Soy Diet Diminish Oxidative Injure and Early Promotional Events Induced by CCl4 in Rat Liver

Husain Khan, Tajdar

INTRODUCTION

Oxidative stress occurs when there is an imbalance between Reactive Oxygen Species (ROS) formation and scavenging by antioxidants. ROS can cause oxidative damage to biomolecules resulting mutagenesis and carcinogenesis (Aggarwal et al., 2009; Rajendran et al., 2008; Jahan et al., 2011). ROS has been implicated in many degenerative diseases, collectively with aging and cancer (Aggarwal et al., 2009; Hamilton, 2007; Morse and Stoner, 1993; Mohamed et al., 2011; Avci et al., 2005). Carbon tetrachloride induced hepatotoxicity via hepatocellular fatty degeneration and centrilobular necrosis (Ha and Lee, 2003). The widely accepted mechanism of CCl₄¯ initiated liver injury involves the bioactivation of CCl₄ by the cytochrome P₄₅₀mediated reactions to the CCl₃ free radical which is further converted to a peroxy radical, CCl₃O₂. These free radicals readily react with polyunsaturated fatty acids to initiate lipid peroxidation. Peroxy radicals can react with polyunsaturated fatty acids in presence of cellular O₂, to initiate series of self-propagating chain reactions that lead to lipid peroxidation (Ha and Lee, 2003; Das et al., 2004; Alisi et al., 2011; Dhanasekaran and Ganapathy, 2011).

Antioxidants protect cells from free radical mediated oxidative damage. Cellular damage initiated by free radicals has been implicated in the development of cancer (Khan et al., 2006; Khan and Sultana, 2006; Khan and Sultana, 2011; Ramesh et al., 2007; Ali et al., 2010; Onocha et al., 2011). It is essential to identify antioxidant, anti-promoting agents present in human diet. Therefore, we have been constantly working on the identification of chemopreventive plants (Khan et al., 2006; Khan and Sultana, 2011, 2004a, 2004b, 2005; Khan et al., 2005; Khan and Sultana, 2009). The significance of nutrition in protecting living organisms from the toxic effects of environmental carcinogens has been realized (Aggarwal et al., 2009; Onocha et al., 2011; Gourineni et al., 2010). Herbal products are gaining increasingly attention due to less toxicity and high efficacy against various diseases (Khan et al., 2006; Hudecava and Ginter, 1992; Gourineni et al., 2010). Soybeans are unique foods, contains complex carbohydrates, protein, dietary fiber, oligosaccharides and isoflavones (Lichtenstein, 1998). Soybean decreases the risk of various diseases and pathological conditions, including various types of cancers, osteoporosis, menopausal symptoms and coronary heart disease (Archer 1988; Barnes, 2010; Nagata, 2010; Steinmetz and Potter, 1991; Pugalendhi and Manoharan, 2010). The chemopreventive effects of soybean and soy containing food may be related to genistein, daidzein and glycitein (Archer, 1988; Nagata, 2010; Messina and Barnes, 1991; Wei et al., 1995). These isoflavones may be conjugated with a 7-O-b-glucoside, a 6’’-O-malonylglucoside or a 6’’-O-acetylglucoside group which play an important role in the prevention atherosclerosis, hypertension (Park et al., 2005), breast cancer (DiSilvestro et al., 2005) and inflammatory bowel syndrome (Wiseman, 2006). Flavonoids have gained importance as scavengers of free radicals and a potent inhibitor of lipidperoxidation (Khan and Sultana, 2006; Bors et al., 1990; Hodgson et al., 1996). Isoflavones show DNA topoisomerase activity, synthesis and release of TGF^β, modulation of apoptosis and potent inhibitory activity of tyrosine specific protein kinases (Barnes, 2010; Nagata, 2010; Birt et al., 2001). It has been reported that population having high intake of isoflavones show lower incidence of cardiovascular diseases, osteoporosis, kidney diseases and cancer risk (Bors et al., 1990; Nagata, 2010).

Keeping in view of complex health promoting potential of soybeans and soy foods we have assumed that the soy diet may inhibit oxidative stress and toxicity induced by CCl₄.

