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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:724-728, April 2004

Biochemical and Molecular Actions of Nutrients

Diallyl Trisulfide Modulates Cell Viability and the Antioxidation and Detoxification Systems of Rat Primary Hepatocytes1

Chih-Chung Wu, Chong-Kuei Lii*, Shun-Jen Tsai{dagger} and Lee-Yan Sheen**,2

Department of Nutrition and Health Science, Fooyin University, Kaohsiung, Taiwan; * Department of Nutrition, Chung Shan Medical University, Taichung, Taiwan; {dagger} Department of Food Science, National Chung-Hsing University, Taichung, Taiwan; and ** Graduate Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan

2To whom correspondence should be addressed. E-mail: lysheen{at}ntu.edu.tw.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
This study investigated the effects of various concentrations of diallyl trisulfide (DATS) and incubation times on cell viability, glutathione (GSH) content, and GSH-related enzyme activity in rat primary hepatocytes. Isolated and cultured primary rat hepatocytes were used as an experimental model. Cells were treated with 0 (control), 0.025, 0.05, or 0.25 mmol/L DATS for 0, 4, 8, or 24 h. After 24 h of treatment, some cells were incubated in fresh medium without DATS for an additional 24 h (48-h incubations). Based on lactate dehydrogenase (LDH) leakage and morphological examination, hepatocytes treated with 0.025 mmol/L DATS did not differ from the control cells at 4, 8, 24, and 48 h of incubation. However, LDH leakage was higher than in the control cells (P < 0.05) when the hepatocytes were treated with 0.05 or 0.25 mmol/L DATS for 4 h or more. The intracellular GSH levels of hepatocytes treated with 0.025 or 0.05 mmol/L DATS were higher than those of the control cells (P < 0.05), whereas those treated with 0.25 mmol/L DATS did not differ. The activity of glutathione reductase (GRd) was higher than in the control cells at 24 h (P < 0.05) when the hepatocytes were treated with 0.025 mmol/L DATS. When the hepatocytes were treated with 0.025 mmol/L DATS, the activity of glutathione S-transferase (GST) was higher than in the control cells at 48 h (P < 0.05). In hepatocytes treated with 0.05 mmol/L DATS, the activity of GST and glutathione peroxidase (GPx) was higher than in the control cells (P < 0.05) at 24 and 48 h of incubation. The results indicate that 0.025 or 0.05 mmol/L DATS could enhance antioxidation and detoxification capabilities by increasing the intracellular GSH level and the activity of GPx, GRd, or GST in rat primary hepatocytes. However, 0.05 or 0.25 mmol/L DATS might adversely affect the viability of hepatocytes.

KEY WORDS: • diallyl trisulfide • hepatocytes • cell viability • detoxification • antioxidation

Garlic (Allium sativum L.) is a commonly used foodstuff. Steam distillation is widely used to extract volatile organosulfur compounds (OSCs)3 from garlic, and the final oily product is known as garlic oil (GO). More than 20 OSCs have been identified in GO by gas chromatography (1). Among these, diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS) are three major components. Garlic oil and its component OSCs are reported to benefit the hepatic antioxidation and detoxification systems and to elicit antibiotic, antiatherosclerotic, immunomodulatory, renoprotective, antimutagenic, and anticarcinogenic properties (2–10).

We previously demonstrated that 1 mmol/L DAS and 0.5 mmol/L DADS might enhance the detoxification and antioxidation capabilities of rat primary hepatocytes (11,12). The symmetrical compound DATS, which contains 2 allyl groups and 3 sulfur atoms, is similar to DAS and DADS in structure. Studies report that DATS shows antiplatelet activity by inhibiting platelet thromboxane formation (13) and inhibits cholesterol biosynthesis by inhibiting the activity of squalene monoxygenase (14). A previous study with mice suggested that treatment with DAS, DADS, and DATS increases both hepatic and forestomach glutathione S-transferase (GST) activity toward anti-7ß,8{alpha}-dihydroxy-9{alpha},10{alpha}-oxy-7,8,9,10- tetrahydrobenzo(a)pyrene, which is the ultimate carcinogen of benzo(a)pyrene (BP), thereby preventing BP-induced cancer in mice (15). Furthermore, treatment with DADS and DATS, which are potent inhibitors of BP-induced forestomach tumorigenesis, greatly increases forestomach NAD(P)H:quinone oxidoreductase (NQO) activity in mice; DADS and DATS increase forestomach NQO activity much more than does DAS (16). In addition, 10 µmol/L DATS causes a marked and progressive increase in intracellular Ca2+ in A549 cells, and exposure to 1 µmol/L DATS for 24 h significantly induces apoptosis, as indicated by increased DNA fragmentation (17). Diallyl trisulfide also augments the activation of T cells and enhances the antitumor function of macrophages, suggesting that it may be useful in tumor therapy (18).

