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Metabolites of Diallyl Disulfide and Diallyl Sulfide in Primary Rat Hepatocytes
Food and Chemical Toxicology 37 (1999) 1139±1146
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Research Section Metabolites of Diallyl Disul®de and Diallyl Sul®de in Primary Rat Hepatocytes L. Y. SHEEN1*, C. C. WU2, C.-K. LII3 and S.-J. TSAI2 Department of Nutrition, China Medical College, Taichung, Taiwan, 2Department of Food Science, National Chung-Hsing University, Taichung, Taiwan and 3Department of Nutrition, Chung Shan Medical College, Taichung, Taiwan, ROC
1
(Accepted 5 May 1999) AbstractÐThe objectives of this study were to analyse and identify the metabolites of diallyl disul®de (DADS) and diallyl sul®de (DAS) in primary rat hepatocytes prepared by collagenase perfusion. According to the results, allyl mercaptan (AM) and allyl methyl sul®de (AMS) were the metabolites of DADS. The highest amount of AMS (0.9320.08 mg/ml at 90 min) was much less than that of AM (46.22 6.6 mg/ml at 60 min). Combined with the Purge and Trap using a gas chromatography±mass spectrometry (GC±MS) system, it is very useful to detect the trace amounts of metabolites in primary rat hepatocytes. Results also showed that AMS was a metabolite of DAS. The highest amount of AMS in the extracellular ¯uid of hepatocytes was 0.6320.16 mg/ml at 30 min of incubation. # 2000 Elsevier Science Ltd. All rights reserved Keywords: garlic; diallyl disul®de; diallyl sul®de; metabolite; primary rat hepatocytes. Abbreviations: AM = allyl mercaptan; AMS = allyl methyl sul®de; BSA = bovine serum albumin; DADS = diallyl disul®de; DAS = diallyl sul®de; DASO = diallyl sulfoxide; DASO2 = diallyl sulfone; FBS = foetal bovine serum; NDMA = N-nitrosodimethylamine; PBS = phosphate buered saline.
INTRODUCTION
Garlic is a commonly used foodstu and contains volatile oils which are the active principles, such as diallyl disul®de (DADS) and diallyl sul®de (DAS), for hypoglycaemia (Jain and Vyas, 1975), hypolipaemia and antiatherosclerosis (Bordia and Bansal, 1973; Bordia et al., 1977). DADS and DAS account for around 40% and 5% of the garlic essential oil, respectively (Sheen et al., 1991). DADS has been shown to have insecticidal (Amonkar and Banerji, 1971), antiplatelet activity (Bordia et al., 1998), and antiproliferative property of human colon tumour cells (Knowles and Milner, 1998), and to lower the levels of plasma and liver cholesterol (Omkumar et al., 1993). DAS has also been shown to inhibit a number of chemically induced forms of cancer (Wargovich et al., 1992); for example, DAS inhibited benzo[a]pyrene-induced forestomach tumours, pulmonary adenoma and skin carcinogenesis (Singh and Shukla, 1998; Sparnins et al., 1988). Some of the mechanisms of the protective eect of DAS *Corresponding author.
against chemically induced carcinogenesis were proposed. For instance, the suppression of vinyl carbamate and N-nitrosodimethylamine (NDMA) induced mutagenesis by DAS correlated with their inhibition of P450 2E1-mediated p-nitrophenol hydroxylation and NDMA N-demethylation (Surh et al., 1995). Albano et al. (1996) also showed that the treatment with DAS of rats receiving ethanol in the alcohol tube-feeding model eectively suppressed the induction of cytochrome P450 2E1 by ethanol. Metabolites of garlic, such as N-acetyl-S-(2-carboxypropyl)-cysteine, N-acetyl-S-allylcysteine and hexahydrohippuric acid, were identi®ed in human urine after consumption of fresh garlic (Jandke and Spiteller, 1987; de Rooij et al., 1996, 1997). Other indications on the metabolism of garlic constituents after ingestion of fresh garlic were obtained by gas chromatography±mass spectrometry (GC±MS) analysis of human breath where allyl mercaptan (AM) and DADS have been identi®ed (Laakso et al., 1989; Minami et al., 1989). Brady et al. (1991) reported that the metabolic conversion of DAS to the diallyl sulfoxide (DASO) and diallyl
0278-6915/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(99)00108-8
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Fig. 1. Total ion chromatograms of extracellular ¯uid of primary rat hepatocytes not treated with diallyl disul®de (A) and treated with 1.0 mM diallyl disul®de (B), and medium containing 1.0 mM diallyl disul®de without hepatocytes (C). All samples were incubated at 378C for 60 min. Peak: 1: ethylene 2: allyl mercaptan 3: ethanol 4: allyl methyl sul®de 5: ammonia 6: diallyl sul®de 7: diallyl disul®de.
