Food and Chemical Toxicology 48 (2010) 33983405

Contents lists available at ScienceDirect

Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

Ameliorative role of conjugated linolenic acid isomers against oxidative DNA damage induced by sodium arsenite in rat model
S.S. Saha, M. Ghosh
Oil Technology Section, Dept. of Chemical Technology, University College of Science and Technology, University of Calcutta, 92, A.P.C. Road, Kolkata 700 009, India

a r t i c l e

i n f o

a b s t r a c t
The present study was undertaken to evaluate the ameliorative role of a-eleostearic acid and punicic acid, isomers of conjugated linolenic (CLnA) acid, against oxidative stress induced DNA damage. Male albino rats were divided into six groups. Group 1 and 2 were normal control and sodium arsenite treated (Sa; 10 mg/kg BW) control respectively. Group 36 were orally treated with different doses of two fatty acids (0.5% and 1.0% of total lipid given for each isomer) along with sodium arsenite (Sa; 10 mg/kg BW). Comet assay of blood leukocytes showed that administration of CLnA reduced DNA damage signicantly (P < 0.05) which was determined by tail DNA percent and olive tail moment. Results showed that activity of antioxidant enzymes viz. catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx) and reduced glutathione (GSH) in plasma, liver and erythrocyte lysate decreased and activity of nitric oxide synthase in plasma and liver increased signicantly due to oxidative stress generated by sodium arsenite. Administration of CLnA isomers restored all the altered parameters and also reduced lipid peroxidation and leakage of transaminase enzymes from liver to blood due to liver injury. a-Eleostearic acid was more efcient antioxidant than punicic acid against oxidative DNA damage. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 12 May 2010 Accepted 7 September 2010

Keywords: Conjugated linolenic acid a-Eleostearic acid Punicic acid DNA damage Oxidative stress Comet assay

1. Introduction Oxygen homeostasis which is vital for proper functioning and uctuations in oxygen metabolism can have detrimental consequences on biological organisms. An imbalance in oxygen levels causes the generation of reactive oxygen species (ROS), which promote oxidative stress by damaging macromolecules, contributing to the pathologies of Alzheimers disease, Parkinsons disease, amyotrophic lateral sclerosis, atherosclerosis, vascular disease, ischemiareperfusion injury, congestive heart failure, stroke, ageing and neurodegenerative diseases (Kulkarni et al., 2007). The most common ROS include superoxide anion, hydrogen peroxide radical, peroxyl radicals and reactive hydroxyl radicals. The nitrogen derived free radicals are nitric oxide and peroxynitryl anion.

Abbreviations: As, arsenic; As(V), arsenate; As(III), arsenite; AST, aspartate aminotransferase; ALT, alanin aminotransferase; CAT, catalase; CLnA, conjugated linolenic acid; CLA, conjugated linoleic acid; EM, erythrocyte membrane; GSH, reduced glutathione; GSSG, oxidized glutathione; GPx, glutathione peroxidase; LMPA, low-melting point agarose; MDA, malondialdehyde; NOS, nitric oxide synthase; PPARc, peroxisome proliferator activated receptor gamma; PUFA, polyunsaturated fatty acid; RBC, red blood cell; ROS, reactive oxygen species; Sa, sodium arsenite; SH-groups, sulfhydryl groups; SOD, superoxide dismutase; TBA, 2-thiobarbituric acid; TCA, trichloro acetic acid. Corresponding author. Tel.: +91 33 2352 0050/51/52x276; fax: +91 33 2351 9755. E-mail addresses: mahuag@gmail.com, mgchemtech@caluniv.ac.in (M. Ghosh). 0278-6915/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.09.011

The link between free radicals and diseases has lead to considerable research into non toxic drugs and natural antioxidants that can scavenge the free radicals. Several plant extracts and plant products have been shown to possess signicant antioxidant activity (Serani et al., 2002). Antioxidant based drug formulations are used for the prevention and treatment of complex diseases like atherosclerosis, cardiovascular disease (CVD), diabetes, Alzheimers disease and cancer (Noonan et al., 2007). A large number of medicinal plants and their puried constituents have shown benecial therapeutic potentials and various herbs and spices have been reported to exhibit antioxidant activity (Huda-Faujan et al., 2009). a-Eleostearic acid and punicic acid, two typical conjugated trienoic fatty acid isomers of conjugated linolenic acid (ClnA), found in seed oils with systematic structure of cis-9, trans-11, trans-13octadecatrienoic and cis-9, trans-11, cis-13-octadecatrienoic acid respectively. Theoretically a-eleostearic acid consists of 33% cis and 66% trans molecular composition and punicic acid has 66% cis and 33% trans molecular conguration. It has been observed that seed fats of Cucurbitaceae family, obtained from common vegetables like karela (Momordica charantia), contains about 3050% a-eleostearic acid and seed fat of snake gourd plant (Trichosanthes anguina) contains about 40% punicic acid (Takagi and Itabashi, 1981). These two conjugated fatty acids are now regarded as natural antioxidant for their oxygen scavenging property (Dhar et al., 1999; Mukherjee et al., 2002). Our earlier study established

S.S. Saha, M. Ghosh / Food and Chemical Toxicology 48 (2010) 33983405

3399

the antioxidant efcacy of a-eleostearic acid and punicic acid at 0.5% level (of total lipid) against arsenite toxicity and results showed that these two isomers have signicant antioxidant activity (Saha and Ghosh, 2009). But the benecial effect of conjugated fatty acids in relation to the prevention and recovery from oxidative stress and genotoxicity is yet to be dened. The present study has been designed to observe the ameliorative role of CLnA isomers against oxidative stress induced DNA damage. Sodium arsenite (Sa) has been used as a stress inducing agent in this experiment as being a potent environmental toxic agent, it leads to development of various hazardous effects on human health. Oxidative stress has been identied as an important mechanism of arsenic toxicity and carcinogenicity and so increased antioxidant level of the body may protect arsenic-induced oxidative stress and genotoxicity.

2. Materials and methods 2.1. Materials Sodium arsenite (NaAsO2) was purchased from Sd Fine-Chem limited, Mumbai, India. The dose of Sa (10 mg/kg BW) was chosen on the basis of the previous studies (Kadirvel et al., 2007; Rodriguez et al., 2001; Flora, 1999). All other chemicals used were of analytical grade and procured from Merck India Ltd., Mumbai, India.