MATERIALS AND METHODS

Chemicals: EDTA, Tris, Reduced glutathione (GSH), oxidized glutathione (GSSG), nicotinamide adenine dinucleotide phosphate reduced (NADPH), Bovine Serum Albumin (BSA), 1,2-dithio-bisnitrobenzoic acid (DTNB), 1-chloro-2, 4-dinitro benzene (CDNB), phenyl methylsulfonylfluoride (PMSF), nitrobluetetrazolium (NBT), phenylmethylsulfonylfluoride (PMSF), Brij-35, Pyridoxal-phosphate, 2-mercaptoethanol, dithiothreitol, Tween 80 were obtained from Sigma chemicals Co (St Louis, MO). DL [¹⁴C] ornithine and [³H] thymidine were purchased from Amersham Corporation (Little Chalfort, UK). All other chemicals were of the highest purity and commercially available.

Animals: Eight week old adult male wistar rats (150-200 g) were obtained from the Central Animal House Facility of Hamdard University, New Delhi and were housed in a ventilated room at 25±2°C under a 12 h light/dark cycle. The animals were acclimatized for one week before the study and had free access to standard laboratory feed (Hindustan Lever Ltd., Bombay, India) and water ad libitum. All procedures using animals were reviewed and approved by the Institutional Animal Ethical Committee that is fully accredited by the Committee for Purpose of Control and Supervision on Experiments on Animals (CPCSEA) Chennai, India.

Experimental design: The treatment regimen for soy diet was based on the preliminary studies carried out in our laboratory. To study the biochemical, serological changes 25 male wistar rats were divided into five groups. Group I served as saline treated control. Group II served as negative control and was administered CCl₄ (1 mL kg^-1 body weight p.o., in corn oil [1:1]). Group III served as positive control and was given higher dose (D₂) of soy diet for seven consecutive days. Groups IV and V were pretreated with soy diet at doses 10 and 15% soy flour as diet for seven consecutive days followed by CCl₄ intoxication on the 7th day. All animals were sacrificed 24 h after CCl₄intoxication. Serum was separated and stored at 4°C for the estimation of GOT, GPT and LDH. Tissue was processed for the estimation of hepatic ornithine decarboxylase (ODC) activity and other biochemical parameters. For [³H] thymidine incorporation study, same treatment regimen was followed except all the animals were given intraperitoneal [³H] thymidine (25 μCi per animal) 2 h before killing. Time of sacrifice was after 48 h of CCl₄ intoxication; liver sections were quickly excised, rinsed with ice-cold saline, freed of extraneous material and processed for the quantification of [³H] thymidine incorporation into the hepatic DNA.

Biochemical estimations: Tissue processing and preparation of Post Mitochondrial Supernatant (PMS) were done as described by Athar and Iqbal (1998). All the biochemical estimations were completed within 24 h of animal sacrifice.

Estimation of reduced glutathione: Reduced glutathione was determined by the method of Jollow et al. (1974). A 1.0 mL sample of PMS was precipitated with 1.0 mL of sulfosalicylic acid (4%). The samples were kept at 4°C for one hour and then centrifuged at 1200 g for 20 min at 4°C. The assay mixture contained 0.4 mL supernatant, 2.6 mL sodium phosphate buffer (0.1 M, pH 7.4) and 0.2 mL DTNB (100 mM) in a total volume of 3.0 mL. The yellow colour developed, was read immediately at 412 nm on a spectrophotometer.

Catalase activity: Catalase activity was measured by the method of Clairborne (1985). The reaction mixture consisted of 2 mL phosphate buffer (0.1 M, pH 7.4), 0.95 mL hydrogen peroxide (0.019 M) and 0.05 mL PMS in a final volume of 3 mL. Changes in absorbance were recorded at 240 nm. Catalase activity was calculated as nmol H₂O₂ consumed min^-1 mg^-1 protein.

