Article

Effects of carnosine, taurine, and betaine pretreatments on diethylnitrosamine-induced oxidative stress and tissue injury in rat liver

Toxicology and Industrial Health 1–10 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233714563432 tih.sagepub.com

C Bas¸ aran-Ku¨c¸u¨kgergin1, I_ Bingu¨l1, M Soluk Tekkes¸ in2, V Olgac¸2, S Dog˘ru-Abbasog˘lu1 and M Uysal1 Abstract Several chemicals such as N-diethylnitrosamine (DEN) promote hepatocellular cancer in rodents and induce hepatocyte injury. DEN affects the initiation stage of carcinogenesis together with enhanced cell proliferation accompanied by hepatocellular necrosis. DEN-induced hepatocellular necrosis is reported to be related to enhanced generation of reactive oxygen species. Carnosine (CAR), taurine (TAU), and betaine (BET) are known to have powerful antioxidant properties. We aimed to investigate the effects of CAR, TAU, and BET pretreatments on DEN-induced oxidative stress and liver injury in male rats. Rats were given CAR (2 g L1 in drinking water), TAU (2.5% in chow), and BET (2.5% in chow) for 6 weeks and DEN (200 mg kg1 intraperitoneally) was given 2 days before the end of this period. Serum alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and -glutamyl transferase activities were determined and a histopathologic evaluation was performed on the liver tissue. Oxidative stress was detected in the liver by measuring malondialdehyde, diene conjugate, protein carbonyl and nitrotyrosine levels, glutathione and glutathione peroxidase levels, and superoxide dismutase and glutathione transferase activities. Pretreatments with CAR, TAU, and BET decreased liver prooxidant status without remarkable changes in antioxidant parameters in DEN-treated rats. Pretreatments with TAU and BET, but not CAR, were also found to be effective to reduce liver damage in DEN-treated rats. In conclusion, TAU, BET, and possibly CAR may have an ameliorating effect on DEN-induced hepatic injury by reducing oxidative stress in rats. Keywords Hepatic injury, diethylnitrosamine, carnosine, taurine, betaine, oxidative stress

Introduction N-Diethylnitrosamine (DEN) is one of the most important hepatotoxins and hepatocarcinogens and is present in tobacco smoke, cosmetics, and various processed foods such as milk and meat products. It can also be generated endogenously from nitrate, nitrite, and secondary amines (Amin et al., 2011; Janani et al., 2010; Jayakumar et al., 2012). Hepatocellular cancer (HCC) development is known to be associated with hepatocyte death and compensatory proliferation (Glauert et al., 2010). Several chemicals such as DEN induce hepatocyte injury and promote HCC in rodents. DEN causes hepatocellular necrosis together with enhanced cell proliferation (Glauert et al., 2010; Maeda et al., 2005; Naugler et al., 2007; Qiu et al.,

2011). DEN-induced hepatic injury is reported to be related to enhanced generation of reactive oxygen species (ROS) (Bansal et al., 2000; Glauert et al., 2010; Kang et al., 2007; Sayed-Ahmed et al., 2010). The generation of ROS following DEN is related to biotransformation

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Department of Biochemistry, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey 2 Department of Pathology, Oncology Institute, Istanbul University, Istanbul, Turkey Corresponding author: Semra Dog˘ru-Abbasog˘lu, Department of Biochemistry, Istanbul Faculty of Medicine, Istanbul University, C ¸ apa, 34093, Istanbul, Turkey. Email: [email protected]