sulfone (DASO2) was observed in vivo and in vitro. Egen-Schwind et al. (1992b) investigated the metabolic and kinetic behaviour of dierent garlic constituents in the isolated perfused rat liver. DADS and AM were identi®ed as metabolites of allicin, whereas DADS probably is the metabolic precursor of AM. In addition, the prehepatic fate of the organosulfur compounds derived from garlic was studied by Lawson and Wang (1993). They determined which sulfur compounds might be present in blood
after consuming garlic or its products and prior to metabolism by the liver or other organs. The results showed that AM was the metabolite of DADS but not of DAS after the reactions of DADS or DAS in the presence of blood. However, the metabolites of DADS and DAS in normal hepatocytes were not well studied. We have successfully applied a Purge and Trap GC±MS system to analyse and identify the trace metabolites of DADS and DAS in hepatocytes which were prepared by collagenase perfusion
Metabolites of DADS and DAS
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Fig. 2. Mass spectra of allyl mercaptan (A) and allyl methyl sul®de (B).
as described by Berry and Friend (1969), Bonney et al. (1974) and our laboratory (Sheen et al., 1996). MATERIALS AND METHODS
Materials Diallyl disul®de (DADS, GC purity 93%) and diallyl sul®de (DAS, GC purity 97%) were purchased from Tokyo Kasei Chemical Co. (Japan) and Fluka Chemical Co. (Switzerland), respectively. HEPES, bovine serum albumin (BSA), trypsin inhibitor, dexamethasone, sodium selenite, potassium chloride, sodium chloride, glucose, phenol red, galactose, potassium dihydrogen phosphate, sodium bicarbonate, pyruvate, Triton X-100, trypan blue, and collagen were obtained from Sigma Chemical (St Louis, MO, USA). Insulin, transferrin, L-15 medium, foetal bovine serum (FBS) and penicillin± streptomycin solution were from Gibco Laboratories (Grand Island, NY, USA). Collagenase (CLS I, 239 U/mg) and Percoll were purchased from Worthington Biochemical Co. and Pharmacia LKB (Piscataway, NJ, USA), respectively. Sodium pentobarbital was from CHRIS KEV Co.
Animals 8-wk-old male Sprague±Dawley rats were used for hepatocyte isolation in the following experiments. The animals were housed in stainless-steel grid cages with an arti®cial 12-hr light/dark cycle. Rats had free access to diets (PMI Feeds, St Louis, MO, USA) and water. Hepatocyte culture Rat hepatocytes were prepared by collagenase perfusion as described by Berry and Friend (1973) and Bonney et al. (1974), and also by our laboratory (Sheen et al., 1996), with some modi®cations. Rats were anaesthetized by ip injection with sodium pentobarbital (100 mg/kg body weight). The liver was perfused via the portal vein at a ¯ow rate of 25 ml/min with 150 ml 25 mM sodium phosphate buer (pH 7.6) containing 3.1 mM KCl, 119 mM NaCl, 5.5 mM glucose, 1.0 g BSA/litre and 5 mg phenol red /litre to remove blood. The buer was replaced with 200 ml of the same buer supplemented with 80 mg collagenase, 40 mM CaCl2 and 5 mg trypsin inhibitor, and the liver was perfused for another 10 min at a rate of 18 ml/min. To produce a single-cell suspension of hepatic paren-
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Fig. 4. Concentrations of intracellular allyl mercaptan (AM) and diallyl disul®de (DADS) in primary rat hepatocytes treated with 1.0 mM diallyl disul®de for 0, 30, 60, 90 and 120 min. External standard was dipropyl disul®de.
each 60-mm collagen-precoated plastic tissue culture dish (Falcon Labware) and incubated in a 378C humidi®ed incubator (NUAIRE, USA) in an atmosphere of air. The medium was changed by the same culture medium but contained 2.0 g BSA/litre instead of FBS, 4 hr after plating. At 20 hr after plating, media were changed once each day. Treatment Fig. 3. Concentrations of extracellular allyl mercaptan (AM), allyl methyl sul®de (AMS) and diallyl disul®de (DADS) in primary rat hepatocytes treated with 1.0 mM diallyl disul®de for 0, 30, 60, 90 and 120 min. External standard was dipropyl disul®de.
chymal cells, the liver was removed, sieved, washed, suspended in buer Percoll and centrifuged (Hitachi himac CR21, Japan) at 48C (Kreamer et al., 1986). Hepatocytes were then resuspended and washed twice with washing medium. Cell viability determined by trypan blue exclusion was above 90%. After ®nal washing, the isolated hepatocytes were resuspended in L-15 cell culture medium (pH 7.6) supplemented with 18 mM HEPES, 2.5% FBS, 5 mg/litre each of insulin and transferrin, 28 mM galactose, 1 mM dexamethasone, 100,000 IU/litre penicillin and 100 mg streptomycin/litre at a density of 5 108 cells/litre. Cells (2.5 106) were plated on
After 20 hr plating, cells were treated with 1 mM DADS or DAS (in propylene glycol) for 0, 30, 60, 90 and 120 min. After each period of incubation, the reaction was stopped by removing medium and washing with cold potassium phosphate buered saline (PBS, pH 7.4). Cells were removed with PBS containing 0.5% Triton X-100 and with a cell scraper for further analysis. Analysis and identi®cation Adding 1 ml 3% silicone into the intra- and extracellular ¯uid samples of hepatocytes at various time periods incubated with 1 mM DADS or DAS, the metabolites were analysed and identi®ed with the combination of Purge and Trap system (Purge and Trap concentrator 3629A, Hewlett Packard, USA) and GC±MS system (G 1800A GCD, Hewlett Packard, USA). GC±MS was performed with a HP GCD. HP GC 5890 instrument was
Metabolites of DADS and DAS
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Fig. 5. Total ion chromatograms of extracellular ¯uid of primary rat hepatocytes not treated with diallyl sul®de (A) and treated with 1.0 mM diallyl sul®de (B), and medium containing 1.0 mM diallyl sul®de without hepatocytes (C). All samples were incubated at 378C for 60 min. Peak: 1: ethylene 2: ethanol 3: allyl methyl sul®de 4: ammonia 5: diallyl sul®de 6: diallyl disul®de.
