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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:149-152, January 2004

Nutrient Interactions and Toxicity
Research Communication

Five Cysteine-Containing Compounds Have Antioxidative Activity in Balb/cA Mice1

Cheng-chin Hsu, Chien-ning Huang*, Yu-chuan Hung and Mei-chin Yin2

Department of Nutritional Science, Chungshan Medical University, Taichung City, Taiwan and * Department of Internal Medicine, Chungshan Medical University Hospital, Taichung City, Taiwan

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


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Balb/cA mice were used to study the in vivo effect of N-acetyl cysteine, S-allyl cysteine, S-ethyl cysteine, S-methyl cysteine and S-propyl cysteine, all derived from garlic, on glutathione (GSH) concentration and catalase and glutathione peroxidase (GPX) activities in plasma, kidney and liver. Cysteine was used for comparison. The effects of these compounds on the levels of fibronectin, triglyceride (TG), cholesterol and {alpha}-tocopherol were also evaluated. Cysteine or cysteine-containing compounds were added to drinking water at 1 g/L. After 4 wk of treatment, GSH levels in kidney and liver were greater (P < 0.05) than in controls. Cysteine decreased catalase and GPX activities in liver, and enhanced both Fe2+- and glucose-induced lipid oxidation in plasma, kidney and liver compared with the control group (P < 0.05). However, the administration of the five cysteine-containing compounds enhanced catalase and GPX activities in kidney and liver, and reduced Fe2+- and glucose-induced lipid oxidation in plasma, kidney and liver compared with the control and cysteine-treated groups (P < 0.05). Intake of the five cysteine-containing compounds reduced fibronectin, TG and cholesterol concentrations in plasma and liver, and increased the {alpha}-tocopherol concentration in plasma, kidney and liver compared with the control and cysteine-treated groups (P < 0.05). The five cysteine-containing compounds derived from garlic had marked effects on antioxidant enzymes and spared {alpha}-tocopherol in mice. Furthermore, these compounds reduced fibronectin, TG and cholesterol concentrations in plasma. These data indicate that these compounds have a range of protective effects for cardiovascular disease prevention or therapy.

KEY WORDS: • cysteine-containing compounds • catalase • glutathione peroxidase • triglyceride

The in vivo effect of garlic extract and related lipophilic organosulfur compounds on antioxidant enzymes such as catalase and glutathione peroxidase (GPX)2 was examined (1–3). These authors indicated that garlic extract and these lipophilic compounds enhanced antioxidant protection in mice or rats. N-Acetyl cysteine (NAC), S-allyl cysteine (SAC), S-ethyl cysteine (SEC), S-methyl cysteine (SMC) and S-propyl cysteine (SPC) are hydrophilic cysteine-containing compounds naturally formed in Allium plants such as garlic and onion (4,5). However, less is known about the effect of these compounds on the activity of antioxidant enzymes. In addition, an inhibitory effect on cholesterol and triglyceride (TG) biosynthesis from SAC, SEC and SPC in cultured cells was also observed (6,7). However, the in vivo effect of these hydrophilic compounds in reducing TG and cholesterol biosynthesis remains unknown.

Several studies reported that total plasma cysteine with potential vascular toxicity is a risk factor for cardiovascular diseases (8–10). Although the above-mentioned five cysteine-containing compounds are structurally similar to cysteine, their roles in the development of cardiovascular pathogenesis should be elucidated.

The major purpose of this study was to evaluate the in vivo antioxidant protection from five cysteine-containing compounds derived from garlic in mice. The effect of these compounds on the activities of catalase and GPX and the influence of these compounds on the concentrations of fibronectin, TG, cholesterol and {alpha}-tocopherol were determined.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Animals and diets. Male Balb/cA mice, 3–4 wk old, were obtained from National Laboratory Animal Center (National Science Council, Taipei City, Taiwan). Mice were housed on a 12-h light:dark schedule, with free access to water and rat and mouse standard diet containing (g/100 g): 64 starch, 23 protein, 3.5 fat, 5 fiber, 1 vitamin mixture and 3 salt mixture (PMI Nutrition International LLC, Brentwood, MO). Use of the mice was reviewed and approved by the Chungshan Medical University animal care committee.

    Organosulfur compound treatment. Cysteine (99.5%), NAC (99.5%), SMC (99%) and SEC (99.5%) were purchased from Sigma Chemical (St. Louis, MO). SAC (99%) and SPC (99%) were supplied by Wakunaga Pharmaceutical (Hiroshima, Japan). Each compound was added to the drinking water of mice at 1 g/L.