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of DEN in cytochrome P450 system (CYPs), especially CYP2E1 (Kang et al., 2007). DEN also alters the structure of DNA and forms alkyl DNA adducts in the rat liver (Amin et al., 2011; Glauert et al., 2010; He et al., 2012; Janani et al., 2010; Jayakumar et al., 2012). Based on these observations, the roles of several antioxidants on oxidative stress-induced tissue damage by wide range of carcinogens including DEN have been investigated (Amin et al., 2011; Bishayee et al., 2010; Ghosh et al., 2012; Glauert et al., 2010; He et al., 2012; Janani et al., 2010; Jayakumar et al., 2012; Ramakrishnan et al., 2006). Carnosine (CAR; -alanyl-L-histidine) is a dipeptide with antioxidant effects (Aldini et al., 2005). Its protective and antioxidant effects are due to its free radical scavenging, membrane protecting, transition metal chelating, and superoxide dismutase (SOD)-like activities (Aldini et al., 2005; Boldyrev, 2005; Hipkiss, 2009). Taurine (TAU; 2-aminoethane sulfonic acid) and betaine (BET; trimethylglycine) also have antioxidant effects and play an important role in the sulfur amino acid metabolism (Craig, 2004; Hansen, 2001). TAU, a nonprotein amino acid, is synthesized from cysteine and methionine and ingested directly with certain foodstuffs (Hansen, 2001). Its antioxidant role has been attributed to its ability to scavenge ROS, to reduce the production of lipid peroxidation end products, and to stabilize biomembranes (Acharya and Lau-Cam, 2010; Balkan et al., 2005; Dog˘ru-Abbasog˘lu et al., 2001; Hansen, 2001; Kim and Kim, 2002). BET is a metabolite of choline and is produced by choline oxidase in the liver. Plants are also a rich dietary source of BET (Craig, 2004). BET participates in the synthesis of methionine from homocysteine and restores S-adenosyl methionine (SAM) levels, which play essential roles in phospholipid metabolism and membrane structure (Ueland, 2011). We and others have reported that CAR (Artun et al., 2010; Mehmetc¸ik et al., 2008; Yan et al., 2009), TAU (Acharya and Lau-Cam, 2010; Balkan et al., 2005; Dog˘ru-Abbasog˘lu et al., 2001; Kim and Kim, 2002), and BET (Balkan et al., 2005; Kim and Kim, 2002) showed protective effects against acute liver damage possibly due to their antioxidant potentials. However, no studies in the literature have investigated the effect of dietary CAR, TAU, and BET pretreatments on liver damage and oxidative stress, which is produced by a necrogenic dose of DEN in rats. In our study, a single necrogenic dose of DEN was administered to rats to produce liver injury. This treatment caused severe increases in serum indices of liver function, hepatocyte necrosis, and prooxidant state in

liver. We wanted to investigate whether CAR, TAU, and BET pretreatments had protective effects against DENinduced oxidative stress and tissue injury.

Materials and methods Chemicals DEN, CAR, TAU, BET, and the chemicals used for biochemical determinations were purchased from SigmaAldrich Chemical Company (St Louis, Missouri, USA).

Animals and diets Male Wistar rats weighing 200–250 g were used in the study. They were obtained from the Experimental Medical Research Institute of Istanbul University. Rats were housed in a light- and temperaturecontrolled room on a 12-h light/12-h dark cycle. They were allowed free access to food and water and were kept in wire-bottomed stainless steel cages (two rats per cage). Daily food and water consumption of rats were determined during the experimental period. The procedure used in this study met the guidelines of the Animal Care and Use Committee of the Istanbul University. Commercial powdered rat chow were mixed with TAU or BET and prepared pellet chows containing 2.5% TAU (w/w) or 2.5 BET (w/w) by M. Barbaros Denizeri Yem Ticaret (Gebze/Kocaeli).

Experimental groups We randomly divided the rats into five groups (n ¼ 6, each) as follows: (a) control group—animals were fed with control diet for 6 weeks; (b) DEN group—these were fed with control diet for 6 weeks and a single dose of DEN (200 mg kg1) was administered by intraperitoneal (i.p.) injection 2 days before the end of this period; (c) CAR þ DEN group—rats were given CAR (2 g L1 in drinking water) for 6 weeks (approximately 120– 150 mg kg1 body weight/day) and DEN was given 2 days before the end of this period; (d) TAU þ DEN group—animals were fed a TAU (2.5%; w/w) containing diet for 6 weeks (approximately 1.0–1.2 g kg1 body weight/day) and DEN was administered as mentioned above; And (e) BET þ DEN group—this group was fed with a BET (2.5%; w/w) containing diet for 6 weeks (approximately 1.0–1.2 g kg1 body weight/day) and DEN was injected as mentioned above. The doses used for pretreatments were calculated according to daily food and water consumption of rats and their concentrations were chosen in accordance with our previous studies (Artun et al., 2010; Balkan et al., 2002, 2004).

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Figure 1. The effects of CAR, TAU, and BET pretreatments on serum ALT, AST, LDH, and GGT activities in DEN); DEN ( ); CAR þ DEN ( ); TAU þ DEN ( ); BET þ DEN ( )). Values treated rats. Groups: control ( are expressed as mean + SD (n ¼ 6, each). ap < 0.05 compared with controls; bp < 0.05 compared with DEN (Kruskal– Wallis; post-hoc Mann–Whitney U). CAR: carnosine; TAU: taurine; BET: betaine; ALT: alanine aminotransferase; AST: aspartate aminotransferase; LDH: lactate dehydrogenase; GGT: -glutamyl transferase; DEN: N-diethylnitrosamine.

