Oxidative and nitrosative stress and apoptosis in the liver of rats fed on high methionine diet: Protective effect of taurine

Nutrition 25 (2009) 436 – 444 www.nutritionjrnl.com

Basic nutritional investigation

Oxidative and nitrosative stress and apoptosis in the liver of rats fed on high methionine diet: Protective effect of taurine Seda Yalçınkaya, M.D.a, Yes¸im Ünlüçerçi, M.D.a, Murat Giris¸, M.S.a, Vakur Olgaç, Ph.D.b, Semra Dog˘ru-Abbasog˘lu, M.D., Ph.D.a, and Müjdat Uysal, M.D.a,* a

Department of Biochemistry, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey Department of Pathology, The Institute of Oncology, Istanbul University, Istanbul, Turkey

b

Manuscript received May 5, 2008; accepted September 22, 2008.

Abstract

Objective: There are few reports about the direct toxic effects of hyperhomocysteinemia on the liver. We investigated oxidative and nitrosative stresses and apoptotic and necrotic changes in the liver of rats fed a high-methionine (HM) diet (2%, w/w) for 6 mo. We also investigated whether taurine, an antioxidant amino acid, is protective against an HM-diet–induced toxicity in the liver. Methods: Lipid peroxide levels, nitrotyrosine formation, and non-enzymatic and enzymatic antioxidants were determined in livers of rats fed an HM diet. In addition, apoptosis-related proteins, proapoptotic Bax and antiapoptotic B-cell lymphoma-2 expressions, apoptotic cell count, histopathologic appearance in the liver, and alanine transaminase and aspartate transaminase activities in the serum were investigated. Results: Plasma homocysteine levels and serum alanine transaminase and aspartate transaminase activities were increased after the HM diet. This diet resulted in increases in lipid peroxide and nitrotyrosine levels and decreases in non-enzymatic and enzymatic antioxidants in liver homogenates in rats. Bax expression increased, B-cell lymphoma-2 expression decreased, and apoptotic cell number increased in livers of rats fed an HM diet. Inflammatory reactions, microvesicular steatosis, and hepatocyte degeneration were observed in the liver after the HM diet. Taurine (1.5%, w/v, in drinking water) administration and the HM diet for 6 mo was found to decrease serum alanine transaminase and aspartate transaminase activities, hepatic lipid peroxide levels, and nitrotyrosine formation without any change in serum homocysteine levels. Decreases in Bax expression, increases in B-cell lymphoma-2 expression, decreases in apoptotic cell number, and amelioration of histopathologic findings were observed in livers of rats fed with the taurine plus HM diet. Conclusion: Our results indicate that taurine has protective effects on hyperhomocysteinemiainduced toxicity by decreasing oxidative and nitrosative stresses, apoptosis, and necrosis in the liver. © 2009 Elsevier Inc. All rights reserved.

Keywords:

Homocysteine; Taurine; Oxidative stress; Nitrosative stress; Apoptosis; Liver

Introduction The composition and amount of diet are effective factors in the pro-oxidant–antioxidant balance in the organism [1,2]. It has been suggested that dietary proteins may influ-

This study was supported by the Research Fund of Istanbul University (project nos. T-694/30062005, UDP-1777/23112007). * Corresponding author. Tel.: ⫹90-212-414-2188; fax: ⫹90-212-6215642. E-mail address: [email protected] (M. Uysal). 0899-9007/09/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2008.09.017

ence pro-oxidant–antioxidant balance and this potential of proteins is related to their amino acid composition, especially their methionine content [1–3]. A diet with highmethionine (HM) dietary content could be detrimental due to its conversion to homocysteine (Hcys), because methionine metabolism is the only known source for Hcys in mammals [2,4,5]. In contrast, methionine residues of proteins are among the amino acids that are most susceptible to oxidation by reactive oxygen species and the sensitivity of proteins to oxidative stress increases as a function of their number of methionine residues [1–3]. Oxidation of methi-