However, the optimum concentration of DATS for the antioxidation and detoxification systems in rat primary hepatocytes is not known. Therefore, the present study investigated the effects of various concentrations of DATS and incubation times on cell viability, glutathione (GSH) content, and GSH-related enzyme activity in rat primary hepatocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Materials. Diallyl trisulfide was purchased from LKT Laboratories. Supplies of L-15 medium, fetal bovine serum (FBS), and penicillin/streptomycin solution were purchased from Gibco Laboratoies. Bovine serum albumin (BSA), HEPES, ß-NADH, ß-NAD(P)H, SDS, 2-thiobarbituric acid, 1,1,3,3-tetraethoxypropane, GSH, oxidized glutathione, 1-chloro-2,4-dinitrobenzene (CDNB), sodium azide (NaN3), glutathione reductase (GRd), and iodoacetic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Collagenase (type I) and Percoll solution were purchased from Worthington Biochemical and Pharmacia LKB, respectively. Antiserums for the glutathione S-transferase (GST) Ya, Yb, and Yc isoforms were purchased from Biotrin.

    Cell isolation and culture. Male Sprague-Dawley rats were purchased from the Taiwan National Animal Breeding and Research Center. At the age of 8 wk, the rats were used for hepatocyte isolation in the following experiments. The rats were housed in stainless steel grid cages with an artificial 12-h light–dark cycle, had free access to food (Laboratory Rodent Diet; PMI Feeds) and water, and were treated in compliance with the 1985 NRC guidelines (19).

Rat hepatocytes were prepared by collagenase perfusion as described by Berry and Friend (20), Bonney et al. (21) and our earlier report (11). The isolated hepatocytes (2.5 x 106) were plated on a 60-mm collagen-precoated plastic tissue culture dish (Falcon Labware) and incubated in a humidified incubator (NUAIRE) at 37°C in an air atmosphere. At 4 h after plating, the culture medium was replaced with fresh medium containing 2.0 g/L of BSA instead of FBS. The medium was then changed daily at 20 h after plating.

    Treatment. At 20 h after plating, cells (2.5 x 106) were treated with 0, 0.025, 0.05 or 0.25 mmol/L DATS (final concentration) for 0, 4, 8, and 24 h. Furthermore, to investigate the potency of DATS, some cells treated for 24 h were then incubated in fresh medium without DATS for an additional 24 h, for a total of 48 h of incubation. At various time intervals, the reaction was stopped by removing the medium and washing with cold PBS. Cells were removed with a cell scraper for further analysis.

    Cell viability. The effect of various concentrations of DATS on the viability of primary hepatocytes was evaluated by lactate dehydrogenase (LDH) leakage and microscopic examination. Intra- and extracellular LDH activity was assayed according to the method of Moldeus et al. (22). Photomicrographs were taken of the monolayer cultures for morphological examination, using a Nikon Diaphot 300 inverted-stage microscope equipped with phase contrast and camera attachments.

    Analysis for GSH and its related enzymes. Samples for GSH assay were prepared by adding 1 mL of 1 mol/L perchloric acid containing 2 mmol/L 1,10-phenanthroline to each dish. The plates were scraped and the contents centrifuged at 10,000 x g for 10 min. The acid-soluble GSH in the hepatocellular supernatant was measured by HPLC (23). Glutathione peroxidase (GPx) activity was assayed spectrophotometrically, using hydrogen peroxide as the substrate (24). Glutathione reductase activity was assayed as described by Bellomo et al. (25). Glutathione S-transferase activity in hepatic cytosol was assayed according to the method of Habig et al. (26), using CDNB as the substrate. Protein concentration of the cells was assayed by the method of Lowry et al. (27).