Blood and tissue samples All rats were killed by taking blood via cardiac puncture under sodium thiopental anesthesia (50 mg kg1, i.p.) two days after the DEN injection. Blood samples were collected in dry tubes by cardiac puncture. The livers were rapidly removed, washed in 0.9% sodium chloride (NaCl) and kept in ice. The materials were stored at 80 C until they were analyzed.

Determinations in serum Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and

-glutamyl transferase (GGT) measurements were performed on a Cobas Integra 800 autoanalyzer (Roche Diagnostics, Mannheim, Germany).

Determination of hepatic lipid peroxides Liver tissue was homogenized in ice-cold 0.15 M potassium chloride (10%; w/v). Lipid peroxidation was assessed using two different methods in the tissue

homogenates. First, the levels of malondialdehyde (MDA) were measured using the thiobarbituric acid test (Ohkawa et al., 1979). The breakdown product of 1,1,3,3-tetraethoxypropane 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 M1 cm1 (Buege and Aust, 1978). Lipids were extracted with chloroform:methanol (2:1) (Folch et al., 1956).

Determination of hepatic PC and NT levels The oxidative protein damage was measured using the quantification of carbonyl groups with 2,4dinitrophenylhydrazine. Protein carbonyl (PC) levels were calculated from the maximum absorbance (360 nm) using a molar extinction coefficient of 22,000 M1 cm1. The results were expressed as nanomoles of carbonyl per milligram protein (Reznick and Packer, 1986). Protein levels were determined using bicinchoninic acid (Smith et al., 1985).

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Figure 2. The effects of CAR, TAU, and BET pretreatments on hepatic MDA, DC, and PC and NT levels in DEN-treated ); DEN ( ); CARþDEN ( ); TAUþDEN ( ); BETþDEN ( )). Values are rats. Groups: control ( expressed as mean + SD (n ¼ 6, each). ap < 0.05 compared with controls; bp < 0.05 compared with DEN (Kruskal–Wallis; post-hoc Mann–Whitney U). CAR: carnosine; TAU: taurine; BET: betaine; MDA: malondialdehyde; DC: diene conjugate; PC: protein carbonyl; NT: nitrotyrosine; DEN: N-diethylnitrosamine.

3-Nitrotyrosine (NT) was measured as a marker of nitrosative injury and peroxynitrite formation in the supernatants using a OxiSelect NT competitive enzyme-linked immunosorbent assay kit (Cell Biolabs, San Diego, California, USA) in accordance with the manufacturer’s instructions. For this reason, liver samples (10% w/vol) were homogenized in a solution containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.5, 150 mM NaCl, 10 mM magnesium chloride, 1 mM ethylenediaminetetraacetic acid, 2% glycerol, aprotinin, leupeptin, and soy bean trypsin inhibitor (1 g mL1 each) at 0–4 C using a polytron homogenizer and homogenates were centrifuged at 12,000g at 4 C for 5 min.

peroxidase (GSH-Px), and glutathione transferase (GST) activities were determined in postmitochondrial fraction of the tissues, which were separated by sequential centrifugation. In brief, tissue homogenates were centrifuged at 600g for 10 min at 4 C to remove crude fractions. Supernatants were then centrifuged at 10,000g for 20 min to obtain the postmitochondrial fraction. SOD activity was assayed by its ability to increase the effect of riboflavin-sensitized photooxidation of o-dianisidine (Mylroie et al., 1986). GSH-Px activity was measured using cumene hydroperoxide as substrate (Lawrence and Burk, 1976). GST activity was determined using 1chloro-2,4-dinitrobenzene as substrate (Habig and Jacoby, 1981).

Determination of hepatic nonenzymatic and enzymatic antioxidants

Histopathologic analysis

Glutathione (GSH) levels were measured in the homogenates with 5,5-dithiobis-(2-nitrobenzoate) at 412 nm (Beutler et al., 1979). SOD, glutathione

Liver tissues were fixed in 10% buffered formalin, processed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for histologic studies.

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Figure 3. The effects of CAR, TAU, and BET pretreatments on hepatic GSH levels and SOD, GSH-Px, and GST activities ); DEN ( ); CARþDEN ( ); TAUþDEN ( ); BET þ DEN ( )). in DEN-treated rats. Groups: control ( Values are expresed as mean + SD (n ¼ 6, each). ap < 0.05: compared with controls; bp < 0.05: compared with DEN (Kruskal–Wallis; post hoc Mann–Whitney U). CAR: carnosine; TAU: taurine; BET: betaine; GSH: glutathione; SOD: superoxide dismutase; GSH-Px: glutathione peroxidase; GST: glutathione transferase; DEN: N-diethylnitrosamine.