    Experimental design. After 1 wk of acclimation, mice (n = 140) were divided into seven groups (n = 20) on the basis of their intake of water, cysteine or each of five cysteine-containing compounds. All mice had free access at all times to feed and water. The amount of water consumed daily was recorded. Body weight was measured every week. Plasma glucose level was measured every other week. After 4 wk, the mice were killed with carbon dioxide. Liver and kidney from each mouse were collected and weighed. Blood was also collected, and plasma was separated immediately from erythrocytes. Each organ (0.2 g) was homogenized on ice in 2 mL PBS, pH 7.2, and the filtrate was collected. The protein concentrations of plasma, kidney filtrate and liver filtrate were determined by the method of Lowry et al. (11) using bovine serine albumin as a standard. In all experiments, the sample was diluted to a final concentration of 1 g protein/L using PBS, pH 7.2.

    Glutathione (GSH) level determination. The GSH concentrations (nmol/mg protein) in plasma, kidney and liver were determined by a colorimetric GSH assay kit (OxisResearch, Portland, OR).

    Catalase and GPX assay. Catalase and GPX activities (U/mg protein) in plasma, kidney and liver were determined by catalase and GPX assay kits (Calbiochem, EMD Biosciences, San Diego, CA).

    Lipid oxidation determination. FeSO4 (50 µmol/L) or 50 mmol/L glucose was used to initiate lipid oxidation in plasma and the filtrate from kidney or liver. Fe2+-induced and glucose-induced lipid oxidation was measured after 3 and 7 d of incubation, respectively, at 37°C. Lipid oxidation was determined by measuring the level of malondialdehyde (MDA, µmol/L) using an HPLC method (12).

    Plasma glucose and fibronectin determination. Plasma glucose (mmol/L) and fibronectin (g/L) concentrations were determined by glucose HK kit (Sigma Chemical) and rabbit anti-rat fibronectin antibody, respectively, and quantified by solid phase immunoenzymatic ELISA (13).

    TG and cholesterol determination. TG and total cholesterol levels (mmol/L) in plasma were determined by TG/GB kit and cholesterol/HP kit (Boehringer Mannheim, Indianapolis, IN), respectively. Total lipids were extracted from liver and TG concentration was quantified by a colorimetric assay (14). Total liver cholesterol was measured using o-phthalaldehyde (15).

    {alpha}-Tocopherol analysis. The concentration of {alpha}-tocopherol in plasma (nmol/L), kidney and liver (nmol/g wet tissue) was measured by an HPLC method (16).

    Statistical analysis. The effect of each treatment was analyzed for the 20 mice in each group. Data were subjected to ANOVA and computed using the SAS General Linear Model (GLM) procedure (17). Differences with P < 0.05 were considered to be significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
The body weights (9.9–11.6 g), daily water volume consumed (4.7–5.4 mL), liver weight (0.68–0.73 g), kidney weight (0.27–0.30 g) and plasma glucose concentrations (6–7.2 mmol/L) of mice were not affected by treatment with cysteine or cysteine-containing compounds. Compared with the control group, intake of cysteine and cysteine-containing compounds increased GSH concentrations in kidney (24.7–65.6%; P < 0.05) and liver (41.1–174.6%; P < 0.05) (Table 1). In plasma, only the SPC group had increased (48.8%) GSH concentration (P < 0.05).


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  		TABLE 1 Glutathione in plasma, kidney and liver from mice treated with cysteine and five cysteine-containing compounds (NAC, SAC, SEC, SMC, SPC)1, 2

 
Compared with the control group, mice treated with cysteine had decreased catalase and GPX activities in liver (P < 0.05), and those treated with the other five cysteine-containing compounds had significantly greater catalase and GPX activities in kidney and liver (Table 2). Cysteine intake significantly elevated Fe2+- and glucose-induced MDA levels in plasma, kidney and liver (Table 3). However, treatment with the other five cysteine-containing compounds reduced MDA levels in plasma, kidney and liver (P < 0.05). Mice treated with the five cysteine-containing compounds had significantly lower fibronectin concentration in plasma (Table 4) compared with the control group. Cysteine intake significantly increased TG and cholesterol levels (P < 0.05) in plasma and liver (data not shown); however, mice treated with the other five cysteine-containing compounds had lower TG and cholesterol levels in plasma and liver than the control and cysteine-treated groups (P < 0.05). These mice also had higher {alpha}-tocopherol concentrations in plasma, kidney and liver compared with the control and cysteine-treated groups (P < 0.05).