    Analysis by SDS-PAGE and Western blot. Levels of GST isozyme expression were assayed by SDS-PAGE and immunoblot analysis. Equal amounts of cytosol protein were treated with 10% polyacrylamine gel and electrophoresed as described by Laemmli (28). After electrophoresis, the proteins separated on SDS-polyacrylamide gel were transferred to polyvinyldiene difluoride membrane. To block the nonspecific binding, the membrane was incubated overnight at 4°C with 5% skim milk, followed by a 30 min incubation at 37°C with an antiserum mixture containing antibodies for GST Ya, Yb, and Yc isoforms. Peroxidase-conjugated secondary antibody (Vector Laboratory) was used to detect the immunoreactive bands. The intensity of each immunoreactive band was quantified by an image analyzer (AlphaImager 2000; Alpha Innotech) equipped with Alphaease software (Alpha Innotech).

    Statistical analysis. Statistical analysis was performed using SAS software (SAS Institute); ANOVA and Duncan’s multiple range test were used to identify significant differences among means (P < 0.05).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Cell viability. Lactate dehydrogenase leakage from cells incubated with 0.025 mmol/L DATS did not differ from that of the control cells during the 48-h incubation (Fig. 1). However, LDH leakage from cells incubated with 0.05 and 0.25 mmol/L DATS for 4, 8, 24, and 48 h was greater than from the control cells (P < 0.05). In addition, microscopic observation showed no changes in the morphology of hepatocytes treated with 0.025 mmol/L DATS, compared with that of the control cells (Fig. 2B, F, J, N), whereas hepatocytes incubated with 0.05 mmol/L (Fig. 2K, O) and 0.25 mmol/L DATS (Fig. 2L, P) for 24 and 48 h did show morphological changes.



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  		FIGURE 1 Lactate dehydrogenase leakage of rat primary hepatocytes that were incubated with 0 (control), 0.025, 0.05, or 0.25 mmol/L DATS at 4, 8, 24, or 48 h incubation times. Values are means ± SD, n = 3 or 4. Means at a time without a common letter differ, P < 0.05.

 


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  		FIGURE 2 Morphology of rat primary hepatocytes that were incubated with 0 (control), 0.025, 0.05, or 0.25 mmol/L DATS at 4, 8, 24, or 48 h incubation times (inverted-stage microscope equipped with phase contrast; 400x). Hepatocytes cultured for 4 h: 0 (A), 0.025 (B), 0.05 (C), or 0.25 mmol/L DATS (D). Hepatocytes cultured for 8 h: 0 (E), 0.025 (F), 0.05 (G), or 0.25 mmol/L DATS (H). Hepatocytes cultured for 24 h: 0 (I), 0.025 (J), 0.05 (K), or 0.25 mmol/L DATS (L). Hepatocytes cultured for another 24 h (48 h incubation total) with fresh medium without DATS (M–P). Bar = 50 µm.

 
    Cell GSH concentration. Hepatocytes treated with 0.025 and 0.05 mmol/L DATS for 4 h or more had higher intracellular GSH levels than the control cells (70–80 and 30–40%, respectively; P < 0.05; Fig. 3). Conversely, intracellular GSH levels of hepatocytes treated with 0.25 mmol/L DATS did not differ from those of the control cells at any incubation time.



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  		FIGURE 3 Intracellular GSH levels in rat primary hepatocytes that were incubated with 0 (control), 0.025, 0.05, or 0.25 mmol/L DATS at 4, 8, 24, or 48 h incubation times. Values are means ± SD, n = 3 or 4. Means at a time without a common letter differ, P < 0.05.

 
    Cell GPx, GRd, and GST activity. The level of GPx activity in hepatocytes treated with 0.025 mmol/L DATS tended to be greater than in the control cells at 24 and 48 h (P = 0.1), and was greater in cells treated with 0.05 mmol/L DATS (P < 0.05; Table 1). Cells treated with 0.25 mmol/L DATS did not differ from untreated hepatocytes.