Statistical analysis

Biochemical analyses

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 Mann–Whitney U test was performed and p < 0.05 was considered to be statistically significant. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) Statistics for Windows software package (version 15.0; SPSS Inc., Chicago, Illinois, USA).

The results obtained in this study are shown in Figures 1 to 3 and summarized as follows: Serum ALT, AST, LDH, and GGT activities showed 100-, 49-, 4.5- and 4.5-fold increases in DEN group compared with the controls. CAR pretreatment did not change serum ALT, AST, LDH, and GGT activities in DEN-induced rats, although these activities tended to decrease. However, TAU and BET pretreatments caused significant decreases in these hepatic markers (Figure 1). Hepatic MDA, DC, PC, and NT levels increased in DEN group. CAR, TAU, and BET pretreatments resulted in significant decreases in hepatic MDA, DC, PC, and NT levels in DEN-treated rats (Figure 2). GSH-Px and GST activities were observed to decrease due to DEN treatment compared with controls. SOD activity did not change, but GSH levels increased. Although there was no change in hepatic SOD and GSH-Px activities, decreased GSH levels

Results Food and water consumption and body weights There were no significant differences in daily food and water consumption and final body weights between control and experimental groups (data not shown).

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Figure 4. The effect of CAR, TAU, and BET pretreatments on histopathologic appearance of liver in DEN-treated rats (H&E 200). (a) Normal hepatic tissue morphology. (b) Parenchymal cell necrosis, erythrocyte extravasation, inflammatory cell infiltration (arrows), and vacuolation of hepatocytes (star) in DEN-treated rats. (c) Parenchymal cell necrosis, erythrocyte extravasation, inflammatory cell infiltration (arrows), and vacuolation of hepatocytes (star) in the CARþDEN group. (d and e) Minimal erythrocyte extravasation and mild inflammatory cell infiltration (arrows) in the TAU þ DEN (d) and BET þ DEN (e) groups. CAR: carnosine; TAU: taurine; BET: betaine; DEN: N-diethylnitrosamine; H&E: hematoxylin and eosin.

and increased GST activities were found in DENtreated rats due to CAR pretreatment. TAU and BET pretreatments did not alter the examined antioxidant parameters in the liver of DEN-treated rats (Figure 3).

Histological examinations Normal liver structure was seen histopathologically in the control group (Figure 4). In the DEN group,

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histopathologic evaluation of male rats showed diffuse parenchymal necrosis, neutrophils, lymphocytes and plasma cells infiltrations, distinct edema and extravasate erythrocytes around the central vein, midzonal, and portal areas. Vacuolar degeneration in parenchymal cells and extensive vacuolation/swelling in cytoplasms were noticed. Blood vessels with thrombus were determined at the periphery. In the CAR þ DEN group, similar histopathologic findings were observed (diffuse parenchymal cell necrosis, erythrocyte extravasation, inflammatory cell infiltration, and vacuolation of hepatocytes). Minimal erythrocyte extravasation, mild inflammatory cell infiltration, and focal parenchymal necrosis were seen in the TAU þ DEN and BET þ DEN groups.

Discussion DEN is known to cause hepatocyte death and stimulate compensatory proliferation in the liver (Glauert et al., 2010; Maeda et al., 2005; Naugler et al., 2007; Qui et al., 2011). The death of DEN-exposed hepatocytes was found to activate adjacent Kupffer cells to produce hepatomitogens that promote compensatory proliferation of surviving hepatocytes. Compensatory proliferation, which is a response triggered by hepatocyte death, appears to have a critical role in DEN-induced hepatocarcinogenesis (Glauert et al., 2010; Maeda et al., 2005; Naugler et al., 2007; Qiu et al., 2011). In the literature, there have been some reports about the effect of a single dose of DEN on oxidative stress and hepatic injury in rats (Bansal et al., 2000; Bansal et al., 2005; Metwally et al., 2011; Sayed-Ahmed et al., 2010) and mice (Kang et al., 2007; Maeda et al., 2005; Naugler et al., 2007; Qiu et al., 2011). However, data obtained show some different results relative to the administered DEN dose and investigation time used in the experiments. In this study, a necrogenic dose of DEN (200 mg kg1) was used. DEN is a strong initiator in triggering oxidative stress and its radical generatingbased hepatic metabolism is presumably the underlying mechanism of the hepatic damage that is evidently observed 48 h following DEN (Bansal et al., 2000; Sayed-Ahmed et al., 2010). Therefore, the experiments were performed 48 h after DEN treatment. Significant increases in serum ALT, AST, LDH, and GGT activities as well as histopathologic necrosis findings were observed. In addition, we observed increased prooxidant parameters such as MDA, DC, PC, and NT and depressed antioxidant system in the liver following DEN treatment. The increase in oxidative stress