equipped with a fused silica capillary column (Carbowax 20 M; 60 m 0.32 mm i.d.) and split ratio was 1:20. The column was temperature programmed from 50 to 2008C at 58C/min and held at the ®nal temperature until the chromatogram was completed. The other conditions were as follows: carrier gas (helium) ¯ow rate, 1 ml/min; temperature of the injection port, 2508C; temperature of the ion source, 2008C; electron voltage, 70 eV; electron multiplier voltage, 1976 V. External standard curve
was made with a solution of dipropyl disul®de dissolved in propylene glycol. Peak identi®cations were based on comparison with ®le spectra of standards or published spectra and relative retention time. Each sample was analysed at least three times. RESULTS
Figure 1 shows the total ion chromatograms of the samples. (A) is total ion chromatogram of the
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Figure 5 shows the total ion chromatograms of the samples. (A) is total ion chromatogram of the extracellular ¯uid of primary rat hepatocytes not treated with DAS. (B) is the extracellular ¯uid of primary rat hepatocytes treated with DAS, while (C) is the medium containing 1.0 mM DAS without hepatocytes. The peak numbers of the total ion chromatograms from 1 to 6 represent ethylene, ethanol, AMS, ammonia, DAS and DADS, respectively. The major dierence between (A) and (B) is that DAS existed in (B) but not in (A). While the distinguished dierence between (B) and (C) is that AMS existed in (B) but not in (C). During the treatment of 1.0 mM DAS for 120 min, the concentrations of extracellular AMS and DAS of hepatocytes at various time intervals are shown in Fig. 6. The amount of DAS decreased with the increasing time intervals, while the amount of AMS increased to the peak of 0.63 2 0.16 mg/ml at 30 min of treatment. DISCUSSION
Fig. 6. Concentrations of extracellular allyl methyl sul®de (AMS) and diallyl sul®de (DAS) in primary rat hepatocytes treated with 1.0 mM diallyl sul®de for 0, 30, 60, 90 and 120 min. External standard was dipropyl disul®de.
extracellular ¯uid of primary rat hepatocytes not treated with diallyl disul®de (DADS). (B) is the extracellular ¯uid of primary rat hepatocytes treated with DADS, while (C) is the medium containing 1.0 mM DADS without hepatocytes. The peak numbers of the total ion chromatograms from 1 to 7 represent ethylene, AM, ethanol, AMS, ammonia, DAS and DADS, respectively. The major dierence between (A) and (B) is that DADS existed in (B) but not in (A). The distinguished dierence between (B) and (C) is that AM and AMS existed in (B) but not in (C). Figure 2 shows the mass spectra of AM (A) and AMS (B) in Fig. 1 (B). During the treatment of 1.0 mM DADS for 120 min, the concentrations of extracellular AM, AMS and DADS of hepatocytes at various time intervals are shown in Fig. 3. The amount of DADS decreased with the increasing time intervals. On the other hand, the amounts of AM and AMS increased to the peak of 46.2 2 6.6 and 0.93 20.08 mg/ml at 60 and 90 min treatment, respectively. Figure 4 shows the concentrations of intracellular AM and DADS at various time intervals in hepatocytes treated with 1.0 mM DADS for 120 min. The highest amounts of AM and DADS at 30 min were 10.03 2 1.8 and 2.14 2 0.51 mg/ml, respectively, then decreased with increasing time of incubation.
A fast decomposition of allicin in the liver has been observed during studies with a liver homogenate (Egen-Schwind et al., 1992a). In the isolated perfused liver, allicin was reduced to DADS which, in turn, was reduced to AM (Egen-Schwind et al., 1992b). Both metabolites (DADS and AM) were detected in the exhaled air of humans after ingestion of fresh garlic (Laakso et al., 1989; Minami et al., 1989). Lawson and Wang (1993) also reported that AM was the metabolite of DADS. According to the results of Fig. 1, AM and AMS were the metabolites of DADS in primary rat hepatocytes. AMS was a new metabolite of DADS in our experimental model and analysis system. As methyl transfer occurred in biological systems (Ridley et al., 1977), AMS and AM may be formed simultaneously through the biomethylation for DADS while its disul®de bond is being broken down. The highest amount of AMS (0.932 0.08 mg/ ml at 90 min) was much less than that of AM (46.22 6.6 mg/ml at 60 min). Such a small amount of AMS is the reason why AMS was not easily detected before. However, since we combined the Purge and Trap system with GC±MS analysis (a closed system) without any additional extraction procedure, we successfully and directly dectected the trace amount of metabolite such as AMS in primary rat hepatocytes. To compare the extra- and intracellular amounts of AM in Figs 3 and 4, it is reasonable that the highest intracellular amount of AM was obtained at 30 min, while the highest extracellular amount of AM at 60 min due to the accumulation. However, owing to the volatility of AM, the amount of AM decreased after 60 min of incubation. Lawson and Wang (1993) reported that there was no metabolite for DAS in the presence of blood.