2.1.4. Animal experiment The work had been done under the supervision of the Animal Ethical Committee of the Department of Chemical Technology (University of Calcutta). Male albino rats of Charles Foster strain (selected for the authenticity of the strain) were housed in individual cages. The animals were divided into six groups (average body weight 100110 g) consisting of six animals in each group. The rst group served as normal control, received only vehicles (sunower oil and deionized water) once per day. Rats in the group 2 were treated with sodium arsenite (Sa; 10 mg/kg BW) along with sunower oil by oral gavages once per day and served as treated control. Rats in the group 3 and 4 were treated with a-eleostearic acid (0.5% and 1.0% of total lipid given, respectively) along with sodium arsenite (Sa; 10 mg/kg BW) by oral gavages once per day. Rats in the group 5 and 6 were treated with punicic acid (0.5% and 1.0% of total lipid given, respectively) along with sodium arsenite (Sa; 10 mg/kg BW) by oral gavage once per day. The rats were fed experimental diets having the following composition: fat-free casein, 18%; fat, 20%; starch, 55%; salt mixture 4% [composition of salt mixture No. 12 (in g): NaCl, 292.5; KH2PO4, 816.6; MgSO4, 120.3; CaCO3, 800.8; FeSO4 7H2O, 56.6; KCl, 1.66; MnSO4 2H2O, 9.35; ZnCl2, 0.5452; CuSO4 5H2O, 0.9988; CoCl2 6H2O, 0.0476] (Joanes and Foster, 1942); cellulose, 3%; and one multivitamin capsule (vitamin A I.P. 10,000 units, thiamine mononitrate I.P. 5 mg, vitamin B I.P. 5 mg, calcium pantothenate USP 5 mg, niacinamide I.P. 50 mg, ascorbic acid I.P. 400 units, cholecalciferol USP 15 units, menadione I.P. 9.1 mg, folic acid I.P. 1 mg, and vitamin E USP 0.1 mg) per kg of diet. The diets were adequate in all nutrients. The animals were sacriced after 21 days under anesthesia and kept in overnight fasting before sacrice. Blood was collected from heart and liver was immediately excised, blotted, and stored frozen (40 C) for analysis. 2.1.5. DNA damage evaluation by comet assay of blood leukocytes DNA damage of blood leukocytes was evaluated by single cell gel electrophoreses (comet assay) using the method of Dhawan et al. (2001) with slight modications described by Singh et al. (1988). Lysis solution (2.5 M NaCl, 100 mM EDTA (Ethylenediamin tetraacetic acid), 10 mM Tris, pH 10, 1% sodium sarcosinate, 1% Triton X-100, and 10% DMSO (Dimethyl sulfoxide)), TrisHCl neutralization buffer (0.4 M, pH 7.5), and electrophoresis buffer (EP) (300 mM NaOH, 1 mM EDTA) were prepared as described by Singh et al. (1988). The blood cells were suspended in 110 lL of low-melting point agarose (LMPA) (0.65% LMPA, w/v in PBS, pH 7.4) and pipetted onto a frosted glass microscope slide precoated with 140 lL of 1% normal melting point agarose (in PBS, pH 7.4). The agarose was allowed to set for 10 min at 4 C and there after the cover slip was removed and the slides were exposed for 24 h to lysis solution. Finally, the slides were rinsed with distilled water and EP buffer to remove salts. These slides were exposed to alkaline EP buffer (pH 13.0) for 40 min and subjected to electrophoresis for 20 min (300 mA, 25 mV). Then the alkali was neutralized with TrisHCl buffer; the slides rinsed with distilled water and methanol and were stained with ethidium bromide. Images of 50 randomly selected cells were analyzed from each sample. The Tail DNA% and Olive Tail Moment [(Tail.meanHead.mean) Tail%DNA/100] were calculated from 50 counted cells. 2.1.6. Plasma peroxidation Plasma peroxidation was measured by the assay of thiobarbituric acid-reactive substances (TBARS) according to the standard method (Wills, 1987). The amount of malondialdehyde (MDA) formed was calculated by taking the extinction coefcient of MDA to be 1.56 105 M1 cm1. 2.1.7. Tissue lipid extraction and peroxidation For lipid peroxide measurement, approximately 1 g of liver tissue was placed into a glass centrifuge tube (70 mL) for 2 min in a solvent mixture consisting of 10 mL of chloroform and 20 mL of methanol, and homogenized on ice. Then, 10 mL of chloroform was added and homogenization continued for another 30s. Finally, 10 mL of redistilled water was added and the mixture was homogenized for 30 s. The tubes were then centrifuged for 20 min at 4000g, and the chloroform layer was separated in a separatory funnel. A 2-thiobarbituric acid (TBA) test in chloroform phase from a Bligh and Dyer extraction (Bligh and Dyer, 1959) was performed

2.1.1. Extraction and quantication of CLnA isomers Authentic karela seeds and snake gourd seeds were obtained from the local market of Kolkata, India. Seeds were crushed and the oil was extracted with solvent petroleum ether. The extracts of the sample were ltered and then concentrated by rotary evaporator. Free fatty acids present in the oil were quantied and then were removed by miscella rening process by adding 10% NaOH solution (20% excess of the required amount) at 40 C for 30 min (Bhattacharyya et al., 1986). The soap formed was removed by centrifugation and the organic phase was washed with water. Deacidied oil was recovered by distillation under vacuum and dried. The rened oil was then bleached with tonsil earth (1% w/w) obtained from P.T. Sud-Chemic (Jakarta, Indonesia) and activated carbon (0.2% w/w), supplied by E. Merck India Pvt. Ltd. (Bombay, India), at 60 C under vacuum for 20 min. After the bleaching operation, the oil was recovered by vacuum ltration and stored at 20 C under nitrogen.

2.1.2. Analysis of fatty acid compositions The fatty acid (FA) compositions of the different dietary oils were determined by gas liquid chromatography (GLC, Agilent Technologies India Pvt. Ltd., Mumbai) techniques after preparing methyl esters of corresponding fatty acids by the method described by Ichihara et al. (1996). The amount of cistrans isomers present in the oil were determined by dissolving the oil in cyclohexane and measuring the absorbance over wavelength range of 200300 nm by UVVis spectrophotometer (Shimadzu corporation, Japan) (Lakshminarayana et al., 1982).

2.1.3. Dietary fat blends Sunower oil was obtained from Corn Agro Limited (Hyderabad, India) and it was mixed with karela seed oil and snake gourd oil to give nal oil blends containing 0.5% and 1.0% by weight of a-eleostearic acid and punicic acid, respectively. Table 1 shows the fatty acid composition of dietary oil mixtures. The dose of CLnA isomers (0.5% and 1.0% of total lipid given) were xed according to the previous studies conducted in the same laboratory (Dhar et al., 1999; Mukherjee et al., 2002).