parameters did not seem to be as dramatic as the increase in serum markers 48 h after administration. Oxidative stress appeared to be way ahead in the route that would lead to liver injury and its markers may have declined to lower levels when injury was perceptible. Only hepatic GSH levels were observed to increase following DEN injection. This increase may be related to adaptive change against stimulated hepatic oxidative stress due to DEN. These findings agree with the results of previous studies in which a similar DEN dose and administration times were used (Bansal et al., 2000; Sayed-Ahmed et al., 2010). CAR is known to be an effective agent to prevent oxidative stress-induced pathologies such as atherosclerosis (Aydın et al., 2010), neurodegeneration (Bellia et al., 2011), and aging (Hipkiss, 2009) including liver damage (Artun et al., 2010; Mehmetc¸ik et al., 2008; Yan et al., 2009). Therefore, we wanted to examine its protective effects on DEN-induced acute liver damage. In this study, CAR pretreatment did not change hepatic markers in the serum of DEN-treated rats; they tended to decrease, but not significantly. There were also no marked differences in hepatic histopathologic findings between DEN and CAR þ DEN groups. CAR pretreatment diminished prooxidant status in the liver of DEN-treated rats. However, GSH levels returned to normal values in DEN-treated rats due to CAR pretreatment. Although there were no changes in SOD and GSH-Px activities, GST activity increased significantly in DEN-treated rats due to CAR pretreatment. Our results are in accordance with our previous results obtained from thioacetamidetreated (Mehmetc¸ik et al., 2008) and ethanol-treated (Artun et al., 2010) rat livers. In addition, we recently reported that CAR treatment did not alter hepatic messenger RNA (mRNA) expressions of SOD and GSH-Px enzymes in normal and galactose-treated rats (Kalaz et al., 2014). CAR pretreatment was effective to decrease DEN-induced oxidative and nitrosative stress by acting as a nonenzymatic antioxidant itself. Therapeutic use of both TAU and BET has also been shown to exhibit protective actions against oxidative tissue damage (Miyazaki and Matsuzaki, 2014; Ueland, 2011). TAU and BET have been found to effectively prevent hepatic oxidative stress due to steatosis (Abdelmalek et al., 2001; Balkan et al., 2004; Barak et al., 1996) and liver necrosis (Acharya and Lau-Cam, 2010; Balkan et al., 2005; Dog˘ruAbbasog˘lu et al., 2001; Kim and Kim, 2002). In this study, TAU and BET pretreatments were observed to reduce hepatic markers in serum of DEN-treated rats.

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The extent of necrotic lesions in the liver was less in TAU þ DEN and BET þ DEN groups. These pretreatments also decreased hepatic prooxidant status but did not restore antioxidant parameters in DEN-treated rats. Similar results were obtained from TAU and BETtreated rats exposed to thioacetamide (Dog˘ru-Abbasog˘lu et al., 2001) and lipopolysaccharide (Balkan et al., 2005). In our previous study, there was no significant change in hepatic mRNA expressions of SOD and GSH-Px enzymes in normal and galactose-treated rats due to TAU treatment (Kalaz et al., 2014).These results indicate that pretreatments with TAU and BET restored liver prooxidant status without affecting antioxidant parameters in DEN-treated rats. For this reason, these protective effects may be related to their scavenging actions. In addition, TAU and BET are reported to act as osmolytes in Kupffer cells and play an important role by inhibiting Kupffer cell functions such as phagocytosis and eicosanoid and tumor necrosis factor formation (Kim and Kim, 2002). Therefore, the inhibitory effect of TAU and BET on Kupffer cell functions may also play an additional role in the decreasing of DEN-induced liver injury. On the other hand, BET treatment has been reported to increase hepatic SAM levels (Barak et al., 1996; Lieber, 2002). Increases in SAM levels due to BET treatment may also contribute to its hepatoprotective effect by restoring hepatocyte membranes and decreasing lipid peroxide levels (Barak et al., 1996; Lieber, 2002). Our results indicate that pretreatments with TAU, BET, and CAR restored hepatic prooxidant status, and TAU, BET, and possibly CAR may be effective to reduce liver damage in DEN-treated rats. Conflict of interest The authors declared no conflicts of interest.

Funding This work was supported by Research Fund of Istanbul University (project no: 16705).

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Effects of carnosine, taurine, and betaine pretreatments on diethylnitrosamine-induced oxidative stress and tissue injury in rat liver.

Several chemicals such as N-diethylnitrosamine (DEN) promote hepatocellular cancer in rodents and induce hepatocyte injury. DEN affects the initiation...
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