Metabolites of DADS and DAS
Brady et al. (1991) reported that the metabolic conversion of DAS to the DASO and DASO2 was observed in vivo and in vitro. But in our study, Fig. 5 shows that AMS was a metabolite of DAS but not DASO or DASO2 in primary rat hepatocytes. This may be due to the dierence of experimental model and analysis system. In our study, a closed system free from being exposed to oxygen was used to analyse the metabolites of DAS. In addition, as methyl transfer occurred in biological systems (Ridley et al., 1977), AMS may also be formed through the biomethylation for DAS while its sulfur±carbon bond is being broken down. However, it is not easy to form AM due to the chemical conformation. The highest amount (0.63 2 0.16 mg/ml) of AMS in extracellular ¯uid of hepatocytes was observed at 30 min of incubation in this study (Fig. 6). Also, due to the volatility of AMS, the amount of AMS decreased after 30 min of incubation. On the other hand, because the amount of intracellular AMS in hepatocytes was too low to be detected, the data of intracellular AMS in hepatocytes was not shown. In conclusion, the trace amount of metabolite in primary rat hepatocytes could be directly detected and determined by Purge and Trap system combined with GC±MS analysis under a closed system. AM and AMS were the metabolites of DADS, while AMS was the metabolite of DAS in primary rat hepatocytes, respectively. AcknowledgementsÐThe authors thank the National Science Council, Taipei, Taiwan for ®nancial support (NSC83-0412-B-039-007).
REFERENCES
Albano E., Clot P., Morimoto M., Tomasi A., IngelmanSundberg M. and French S. W. (1996) Role of cytochrome P450 2E1-dependent formation of hydroxyethyl free radical in the development of liver damage in rats intragastrically fed with ethanol. Hepatology 23, 155± 163. Amonkar S. V. and Banerji A. (1971) Isolation and characterization of larvicidal principle of garlic. Science 174, 1343±1344. Berry M. N. and Friend D. S. (1969) High yield production of isolated rat liver parenchymal cells: a biochemical and ®ne structural study. Journal of Cell Biology 43, 506±520. Bonney V. R., Becker J. E., Walker P. R. and Potter V. R. (1974) Primary monolayer cultures of rat liver parenchymal cells suitable for study of regulation of enzyme synthesis. In Vitro 9, 399±413. Bordia A. and Bansal H. C. (1973) Essential oil of garlic in prevention of atherosclerosis. Lancet ii, 1491±1492. Bordia A., Verma S. K. and Srivastava K. C. (1998) Eect of garlic (Allium sativum) on blood lipids, blood sugar, ®brinogen and ®brinolytic activity in patients with coronary artery disease. Prostaglandins Leukotrienes and Essential Fatty Acids 58, 257±263. Bordia A., Verma S. K., Vyas A. K., Khabya B. L., Rathore A. S., Bhu N. and Bedi H. K. (1977) Eect of
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essential oil of onion and garlic on experimental atherosclerosis in rabbits. Atherosclerosis 26, 379±386. Brady J. F., Ishizaki H., Fukuto J. M., Lin M. C., Fadel A., Gapac J. M. and Yang C. S. (1991) Inhibition of cytochrome P-450 2E1 by diallyl sul®de and its metabolites. Chemical Research in Toxicology 4, 642±647. de Rooij B. M., Boogaard P. J., Rijksen D. A., Commandeur J. N. and Vermeulen N. P. (1996) Urinary excretion of N-acetyl-S-allyl-L-cysteine upon garlic consumption by human volunteers. Archives of Toxicology 70, 635±639. de Rooij B. M., Boogaard P. J., Commandeur J. N., van Sittert N. J. and Vermeulen N. P. (1996) Allylmercapturic acid as urinary biomarker of human exposure to allyl chloride. Occupational and Environmental Medicine 54, 653±661. Egen-Schwind C., Eckard R., Jekat F. W. and Winterho H. (1992a) Pharmacokinetics of vinyldithiins, transformation products of allcin. Planta Medica 58, 8±13. Egen-Schwind C., Eckard R. and Kemper F. H. (1992b) Metabolism of garlic constituents in the isolated perfused rat liver. Planta Medica 58, 301±305. Jain R. C. and Vyas C. R. (1975) Garlic in alloxaninduced diabetic rabbits. American Journal of Clinical Nutrition 28, 684±685. Jandke J. and Spiteller G. (1987) Unusual conjugates in biological pro®les originating from consumption of onions and garlic. Journal of Chromatography 421, 1±8. Knowles L. M. and Milner J. A. (1998) Depressed p34cdc2 kinase