Glutathione-S-transferase activity: Glutathione-S-transferase activity was estimated by the method of Habig et al. (1974).The reaction mixture consisted of 1.425 mL sodium phosphate buffer (0.1 M, pH 7.4), 0.2 mL reduced glutathione (1 mM), 0.025 mL CDNB (1 mM) and 0.3 mL PMS (10% w/v) in a total volume of 2.0 mL. The changes in the absorbance were recorded at 340 nm and enzymes activity was calculated as nmol CDNB conjugate formed min^-1 mg^-1 protein using a molar coefficient of 9.6x10³ M^-1 cm^-1.

Glutathione reductase activity: Glutathione reductase activity was assayed by the method of Carlberg and Mannervik (1975), as modified by Mohandas et al. (1984). The assay system consisted of 0.1 M, pH 7.4 sodium phosphate buffer, 0.5 mM EDTA, 1 mM GSSG, 0.1 mM NADPH and PMS (10% w/v) in a total mixture of 2.0 mL. The enzyme activity was measured at 340 nm and calculated using a molar extinction coefficient of 6.22x10³ M cm^-1.

Glutathione peroxidase activity: Glutathione peroxidase activity was assayed by the method of Mohandas et al. (1984), as described by Athar and Iqbal (1998). The assay mixture consisted of 0.1 M, pH 7.4 sodium phosphate buffer, 1 mM EDTA, 0.2 mM NADPH, 1 mM sodium azide, 1 IU mL^-1 glutathione reductase, 0.25 mM H₂O₂ and PMS (10% w/v) in a total volume of 2.0 mL. The activity was recorded at 340 nm and calculated using a molar extinction coefficient of 6.22x103 M^-1 cm^-1.

Glucose-6-phosphate dehydrogenase activity: The activity of glucose-6-phosphate dehydrogenase was assayed by the method of Zaheer et al. (1965). The reaction mixture consisted of 0.3 mL tris-HCl buffer (0.05 M, pH 7.6), 0.1 mL NADP (0.1 mM), 0.1 mL glucose-6-phosphate (0.8 mM), 0.1 mL MgCl₂ (8 mM), 0.3 mL PMS and 2.4 mL distilled water in a total volume of 3 mL. The changes in absorbance were recorded at 340 nm and enzyme activity was calculated as nmol NADPH oxidized min^-1 mg^-1 protein using a molar extinction coefficient of 6.22x10³M^-1 cm^-1.

Quinone reductase activity: Quinone reductase activity was measured by the method of Benson et al. (1980). The assay mixture consisted of 0.1 mL cytosolic fraction (10%), 0.7 mL of BSA (0.1%), 0.02 mL tween-20 (1%), 0.1 mL of FAD (150 M), 0.02 mL of NADPH (0.2 mM), 0.05 mL of 2,6, DCIP (0.29%) and 2 mL of tris-HCl buffer (25 mM, pH 7.4) with a final volume of 3 mL and the optical density was read at 600 nm for 3 min. The enzyme activity was calculated as nmol 2,6, DCIP reduced min^-1 mg^-1 protein.

Xanthine oxidase activity: The activity of xanthine oxidase was assayed by the method of Stirpe and Corte (1969). The reaction mixture consisted of 0.2 mL PMS which was incubated for five minutes at room temperature with 0.8 mL phosphate buffer (0.1 M, pH 7.4). Then 0.1 mL xanthine (9 mM) was added to the reaction mixture and kept at 37°C for 20 min which was followed by the addition of 0.5 mL of 10% perchloric acid and 2.4 mL of double distilled water in a total volume of 4 mL. After 10 min, the mixture so obtained was centrifuged at 4000-rev min^-1 for 10 min and μg uric acid formed min^-1 mg^-1 protein was recorded at 290 nm.