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onine residues generates methionine sulfoxide in proteins and may lead to loss of their biological activity [3]. Although it is debatable whether the harmful effects of an HM diet on tissues is related to hyperhomocysteinemia (HHcys) and/or to HM [1–3,5], an HM diet is usually used to produce HHcys in experimental animals [2]. The role of HHcys in the development of atherosclerosis has been disputed. HHcys is thought to be a risk factor for atherosclerotic cardiovascular diseases [4 – 6]. Indeed, some investigators have found that an HM diet caused atherosclerotic changes in experimental animals [7–9]. Excessive methionine uptake also has been reported to produce direct toxic effects in some tissues such as the heart [10 –12], brain [13,14], and liver [15,16]. Although the mechanisms are not clearly understood, oxidative stress has been proposed to play a role in the development of these toxic effects [7–16]. Some investigators have shown that an HM diet induces a pro-oxidant state in the liver of experimental animals [15–20] and results in steatosis, inflammatory reactions, and hepatocyte damage [15,16]. The induction of apoptosis and/or necrosis is known to lead to tissue damage [21]. Indeed, some investigators have suggested that HHcys may stimulate proapoptotic mechanisms, but findings showing a pro-apoptotic role of HHcys usually depend on in vitro experiments [22–25]. In vivo efficiency of apoptotic mechanisms in HHcys have not been confirmed sufficiently. Although an HM diet has been found to induce apoptosis in neuronal cells of rats [14], apoptosis has not been detected in the liver of mice with severe HHcys showing cystathionine ␤-synthase deficiency [26]. Therefore, we investigated oxidative and nitrosative stresses, apoptosis, and necrosis in livers of rats fed an HM diet (2%, w/w) for 6 mo to produce HHcy. For this reason, lipid peroxide levels, nitrotyrosine (NT) formation, and non-enzymatic and enzymatic antioxidants were assessed in the livers of rats fed an HM diet. In addition, apoptosisrelated proteins, proapoptotic Bax and antiapoptotic B cell lymphoma-2 (Bcl-2) expressions, and apoptotic cell count were investigated. Histopathologic appearance in the liver and alanine transaminase (ALT) aspartate transaminase (AST) activities in the serum were also determined. Moreover, taurine (2-aminoethanesulfonic acid) is the major intracellular free ␤-amino acid, which is normally present in most mammalian tissues [27]. It has various cytoprotective properties such as antioxidation, osmoregulation, membrane stabilization, and neurotransmission [27,28]. Taurine also has been reported to have antiapoptotic properties [29 –32]. Taurine was found to be a protective agent against oxidative stress–induced hepatic pathologies [33–37]. However, there was only one study about the effect of taurine on HHcys-induced tissue damage [11]. The present study was undertaken to investigate the effect of taurine on oxidative and nitrosative stresses, apoptosis, and necrosis in the liver tissue of rats fed an HM diet.

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Materials and methods Animal and treatments Male Wistar rats weighing 200 –250 g were used in the study. They were obtained from the Experimental Medical Research Institute of Istanbul University. All animals were housed in wire-mesh– bottomed cages in a 12-h light/dark cycle and kept in a room at a constant temperature 22 ⫾ 2°C. They were allowed free access to tap water and standard chow diet. Food and water consumption were measured periodically. The experimental procedure used in this study met the guidelines of the animal care and use committee of Istanbul University. L-methionine, taurine, and other chemicals were supplied from Sigma (St. Louis, MO, USA). The animals were divided into four groups: 1) the control (C) group was fed commercial rat chow, 2) the taurine (T) group was fed the C diet plus taurine (15 g/L) in drinking water for 6 mo, 3) the HM group was fed the C diet enriched with methionine (2%; w/w) for 6 mo, and 4) the taurine plus HM (T ⫹ HM) group received taurine (15 g/L) in drinking water and the HM diet for 6 mo. Blood and tissue samples At the end of the 6-mo feeding period, animals were fasted overnight and then anesthetized with diethyl ether. Blood samples were collected by cardiac puncture into dry tubes containing ethylene-diaminetetra-acetic acid. Serum and ethylene-diaminetetra-acetic acid/plasma were obtained by centrifugation. Liver tissue was rapidly removed, washed in ice-cold saline, and kept in ice. The materials were stored at ⫺80°C until they were analyzed. Laboratory analysis in plasma/serum Plasma Hcys levels were measured with high pressure liquid chromatography [38]. Serum ALT and AST activities were determined with enzymatic methods using a Roche autoanalyzer (Cobas Integra 800, Roche Diagnostics, Manneheim, Germany). Determination of lipid peroxides and non-enzymatic and enzymatic antioxidants in the liver Liver tissues were homogenized in ice-cold 0.15 mol/L of KCl (10%, w/v). Lipid peroxidation was assessed by two different methods in the tissue homogenates. First, levels of malondialdehyde (MDA) were measured by a thiobarbituric acid test [39]. The breakdown product of 1,1,3,3tetraethoxypropane was used as a standard. Second, diene conjugate (DC) levels were determined in tissue lipid extracts at 233 nm spectrophotometrically and calculated using a molar extinction coefficient of 2.52 ⫻ 104 mol · L⫺1 · cm⫺1 [40]. Glutathione (GSH) levels were measured in liver homoge-