activity and G2/M phase arrest induced by diallyl disul®de in HCT-15 cells. Nutrition and Cancer 30, 169±174. Kreamer B. L., Staecker J. L., Sawada N., Sattler G. L., Hsia M. T. S. and Pitot H. C. (1986) Use of low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations. In Vitro Cellular and Developmental Biology 22, 201± 211. Laakso I., Seppanen-Laakso T., Hiltunen R., Muller B., Jansen H. and Knobloch K. (1989) Volatile garlic odor components: gas phases and adsorbed exhaled air analysed by headspace gas chromatography-mass spectrometry. Planta Medica 55, 257±261. Lawson L. D. and Wang Z. J. (1993) Pre-hepatic fate of the organosulfur compounds derived from garlic (Allium sativum). Planta Medica 59, A688±689. Minami T., Boku T., Inada K., Morita M. and Okazaki Y. (1989) Odor components of human breath after the ingestion of grated raw garlic. Journal of Food Science 54, 763±765. Omkumar R. V., Kadam S. M., Banerji A. and Ramasarma T. (1993) On the involvement of intramolecular protein disul®de in the irreversible inactivation of 3-hydroxy-3-methylglutaryl-CoA reductase by diallyl disul®de. Biochimica et Biophysica Acta 1164, 108±112. Ridley W. P., Dizikes L. J. and Wood J. M. (1977) Biomethylation of toxic elements in the environment. Science 197, 329±332. Sheen L. Y., Lii C-K., Sheu S. F., Meng R. H. C. and Tsai S-J. (1996) Eect of the active principle of garlic± diallyl sul®de±on cell viability, detoxi®cation capability and the antioxidation system of primary rat hepatocytes. Food and Chemical Toxicology 34, 971±978. Sheen L. Y., Lin S. Y. and Tsai S-J. (1991) Preparation of spray-dried microcapsules with various amounts of basil, garlic and ginger essential oils and changes in oil compositions during spray drying. Food Science 18, 344±355. Singh A. and Shukla Y. (1998) Antitumour activity of diallyl sul®de on polycyclic aromatic hydrocarbon-induced mouse skin carcinogenesis. Cancer Letters 131, 209±214. Sparnins V. L., Barany G. and Wattenberg L. W. (1988) Eects of organosulfur compounds from garlic and
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onions on benzo[a]pyrene-induced neoplasia and glutathione S-transferase activity in the mouse. Carcinogenesis 9, 131±134. Surh Y. J., Lee R. C., Park K. K., Mayne S. T., Liem A. and Miller J. A. (1995) Chemoprotective eects of capsaicin and diallyl sul®de against mutagenesis or
tumorigenesis by vinyl carbamate and N-nitrosodimethylamine. Carcinogenesis 16, 2467±2471. Wargovich M. J., Imada O. and Stephens L. C. (1992) Initiation and postinitiation chemopreventive eects of diallyl sul®de in esophageal carcinogenesis. Cancer Letters 64, 39±42.
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Research Section Metabolites of Diallyl Disul®de and Diallyl Sul®de in Primary Rat Hepatocytes L. Y. SHEEN1*, C. C. WU2, C.-K. LII3 and S.-J. TSAI2 Department of Nutrition, China Medical College, Taichung, Taiwan, 2Department of Food Science, National Chung-Hsing University, Taichung, Taiwan and 3Department of Nutrition, Chung Shan Medical College, Taichung, Taiwan, ROC
1
(Accepted 5 May 1999) AbstractÐThe objectives of this study were to analyse and identify the metabolites of diallyl disul®de (DADS) and diallyl sul®de (DAS) in primary rat hepatocytes prepared by collagenase perfusion. According to the results, allyl mercaptan (AM) and allyl methyl sul®de (AMS) were the metabolites of DADS. The highest amount of AMS (0.9320.08 mg/ml at 90 min) was much less than that of AM (46.22 6.6 mg/ml at 60 min). Combined with the Purge and Trap using a gas chromatography±mass spectrometry (GC±MS) system, it is very useful to detect the trace amounts of metabolites in primary rat hepatocytes. Results also showed that AMS was a metabolite of DAS. The highest amount of AMS in the extracellular ¯uid of hepatocytes was 0.6320.16 mg/ml at 30 min of incubation. # 2000 Elsevier Science Ltd. All rights reserved Keywords: garlic; diallyl disul®de; diallyl sul®de; metabolite; primary rat hepatocytes. Abbreviations: AM = allyl mercaptan; AMS = allyl methyl sul®de; BSA = bovine serum albumin; DADS = diallyl disul®de; DAS = diallyl sul®de; DASO = diallyl sulfoxide; DASO2 = diallyl sulfone; FBS = foetal bovine serum; NDMA = N-nitrosodimethylamine; PBS = phosphate buered saline.