Table 1 Fatty acid composition of dietary oils and oil blends. Dietery Fats FA composition (% w/w) 16:0 Sunower oil Karela seed oil Snake gourd oil Sunower + Karela seed oil (99:1, w/w) Sunower + Karela seed oil (98:2, w/w) Sunower + Snake gourd oil (98.75:1.25, w/w) Sunower + Snake gourd oil (97.5:2.5, w/w) 6.3 1.8 6.2 6.3 6.3 6.3 6.3 18:0 3.5 31.6 6.1 3.8 4.0 3.5 3.6 18:1 32.4 7.5 26.6 32.2 31.9 32.3 32.3 18:2 57.8 6.8 21.0 57.2 56.8 57.4 56.9 18:3 (CLnA, 9c,11t,13t-18:3) 52.3 0.5 1.0 18:3 (CLnA, 9c,11t,13c-18:3) 40.1 0.5 1.0

3400

S.S. Saha, M. Ghosh / Food and Chemical Toxicology 48 (2010) 33983405

according to the method described by Schmedes and Hlmer (1989). Chloroform (2.5 mL) from the Bligh and Dyer extraction was pipetted into a 30-mL autoclavable glass culture tube (Kimax; Kimble/Kontes, Vineland, NJ) having a Teon-lined screw cap; 4 mL of the TBA reagent was added and the tube was capped tightly. Safetyshielded tubes were heated for 30 min in a boiling water bath and cooled in tap water. After cooling, 3.5 mL 5% trichloro acetic acid (TCA) was added to each tube, mixed thoroughly, and the tubes were then centrifuged at 3000g. 2.1.8. Preparation and oxidative sensitivity of erythrocyte membrane (EM) ghost After plasma separation, the red blood cells (RBC) were washed three times by centrifugation at 3000g for 10 min with three volume of a cooled isotonic solution containing 0.15 M NaCl and 105 M EDTA. RBC was hemolyzed using hypotonic solution and centrifuged at 20,000g for 40 min at 4 C. The supernatant was removed carefully with a Pasteur pipette. The process was repeated two more times. After the last wash step, the supernatant was removed as much as possible and the loosely packed milky-looking membrane pellet was resuspended at the bottom of the tube using 0.89% NaCl solution. The concentrated membrane solution was taken into a 2 mL screw-capped vial and stored at 40 C (Rose and Oklander, 1996). A modication of the 2-thiobarbituric acid (TBA) test (Mezick et al., 1970) was used to measure the lipid peroxides. A 0.5 mL aliquot of the red blood cell membrane suspension was mixed with 1.0 mL of 10% TCA and 2.0 mL of 0.67% 2-TBA. The mixture was heated at 95 C for 15 min, cooled, and centrifuged. The absorbance of the supernatant was measured at 534 nm in a spectrophotometer (Shimadzu, Tokyo, Japan), and the relative amounts of lipid peroxides were expressed in absorbance units, A534 nm (Brownlee et al., 1977). 2.1.9. Preparation of erythrocyte lysate Two ml of heparinized blood was centrifuged for 10 min at 1000g and the plasma was removed by suction. The erythrocytes were washed twice with phosphate buffered saline (PBS), pH 7.4. Distilled water (10 mL) was then added and the erythrocytes were resuspended by agitation and lysed for 2 h at 4 C. A mixture of chloroformethanol (3:5, v/v, 0.8 mL) and 0.3 mL of water was added to the lysate so as to precipitate the haemoglobin, which was centrifuged at 3000g for 10 min at 4 C (Dani and Dhawan, 2005). 2.1.10. Measurement of antioxidative enzymes activity A part of the liver tissue was homogenized in 0.1 M Tris buffer (pH 7.0) and the homogenate was centrifuged at 105,000g for 1 h. Superoxide dismutase (SOD) activity was assayed in plasma and liver homogenate and erythrocyte lysate according to as previously described (Martin et al., 1987) and expressed as units/ mg of protein. Catalase (CAT) activity in plasma and liver homogenates and erythrocyte lysate was assayed as described previously (Yumoto et al., 2000) and expressed as mmol of hydrogen peroxide decomposed/min/mg of protein. Reduced glutathione (GSH) levels in liver homogenate, erythrocyte lysate and plasma were quantied as described previously (Beutlar et al., 1967) and expressed as lg/mg of protein. Glutathione peroxidase (GPx) activity was assayed according to as previously described (Mohandas et al., 1984) and expressed as units/mg of protein. 2.1.11. Measurement of prooxidative enzymes activity Nitric oxide synthase (NOS) in liver homogenate and blood was determined by standard method (Hevel and Marletta, 1994) and expressed as nanomoles/mg of protein. 2.1.12. Measurement of AST and ALT activity Activity of Aspartate aminotransferase (AST) and Alanin aminotransferase (ALT) was analyzed using enzymatic kit supplied by Span Diagnostics Ltd. (Surat, India). 2.1.13. Measurement of protein content Protein content was measured by the Lowry method (Lowry et al., 1951). 2.1.14. Statistical analysis The data were expressed as mean SEM. A one-way ANOVA was also used for statistical analysis between groups. The F ratio of one-way ANOVA is signicant when the P value <0.05. Tukeys multiple range method (Scheffe, 1961) was used for comparison. The statistical program was Minitab release 13.31 (Minitab, State College, PA).

3. Results The purpose of the present study was to nd out the ameliorative role of conjugated linolenic acids, a-eleostearic acid and punicic acid, at different levels on oxidative DNA damage. The alkaline comet assay was used to measure the DNA single-strand breaks in blood leukocytes of control, arsenic-treated and CLnA supple-