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  		TABLE 2 Catalase and glutathione peroxidase (GPX) activities in plasma, kidney, liver from mice treated with cysteine and five cysteine-containing compounds (NAC, SAC, SEC, SMC, SPC)1

 

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  		TABLE 3 Iron- and glucose-induced MDA concentrations in plasma, kidney, liver from mice treated with cysteine and five cysteine-containing compounds (NAC, SAC, SEC, SMC, SPC)1

 

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  		TABLE 4 Fibronectin, triglyceride and cholesterol concentrations in plasma, and {alpha}-tocopherol concentration in plasma, kidney, liver from mice treated with cysteine and five cysteine-containing compounds (NAC, SAC, SEC, SMC, SPC)1

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The effect of NAC on GSH level, GPX and catalase activities in mice was examined (18,19). The current work not only agrees with those earlier studies but also found that the administration of four other cysteine-containing compounds could effectively elevate GSH concentration and enhance GPX and catalase activities. Furthermore, we observed that these cysteine-containing compounds spared {alpha}-tocopherol in mice in vivo, suggesting that the spared {alpha}-tocopherol could also contribute to antioxidant protection. One of our previous studies indicated that NAC and SEC possessed nonenzymatic antioxidant activities such as reducing power and metal chelating capability (20), suggesting that these compounds may enhance antioxidant protection via both enzymatic and nonenzymatic activities. The five cysteine-containing compounds are compounds naturally formed in Allium food such as garlic. On the basis of their natural, hydrophilic and amino acid-based properties, the use of these compounds as antioxidative compounds should be safe and acceptable.

Several studies have indicated that cysteine possesses autooxidation and cytotoxicity properties (8–10), suggesting that cysteine should be considered to be a risk factor for vascular disease. Our current work also found several adverse effects from cysteine, such as increased TG, cholesterol and fibronectin concentrations in plasma and decreased catalase and GPX activities in liver. These adverse effects from cysteine may favor the pathogenic development of cardiovascular diseases. Although the five cysteine-containing compounds are structurally similar to cysteine, these compounds appear to have different biochemical activity. It is highly likely that the noncysteine part in each cysteine-containing molecule plays an important role in its antioxidant activity, and TG- and cholesterol-reducing actions.

Ab inhibitory effect on cholesterol and TG biosynthesis of SAC, SPC and SEC in cultured cells was observed (6,7). The current work provided in vivo evidence to substantiate the effectiveness of these cysteine-containing compounds in reducing TG and cholesterol levels in plasma and liver. Thus, these compounds may be of benefit in hyperlipidemic and/or hypercholesterolemic prevention and therapy. The TG- or cholesterol-reducing effects from these compounds may be due to suppression of biosynthesis in liver or absorption interference in the small intestine. Further study is warranted to elucidate the mode of action of these compounds in reducing TG and cholesterol levels.

Our previous study found that NAC and SEC could protect LDL against glycation (21). The current work demonstrated that these compounds could also reduce fibronectin biosynthesis and inhibit glucose-induced oxidation. The plasma fibronectin level is markedly elevated in diabetic patients, which further contributes to the development of diabetic complications (13,22,23). Thus, these compounds might benefit diabetic patients in preventing the development of hyperfibronectin- and glycative-associated pathogenesis. Further in vivo study on the association with diabetes is required to verify the protection offered by these compounds.

In conclusion, five cysteine-containing compounds derived from garlic exhibited marked enzymatic antioxidant protection and spared {alpha}-tocopherol in mice. Furthermore, these compounds reduced fibronectin, TG and cholesterol concentrations in plasma and liver. These data indicate that these compounds have a range of protective effects for cardiovascular disease prevention or therapy.


   ACKNOWLEDGMENTS
 
The authors would like to thank Wakunaga Pharmaceutical (Hiroshima, Japan) for kindly supplying S-allyl cysteine and S-propyl cysteine.


   FOOTNOTES
 
1 Supported by grants from the National Science Council, ROC (NSC 92–2320-B-040–030). Back

3 Abbreviations used: GPX, glutathione peroxidase; GSH, glutathione; MDA, malondialdehyde; NAC, N-acetyl cysteine; SAC, S-allyl cysteine; SEC, S-ethyl cysteine; SMC, S-methyl cysteine; SPC, S-propyl cysteine; TG, triglyceride. Back

Manuscript received 27 August 2003. Initial review completed 2 October 2003. Revision accepted 14 October 2003.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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1. Chen, L., Hong, J., So, E., Husin, A. H., Cheng, W. F. & Yang, C. S. (1999) Decrease of hepatic catalase level by treatment with diallyl sulfide and garlic homogenates in rats and mice. J. Biochem. Toxicol. 13:127-134.