INTRODUCTION
Garlic is a commonly used foodstu and contains volatile oils which are the active principles, such as diallyl disul®de (DADS) and diallyl sul®de (DAS), for hypoglycaemia (Jain and Vyas, 1975), hypolipaemia and antiatherosclerosis (Bordia and Bansal, 1973; Bordia et al., 1977). DADS and DAS account for around 40% and 5% of the garlic essential oil, respectively (Sheen et al., 1991). DADS has been shown to have insecticidal (Amonkar and Banerji, 1971), antiplatelet activity (Bordia et al., 1998), and antiproliferative property of human colon tumour cells (Knowles and Milner, 1998), and to lower the levels of plasma and liver cholesterol (Omkumar et al., 1993). DAS has also been shown to inhibit a number of chemically induced forms of cancer (Wargovich et al., 1992); for example, DAS inhibited benzo[a]pyrene-induced forestomach tumours, pulmonary adenoma and skin carcinogenesis (Singh and Shukla, 1998; Sparnins et al., 1988). Some of the mechanisms of the protective eect of DAS *Corresponding author.
against chemically induced carcinogenesis were proposed. For instance, the suppression of vinyl carbamate and N-nitrosodimethylamine (NDMA) induced mutagenesis by DAS correlated with their inhibition of P450 2E1-mediated p-nitrophenol hydroxylation and NDMA N-demethylation (Surh et al., 1995). Albano et al. (1996) also showed that the treatment with DAS of rats receiving ethanol in the alcohol tube-feeding model eectively suppressed the induction of cytochrome P450 2E1 by ethanol. Metabolites of garlic, such as N-acetyl-S-(2-carboxypropyl)-cysteine, N-acetyl-S-allylcysteine and hexahydrohippuric acid, were identi®ed in human urine after consumption of fresh garlic (Jandke and Spiteller, 1987; de Rooij et al., 1996, 1997). Other indications on the metabolism of garlic constituents after ingestion of fresh garlic were obtained by gas chromatography±mass spectrometry (GC±MS) analysis of human breath where allyl mercaptan (AM) and DADS have been identi®ed (Laakso et al., 1989; Minami et al., 1989). Brady et al. (1991) reported that the metabolic conversion of DAS to the diallyl sulfoxide (DASO) and diallyl
0278-6915/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(99)00108-8
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Fig. 1. Total ion chromatograms of extracellular ¯uid of primary rat hepatocytes not treated with diallyl disul®de (A) and treated with 1.0 mM diallyl disul®de (B), and medium containing 1.0 mM diallyl disul®de without hepatocytes (C). All samples were incubated at 378C for 60 min. Peak: 1: ethylene 2: allyl mercaptan 3: ethanol 4: allyl methyl sul®de 5: ammonia 6: diallyl sul®de 7: diallyl disul®de.
sulfone (DASO2) was observed in vivo and in vitro. Egen-Schwind et al. (1992b) investigated the metabolic and kinetic behaviour of dierent garlic constituents in the isolated perfused rat liver. DADS and AM were identi®ed as metabolites of allicin, whereas DADS probably is the metabolic precursor of AM. In addition, the prehepatic fate of the organosulfur compounds derived from garlic was studied by Lawson and Wang (1993). They determined which sulfur compounds might be present in blood
after consuming garlic or its products and prior to metabolism by the liver or other organs. The results showed that AM was the metabolite of DADS but not of DAS after the reactions of DADS or DAS in the presence of blood. However, the metabolites of DADS and DAS in normal hepatocytes were not well studied. We have successfully applied a Purge and Trap GC±MS system to analyse and identify the trace metabolites of DADS and DAS in hepatocytes which were prepared by collagenase perfusion
Metabolites of DADS and DAS
1141
Fig. 2. Mass spectra of allyl mercaptan (A) and allyl methyl sul®de (B).
as described by Berry and Friend (1969), Bonney et al. (1974) and our laboratory (Sheen et al., 1996). MATERIALS AND METHODS
Materials Diallyl disul®de (DADS, GC purity 93%) and diallyl sul®de (DAS, GC purity 97%) were purchased from Tokyo Kasei Chemical Co. (Japan) and Fluka Chemical Co. (Switzerland), respectively. HEPES, bovine serum albumin (BSA), trypsin inhibitor, dexamethasone, sodium selenite, potassium chloride, sodium chloride, glucose, phenol red, galactose, potassium dihydrogen phosphate, sodium bicarbonate, pyruvate, Triton X-100, trypan blue, and collagen were obtained from Sigma Chemical (St Louis, MO, USA). Insulin, transferrin, L-15 medium, foetal bovine serum (FBS) and penicillin± streptomycin solution were from Gibco Laboratories (Grand Island, NY, USA). Collagenase (CLS I, 239 U/mg) and Percoll were purchased from Worthington Biochemical Co. and Pharmacia LKB (Piscataway, NJ, USA), respectively. Sodium pentobarbital was from CHRIS KEV Co.