Estimation of lipid peroxidation: The assay of microsomal lipid peroxidation was done according to the method of Wright et al. (1981). The reaction mixture consisted of 0.58 mL phosphate buffer (0.1 M, pH 7.4), 0.2 mL microsome, 0.2 mL ascorbic acid (1 mM) and 0.02 mL ferric chloride (100 mM) in a total volume of 1 mL. The mixture was incubated at 37°C in a shaking water bath for 1 h. Then 1 mL 10% trichloroacetic acid and 1 mL 0.67% TBA was added. All the tubes were placed in a boiling water bath for 20 min. The tubes were placed in an ice bath and then centrifuged at 2500 g for 10 min. The amount of malanodialdehyde (MDA) formed in each of the samples was assayed by measuring the optical density of the supernatant at 535 nm.The results were expressed as nmol MDA formed h^-1 g^-1 tissue at 37°C using a molar extinction coefficient of 1.56x10⁵M^-1 cm^-1.

Lactate dehydrogenase (LDH) activity: Lactate dehydrogenase activity was estimated in serum by the method of Kornberg (1955). The reaction mixture consisted of serum, NADH (0.02 M), Sodium pyruvate (0.01 M), sodium phosphate buffer (0.1 M, pH 7.4) and distilled water in a total volume of 3.0 mL. The changes in the absorbance were recorded at 340 nm and enzyme activity was calculated as nmol NADH oxidized min^-1 mg^-1 protein.

Determination of serum oxaloacetate and pyruvate transaminases (GOT and GPT): Serum GOT and GPT were determined by the method of Reitman and Frankel (1957). Ach substrate (0.5 mL) (2 mM α-ketoglutarate and either 200 mM α L-Alanine or L-Aspartate was incubated for 5 min at 37°C in a water bath. Serum (0.1 mL) was then added and the volume was adjusted to 1.0 mL with sodium phosphate buffer. The reaction mixture was incubated for exactly 30 and 60 min for GPT and GOT, respectively. Then to the reaction mixture, 0.5 mL of DNPH (1 mM) was added and left for another 30 min at room temperature. Finally, the colour was developed by addition of 5.0 mL of NaOH (0.4 N) and product read at 505 nm.

Estimation for tumor markers
Ornithine decarboxylase activity: ODC activity was determined using 0.4 mL hepatic 105,000 g supernatant fraction per assay tube by measuring the release of CO₂ from DL-[¹⁴C] ornithine by the method of O’Brien et al. (1975). The liver was homogenized in Tris-HCl buffer (pH 7.5, 50 mM) containing EDTA (0.4 mM), pyridoxal phosphate (0.32 mM), PMSF (0.1 mM), 2-mercaptoethanol (1.0 mM), dithiothreitol (4.0 mM) and Tween 80 (0.1%) at 4°C using a Teflon-glass homogenizer. In brief, the reaction mixture contained 400 μL enzymes and 0.095 mL co-factor mixture containing pyridoxal phosphate (0.32 mM), EDTA (0.4 mM), dithiothreitol (4.0 mM), ornithine (0.4 mM), Brij 35 (0.02%) and DL-[¹⁴C] ornithine (0.05 μCi) in total volume of 0.495 mL. After adding buffer and cofactor mixture to blank and others tubes, the tubes were closed immediately with a rubber stopper containing 0.2 mL ethanolamine and methoxyethanol mixture (2:1) in the central well and kept in water-bath at 37°C. After 1 h of incubation, injecting 1.0 mL citric arrested the enzyme activity acid solution (2.0 M) along the sides of glass tubes and the solution was continued for 1 h to ensure complete absorption of CO₂. Finally, the central well was transferred to a vial containing 2 mL ethanol and 10 mL toluene based scintillation fluid. Radioactivity was counted in liquid scintillation counter (LKB Wallace-1410). ODC activity was expressed as pmol CO₂ released h^-1 mg^-1 protein.

Hepatic DNA synthesis: The isolation of hepatic DNA and incorporation of [³H] thymidine in DNA was done by the method of Smart et al. (1986). Liver was quickly removed cleaned free of extraneous material and homogenate (10% w/v) was prepared in ice-cold water. The precipitate thus obtained was washed with cold trichloroacetic acid (TCA) (5%) and incubated with cold perchloric acid (PCA) (10%) at 4°C for overnight. After the incubation it was centrifuged and the precipitate was washed with cold PCA (5%). The precipitate was dissolved in warm PCA (10%) followed by incubation in boiling water bath for 30 min and filtered through Whatman 50. The filtrate was used for [³H] thymidine counting in liquid scintillation counter (LKB-Wallace-1410) by adding the scintillation fluid. The amount of DNA in the filtrate was estimated by diphenylamine method of Giles and Myers (1965). The amount of [³H] thymidine incorporated was expressed as DPM μg^-1 DNA.

