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Indian Journal of Experimental Biology Vol. 48, April 2010, pp. 373-377 Effect of antioxidant vitamins A, C, E and their analogues on azo-dye binding protein in liver of rats treated with p-dimethylaminoazobenzene A Antony Joseph Velanganni* Department of Biochemistry, J.J. College of Arts and Science, Pudukkottai 622 422, India and C Balasundaram Department of Animal Science, Bharathidasan University, Tiruchirappalli 620 024, India Received 21 July 2009; revised 19 November 2009 p-Dimethylaminoazobenzene (DAB) is an azo-dye and known to cause liver tumour in rats. Azo-dye binding protein is a specific cytosolic protein involved in the translocation of azo-dye carcinogen metabolites from liver cytoplasm into the nucleus. Administration of vitamin A (40,000 and 50,000 IU), L-ascorbic acid (500 and 1,000 mg) and vitamin E succinate (200–500 mg) reduced the amount of azo-dye binding protein in liver of rats treated with DAB. Supplementation of high doses of vitamin A acetate, vitamin A palmitate, sodium ascorbate, ascorbyl palmitate and vitamin E acetate had no effect on the quantity of azo-dye binding protein in liver. When the vitamin mixture was given, the level of azo-dye binding protein decreased in the liver at all the studied doses, which may be due to their synergistic effect. Keywords: Antioxidant vitamins A, C and E, Azo-dye binding protein, p-Dimethylaminoazobenzene The pioneering work of Miller and Miller1 established that aminoazo-dye, a known hepato carcinogen, binds to liver protein in rats; further in vivo studies on various animal models proved that the chosen carcinogens bound to cellular protein in tissues rendered them tissues susceptible to carcinogenic action. Azo-dye binding protein is a specific cytosolic protein involved in the translocation of azo-dye carcinogen metabolites from liver cytoplasm into the nucleus. The binding of azo-dye metabolites with nuclear macromolecule necessitate further prior processing that occur in the nucleus. The azo-dye binding protein is found in all the highlydifferentiated hepatomas. It is not found in poorly differentiated hepatomas, anaplastic carcinomas and adenocarcinomas2. Thus the presence of azo-dye binding protein in tumour indicates the degree of cell differentiation. The hepatic binding protein is involved in the translocation of azo-dye carcinogen metabolites to the nucleus from liver cytoplasm. Interaction of azo-dye metabolites with nuclear macromolecules facilitates carcinogenesis that occurs in the nucleus3. Covalent binding of azo-dye metabolites to DNA occurs when the azo-dye metabolites are incubated with liver nuclei and not with isolated liver DNA. This is due to the specific soluble protein that controls the translocation form cytosol to the nucleus4. The amount of protein-bound azo-dye is three times more in mice after 2-3 weeks of DAB feeding; in hamster, azo-dye binding to proteins increased gradually up to 6 weeks, and then remained constant5. Sub-fractionation of liver homogenates on administration of azo-dyes shows that the dye is bound principally with the microsomal fraction (insoluble) and the soluble supernatant6-8. The soluble fraction subjected to zone electrophoresis has shown three protein fractions, namely a major basic protein fraction and two minor mono-acidic protein fractions9. The present study is concerned with the soluble proteins that bind specifically to the azo-dye so as to evaluate the role of antioxidant vitamins A, C, E and their analogues in preventing the binding of azo-dyes in DAB-treated rats. —————— *Correspondent author Telephone: +91-04322-260103 Fax: +91-04322-260103 E-mail: [email protected] Materials and Methods Animals—Male albino rats of Wistar strain (170 ± 20 g), reared and maintained in the animal house of 374 INDIAN J. EXP BIOL., APRIL 2010 the Department of Biochemistry (under