mented groups at different levels (Fig. 1). A signicant (P < 0.05) increase in the levels of DNA strand breaks as calculated by the tail DNA% and Olive Tail Moment in arsenic-treated animals compared to control animals. Arsenic exposure caused over a twofold increase in the level of DNA damage in leukocytes. Simultaneous administration of 0.5% and 1.0% (w/w, of total lipid) of a-eleostearic acid and punicic acid each to arsenic-treated rats markedly lowered the DNA damage. Treatment of 0.5% of a-eleostearic acid and punicic acid (w/w, of total lipid) signicantly (P < 0.05) reduced DNA damage when compared to 1.0% treated rats. a-Eleostearic acid was more efcient in reducing oxidative DNA damage than punicic acid at 0.5% level as seen by the difference in tail DNA% and Olive tail moment (Table 2). The activities of antioxidant enzymes such as SOD, CAT and GPx in plasma and liver were found to be signicantly (P < 0.05) decreased as compared to the control due to Sa administration. The decreased antioxidant enzyme activity in plasma and liver appeared to be completely restored to control value by CLnA supplementation (Tables 3 and 4). All the antioxidant enzyme activities were higher at 0.5% level in both the cases; but it was more in case of 0.5% a-eleostearic acid group than 0.5% punicic acid group. Sa treated rats showed a signicant reduction in cellular GSH levels in liver and plasma compared to respective controls which has been completely restored by CLnA administration (Tables 3 and 4). In case of both the isomers GSH content in plasma and liver was signicantly higher in case of 0.5% treated groups compared to 1.0% treated groups. GSH content in plasma and liver was significantly higher in case of 0.5% a-eleostearic acid treated rats than 0.5% punicic acid treated rats. NOS activity was signicantly increased in liver and plasma as compared to the control due to Sa administration. Increased NOS activity also restored to normal value or below the normal value in some cases in plasma and liver due to supplementation of CLnA isomers (Tables 3 and 4). Results showed that administration of 0.5% of both the CLnA isomers causes maximum reduction in NOS activity in plasma and liver compared to 1.0% level. There was no signicant difference in NOS activity in plasma at 0.5% level between a-eleostearic acid and punicic acid treated group (Table 3). In case of liver, NOS activity was signicantly lower in case of 0.5% a-eleostearic acid group than 0.5% punicic acid group (Table 4). Overall antioxidant activity of a-eleostearic acid was more prominent than punicic acid at 0.5% level. SOD, CAT, GPx activity and GSH content in erythrocyte lysate (Table 5) were also decreased signicantly (P < 0.05) compared to respective control due to Sa administration. But administration of a-eleostearic acid and punicic acid cause complete restoration of SOD, CAT, GPx activity and GSH content. At 0.5% level antioxidant enzyme activities in erythrocyte lysate were higher in case of both the isomers. Improvement in antioxidant enzyme activities and GSH content in erythrocyte lysate were more prominent in case of 0.5% a-eleostearic acid treated group than 0.5% punicic acid treated group. Administration of Sa increased lipid peroxidation (Table 6) signicantly (P < 0.05) in plasma, liver and erythrocyte membrane ghost compared to respective control. Supplementation of CLnA isomers caused signicant reduction in lipid peroxidation and restored them almost to control value. In case of both the isomers maximum reduction in lipid peroxidation occurred in case of 0.5% level and lipid peroxidation was signicantly lower in case of 0.5% a-eleostearic acid treated group than 0.5% punicic acid treated group. In the present study, the activities of AST and ALT were signicantly increased in plasma (Table 7) and decreased in liver of rats treated with Sa compared to respective control and this was an indication of liver damage. The present study indicated that supplementation of CLnA isomers caused complete restoration of AST and ALT activity in plasma and liver compared to Sa treated

S.S. Saha, M. Ghosh / Food and Chemical Toxicology 48 (2010) 33983405

3401

Fig. 1. A photomicrograph of comet assay showing blood leukocytes of different experimental groups. Control (CONTROL); 10 mg/kg sodium arsenite treated (Sa); 0.5% aeleostearic acid fed along with 10 mg/kg sodium arsenite treated (0.5% EA + Sa); 1.0% a-eleostearic acid fed along with 10 mg/kg sodium arsenite treated (1.0% EA + Sa); 0.5% punicic acid fed along with 10 mg/kg sodium arsenite treated (0.5%PA + Sa); 1.0% punicic acid fed along with 10 mg/kg arsenite treated (1.0%PA + Sa) (original magnication 40).

Table 2 Comparison of tail DNA and olive tail moment levels in different dietery Groups. Experimental group Parameter Tail DNA (%) Control(C) Sa Sa + 0.5% a-eleostearic acid Sa + 1.0% a-eleostearic acid Sa + 0.5% Punicic Acid Sa + 1.0% Punicic Acid 3.18 0.32a 43.69 2.76b 3.39 0.31c 6.70 0.58d 8.45 0.64e 10.91 0.77f Olive tail moment 0.25 0.017a 5.24 0.23b 0.26 0.02c 0.42 0.073d 0.68 0.04e 0.92 0.07f

4. Discussion The focus of the present study was to establish the ameliorative role of CLnA isomers against oxidative DNA damage caused by oxidative stress. DNA damage has been recognized as the onset of many diseases including cancer and could be a useful indicator of the oxidative status and antioxidant defense system of organism (Thirunavukkarasu and Sakthisekaran, 2003). Unlike most of the carcinogens, arsenic does not directly react with DNA to cause genetic damages. In the present study, arsenic-exposed rats showed increased level of DNA single strand breakage and alkali-labile sites in leucocytes as measured by the comet assay. Free radicals including hydroxyl radicals, singlet oxygen, peroxyl radicals and peroxynitrite are able to produce modications to the DNA bases, strand breakage and various other DNA damages (Imaeda et al., 2002). In addition, ROS can attack protein or lipid molecules (process of lipid peroxidation) and generate intermediates that can react with DNA to form adducts. Malondialdehyde (MDA), a carcinogenic compound, is produced by lipid peroxidation and can react with nucleic acid bases to form multiple adducts and causes DNA damage (Lawrence, 1999). 8-Hydroxy-20 -deoxyguanosine is considered as one of the major products of ROS induced DNA damage and increased levels of hydroxyl guanosine have been observed in animal models and humans exposed to arsenic (Yamauchi et al., 2004). Evidences support the ability of arsenic to inhibit

C, control; Sa, sodium arsenite treated group; Sa + 0.5% EA, sodium arsenite treated group fed sunower oil containing 0.50% a-eleostearic acid; Sa + 1.0% EA, sodium arsenite treated group fed sunower oil containing 1.0% a-eleostearic acid; Sa + 0.5% EA, sodium arsenite treated group fed sunower oil containing 0.50% punicic Acid; Sa + 1.0% EA, sodium arsenite treated group fed sunower oil containing 1.0% punicic acid. All values are mean SEM, n = 6. a C Vs Sa (P < 0.05). b Sa + 0.5% EA Vs Sa (P < 0.05). c Sa + 0.5% EA Vs Sa + 0.5%PA (P < 0.05). d Sa + 1.0% PA Vs Sa (P < 0.05). e Sa + 0.5% PA Vs Sa (P < 0.05). f Sa + 1.0% PA Vs Sa (P < 0.05).

rats. The ameliorative activity of CLnA was best showed in 0.5% level compared to 1% level and a-eleostearic acid was found to be more effective than punicic acid.

