced by hydrogen sulfide as well as by forming mixed protein disulfide adducts of critical energetic proteins found in the cytosol and mitochondria that resulted in cellular death. Using a primary hepatocyte culture, Sheen et al. also showed that 2 mM DADS caused a significant increase in TBARS formation, indicative of an oxidative stress mediated toxic pathway [40]. Scheme 1 summarizes the proposed toxic mechanism for DADS induced hepatocyte cytotoxicity. Significant hepatocyte toxicity caused by NaHS correlated with a mitochondrial membrane potential decrease at 30 min of exposure and continued to 120 min. In a cell-free mitochondrial respiration assay, NaHS was more effective than DADS at inhibiting cytochrome c oxidase. DADS inhibition of cytochrome c oxidase

Scheme 2. H2 S production from diallyl disulfide (DADS) (adapted from Benavides et al., 2007).

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is speculated to be due to H2 S formation when mitochondrial matrix GSH reacts with DADS. H2 S was detected by the use of a polarographic H2 S sensor when GSH was added to DADS [41]. Adding NaHS to hepatocytes caused a significant increase in ROS and lipid peroxidation thiobarbituric acid reactive species. Inhibition of catalase heme-Fe by NaHS could also contribute to the elevated ROS levels in NaHS treated hepatocytes. Furthermore, ROS scavengers TEMPOL or quercetin added to NaHS treated hepatocytes prevented ROS and TBARS formation, and correlated with decreased hepatocyte toxicity. This result supports the proposed ROS mechanism for H2 S induced hepatocyte cytotoxicity [33]. Ruiz et al. showed that human subjects consuming garlic had increased level of H2 S, and DADS in their expired breath, as well as DAS and AM [13]. A nucleophilic substitution at the ˛ carbon of DADS by GSH yielded S-allyl-glutathione and allyl perthiol, a key intermediate in the formation of H2 S (see Scheme 2). Mitochondria are known to maintain a higher GSH reductive state than in the cytosol [42] probably because GSH reduction is better maintained. GSH reduced by reduced thioredoxin (Trx2) could be catalyzed by a unique mitochondrial thioredoxin reductase (TR2) (utilizing NADPH formed by isocitrate dehydrogenase of the citric acid cycle). This would explain the availability of GSH in the mitochondria to react with DADS to form H2 S. Adding the H2 S scavenger hydroxocobalamin significantly protected hepatocytes treated with DADS. Approximately 35% of H2 S was detected when GSH was added to a phosphate buffer solution containing DADS [41]. This supports the research described here as the amount of H2 S generated from 1.0 mM DADS could be enough to affect mitochondrial respiration activity. In summary, we propose that DADS forms H2 S that binds to the cytochrome c oxidase heme-Fe and inhibits hepatocyte mitochondrial respiration. DADS can also form mixed protein disulfides. DAS on the other hand may form various reactive metabolites: DASO epoxide, DASO2 epoxide, allyl sulfenic acid, and acrolein, all of which could contribute to DAS induced hepatocyte cytotoxicity [17]. Acrolein is also a mitochondrial toxin [38,43–45]. Conflict of interest None declared. References [1] C. Dwivedi, S. Rohlfs, D. Jarvis, F.N. Engineer, Chemoprevention of chemically induced skin tumor development by diallyl sulfide and diallyl disulfide, Pharm. Res. 9 (1992) 1668–1670. [2] R. Gebhardt, H. Beck, Differential inhibitory effects of garlic-derived organosulfur compounds on cholesterol biosynthesis in primary rat hepatocyte cultures, Lipids 31 (1996) 1269–1276. [3] B.H. Cho, S. Xu, Effects of allyl mercaptan and various allium-derived compounds on cholesterol synthesis and secretion in Hep-G2 cells, Comp. Biochem. Physiol. C: Toxicol. Pharmacol. 126 (2000) 195–201. [4] S. Xu, B.H. Simon Cho, Allyl mercaptan, a major metabolite of garlic compounds, reduces cellular cholesterol synthesis and its secretion in Hep-G2 cells, J. Nutr. Biochem. 10 (1999) 654–659. [5] L. Liu, Y.Y. Yeh, Inhibition of cholesterol biosynthesis by organosulfur compounds derived from garlic, Lipids 35 (2000) 197–203. [6] J.S. Yang, L.F. Kok, Y.H. Lin, T.C. Kuo, J.L. Yang, C.C. Lin, G.W. Chen, W.W. Huang, H.C. Ho, J.G. Chung, Diallyl disulfide inhibits WEHI-3 leukemia cells in vivo, Anticancer Res. 26 (2006) 219–225. [7] J.F. Brady, M.H. Wang, J.Y. Hong, F. Xiao, Y. Li, J.S. Yoo, S.M. Ning, M.J. Lee, J.M. Fukuto, J.M. Gapac, et al., Modulation of rat hepatic microsomal monooxygenase enzymes and cytotoxicity by diallyl sulfide, Toxicol. Appl. Pharmacol. 108 (2006) 342–354. [8] R. Munday, C.M. Munday, Induction of phase II enzymes by aliphatic sulfides derived from garlic and onions: an overview, Methods Enzymol. 382 (2004) 449–456. [9] G. Griffiths, L. Trueman, T. Crowther, B. Thomas, B. Smith, Onions—a global benefit to health, Phytother. Res. 16 (2002) 603–615. [10] K. Rahman, Garlic and aging: new insights into an old remedy, Ageing Res. Rev. 2 (2003) 39–56. [11] M. [Ali, K.K. Al-Qattan, F. Al-Enezi, R.M. Khanafer, T. Mustafa, Effect of allicin from garlic powder on serum lipids and blood pressure in rats fed with a

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