the supervision of Institutional Animal Ethics Committee), J. J. College of Arts and Science, Pudukkottai, were used. DAB and vitamins—The hepato carcinogen p-dimethylaminoazobenzene (p-DAB; CAS No. 60-11-7) was purchased from Sigma Chemical Co., St. Louis, USA. Vitamin A, C and E and their analogues vitamin A acetate, vitamin A palmitate, sodium ascorbate, ascorbyl palmitate, vitamin E acetate and vitamin E succinate with research grade purity were used for the present work. Individual vitamin treatment —Rats (275) were divided into 3 groups. First group (n = 25) served as normal control. The second group (n = 25) received 0.06% of DAB2 for 4 months followed by basal diet alone for an additional 2 months and served as DAB control. The third group consisting of the remaining 225 rats was further subdivided into subgroups A, C and E (n = 75) based on the vitamins used. The subgroups A, C and E were further divided into subsets of A1, A2 and A3, C1, C2 and C3 and E1, E2 and E3 respectively consisting of 25 rats each. Finally, all the subsets were further divided into 5 sub-subsets (Sub-subsets I to V) of 5 rats each. All the rats were acclimatized to laboratory conditions for one week and fed ad libitum with a basal diet [containing 660 g glucose, 180 g milk casein, 40 g salt mixture10, 100 g corn oil, 20 g cod liver oil, 1.5 g choline chloride, 50 mg vitamin K3, 20 mg riboflavin, 20 mg thiamine, 20 mg pyridoxine, 60 mg calcium pantothenate, 50 mg nicotinamide, 1.8 mg folic acid, 0.6 mg biotin, 100 mg inositol, 50 mg p-aminobenzoate, 40 μg cyanocobalamin per kg diet11. Animals of second and third groups were fed with 0.06% DAB supplemented diet2. Control rats were continued to be fed on the basal diet for 6 months. The third group was administered chosen doses of vitamins (per kg body wt) based on a previous study12, once in a week by gavage route {A1: DAB + vit. A (10,000-50,000 IU), A2: DAB + vit. A acetate (10,000-50,000 IU), A3: DAB + vit. A palmitate (10,000-50,000 IU), C1: DAB + L-ascorbic acid (75-1000 mg), C2: DAB + sodium ascorbate (75-1000 mg), C3: DAB + ascorbyl palmitate (75-1000 mg) and E1: DAB + vit. E (50-500 mg), E2: DAB + vit. E acetate (50-500 mg) and E3: DAB + vit. E succinate (50-500 mg)} for a period of 4 months. After this period, the vitamin enriched basal diet was offered for an additional period of 2 months. Multiple vitamin treatment—Multiple vitamin treatment study was performed with mixture13-15 of vitamin A, L-ascorbic acid and vitamin E succinate. For this study, 35 rats were divided into 7 groups of 5 rats each for normal, DAB, DAB + vitamin A (10,000 IU) + vitamin C (75 mg) + vitamin E (50 mg), DAB + vitamin A (20,000 IU) + vitamin C (150 mg) + vitamin E (100 mg), DAB + vitamin A (30,000 IU) + vitamin C (250 mg) + vitamin E (200 mg), DAB + vitamin A (40,000 IU) + vitamin C (500 mg) + vitamin E (400 mg), DAB + vitamin A (50,000 IU) + vitamin C (1000 mg) + vitamin E (500 mg). The mixture of vitamins was given to all the rats by gavage route once in a week for 6 months. Isolation of basic azo-dye binding protein— Animals administered with 0.06% of DAB were sacrificed with ether after 6 months and used for the isolation of basic azo-dye binding protein. Immediately, excised livers of rats were perfused with 20 ml cold 0.25 M sucrose. All the sub cellular particles were removed by centrifuging at 30,000 rpm (65,390 g) for 120 min. The resulting soluble cell supernatant was used to isolate azo-dye binding protein as per Ketterer et al16. Amino acid analysis—Azo-dye binding protein was hydrolysed in constant boiling HCl at 105oC for 16 h and hydrolysate was analysed by chromatography on Whatman no.1 filter paper with the solvent butanol-acetic acid-water (12:3:5). Ninhydrin reagent (100 mg/ml of acetone) was used to spot the amino acids and the amino acids present in the sample were then identified by comparing the Rf