3402

S.S. Saha, M. Ghosh / Food and Chemical Toxicology 48 (2010) 33983405 Table 3 Effect of CLnA isomers on some oxidative stress indicative biochemical parameters in plasma of rats fed blended oils. Experimental group Parameters SOD Control (C) Sa Sa + 0.5% a-eleostearic acid Sa + 1.0% a-eleostearic acid Sa + 0.5% punicic acid Sa + 1.0% punicic acid 1.83 0.09a 0.98 0.12b 2.25 0.07c 1.61 0.12d 1.82 0.08e 1.53 0.09f CAT 9.14 0.69a 3.12 0.64b 12.75 0.71c 6.73 0.81d 7.96 0.83e 5.98 0.62 GPx 0.005 0.0002a 0.002 0.0002b 0.006 0.0002c 0.005 0.0002d 0.005 0.0002e 0.004 0.0002f GSH 24.24 1.34a 8.90 1.04b 25.29 1.70c 18.65 1.22d 18.46 1.67e 16.85 1.59f NOS 2.77 0.20a 5.07 0.25b 2.61 0.33 2.87 0.24d 2.85 0.25e 3.08 0.36f

SOD (units/mg protein); CAT (mmol/min/mg protein); Gpx (units/mg protein); GSH (lg/mg protein); NOS (nmols/mg protein). For other abbreviations see Table 2. All values are mean SEM, n = 6. a C Vs Sa (P < 0.05). b Sa + 0.5% EA Vs Sa (P < 0.05). c Sa + 0.5% EA Vs Sa + 0.5% PA (P < 0.05). d Sa + 1.0% eA Vs Sa (P < 0.05). e Sa + 0.5% PA Vs Sa (P < 0.05). f Sa + 1.0% PA Vs Sa (P < 0.05).

Table 4 Effect of CLnA isomers on some hepatic oxidative stress indicative biochemical parameters in of rats fed blended oils. Experimental group Parameters SOD Control (C) Sa Sa + 0.5% a-eleostearic acid Sa + 1.0% a-eleostearic acid Sa + 0.5% punicic acid Sa + 1.0% punicic acid 21.87 2.1 4.98 2.58b 30.07 2.21 23.05 2.23d e 19.80 1.84 14.24 1.01
a

CAT 41.11 2.54 14.08 2.59b 54.56 3.16c 34.79 4.28d e 38.17 4.37 32.19 1.82f
a

GPx 0.038 0.004 0.011 0.003b 0.048 0.005c 0.03 0.003d 0.033 0.003e 0.027 0.003f
a

GSH 321.74 7.35 197.86 8.57b 354.04 9.5c 305.05 7.15d e 319.73 7.92 298.41 6.84f
a

NOS 22.1 1.91a 47.25 2.82b 18.21 1.15c 27.92 2.18d 26.63 1.18e 28.65 1.60f

SOD (units/mg protein); CAT (mmol/min/mg protein); Gpx (units/mg protein); GSH (lg/mg protein); NOS (nmols/mg protein). For other abbreviations see Table 2. All values are mean SEM, n = 6. a C Vs Sa (P < 0.05). b Sa + 0.5% EA Vs Sa (P < 0.05). c Sa + 0.5% EA Vs Sa + 0.5% PA (P < 0.05). d Sa + 1.0% eA Vs Sa (P < 0.05). e Sa + 0.5% PA Vs Sa (P < 0.05). f Sa + 1.0% PA Vs Sa (P < 0.05).

Table 5 Effect of CLnA isomers on antioxidant enzyme activity in erythrocyte lysate of rats fed blended oils. Experimental Group Parameters SOD Control (C) Sa Sa + 0.5% a-eleostearic acid Sa + 1.0% a-eleostearic acid Sa + 0.5% punicic acid Sa + 1.0% punicic acid 631.74 11.99 444.93 10.47b 676.40 9.81c 600.85 11.01d 614.22 13.14e 550.60 10.47f
a

CAT 542.30 12.77 335.41 12.55b 587.64 8.77c 514.14 10.23d 538.68 10.20e 474.63 11.52f
a

GPx 7.78 0.61 2.13 0.52b 9.16 0.78 6.32 0.75d 8.05 0.80e 5.48 0.77f
a

GSH 2131.80 73.29a 1446.56 63.76b 2308.60 77.22c 1942.72 58.34d 1993.31 43.99e 1786.26 48.34f

SOD (units/mg protein); CAT (mmol/min/mg protein); Gpx (units/mg protein); GSH (lg/mg protein). For other abbreviations see Table 2. All values are mean SEM, n = 6. a C Vs Sa (P < 0.05). b Sa + 0.5% EA Vs Sa (P < 0.05). c Sa + 0.5% EA Vs Sa + 0.5% PA (P < 0.05). d Sa + 1.0% eA Vs Sa (P < 0.05). e Sa + 0.5% PA Vs Sa (P < 0.05). f Sa + 1.0% PA Vs Sa (P < 0.05).

the DNA-repair machinery, which is likely to enhance the genotoxicity and mutagenicity of other directly genotoxic compounds as a part of cocarcinogenic mechanism of action (Andrew et al., 2006). Results of the present investigation strongly suggest that arsenic-induced DNA damage is mainly mediated by free radicals

as these alterations are signicantly reduced on the supplementation of antioxidants to arsenic-exposed rats. Supplementation of CLnA isomers to the arsenite exposed rats reduces DNA damage as indicated by the comet assay data (Table 7). This is due to the free radical scavenging and lipid peroxidation reducing activity of

S.S. Saha, M. Ghosh / Food and Chemical Toxicology 48 (2010) 33983405 Table 6 Lipid Peroxidation of plasma, liver and erythrocyte membrane (EM) ghost of rats fed blended oils. Experimental group Parameters Plasma lipid peroxidation (nmole of MDA/ml of plasma) Control (C) Sa Sa + 0.5% a-eleostearic acid Sa + 1.0% a-eleostearic acid Sa + 0.5% punicic acid Sa + 1.0% punicic acid 15.46 0.80a 36.37 1.21b 11.63 1.11c 16.91 1.76d 16.99 0.78e 25.70 1.57f Liver lipid peroxidation (nmole of MDA/mg of liver lipid) 1.99 0.16a 5.38 0.51b 1.94 0.28 2.83 0.24d 2.49 0.17e 3.23 0.51f EM lipid peroxidation (nmole of MDA/mg of protein) 21.24 1.22a 36.05 1.15b 17.89 0.92c 22.68 1.40d 23.29 1.48e 27.10 1.18f

3403

For other abbreviations see Table 2. All values are mean SEM, n = 6. a C Vs Sa (P < 0.05). b Sa + 0.5% EA Vs Sa (P < 0.05). c Sa + 0.5% EA Vs Sa + 0.5% PA (P < 0.05). d Sa + 1.0% eA Vs Sa (P < 0.05). e Sa + 0.5% PA Vs Sa (P < 0.05). f Sa + 1.0% PA Vs Sa (P < 0.05).