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nates with 5,5-dithiobis-(2-nitrobenzoate) at 412 nm [41]. Vitamin E and vitamin C levels were measured in tissue homogenates by the methods of Desai [42] and Omaye et al. [43], respectively. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione transferase (GST) activities were determined in postmitochondrial fraction of the liver, which was separated by sequential centrifugation. In brief, tissue homogenates were centrifuged at 600 ⫻ g for 10 min at 4°C to remove crude fractions. Then, supernatants were centrifuged at 10 000 ⫻ g for 20 min to obtain the postmitochondrial fraction. SOD activity was assayed by its ability to increase the effect of riboflavinsensitized photo-oxidation of o-dianisidine [44]. GSH-Px [45] and GST [46] activities were measured using cumene hydroperoxide and 1-chloro-2,4-dinitrobenzene as substrates, respectively. Protein levels were determined using bicinchoninic acid [47]. Western blot analysis for NT Liver samples (10%, w/vol) were homogenized in a solution containing 50 mmol/L of Tris-HCl (pH 7.4); 1% Igepal 630; 0.25% sodium deoxycholate; 150 mmol/L of NaCl; 1 mmol/L of EGTA; 1 mmol/L of NaF; aprotinin, leupeptin, and soybean trypsin inhibitor (1 ␮g/mL each) at 0 – 4°C using a polytron homogenizer and homogenates were centrifuged at 12 000 ⫻ g at 4°C for 5 min. Liver NT formation was determined by western blot as previously described [48]. Briefly, 25 ␮g of proteins was mixed with Laemmli buffer, loaded without heating, separated by 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis and transferred to the polyvinylidene diflouride membranes (Millipore) [49]. Rabbit anti-NT polyclonal antibody (Upstate Biotechnology) was incubated with the membranes for 16 h at 4°C and then exposed to peroxidase-conjugated goat immunoglobulin G as a second antibody (Upstate Biotechnology) at 1:3000 dilutions. After washing, immunodetection was carried out using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, NJ, USA). Western blot analysis indicated that there was a major group of NTcontaining proteins recognized by the anti-NT antibody in the hepatic tissues of all rats. This group was detected as

having a band with a molecular mass of 66 –215 kDa. The band was quantitated using image analysis software (Vilber Lourmat Biotechnology-Bio-Profil). Immunohistochemistry for Bcl-2 and Bax All specimens were fixed in 10% buffered formalin. Paraffin blocks prepared from routinely processed specimens were cut into 5-␮m slices and deparaffinized. Antigen retrieval was performed after this process. After microwave incubation of the peroxy block, followed by the ultra V block procedure, primary antibodies Bax mouse monoclonal immunoglobulin G2b (1/100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bcl-2␣ Ab-1 (100/D5), and MS-123-R7 (ready-to-use Neomarkers) were applied. After this process biotinylated secondary antibody (goat anti-mouse immunoglobulin G/horseradish peroxidase; Santa Cruz Biotechnology), streptavidin peroxidase and substratechromogen (AEC) solution were applied, respectively. Nuclear staining was done with hematoxylin. The samples of liver tissue were fixed for histopathologic examination. Five samples for each group were selected randomly and their paraffin blocks were prepared. At least four paraffin blocks were investigated. The pathologic changes in tissues were observed and evaluated by two independent researchers with a modified histopathologic score formula. Terminal deoxyuridine triphosphate biotin nick-end labeling assay Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling (TUNEL) staining was performed using an ApopTag R Peroxidase in situ apoptosis detection kit (S7101; Chemicon, Temecula, CA, USA) according to the manufacturer’s protocol, with minor modifications. The paraffin-embedded liver sections were deparaffinized and rehydrated and then treated with proteinase K at 20 ␮g/mL for 15 min at room temperature. Sections were incubated with reaction buffer containing terminal deoxynucleotidyl transferase enzyme at 37°C for 1 h. After wash-

Table 1 Plasma Hcys levels and serum ALT and AST activities and hepatic MDA and DC levels in rats fed an HM diet with and without taurine treatment* C (n ⫽ 6) Hcys (␮mol/L) ALT (U/L) AST (U/L) MDA (nmol/g) DC (␮mol/g)