values with that of the authentic amino acids, co-chromatographed. Determination of molecular weight by SDSPAGE—Sodium dodecyl sulphate-poly acrylamide gel electrophoresis (SDS-PAGE) was performed with 10% poly acrylamide gel following the procedure of Laemmli17. Solution of basic azo-dye binding protein in PBS (10mg/2 ml) was prepared and applied in SDS-PAGE. The molecular weight of azo-dye binding protein was determined using SDS-PAGE in which protein marker of known molecular weight and the azo-dye binding protein fraction were run simultaneously. Sample containing 100 µg protein was mixed with an equal volume of sample buffer and loaded in the wells of stacking gel. Electrophoresis was performed at 50 V initially and then at 90 V until bromophenol blue moved nearer to the bottom of the gel. The VELANGANNI & BALASUNDARAM: ANTIOXIDANT VITAMINS ETC. & AZO-DYEBINDING PROTEIN proteins present in the gel were stained for 4 h, using 0.2% Coomassie brilliant blue R-250 (Sigma) in 50% methanol and 7% acetic acid. The gel was later destained using destaining solution (7% acetic acid and 30% methanol) until the background was clear. Estimation of bound azo-dye—The liver of rats administered 0.06% DAB were treated in a blender with 4 times its weight of distilled water until the tissue became a frothy pulp. The liver pulp was boiled for 5 min in acetate buffer and adjusted to pH 5 with acetic acid (5 ml for buffer /g of liver). After cooling, the coagulated protein was centrifuged and washed with buffer and then with 95% ethanol until no further colour could be extracted. Any remaining unbound azo-dye and lipid material was removed from the protein by extraction in a soxhlet apparatus with 95% alcohol for 48 h at 60oC. The protein mass was broken up and the ethanol was allowed to evaporate from the powder, which was then dried over P2O5 in a vacuum desicator2,18. Chemical assay—The liver protein powder was hydrolysed to release bound azo-dye and the released azo-dye was extracted with pentan-1-ol. The extracts were poured into 5 volumes of light petroleum and 10 ml of a freshly prepared mixture of concentratedHCl and acetone (2:1, v/v) was added. The mixture was shaken and allowed to stand for 30 min. The lower layer containing the dye was transferred to a measuring cylinder and its extinction was measured at 520 nm in a spectrophotometer. The colour intensity became maximal after 30 min and remained constant for several hours. The extraction was corrected to a final volume of 10 ml and the bound azo-dye was expressed in arbitrary units of E520/100 mg of protein18. Statistical analysis of the data was performed with Student’s ‘t’ test. Results The amino acid analysis of hydrolysates of the basic azo-dye binding protein shows the presence of following 17 amino acids: alanine (Ala), arginine (Arg), aspartic acid (Asp), cysteine (Cys), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), tyrosine (Tyr), threonine (Thr) and valine (Val). Amino acids such as Asn, Gln and Trp were not found in azo-dye binding protein (since tryptophan would have been destroyed during acid hydrolysis16, it is not been included in the list). The amino acid analysis of hydrolysates of basic azo-dye binding peptide after 375 being digested with protease contains Ala, Asp, Glu, Gly, Ser, Thr, other amino acids were destroyed during digestion with protease. Isolated azo-dye binding protein had a molecular weight of 45 KDa as evident from the SDS-PAGE study (Fig. 1). The amount of bound azo-dye in DAB-treated rats was found to be 0.41 arbitrary units/mg protein18. However, administration of vitamin A (40,000 and 50,000 IU), L-ascorbic acid (500 and 1,000 mg) and vitamin E succinate (200–500 mg) reduced the amount of azo-dye binding protein in liver (Fig. 2 to 4). Individual supplementation of high doses of vitamin A acetate, vitamin A palmitate, sodium ascorbate, ascorbyl palmitate