Animals 8-wk-old male Sprague±Dawley rats were used for hepatocyte isolation in the following experiments. The animals were housed in stainless-steel grid cages with an arti®cial 12-hr light/dark cycle. Rats had free access to diets (PMI Feeds, St Louis, MO, USA) and water. Hepatocyte culture Rat hepatocytes were prepared by collagenase perfusion as described by Berry and Friend (1973) and Bonney et al. (1974), and also by our laboratory (Sheen et al., 1996), with some modi®cations. Rats were anaesthetized by ip injection with sodium pentobarbital (100 mg/kg body weight). The liver was perfused via the portal vein at a ¯ow rate of 25 ml/min with 150 ml 25 mM sodium phosphate buer (pH 7.6) containing 3.1 mM KCl, 119 mM NaCl, 5.5 mM glucose, 1.0 g BSA/litre and 5 mg phenol red /litre to remove blood. The buer was replaced with 200 ml of the same buer supplemented with 80 mg collagenase, 40 mM CaCl2 and 5 mg trypsin inhibitor, and the liver was perfused for another 10 min at a rate of 18 ml/min. To produce a single-cell suspension of hepatic paren-
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Fig. 4. Concentrations of intracellular allyl mercaptan (AM) and diallyl disul®de (DADS) in primary rat hepatocytes treated with 1.0 mM diallyl disul®de for 0, 30, 60, 90 and 120 min. External standard was dipropyl disul®de.
each 60-mm collagen-precoated plastic tissue culture dish (Falcon Labware) and incubated in a 378C humidi®ed incubator (NUAIRE, USA) in an atmosphere of air. The medium was changed by the same culture medium but contained 2.0 g BSA/litre instead of FBS, 4 hr after plating. At 20 hr after plating, media were changed once each day. Treatment Fig. 3. Concentrations of extracellular allyl mercaptan (AM), allyl methyl sul®de (AMS) and diallyl disul®de (DADS) in primary rat hepatocytes treated with 1.0 mM diallyl disul®de for 0, 30, 60, 90 and 120 min. External standard was dipropyl disul®de.
chymal cells, the liver was removed, sieved, washed, suspended in buer Percoll and centrifuged (Hitachi himac CR21, Japan) at 48C (Kreamer et al., 1986). Hepatocytes were then resuspended and washed twice with washing medium. Cell viability determined by trypan blue exclusion was above 90%. After ®nal washing, the isolated hepatocytes were resuspended in L-15 cell culture medium (pH 7.6) supplemented with 18 mM HEPES, 2.5% FBS, 5 mg/litre each of insulin and transferrin, 28 mM galactose, 1 mM dexamethasone, 100,000 IU/litre penicillin and 100 mg streptomycin/litre at a density of 5 108 cells/litre. Cells (2.5 106) were plated on
After 20 hr plating, cells were treated with 1 mM DADS or DAS (in propylene glycol) for 0, 30, 60, 90 and 120 min. After each period of incubation, the reaction was stopped by removing medium and washing with cold potassium phosphate buered saline (PBS, pH 7.4). Cells were removed with PBS containing 0.5% Triton X-100 and with a cell scraper for further analysis. Analysis and identi®cation Adding 1 ml 3% silicone into the intra- and extracellular ¯uid samples of hepatocytes at various time periods incubated with 1 mM DADS or DAS, the metabolites were analysed and identi®ed with the combination of Purge and Trap system (Purge and Trap concentrator 3629A, Hewlett Packard, USA) and GC±MS system (G 1800A GCD, Hewlett Packard, USA). GC±MS was performed with a HP GCD. HP GC 5890 instrument was
Metabolites of DADS and DAS
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Fig. 5. Total ion chromatograms of extracellular ¯uid of primary rat hepatocytes not treated with diallyl sul®de (A) and treated with 1.0 mM diallyl sul®de (B), and medium containing 1.0 mM diallyl sul®de without hepatocytes (C). All samples were incubated at 378C for 60 min. Peak: 1: ethylene 2: ethanol 3: allyl methyl sul®de 4: ammonia 5: diallyl sul®de 6: diallyl disul®de.
equipped with a fused silica capillary column (Carbowax 20 M; 60 m 0.32 mm i.d.) and split ratio was 1:20. The column was temperature programmed from 50 to 2008C at 58C/min and held at the ®nal temperature until the chromatogram was completed. The other conditions were as follows: carrier gas (helium) ¯ow rate, 1 ml/min; temperature of the injection port, 2508C; temperature of the ion source, 2008C; electron voltage, 70 eV; electron multiplier voltage, 1976 V. External standard curve
was made with a solution of dipropyl disul®de dissolved in propylene glycol. Peak identi®cations were based on comparison with ®le spectra of standards or published spectra and relative retention time. Each sample was analysed at least three times. RESULTS
Figure 1 shows the total ion chromatograms of the samples. (A) is total ion chromatogram of the
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Figure 5 shows the total ion chromatograms of the samples. (A) is total ion chromatogram of the extracellular ¯uid of primary rat hepatocytes not treated with DAS. (B) is the extracellular ¯uid of primary rat hepatocytes treated with DAS, while (C) is the medium containing 1.0 mM DAS without hepatocytes. The peak numbers of the total ion chromatograms from 1 to 6 represent ethylene, ethanol, AMS, ammonia, DAS and DADS, respectively. The major dierence between (A) and (B) is that DAS existed in (B) but not in (A). While the distinguished dierence between (B) and (C) is that AMS existed in (B) but not in (C). During the treatment of 1.0 mM DAS for 120 min, the concentrations of extracellular AMS and DAS of hepatocytes at various time intervals are shown in Fig. 6. The amount of DAS decreased with the increasing time intervals, while the amount of AMS increased to the peak of 0.63 2 0.16 mg/ml at 30 min of treatment. DISCUSSION
Fig. 6. Concentrations of extracellular allyl methyl sul®de (AMS) and diallyl sul®de (DAS) in primary rat hepatocytes treated with 1.0 mM diallyl sul®de for 0, 30, 60, 90 and 120 min. External standard was dipropyl disul®de.