T (n ⫽ 6)

6.16 ⫾ 0.88 41.2 ⫾ 11.9a 118.8 ⫾ 13.8a 262.8 ⫾ 20.7a 2.21 ⫾ 0.21a a

HM (n ⫽ 8)

5.75 ⫾ 0.93 41.0 ⫾ 10.9a 117.5 ⫾ 10.8a 256.0 ⫾ 15.0a 2.13 ⫾ 0.40a a

T ⫹ HM (n ⫽ 8)

75.0 ⫾ 39.5 84.2 ⫾ 16.0b 194.8 ⫾ 45.8b 339.0 ⫾ 40.1b 2.98 ⫾ 0.55b b

71.5 ⫾ 27.8b 50.9 ⫾ 12.8ac 139.6 ⫾ 17.0c 296.7 ⫾ 19.3c 2.46 ⫾ 0.67ab

ALT, alanine transaminase; AST, aspartate transaminase; C, control; DC, diene conjugate; Hcys, homocysteine; HM, high methionine; MDA, malondialdehyde; T ⫹ HM, taurine plus high methionine; T, taurine * All values are presented as mean ⫾ SD. Values not sharing a common letter are significantly different by Kruskal-Wallis test followed by Mann-Whitney U test (P ⬍ 0.05).

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Table 2 GSH, vitamin E, and vitamin C levels and SOD, GSH-Px, and GST activities in livers of rats fed an HM diet with and without taurine treatment* C (n ⫽ 6) GSH (␮mol/g) Vitamin E (nmol/g) Vitamin C (␮mol/g) SOD (U/mg protein) GSH-Px (nmol · min⫺1 · mg⫺1 protein) GST (nmol · min⫺1 · mg⫺1 protein)

T (n ⫽ 6)

6.11 ⫾ 0.70 46.3 ⫾ 9.45ab 1.23 ⫾ 0.13a 31.7 ⫾ 1.91a 995.0 ⫾ 99.2a 510.2 ⫾ 34.6a a

HM (n ⫽ 8)

5.66 ⫾ 0.74 46.5 ⫾ 5.16b 1.22 ⫾ 0.15a 31.3 ⫾ 2.55a 970.8 ⫾ 95.6ab 513.3 ⫾ 52.0a

ab

T ⫹ HM (n ⫽ 8)

4.93 ⫾ 0.52 35.2 ⫾ 7.62a 1.15 ⫾ 0.15a 26.6 ⫾ 3.93a 675.4 ⫾ 179.1c 442.5 ⫾ 64.7b b

5.86 ⫾ 0.66ac 47.2 ⫾ 4.92b 1.24 ⫾ 0.14a 31.0 ⫾ 5.77a 827.0 ⫾ 170.2bc 434.5 ⫾ 62.8b

C, control; GSH, glutathione; GSH-Px, glutathione peroxidase; GST, glutathione transferase; HM, high methionine; SOD, superoxide dismutase; T ⫹ HM, taurine plus high methionine; T, taurine * All values are presented as mean ⫾ SD. Values not sharing a common letter are significantly different by Kruskal-Wallis test followed by Mann-Whitney U test (P ⬍ 0.05).

ing with stop/wash buffer, sections were treated with an anti-digoxigenin conjugate for 30 min at room temperature and subsequently developed color with a peroxidase substrate. The nuclei were lightly counterstained with 0.5% methyl green. The cells showing nuclear dark-brown staining pattern were accepted as having positive immunoreactivity. Microscopic analysis was performed under highpower magnification (40⫻) in a blinded fashion and 10 random high-power fields were counted for each TUNELstained tissue sample (Olympus Bx51-P). Histopathologic analysis Liver tissues were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histologic studies.

3. The GSH and vitamin E levels and GSH-Px and GST activities were found to decrease in the livers of rats in the HM compared with the C group, but vitamin C levels and SOD activity remained unchanged. The T ⫹ HM diet caused increases in hepatic GSH and vitamin E levels, but no changes were observed in the levels of other antioxidants (Table 2). 4. A significant increase in NT formation expression was observed in the livers of rats after the HM diet. NT expression decreased in the livers of rats in the T ⫹ HM group (Fig. 1). 5. Bax expression was increased and Bcl-2 expression was decreased in the livers of rats in the HM group. The T ⫹ HM diet caused decreases in Bax expression and increases in Bcl-2 expression in the livers of rats (Fig. 2).