Table 7 Effect of CLnA isomers on AST and ALT enzyme activity in plasma and liver of rats fed blended oils. Experimental group Parameter Plasma (IU/L) AST Control (C) Sa Sa + 0.5% a-eleostearic acid Sa + 1.0% a-eleostearic acid Sa + 0.5% punicic acid Sa + 1.0% punicic acid 49.44 1.75 73.58 2.44b 48.06 2.16 56.65 1.78d 56.34 2.14e 59.17 1.49f
a

Liver (IU/mg of protein) ALT 25.91 0.90 48.48 2.84b 22.41 1.17c 28.52 0.91d 29.63 0.93e 31.42 1.29f
a

AST 0.032 0.001 0.014 0.001b 0.03 0.002 0.026 0.001d 0.028 0.002e 0.023 0.002f
a

ALT 0.304 0.020a 0.133 0.009b 0.289 0.013c 0.226 0.014d 0.227 0.011e 0.200 0.013f

For other abbreviations see Table 2. All values are Mean SEM, n = 6. a C Vs Sa (P < 0.05). b Sa + 0.5% EA Vs Sa (P < 0.05). c Sa + 0.5% EA Vs Sa + 0.5% PA (P < 0.05). d Sa + 1.0% eA Vs Sa (P < 0.05). e Sa + 0.5% PA Vs Sa (P < 0.05). f Sa + 1.0% PA Vs Sa (P < 0.05).

CLnA. Thus, it reduces DNA adduct formation by MDA and free radicals. It is suggested that arsenic induced lipid peroxidation in liver could possibly result from an enhanced microsomal oxidation capacity induced by arsenic. CLnA isomers could possibly reduce lipid peroxyl radical formation by interrupting the radical chain thereby preventing further lipid peroxidation. The study also shows signicant decrease in GSH level along with signicant fall in activity of scavenging enzymes (SOD, CAT and GPx) and signicant increase in NOS activity due to Sa administration. Such alteration of oxidative stress marker is suggested to be due to overuse failure of the antioxidant defense system as evidenced by previous studies (Ramanathan et al., 2002). But supplementation of CLnA isomers caused improvement in antioxidant status and reduction in lipid peroxidation. CLnA isomers could reduce generation of free radicals by scavenging the peroxyl radicals. In the present investigation of exposure to arsenic, the possible accumulation of H2O2 in liver as a result of diminished activity of catalase particularly on CLnA administration circumvented by the increased GSH concentrations which leads to the glutathione peroxidase-mediated reduction of H2O2 and organic hydro-peroxides. Decreased GSH concentration in the liver of Sa treated rats is due to higher arsenic accumulation in the liver (Table 4). Arsenite has strong afnity towards GSH. GSH itself may be oxidized due to the interaction with the free radicals induced by arsenic. GSH depletion leads to the production of free radicals. These free radicals interact with membrane lipids leading to the production of lipid hydro-peroxides.

Some researchers reported that the treatment with sodium arsenite induced signicant increases of hydroxyl radical formation in male and female rats (Garcia-Shavez et al., 2006). Thus SOD can act as a primary defense and prevents further generation of free radicals. The decreased SOD activity on arsenic administration suggests that the accumulation of superoxide anion radical might be responsible for increased lipid peroxidation in the tissue and membrane and this kind of suppressing action of arsenite on SOD activity has been previously reported by Zaman et al. (1995). Reduced activity of catalase after exposure to sodium arsenite in the present nding could be correlated to increased generation of H2O2 formed during the reaction catalyzed by SOD (Ramanathan et al., 2002). Erythrocytes are considered as early models for conducting studies on oxidative stress enzymes as these are highly prone to oxidative reactions because of relatively high oxygen tensions, the presence of haemoglobin, and a plasma membrane rich in polyunsaturated lipids. In the current study, decreased levels of catalase and other antioxidant enzyme activities were observed in erythrocytes. The decrease in the catalase activity could possibly be due to utilization of this enzyme in converting H2O2 to H2O. Results of the present study further demonstrated that the lipid peroxidation increased signicantly due to arsenic treatment but CLnA administration restored it mostly to control value in plasma, liver and erythrocyte membrane. SOD, CAT, GSH activities in mice treated with a-eleostearic acid (0.5% of total lipid given) along with