and vitamin E acetate have no effect on quantity of azo-dye binding protein in liver (Fig. 5). When the vitamin mixture was given, the level of azo-dye binding protein decreased in the liver at all the studied doses (Fig. 6). Discussion Amino acid analysis of azo-dye binding peptide revealed the presence of Ala, Asp, Glu, Gly, Ser and Thr. The earlier studies on azo-dye bound peptide revealed that it contained Pro, Leu/Ile, Val and Gly as major components and Glu, Phe, Ser, Ala and Asp as minor components19. Terayama and Takeuchi20 isolated a fraction on hydrolysis of azo-dye binding protein which was found to have Phe, Ser, Gly, Pro, Val, Glu and Asp. The discrepancies between the earlier results and the present study may be due to the fact that more than one azo-dye binding protein exists and the possibility of the azo-dye to bind with different types of amino acid sequences in each protein gives several different azo-dye bound peptides16. Fig. 1 — SDS-Page profile of purified basic azo-dye-binding protein [Lane 1: marker proteins, Lane 2: protein bound azo-dye] 376 INDIAN J. EXP BIOL., APRIL 2010 Figs 2-6 — Protein bound azo-dye in liver [2—a: DAB, b: DAB + vit. A (10,000 IU), c: DAB + vit. A (20,000 IU), d: DAB + vit. A (30,000 IU), e: DAB + vit. A (40,000 IU), f: DAB + vit. A (50,000 IU). 3—a: DAB, b: DAB + vit. C (75 mg), c: DAB + vit. C (150 mg), d: DAB + vit. C (250 mg), e: DAB + vit. C (500 mg), f: DAB + vit. C (1,000 mg). 4—a: DAB, b: DAB + vit. E (50 mg), c: DAB + vit. E (100 mg), d: DAB + vit. E (200 mg), e: DAB + vit. E (400 mg), f: DAB + vit. E (500 mg). 5— a: DAB, b: DAB + vit. A (50,000), c: DAB + vit. A acetate (50,000), d: DAB + vit. A palmitate (50,000), e: L-ascorbic acid (1,000), f: DAB + sodium ascorbate (1,000), g: DAB + ascorbyl palmitate (1,000), h: DAB + vit. E (500), i: DAB + vit. E acetate (500), j: DAB + vit. E succinate (500). 6— a: DAB, b: vit. A (10,000 IU) + vit. C (75 mg) + vit. E (50 mg), c: vit. A (20,000 IU) + vit. C (150 mg) + vit. E (100 mg), d: vit. A (30,000 IU) + vit. C (250 mg) + vit. E (200 mg), e: vit. A (40,000 IU) + vit. C (500 mg) + vit. E (400 mg), f: vit. A (50,000 IU) + vit.C (1,000 mg) + vit. E (500 mg).*P<0.05 vs DAB] The diet rich in riboflavin increased the induction period of liver tumours by azo-dyes21. Similary, rats fed with both riboflavin and cupric oxyacetate hexahydrate delayed the formation of liver tumour induced by azo-dyes18. The results of the present study also indicate that supplementation of vitamin A, C and E offer protection against DAB by delaying the binding of the azo-dye. The binding of protein with azo-dye was lowered in vitamin treated rats. DAB can be inactivated in the liver by an enzyme system, azo-dye reductase, that reductively cleaves the molecule to non-carcinogenic amine moieties and that the coenzyme required is FAD5,22. The protection given by vitamins is through the increased enzymatic destruction of the azo-dye. Dyes were bound with the proteins from the high-sulfur-protein area or hightyrosine-protein area, depending on the level of dye dissolution23. The passage of azo-dye binding protein through an immobilized protease column reduced the azo-dye metabolite translocation by 65%, concomitant with the degradation of proteins24. Translocation from the cytoplasm into nucleus was not observed with protein-free metabolites and addition of protein promotes the metabolite translocation in rat liver24. In the present study, supplementation of vitamin mixture inhibits the translocation of azo-dye binding proteins in liver of rats treated with DAB. In conclusion, antioxidant vitamin mixture prevents the binding of azo-dye in the liver by reducing the level of azo-dye binding protein. References 1 2 3 4 5 6 7 Miller J A & Miller E C, The carcinogenicity of certain derivatives of p-dimethylaminoazobenzene in the rat, J Exp Med, 87 (1947) 139. Bannikov G A, Guelstein