Protein estimation: Protein content in all samples was estimated by the method of Lowry et al. (1951) using bovine serum albumin as standard.

Statistical analysis: The level of significance between different groups is based on ANOVA test followed by Dunnett's t test.

RESULTS

CCl₄ intoxication leads to depletion of hepatic glutathione, its metabolizing enzymes GST and GR and antioxidant enzymes CAT, GPx, QR and G6PD by (p<0.001), respectively as compared with the saline treated control group. CCl₄ also caused elevation in the activity of XO and H₂O₂content and increase in the levels of MDA formation and hepatic toxicity markers SGOT and SGPT by (p<0.001). Pretreatment with soy diet (10 and 15%) restored hepatic glutathione content and its dependent enzymes GST and GR significantly (p<0.001) as shown in Table 1. Other antioxidant enzymes like CAT GPx, G6PD and QR were significantly restored at (p<0.001) as shown in Table 2.

There was a marked depletion in levels of XO and MDA formation at (p<0.001) and concomitant down regulation of release of SGOT and SGPT in serum at (p<0.001). Figure 1 and 2 show the significant inhibition of early markers of tumor promotion like ODC activity (p<0.001) and hepatic DNA synthesis (p<0.005) in rat. The prophylactic treatment of animals with soy diet against CCl₄ induced elevation in XO level, H₂O₂ and MDA formation is shown in Table 3.


Fig. 1:	Effect of pretreatment of SF on ³[H] thymidine incorporation in hepatic DNA

Table 1:	Effect of pretreatment of soy diet on the CCl₄ mediated depletion in the activity of glutathione content and its metabolizing enzymes in the liver of wistar rats

p≤0.05, p≤0.01 and **p≤0.001 on comparison of normal control with CCl₄treated group and only soy group. ^#p≤0.05 ^##p≤0.01 and ^###p≤0.001 on comparison of soy diet treated groups with only CCl₄treated group

Table 2:	Effect of pretreatment of soy diet on the CCl₄ mediated depletion in the activity of antioxidant enzymes in the liver of wistar rats

p≤0.05, p≤0.01 and **p≤0.001 on comparison of normal control with CCl₄treated group and only soy group. ^#p≤0.05, ^##p≤0.01 and ^###p≤0.001 on comparison of soy diet treated groups with only CCl₄treated group

Table 3:	Effect of pretreatment of soy diet on the CCl₄ mediated alteration in the activities of xanthine oxidase and enhancement of lipid peroxidation in the liver of wistar rats

p≤0.05, p≤0.01 and **p≤0.001 on comparison of normal control with CCl₄treated group and only soy group. ^#p≤0.05, ^##p≤0.01 and ^###p≤0.001 on comparison of soy diet treated groups with only CCl₄treated group

Table 4:	Effect of pretreatment of soy diet on the CCl₄ mediated enhancement of liver markers in wistar rats

p≤0.05, p≤0.01 and **p≤0.001 on comparison of normal control with CCl₄treated group and only soy group. ^#p≤ 0.05, ^##p≤ 0.01 and ^###p≤0.001 on comparison of soy diet treated groups with only CCl₄treated group