Statistical analysis The results were expressed as mean ⫾ SD. Experimental groups were compared using Kruskal-Wallis variance analysis test. Where significant effects were found, post hoc analysis using the Mann-Whitney U test was performed, and P ⬍ 0.05 was considered statistically significant. Results The weight gain of rats during the 6-mo period was not significantly different among groups. Liver weights and the ratios of liver weights to body weights also were not found to be significantly different (data not shown). The results obtained in this study are presented in Tables 1 and 2 and Figures 1– 4. Accordingly: 1. Plasma Hcy levels and serum ALT and AST activities were detected to be increased in rats fed an HM diet compared with controls. Plasma Hcy levels did not alter, but serum ALT and AST activities were decreased in rats given the T ⫹ HM diet (Table 1). 2. Hepatic MDA and DC levels were increased in the HM group. These levels decreased after the T ⫹ HM diet (Table 1).

Fig. 1. Western blot analysis of (a) NT formation, (b) ␤-tubulin expression, and (c) densitometric quantification of NT formation in the livers of rats fed on an HM diet with and without taurine. Results are average values of four or six animals. Values not sharing a common superscript letter are significantly different by the Kruskal-Wallis test (P ⬍ 0.05). C, control; HM, high methionine; NT, nitrotyrosine; T ⫹ HM, taurine plus high methionine; T, taurine.

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Fig. 2. Immunohistochemistry of Bcl-2 and Bax expressions in rats fed an HM diet with and without taurine (magnification 400⫻). Bcl-2, B-cell lymphoma-2; C, control; HM, high methionine; T ⫹ HM, taurine plus high methionine; T, taurine.

6. In the HM group, the apoptotic cell count was increased in the livers of rats. A decrease in the apoptotic cell count was observed in the livers in the T ⫹ HM group (Fig. 3). 7. Histopathologic examination showed increases in lymphocyte and neutrophil infiltration in the central and portal areas in the livers of the HM group. In some areas, microvesicular steatosis and hepatocyte degeneration also were observed. In the T ⫹ HM group, inflammatory reactions decreased and other changes were almost absent in sections of livers (Fig. 4).

Discussion Oxidative stress is defined as a disturbance in the prooxidant/antioxidant balance within tissues [21]. It has been thought that oxidative stress is one of the causative factors in HHcys-induced tissue damage [50]. Methionine feeding has often been used to elevate serum and tissue Hcys levels to study the pathogenesis of HHcys-related metabolic disturbances. Lipid peroxide levels have been observed to increase together with changes in antioxidant system parameters in livers of experimental animals fed with an HM diet [15–20]. However, there are some differences in results

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Fig. 3. Apoptotic cell death detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling method in rats fed an HM diet with and without taurine (arrows indicate apoptotic cells, magnification 400⫻). C, control; HM, high methionine; T ⫹ HM, taurine plus high methionine; T, taurine.

obtained from these studies. This situation may be related to te methionine content of the diet, application time, and animals used in these experiments. In the present study, daily methionine supplementation at 1 g/kg of body weight was achieved for 6 mo in rats and plasma Hcys was detected to be strongly increased after an HM diet. Hepatic MDA and DC levels were found to increase, but GSH and vitamin E

levels and GSH-Px and GST activities decreased in the liver after the HM diet. This diet did not alter vitamin C levels and SOD activity in the liver after the HM diet. These results indicate that the HM diet induces a pro-oxidant state in the liver and are in accordance with previous studies [15–20]. As for nitrosative stress, there are two studies in the literature reporting NT levels, an indicator of peroxyni-

Fig. 4. Hepatic histopathology of rats fed an HM diet with and without taurine (hematoxylin and eosin staining, magnifications 200⫻ for C and T and 400⫻ for HM and T ⫹ HM). C, control; HM, high methionine; T ⫹ HM, taurine plus high methionine; T, taurine.