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  		TABLE 1 Levels of GPx, GRd, and GST activity in rat primary hepatocytes that were incubated with 0 (control), 0.025, 0.05, or 0.25 mmol/L DATS at 24 and 48 h1, 2

 
The level of GRd activity in hepatocytes treated with 0.025 mmol/L DATS was greater than in the control cells at 24 h (P < 0.05; Table 1). Cells treated with 0.25 mmol/L DATS did not differ from untreated hepatocytes.

The level of GST activity in hepatocytes treated with 0.025 or 0.05 mmol/L DATS was greater than in the control cells s at 24 and 48 h (P < 0.05; Table 1). Cells treated with 0.25 mmol/L DATS did not differ from untreated hepatocytes.

    Cell GST isozyme expression. The band intensity of GST Ya, Yb, and Yc expression (Fig. 4), quantified by an image analyzer (Table 2), showed that the expression of all 3 GST isozymes was greater in hepatocytes treated with 0.025, 0.05, and 0.25 mmol/L DATS than in the control cells at 24 and 48 h (P < 0.05). The expression of GST was greatest in hepatocytes treated with 0.05 mmol/L DATS for 48 h. The expression levels of GST Ya, Yb, and Yc in hepatocytes treated with 0.025, 0.05, and 0.25 mmol/L DATS were ~1.9–3.0, 2.5–4.4, and 2.1–4.0 times the control values at 24 h and 2.0–3.9, 2.4–4.8, and 2.0–4.0 times the control values at 48 h.



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  		FIGURE 4 Expression of GST isoforms in rat primary hepatocytes that were incubated with 0 (control), 0.025, 0.05, or 0.25 mmol/L DATS at 24 and 48 h. Cytosol proteins (2 µg) were electrophoresed on a 10% polyacrylamide gel. After electrophoresis, the proteins were transferred to polyvinylidene difluoride membrane and immunostained. The expression of protein levels was evaluated in 3 separate experiments. Lanes 1 to 4 show hepatocytes treated with 0, 0.025, 0.05, or 0.25 mmol/L DATS, respectively, at 24 and 48 h of incubation.

 

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  		TABLE 2 Immunoblot band intensity of GST Ya, Yb, and Yc isozymes in rat primary hepatocytes that were incubated with 0 (control), 0.025, 0.05, or 0.25 mmol/L DATS at 24 and 48 h1, 2

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Isolated and cultured rat primary hepatocytes provide a useful model for studies of liver cell function in vitro (29). Cell viability was determined by LDH leakage assay and microscopic examination. The LDH leakage assay is both sensitive as an indicator of membrane damage and easy to perform (22). The more LDH leakage, the more plasma membrane damage. Results indicated that incubation with 0.025 mmol/L DATS for 48 h did not affect the viability of hepatocytes, but treatment with 0.05 and 0.25 mmol/L DATS for 24 h increased LDH leakage (P < 0.05). However, after 24 h of incubation, LDH leakage decreased when the culture medium was replaced with fresh medium without DATS.

These results indicated that the viability of hepatocytes might recover in the absence of treatment with higher concentrations of DATS. Hepatocyte LDH leakage was dose-dependent on the DATS concentration. However, treatment with 0.05 and 0.25 mmol/L DATS caused marked morphological changes in hepatocytes after 24 and 48 h of incubation. Microscopic examination showed close correlation with the LDH leakage data.