3404

S.S. Saha, M. Ghosh / Food and Chemical Toxicology 48 (2010) 33983405

sodium arsenite are slightly higher than the control due to maximum antioxidant activity and inhibition of free radical generation which causes slight accumulation of antioxidant enzymes. Thus, it may be possible that in the in vivo conditions these CLnA isomers may have reduced the formation of hydro-peroxides by scavenging the free radicals and peroxidation of PUFA occurs in erythrocyte membrane (EM) and other lipids (Dhar et al., 1999). Another possible explanation may be that the biohydrogenation or free radical addition to one of the conjugated double bonds of CLnA might have taken place, resulting in the formation of conjugated dienes that could have possibly acted as antioxidants (Ip et al., 1994). The possible mechanism concludes in the hydroperoxide formation and biohydrogenation of ClnA. In different studies it has been demonstrated that conjugated linoleic acid (CLA) is a potent antioxidant and that c-9, t-11 isomer is selectively incorporated into cellular phospholipid, which may, at least, in part, explain the anticarcinogenic activity of CLA as antioxidant mechanism and it may represent a heretofore-unrecognized in situ defense mechanism against membrane attack by oxygen radicals (Pariza and Ha, 1990). It appears to act as a chain-breaking antioxidant by trapping chain propagating free radicals (Brown et al. 2004). Some researchers showed that CLA could play its antioxidative roles by directly acting with free radicals to terminate the radical chain reaction or chelating transition metals to suppress the initiation of radical formation (Yu et al., 2002a). Studies showed that through activation of peroxisome proliferator activated receptor gamma (PPARc), CLA decreased inducible nitric oxide synthase (Yu et al., 2002b). Thus these conjugated fatty acid isomers could act as antioxidant in in vivo system. Administration of Sa causes injury to the hepatic cells which is indicated by the increase in activity of transaminases in plasma and decrease in liver. This may be due to the leakage of these enzymes from the liver cytosol into the blood stream and liver dysfunction and disturbance in the biosynthesis of these enzymes with alteration in the permeability of liver membrane takes place. Several of soluble enzymes of blood serum have been considered as indicators of the hepatic dysfunction and damage. The increase in the activities of these enzymes in plasma is indicative for liver damage and thus causes alteration in liver function. The activity of AST is signicantly increases in such cases and escapes to the plasma from the injured hepatic cells. In addition, ALT level is also an indicator of the existence of liver diseases, as this enzyme is present in large quantities in the liver. It increases in serum when cellular degeneration or destruction occurs in this organ (Hassoun and Stohs, 1995). Unavailability of excess GSH may be one of the probable causes for the enhanced cytotoxicity in liver tissues which is found in terms of enhanced lipid peroxidation, and diminished catalase and SOD activity. Present study showed that administration of CLnA isomers restored activities of transaminase enzymes to control value in liver and blood. Administration of CLnA preserved the structural integrity of the hepatocellular membrane. It may be due to decrease in lipid peroxidation and ROS generation. This was evident from the reduction in the activities of transaminase enzymes against the arsenic induced rise in the enzyme levels in plasma (Table 7). It could be suggested that the leakage of enzymes because of liver injury is prevented by the liver cell membrane stabilizing action of CLnA. From these ndings, it appears that CLnA isomers possesses optimum antioxidant activity at 0.5% of total lipid level which means that they can act as potent antioxidant at lower level. From these results it is clear that a-eleostearic acid is more efcient in reducing oxidative stress and DNA damage than punicic acid at different doses which is due to their antagonistic cistrans molecular arrangement. a-Eleostearic is more stable than punicic acid due to its high trans content.

5. Conclusion In conclusion, our result suggested that a-eleostearic acid and punicic acid could act as effective antioxidant agent against arsenic-induced biochemical and molecular alterations through their free radical scavenging property and also by reducing lipid peroxidation. They signicantly improved the activities of antioxidant enzymes and reduced activity of NOS in plasma, erythrocyte lysate and liver homogenate to combat against oxidative stress. Thus, these isomers succeeded in reducing DNA damage induced by oxidative stress. At 0.5% of total lipid level both the isomers showed their optimum antioxidant activity. Overall there was some difference in the effectiveness of two isomers, which was due to their antagonistic cistrans molecular arrangement and a-eleostearic was more effective than punicic acid against induced oxidative DNA damage due to its high trans content. Conict of Interest None declared. Acknowledgements The work was supported by University of Calcutta under University with Potential for Excellence scheme. Authors like to acknowledge Dr. Jaydeep Biswas, Director and Dr. Madhumita Roy, Sct., Cittaranjan National Cancer Institute, Kolkata for their help in doing comet assay. The help and support received from Dr. S. Ghosh, Department of Chemical Technology is also gratefully acknowledged. References
Andrew, A.S., Burgess, J.L., Meza, M.M., Demidenko, E., Waugh, M.G., Hamilton, J.W., Karagas, M.R., 2006. Arsenic exposure is associated with decreased DNA repair in vitro and in individuals exposed to drinking water arsenic. Environ. Health Perspect. 114, 11931198. Beutlar, E., Duron, O., Kelly, B.M., 1967. Improved method for the determination of the blood glutathion. J. Lab. Clin. Med. 61, 882888. Bhattacharyya, A.C., Majumdar, S., Bhattacharyya, D.K., 1986. Edible quality rice bran oil from high FFA rice bran oil by miscella rening. J. Am. Oil Chem. Soc. 63, 11891191. Bligh, E.H., Dyer, W.J., 1959. A rapid method of total lipid extraction and purication. Can. J. Biochem. Physiol. 37, 911917. Brown, J.M., Boysen, M.S., Chung, S., Fabiyi, O., Morrison, R.F., Mandrup, S., Mcintosh, M.K., 2004. Conjugated linoleic acid induces human adipocyte delipidation Autocrine/paracrine regulation of MEK/ERK signaling by adipocytokines. J. Biol. Chem. 279, 2673526747. Brownlee, N.R., Huttner, J.J., Panganamala, R.V., Cornwell, D.G., 1977. Role of vitamin E in glutathione induced oxidant stress: methemoglobin, lipid peroxidation, and hemolysis. J. Lipid Res. 18, 635644. Dani, V., Dhawan, D.K., 2005. Radioprotective role of zinc following single dose radioiodine (131I) exposure to red blood cells of rats. Ind. J. Med. Res. 122, 338 342. Dhar, P., Ghosh, S., Bhattacharyya, D.K., 1999. Dietary effects of conjugated octadecatrienoic fatty acid (9 cis, 11 trans, 13 trans) levels on blood lipids and nonenzymatic in vitro lipid peroxidation in rats. Lipids 34, 109114. Dhawan, A., Mathur, N., Seth, P.K., 2001. The effect of smoking and eating habits on DNA damage in Indian population as measured in the Comet assay. Mutat. Res. 474, 121128. Flora, J.S., 1999. Arsenic-induced oxidative stress and its reversibility following combined administration of n-acetylcysteine and meso 2,3-dimercaptosuccinic acid in rats. Clin. Exp. Pharma. Phys. 26, 865869. Garcia-Shavez, E., Jimenez, I., Segura, B., Razo, L.M.D., 2006. Lipid peroxidative damage and distribution of inorganic and its metabolite in the rat nervous system after arsenite exposure: inuence of alpha tocopherol. Neurotoxicolology 27, 10241031. Hassoun, E.A., Stohs, S.J., 1995. Comparative studies on oxidative stress as a mechanism for the fetotoxic of TCDD, endrin and lindane in C57BL/6J and DBA/ 2J mice. Teratology 51, 186192. Hevel, J.M., Marletta, M.A., 1994. Nitric-oxide synthase assays. Meth. Enzymol. 233, 250258. Huda-Faujan, N., Noriham, A., Norrakiah, A.S., Babji, A.S., 2009. Antioxidant activity of plants methanolic extracts containing phenolic compounds. Afr. J. Biotechnol. 8, 484489.