extracellular ¯uid of primary rat hepatocytes not treated with diallyl disul®de (DADS). (B) is the extracellular ¯uid of primary rat hepatocytes treated with DADS, while (C) is the medium containing 1.0 mM DADS without hepatocytes. The peak numbers of the total ion chromatograms from 1 to 7 represent ethylene, AM, ethanol, AMS, ammonia, DAS and DADS, respectively. The major dierence between (A) and (B) is that DADS existed in (B) but not in (A). The distinguished dierence between (B) and (C) is that AM and AMS existed in (B) but not in (C). Figure 2 shows the mass spectra of AM (A) and AMS (B) in Fig. 1 (B). During the treatment of 1.0 mM DADS for 120 min, the concentrations of extracellular AM, AMS and DADS of hepatocytes at various time intervals are shown in Fig. 3. The amount of DADS decreased with the increasing time intervals. On the other hand, the amounts of AM and AMS increased to the peak of 46.2 2 6.6 and 0.93 20.08 mg/ml at 60 and 90 min treatment, respectively. Figure 4 shows the concentrations of intracellular AM and DADS at various time intervals in hepatocytes treated with 1.0 mM DADS for 120 min. The highest amounts of AM and DADS at 30 min were 10.03 2 1.8 and 2.14 2 0.51 mg/ml, respectively, then decreased with increasing time of incubation.
A fast decomposition of allicin in the liver has been observed during studies with a liver homogenate (Egen-Schwind et al., 1992a). In the isolated perfused liver, allicin was reduced to DADS which, in turn, was reduced to AM (Egen-Schwind et al., 1992b). Both metabolites (DADS and AM) were detected in the exhaled air of humans after ingestion of fresh garlic (Laakso et al., 1989; Minami et al., 1989). Lawson and Wang (1993) also reported that AM was the metabolite of DADS. According to the results of Fig. 1, AM and AMS were the metabolites of DADS in primary rat hepatocytes. AMS was a new metabolite of DADS in our experimental model and analysis system. As methyl transfer occurred in biological systems (Ridley et al., 1977), AMS and AM may be formed simultaneously through the biomethylation for DADS while its disul®de bond is being broken down. The highest amount of AMS (0.932 0.08 mg/ ml at 90 min) was much less than that of AM (46.22 6.6 mg/ml at 60 min). Such a small amount of AMS is the reason why AMS was not easily detected before. However, since we combined the Purge and Trap system with GC±MS analysis (a closed system) without any additional extraction procedure, we successfully and directly dectected the trace amount of metabolite such as AMS in primary rat hepatocytes. To compare the extra- and intracellular amounts of AM in Figs 3 and 4, it is reasonable that the highest intracellular amount of AM was obtained at 30 min, while the highest extracellular amount of AM at 60 min due to the accumulation. However, owing to the volatility of AM, the amount of AM decreased after 60 min of incubation. Lawson and Wang (1993) reported that there was no metabolite for DAS in the presence of blood.
Metabolites of DADS and DAS
Brady et al. (1991) reported that the metabolic conversion of DAS to the DASO and DASO2 was observed in vivo and in vitro. But in our study, Fig. 5 shows that AMS was a metabolite of DAS but not DASO or DASO2 in primary rat hepatocytes. This may be due to the dierence of experimental model and analysis system. In our study, a closed system free from being exposed to oxygen was used to analyse the metabolites of DAS. In addition, as methyl transfer occurred in biological systems (Ridley et al., 1977), AMS may also be formed through the biomethylation for DAS while its sulfur±carbon bond is being broken down. However, it is not easy to form AM due to the chemical conformation. The highest amount (0.63 2 0.16 mg/ml) of AMS in extracellular ¯uid of hepatocytes was observed at 30 min of incubation in this study (Fig. 6). Also, due to the volatility of AMS, the amount of AMS decreased after 30 min of incubation. On the other hand, because the amount of intracellular AMS in hepatocytes was too low to be detected, the data of intracellular AMS in hepatocytes was not shown. In conclusion, the trace amount of metabolite in primary rat hepatocytes could be directly detected and determined by Purge and Trap system combined with GC±MS analysis under a closed system. AM and AMS were the metabolites of DADS, while AMS was the metabolite of DAS in primary rat hepatocytes, respectively. AcknowledgementsÐThe authors thank the National Science Council, Taipei, Taiwan for ®nancial support (NSC83-0412-B-039-007).
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