2. Thabrew, M. I., Samarawickrema, N. A., Chandrasena, L. G. & Jayasekera, S. (2000) Protection by garlic against adriamycin induced alterations in the oxido-reductive status of mouse red blood cells. Phytother. Res. 14:215-217.[Medline]

3. Wang, E. J., Li, Y., Lin, M., Chen, L., Stein, A. P., Reuhl, K. R. & Yang, C. S. (1996) Protective effects of garlic and related organosulfur compounds on acetaminophen-induced hepatotoxicity in mice. Toxicol. Appl. Pharmacol. 136:146-154.[Medline]

4. Sheela, C. G., Kumud, K. & Augusti, K. T. (1995) Anti-diabetic effects of onion and garlic sulfoxide amino acids in rats. Planta Med. 61:356-357.[Medline]

5. Hatono, S., Jimenez, A. & Wargovich, M. J. (1996) Chemopreventive effect of S-allylcysteine and its relationship to the detoxification enzyme glutathione S-transferase. Carcinogenesis 17:1041-1044.[Abstract/Free Full Text]

6. Han, S., Hu, Y., Anno, T. & Yanagita, T. (2002) S-propyl cysteine reduces the secretion of apolipoprotein B100 and triacylglycerol by HepG2 cells. Nutrition 18:505-509.[Medline]

7. Liu, L. & Yeh, Y. (2000) Inhibition of cholesterol biosynthesis by organosulfur compounds derived from garlic. Lipids 35:197-203.[Medline]

8. El-Khairy, L., Ueland, P., Refsum, H., Graham, I. & Vollset, S. E. (2001) Plasma total cysteine as a risk factor for vascular disease: the European concerted action project. Circulation 103:2544-2549.[Abstract/Free Full Text]

9. Jacob, N., Bruckert, E., Giral, P., Foglietti, M. J. & Turpin, G. (1999) Cysteine is a cardiovascular risk factor in hyperlipidemic patients. Atherosclerosis 146:53-59.[Medline]

10. Saez, G., Thornalley, P. J., Hill, H.A.O., Hems, R. & Bannister, J. V. (1982) The production of free radicals during the autoxidation of cysteine and their effects on isolated rat hepatocytes. Biochim. Biophys. Acta 719:24-31.[Medline]

11. Lowry, O. H., Rosebrough, N. J. & Farr, A. L. (1951) Protein determination with the Folin phenol reagent. J. Biol. Chem. 193:265-275.[Free Full Text]

12. Jain, S. K. & Palmer, M. (1997) The effect of oxygen radical metabolites and vitamin E on glycosylation or proteins. Free Radic. Biol. Med. 22:593-596.[Medline]

13. Kanters, S. D., Banga, J. D., Algra, A., Prinis, R. C., Beutler, J. J. & Fijnheer, R. (2001) Plasma levels of cellular fibronectin in diabetes. Diabetes Care 24:323-327.[Abstract/Free Full Text]

14. Biggs, H. G., Erikson, J. M. & Moorehead, W. R. (1975) A manual colorimetric assay of triglycerides in serum. Clin. Chem. 21:437-441.[Medline]

15. Rudel, L. L. & Morris, M. D. (1973) Determination of cholesterol using-o-phthalaldehyde. J. Lipid Res. 14:164-166.

16. Palozza, P. & Krinsky, N. I. (1992) The inhibition of radical-initiated peroxidation of microsomal lipids by both {alpha}-tocopherol and ß-carotene. Free Radic. Biol. Med. 11:407-414.

17. SAS Institute Inc (1990) SAS/STAT User’s Guide, version 6 1990 SAS Institute Cary, NC.

18. Demir, S. & Inal-Erden, M. (1998) Pentoxifylline and N-acetylcysteine in hepatic ischemia/reperfusion injury. Clin. Chim. Acta 275:127-135.[Medline]

19. Labib, R., Abdel-Rahman, M. S. & Turkall, R. (2003) N-Acetyl cysteine pretreatment decreases cocaine and endotoxin-induced hepatoxicity. J. Toxicol. Environ. Health 66:223-239.

20. Yin, M. C., Hwang, S. W. & Chan, K. C. (2002) Nonenzymatic antioxidant activity of four organosulfur compounds derived from garlic. J. Agric. Food Chem. 50:6143-6147.[Medline]

21. Ou, C. C., Tsao, S. M., Lin, M. C. & Yin, M. C. (2003) Protective action on human LDL against oxidation and glycation by four organosulfur compounds derived from garlic. Lipids 38:219-224.[Medline]

22. Hill, R. E. & Williams, R. E. (2002) A quantitative analysis of perineurial cell basement membrane collagen IV, laminin and fibronectin in diabetic and non-diabetic human sural nerve. J. Anat. 201:185-192.[Medline]

23. Jackle-Meyer, I., Szukics, B., Neubauer, K., Metze, V., Petzoldt, R. & Stolte, H. (1995) Extracellular matrix proteins as early markers in diabetic nephropathy. Eur. J. Clin. Chem. Clin. Biochem. 33:211-219.[Medline]




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