The organosulfur compounds DAS, DADS, and DATS are the major components of garlic oil (1). The LDH leakage data from the present study indicate that the cytoxicity of DATS is much higher than that of DAS and DADS, in contrast to the data published by Sheen et al. (11,12). The present results support the data published by Sundaram and Milner (30), which showed that DAS, DADS, and DATS markedly inhibited the growth of canine mammary tumor cells (CMT-13). The effectiveness of DAS, DADS, and DATS was related to the number of sulfur atoms and was ranked in the following order: DATS > DADS > DAS. Diallyl trisulfide is more reactive with the thiol group of cysteine than DADS (31). Sakamoto et al. (17) showed that the exposure of cultured human A549 lung tumor cells to 1 µmol/L DATS for 24 h significantly induced apoptosis, as indicated by increased DNA fragmentation, and also showed that DATS was more cytoxic to human A549 lung tumor cells than DADS. Interestingly, 1 µmol/L DATS could markedly inhibit the growth of human lung neoplastic cells (17), but in the present study, treatment with 25 µmol/L (0.025 mmol/L) DATS did not affect normal liver cells. The selective toxicity of DATS to lung neoplastic cells and normal liver cells is notable. There is great potential for DATS as an anti–lung cancer phytochemical.

Glutathione is the main intracellular thiol-containing molecule that plays an important role in hepatic antioxidation and drug-metabolism (32). Glutathione protects hepatocytes from the toxic effects of reactive oxygen species such as free radicals and peroxides (32,33). In the present study, the intracellular GSH concentration of hepatocytes treated with 0.025 or 0.05 mmol/L DATS was markedly higher than the control values (P < 0.05). This result enhances the antioxidation and detoxification capabilities of hepatocytes. Hattori et al. (34) also showed that ajoene, another active principle of garlic oil, protects against hepatic injury induced by acetaminophen administration by suppressing the depletion of hepatic GSH content.

In the GSH redox system, GSH is a substrate for GPx, which is an antioxidant enzyme that protects cellular constituents from the damaging effects of peroxides formed through metabolism or through other reactive oxidant species reactions. Glutathione peroxidase plays an important role in protecting cell proteins and cell membranes against oxidation. Glutathione reductase is a flavoprotein that regenerates GSH and provides reducing power for various thiol-coupled transferases and peroxidases (33). In the present study, GPx activity in hepatocytes treated with 0.025 or 0.05 mmol/L DATS was higher than the control values at 24 and 48 h. Treatment with 0.025 mmol/L DATS also significantly increased GRd activity at 24 h. Therefore, DATS may modulate the GSH redox system by regulating the activity of GPx and GRd.

Glutathione S-transferase catalyzes the interaction of GSH with many types of compounds. Glutathione S-transferase is a family of multifunctional enzyme proteins that detoxifies a wide variety of electrophilic xenobiotics (35). The GST family is composed of several isozymes in a homo- or heterodimer (36). Glutathione S-transferase expression is highly inducible in the presence of various drugs and toxicants (37). In the present study, except for 0.025 mmol/L DATS treatment at 24 h, the GST activity of hepatocytes treated with 0.025 or 0.05 mmol/L DATS was markedly greater than the control values at 24 and 48 h of incubation (P < 0.05). These results also showed beneficial effects on the hepatocyte detoxification system. In addition, accompanying the increased GST activity, 0.025 or 0.05 mmol/L DATS treatment also increased the protein levels of GST Ya, Yb and Yc in hepatocytes at 24 and 48 h of incubation (P < 0.05). The increases in GST expression and activity contribute to the anticarcinogenic, antitumorigenic activity (10,35,38).

In conclusion, 0.025 or 0.05 mmol/L DATS could enhance the antioxidation and detoxification capabilities of rat primary hepatocytes by increasing the intracellular GSH level and the activity of GPx, GRd, and GST. However, 0.05 or 0.25 mmol/L DATS could adversely affect the viability of hepatocytes.


   FOOTNOTES
 
1 Supported by a grant from the National Science Council of Taiwan (NSC 90–2815-C-039–006-B). Back

3 Abbreviations used: BP, benzo(a)pyrene; BSA, bovine serum albumin; DADS, diallyl disulfide; DAS, diallyl sulfide; DATS, diallyl trisulfide; GO, garlic oil; GPx, glutathione peroxidase; GRd, glutathione reductase; GSH, glutathione; GST, glutathione S-transferase; LDH, lactate dehydrogenase; NQO, NAD(P)H:quinone oxidoreductase; OSC, organosulfur compound. Back

Manuscript received 20 August 2003. Initial review completed 22 September 2003. Revision accepted 16 January 2004.


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