S.S. Saha, M. Ghosh / Food and Chemical Toxicology 48 (2010) 33983405 Ichihara, K., Shibahara, A., Yamamoto, K., Nakayama, T., 1996. An improved method for rapid analysis of the fatty acids of glycerolipids. Lipids 31 (5), 535539. Imaeda, A., Kaneko, T., Aoki, T., Kondo, Y., Nakamura, N., Nagase, H., Yoshikawa, T., 2002. Antioxidative effect of uvastatin and its metabolites against DNA damage in streptozotocin-treated mice. Food Chem. Toxicol. 40, 14151422. Ip, C., Scimeca, J.A., Thompson, H.J., 1994. Conjugated linoleic acid. A powerful anticarcinogen from animal fat sources. Cancer 74, 10501054. Joanes, J.H., Foster, C.A., 1942. Salt mixture for use with basal diet either low or high in phosphorus. J. Nutr. 24, 245256. Kadirvel, R., Sundaram, K., Mani1, S., Samuel, S., Elango, N., Panneerselvam, C., 2007. Supplementation of ascorbic acid and a-tocopherol prevents arsenic-induced protein oxidation and DNA damage induced by arsenic in rats. Hum. Exp. Toxicol. 26, 939946. Kulkarni, A.C., Kuppusamy, P., Parinandi, N., 2007. Oxygen, the lead actor in the pathophysiologic drama: enactment of the trinity of normoxia, hypoxia, and hyperoxia in disease and therapy. Antioxid. Redox Signal. 9, 17171730. Lakshminarayana, G., Kaimal, T.N.B., Mani, V.V.S., Sita Devi, K., Chandrasekhar Rao, T., 1982. Fatty acid changes during maturation of Momordica charantia and Trichosanthes anguina seeds. Phytochemistry 21, 303305. Lawrence, J.M., 1999. Lipid peroxidation DNA damage by malondialdehyde. Mutat. Res. 424, 8395. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193, 265275. Martin Jr., J.P., Dailey, M., Sugarman, E., 1987. Negative and positive assays of superoxide dismutase based on hematoxylin autoxidation. Arch. Biochem. Biophys. 255, 329336. Mezick, J.A., Settlemire, C.T., Brierley, G.P., Bareeld, K.P., Jensen, W.N., Cornwell, D.G., 1970. Erythrocyte membrane interactions with menadione and menadione-induced hemolysis. Biochim. Biophys. Acta. 219, 361371. Mohandas, J., Marshall, J.J., Duggin, G.G., Horvath, J.S., Tiller, D.J., 1984. Low activities of glutathione-related enzymes as factors in the genesis of urinary bladder cancer. Cancer Res. 44, 50865091. Mukherjee, C., Bhattacharyya, S., Ghosh, S., Bhattacharyya, D.K., 2002. Dietary effects of punicic acid on the composition and peroxidation of rat plasma lipid. J. Oleo Sci. 51, 513522. Noonan, D.M., Benelli, R., Albini, A., 2007. Angiogenesis and cancer prevention: a vision. Recent Results. Cancer Res. 174, 219224. Pariza, M.W., Ha, Y.L., 1990. Newly recognized anticarcinogenic fatty acids. Basic Life Sci. 52, 167170. Ramanathan, K., Balakumar, B.S., Panneerselvam, C., 2002. Effects of ascorbic acid and alpha tocopherol on arsenic-induced oxidative stress. Human Exp. Toxicol. 21, 675680.

3405

Rodriguez, V.M., Carrizales, L., Jimenez-Capdeville, M.E., Dufour, L., Giordano, M., 2001. The effects of sodium arsenite exposure on behavioral parameters in the rat. Brain Res. Bull. 55, 301308. Rose, H.G., Oklander, M.J., 1996. Improved procedures for the extraction of lipids from human erythrocytes. Lipid Res. 6, 428431. Saha, S.S., Ghosh, M., 2009. Comparative study of antioxidant activity of aeleostearic acid and punicic acid against oxidative stress generated by sodium arsenite. Food Chem. Toxicol. 47, 25512556. Scheffe, H., 1961. The Analysis of Variance. John Wiley and Sons, New York. Sec. 2.2 (pp. 2728), Sec. 3.6 (pp. 7374).. Schmedes, A., Hlmer, G., 1989. New thiobarbituric acid (TBA) Method for determining free malondialdehyde (MDA) and hydroperoxides selectivity as a measure of lipid peroxidation. J. Am. Oil Chem. Soc. 66, 813817. Serani, M., Bellocco, R., Wolk, A., Ekstrom, A.M., 2002. Total antioxidant potential or fruits and vegetables and risk of gastric cancer. Gastroenterology 123, 985991. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell. Res. 175, 184191. Takagi, T., Itabashi, Y., 1981. Occurrence of mixtures of geometrical isomers of conjugated octadecatrienoic acids in some seed oils: analysis by open-tubular gas chromatography and HPLC. Lipids 16, 546551. Thirunavukkarasu, C., Sakthisekaran, D., 2003. Sodium selenite, dietary micronutrient, prevents the lymphocyte DNA damage induced by Nnitrosodiethylamine and phenobarbital promoted experimental hepatocarcinogenesis. J. Cell Biochem. 88, 578588. Wills, E.D., 1987. Evaluation of lipid peroxidation in lipids and biological membranes, in Biochemical Toxicology: A Practical Approach (Snell. K. and Mullock, B., ed.). IRL Press. Oxford. Washington DC. pp. 127151. Yamauchi, H., Aminaka, Y., Yoshida, K., Sun, G., Pi, J., Waalkes, M.P., 2004. Evaluation of DNA damage in patients with arsenic poisoning: urinary 8hydroxydeoxyguanine. Toxicol. Appl. Pharmacol. 198, 291296. Yu, L., Adams, D., Gabel, M., 2002a. Conjugated linoleic acid isomers differ in their free radical scavenging properties. J. Agric. Food Chem. 50, 41354140. Yu, Y., Correll, P.H., Vanden Heuvel, J.P., 2002b. Conjugated linoleic acid decreases production of pro-inammatory products in macrophages: evidence for a PPAR gamma-dependent mechanism. Biochim. Biophys. Acta. 1581, 8999. Yumoto, I., Ichihashi, D., Iwata, H., Istokovics, A., Ichise, N., Matsuyama, H., Okuyama, H., Kawasaki, K., 2000. Purication and characterization of a Catalase from the facultatively psychrophilic bacterium Vibrio rumoiensis S-1 T exhibiting high catalase activity. J. Bacteriol. 182, 19031909. Zaman, K., MacGill, R.S., Johnson, J.E., Ahmad, S., Pardini, R.S., 1995. An insect model for assessing oxidative stress related to arsenic toxicity. Arch. Insect Biochem. Physiol. 29, 199209.