V I & Tchipysheva T A, Distribution of basic azo-dye binding protein in normal rat tissues and carcinogen-induced liver tumours, Int J Cancer, 11 (1973) 398. Srinivasan K, Levine W G & Bhargava M M, Non-covalent binding of 3’-methyl N, N-dimethyl-4-aminoazobenze and its metabolites to liver cytosolic proteins and its role in their nuclear translocation, Drug Metab Dispos, 15 (1987) 505. Srinivasan K, Levine W G & Bhargava M M, Protein binding, nuclear translocation & biliary secretion of metabolites of 3’-methyl-N, N-dimethyl-4-aminoazobenzene during hepatocarcinogenesis in rats, Xenobiotica, 21 (1991) 961. Decloitre F & Menuier M, Azo-dye binding to proteins and detoxication of p.dimethylaminoazobenzene in mouse and hamster liver, Int J Cancer, 6 (1970) 481. Hultin T, The intracellular distribution of protein-bound azo dye in rat liver, Exp Cell Res, 10 (1956) 71. Gelboin H V, Miller J A & Miller E C, Studies on hepatic protein-bound dye formation in rats given single large doses VELANGANNI & BALASUNDARAM: ANTIOXIDANT VITAMINS ETC. & AZO-DYEBINDING PROTEIN 8 9 10 11 12 13 14 15 of 3’methyl-4-dimethylaminoazobenzene, Cancer Res, 18 (1958) 608. Fiala S & Fiala A E, Intracellular localization of carcinogen and its relationship to the mechanism of carcinogenesis in rat liver, Br J Cancer, 13 (1959) 236. Sorof S, Young E M, McCue M M & Fetterman P L, Zonal electrophoresis of the soluble proteins of liver and tumour in azo dye carcinogenesis, Cancer Res, 23 (1963) 864. Hawk P B, Oser B L & Summerson W H, Practical physiological Chemistry, 13th edn. (Blackiston Co., New York), 1954, 1071. Oh S H & Lee M H, Effect of p-dimethylaminoazobenzene and 2(3)-tert-Butyl-4-hydroxyanisole on lipid peroxidation, glutathione-s-transferase, peroxidase and reductase in rat liver, Yonsei Med J, 22 (1981) 95. Velanganni A A J & Balasundram C, Protective effect of vitamin A, ascorbic acid and α-tocopherol on 2,4dimethylaminoazobenzene-induced hepatoma in rats, Curr Sci, 85 (2003) 201. Prasad K N, Hernandez C, Edwards-Prasad J, Nelson J, Borus T & Robinson W A, Modification of the effect of tamoxifen, cis-plastin, DTIC and interferon-a2b on human melanoma cells in culture by a mixture of vitamins, Nutr Cancer 22 (1994) 233. Prasad K N & Kumar R, Effect of individual and multiple antioxidant vitamins on growth and morphology of human nontumorgenic and tumorgenic parotid acinar cells in culture, Nutr Cancer 26 (1996) 11. Velanganni A A J, Dharaneedharan S, Geraldine P & Balasundram C, Dietary supplementation of vitamin A, C 16 17 18 19 20 21 22 23 24 377 and E prevents p-dimethylaminoazobenzene induced hepatic DNA damage in rats, Indian J Biochem Biophy, 44 (2007) 157. Ketterer B, Ross-Mansell P & Whitehead J K, The isolation of carcinogen-binding protein from livers of rats given 4dimethylaminoazobenzene, Biochem J, 103 (1961) 316. Laemmli U K, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680. Fare G, The effect of cupric oxyacetate on the binding of azo-dye by protein during the induction of liver tumours in the rat, Biochem J, 91 (1964) 473. Hughes P E, Fraction of azopeptides in tryptic digests of rat liver, Cancer Res, 19 (1959) 472. Terayama H & Takeuchi M, Studies on the nature of binding of carcinogenic aminoazo dye with liver proteins VIII: Some chemical properties of purified polar dyes, Gann, 53 (1962) 293. Kensler C J, Sugiura K, Young N F, Halter C R & Rhoads C P, Partial protection of rats by riboflavin with casein against liver cancer caused by dimethylaminoazobenzene, Science, 93 (1941) 308. Miller J A & Miller E C, The carcinogenic aminoazo dyes, Adv Cancer Res, 1 (1953) 339. Pielesz A, Characteristics of azo-dye binding sites on woolfiber keratin, J Appl Pol Sci, 91(2004) 2629. Srinivasan K & Bhargava M M, Hepatic binding proteins translocating azo dye carcinogen metabolites from cytoplasm into nucleus in rats, Food Chem Toxicol, 42 (2004) 503.