Fig. 2:	Effect of pretreatment of SF on ODC activity in liver

Prophylactic administration of soy diet prior to CCl₄ intoxication significantly decreased the MDA levels at significance (p<0.001), respectively in a dose dependent manner. However, soy diet alone group produced results near to saline control values. Soy diet showed a significant protection of GSH content by p<0.001 in a dose dependent manner is shown in Table 1. The effect of prophylactic administration of soy diet on CCl₄ mediated leakage of liver marker enzymes in serum is shown in Table 4. Marked inhibition was recorded in serum toxicity markers in soy diet treated groups at (p<0.001). The effect of prophylactic treatment of rats with soy diet on CCl₄ induced rate of [³H] thymidine incorporation into hepatic DNA has been shown in Fig. 2. Intoxication with CCl₄ resulted in significant (p<0.005) increase in the rate of [³H] thymidine incorporation into hepatic DNA and significant (p<0.001) increase in ODC activity which is an early marker of tumor promotion. The prophylactic treatment of rats with soy diet showed a marked (p<0.001) suppressing effect on the rate [³H] thymidine incorporation into hepatic DNA of treated control and simultaneous and significant (p<0.001) inhibition of ODC activity was recorded in a dose dependent manner as shown in Fig. 1 and 2.

DISCUSSION

Single oral CCl₄ dose induced significant depletion in the hepatic GSH content and its metabolizing enzymes. Glutathione is often considered as first line of defense against oxidative damage (Ali-Osman, 1989; Patrick-Iwuanyanwu and Wegwu, 2008). There was significant restoration of glutathione and dependent enzymes in soy diet pretreated groups. The restoration of depleted GSH and GST on pretreatment of animals with soy diet indicate its role as chemopreventive measure. CCl₄ causes increased formation of pro-oxidants and a concomitant decrease in the antioxidant status of the cells. Hepatotoxic effects of CCl₄ depend on the major unstable radical trichloromethyl radical which reacts with oxygen molecule and form trichloromethylperoxy radical which is reported to be highly reactive. These free radicals covalently bind to macromolecules and proteins and thus initiating a site for secondary biochemical process which ultimately leads to several pathological conditions of CCl₄ metabolism (Singh et al., 2005; Hassan et al., 2008). It has been suggested that Soy diet protects by suppressing CCl₄mediated toxicity through decrease in levels of MDA formation and inhibition in hepatic XO levels. GST is responsible for GSH conjugation with various electrophiles and plays an important role in detoxification of various xenobiotics (Touliatos et al., 2000). Induction of GST by Soy diet could be one possible mechanism to combat hepatic oxidative stress (Singh et al., 2000; Dhanasekaran and Ganapathy, 2011). QR catalyzes hydroquinone formation from quinones, preventing cyt P₄₅₀dependent activation to toxic semiquinones and ultimate generation of Reactive Oxygen Species (ROS) (Khan and Sultana, 2005). Well documented correlation exists between its modulation and chemoprevention of cancer Substantial depression in hepatic GSH with concomitant decrease in GR, GPx, QR, GST and G6PD levels on CCl₄administration; however, dose dependent marked restoration was observed with prophylactic treatment of Soy diet.

The mechanisms by which Soy diet attenuates hepatic damage induced by CCl₄ can be attributed to recovery of antioxidant status. Previous studies have shown that Soy potential to increase cellular antioxidants like GSH and other phase II metabolizing enzymes (Khan and Sultana, 2004a, b; Khan et al., 2005; Borras et al., 2010). Soy diet prior to CCl₄intoxication restored the levels of serum toxicity markers viz., SGOT and SGPT as compared to negative control. CCl₄administration resulted in significant induction in hepatic ODC and [³H] thymidine uptake into hepatic DNA, There is enough evidence that indicates that the polyamines play essential role in the regulation of various cellular and metabolic functions including DNA synthesis, modulation of membranous functions and numerous enzyme activities (Wallon and O'Brien, 2005). Prophylactic treatment with Soy diet prior to CCl₄intoxication showed profound suppression ODC activity and rate of [³H] thymidine incorporation.

CONCLUSION

Modulatory action of Soy diet is observed by several mechanisms, it reduces the risk of acute hepatic injury induced by CCl₄and thus protecting against post necrotic hepatic injury. In conclusion present study give some insight into mechanisms involved in modulatory action of Soy diet against CCl₄ induced hepatic toxicity in rats. Thus, we suggest it may further be used as potent cancer chemopreventive agent.

ACKNOWLEDGEMENT

Dr Tajdar Husain Khan is thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi, India, for providing the funds to carry out this study.