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trite formation in tissues in HHcys. NT levels determined using western blot analysis were found to increase in the myocardia of HHcys rats [51]. In another study, NT formation detected by an immunohistochemical procedure was found to increase in the liver after an HM diet [16]. In our study, we also detected that NT formation increased in the livers of rats fed an HM diet. These results indicate that nitrosative stress was also induced in the liver after an HM diet. Although several investigators have reported that an HM diet induces a pro-oxidant state in the livers of experimental animals, there are only two studies in the literature about the resulting steatosis, inflammatory reactions and hepatocyte damage in hyperhomocysteinemic conditions [15,16]. Oxidative and nitrosative stresses are known to induce apoptotic and necrotic events in tissues and this induction leads to tissue damage [21,52]. Therefore, the first aim of this study was to investigate whether there is induction of apoptosis and necrosis in livers of rats fed an HM diet. In our study, the effect of an HM diet on liver necrosis was assessed by biochemical and histopathologic findings. Serum ALT and AST activities increased significantly and histopathologic examination showed increases in lymphocyte and neutrophil infiltration, microvesicular steatosis, and hepatocyte degeneration after an HM diet. These results clearly show that an HM diet causes hepatocyte damage. Apoptosis also leads to cell death and differs from necrosis by distinct morphologic and biochemical features [21,52]. There are two classes of regulatory proteins in the Bcl-2 family that have opposite effects on apoptosis: the proapoptotic members (Bax, Bcl-XS) promote programmed cell death, whereas the antiapoptotic members (Bcl-2, BclXL) protect cells against apoptosis. Members of the Bcl-2 family play a key role in apoptosis, because they decrease mitochondrial membrane permeability and prevent the release of cytochrome c into the cytoplasm. Therefore, the ratio of Bax to Bcl-2 is accepted to be a parameter of apoptotic cell death [21,52]. In the literature, there are only two studies that investigated the in vivo effect of HHcys on apoptosis [14,26]. It has been reported that an HM diet causes increases in proapoptotic Bax levels and decreases in antiapoptotic Bcl-2 levels [14]. The investigators also found activations of caspase-3 and caspase-9, release of cytochrome c from mitochondria to the cytosol, and fragmentation of DNA in neuronal cells of rats [14]. However, Robert et al. [26] reported that HHcys caused liver injury by increasing oxidative stress and damaging mitochondria in mice with severe HHcys showing cystathionine ␤-synthase deficiency. In these mice, increases in proapoptotic Bax levels and decreases in antiapoptotic Bcl-2 levels were observed in the liver, but caspase-3 activity and DNA fragmentation remained unchanged [26]. However, in our study, Bax expression was found to be increased, but Bcl-2 expression decreased together with an increased number of apoptotic cells, indicating DNA fragmentation, in the livers

of rats fed an HM diet. Therefore, our results show that apoptosis is also involved in HM-diet–induced liver injury. The second aim of this study was to investigate the effect of taurine treatment on HHcys-induced pro-oxidant state together with apoptotic and necrotic events in the liver. There is one study about the effect of taurine treatment on HHcys-induced tissue damage in the literature and this study was done in myocardial tissue [11]. These investigators reported that taurine effectively attenuated the HHcysinduced reactive oxygen species production and inhibited manganese-SOD and catalase activities in the myocardial mitochondria in rats fed an HM diet [11]. In our study, the effect of taurine on HM-diet–induced liver injury was tested. The daily amount of taurine received by rats was approximately 250 mg/rat according to average water intake. The given taurine concentration is a pharmacologic dose and similar doses were used by several investigators as previously reported [32,35,37]. We found that taurine treatment plus an HM diet reduced MDA and DC levels and NT formation and increased hepatic GSH and vitamin E levels, without affecting changes in other antioxidants. Several mechanisms may play a role in taurine-mediated reduction in oxidative stress. Taurine was reported to protect cells by scavenging oxygen free radicals, by upregulating the antioxidant defenses, forming chloramines with HOCl, or binding free metal ions such as Fe2⫹ by its sulfonic acid group [27,28]. Because cysteine is a precursor of taurine and GSH, taurine supplementation may cause enhancement in GSH levels by directing cysteine into the GSH synthesis pathway. Therefore, increased GSH levels after taurine treatment may play an additional role in decreasing oxidative stress. Conversely, the T ⫹ HM diet ameliorated the changes in Bax and Bcl-2 expressions and decreased apoptotic cell counts in the livers of rats. Therefore, it is considered that taurine treatment ameliorates Hcys-induced apoptosis by reducing proapoptotic pathway activation and preventing the loss of the antiapoptotic way through inhibition of oxidative and nitrosative stresses in the liver. In addition, taurine treatment was found to decrease serum ALT and AST activities and to ameliorate histopathologic changes in the liver due to the HM diet. In conclusion, this study is the first in the literature showing an in vivo preventive effect of taurine in HHcysinduced liver injury. Our results show that taurine administration may exert beneficial effects in Hcys-induced liver toxicity by decreasing oxidative and nitrosative stresses, apoptosis, and necrosis.

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