Redalyc.The Effects of Dietary Copper Supplementation on Oxidative And

Archivos de Zootecnia ISSN: 0004-0592 [email protected] Universidad de Córdoba España

Ajuwon, O.R.; Idowu, O.M.O.; Afolabi, S.A.; Kehinde, B.O.; Oguntola, O.O.; Olatunbosun, K.O. The effects of dietary copper supplementation on oxidative and antioxidant systems in broiler chickens Archivos de Zootecnia, vol. 60, núm. 230, junio, 2011, pp. 275-282 Universidad de Córdoba Córdoba, España

Available in: http://www.redalyc.org/articulo.oa?id=49520779012

How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative THE EFFECTS OF DIETARY COPPER SUPPLEMENTATION ON OXIDATIVE AND ANTIOXIDANT SYSTEMS IN BROILER CHICKENS

EFECTOS DE LA SUPLEMENTACIÓN CON COBRE SOBRE LOS SISTEMAS OXIDATIVO Y ANTIOXIDANTE EN BROILERS

Ajuwon, O.R.1*, Idowu, O.M.O.3, Afolabi, S.A.2, Kehinde, B.O.2, Oguntola, O.O.2 and Olatunbosun, K.O.2

1Oxidative Stress Research Centre. Faculty of Health and Wellness Sciences. Cape Peninsula University of Technology. Bellville. South Africa. *[email protected] 2Department of Environmental Management and Toxicology. University of Agriculture. Abeokuta. Nigeria. 3Department of Animal Nutrition. University of Agriculture. Abeokuta. Nigeria.

ADDITIONAL KEYWORDS PALABRAS CLAVE ADICIONALES Oxidative damage. Erythrocytes. Liver. Blood. Daños oxidativos. Eritrocitos. Hígado. Sangre.

SUMMARY

This study was designed to investigate the in de los eritrocitos y el hígado de broilers. Las aves vivo effects of dietary copper supplementation on fueron sometidas a una dieta comercial (grupo the oxidative and antioxidant systems in the control, n= 20) o suplementada con 250 mg CuSO4/ erythrocyte and liver of broiler chickens. Birds kg dieta (grupo experimental, n= 20) durante 6 were exposed to commercially prepared diet (con- semanas. En los eritrocitos y tejido hepático, se trol group, n= 20) or commercial diet supplemented evaluó, empleando técnicas consolidadas, la so- with 250 mg CuSO4/kg diet (experimental group, n= brecarga de cobre, la peroxidación oxidativa de 20) for 6 weeks. The copper burdens, oxidative los lípidos (LPO), concentración de glutatión (GSH), lipid peroxidation (LPO), glutathione (GSH) actividad de los enzimas antioxidantes, super concentration, activity of antioxidant enzymes, oxido dismutasa (SOD) y catalasa (CAT). La superoxide dismutase (SOD) and catalase (CAT) exposición oral al cobre, durante 6 semanas, were evaluated in the erythrocyte and hepatic aumentó (p<0,05) la carga de cobre y la tissues using well established techniques. Oral peroxidación lipídica respectivamente en sangre exposure to copper for a period of 6 weeks e hígado de las aves tratadas respecto del control. significantly (p<0.05) increased the copper burden Se concluye, que la suplementación dietética con and lipid peroxidation respectively in the blood and cobre (250 mg CuSO4/kg dieta) indujo estrés liver of treated birds compared with control. oxidativo en los eritrocitos e hígado de los broilers. Concomitantly, there was a significant reduction (p<0.05) in the activities of SOD, CAT, and the INTRODUCTION concentration of GSH in the exposed birds compared with control. It is concluded from this Copper is present in all tissues and is a study that dietary copper supplementation (250 functional constituent of all living cells. mg CuSO /kg diet) induced oxidative stress in the 4 Because of its significance in most of the erythrocyte and liver of broiler chickens. enzymatic reactions and cellular metabolism, Cu is an essential component of human RESUMEN nutrition and the diets of birds, including El estudio fue diseñado para estudiar los poultry. efectos in vivo de la suplementación dietética con Commercial organic copper sources and cobre sobre los sistemas oxidativos y antioxidantes reagent-grade inorganic copper salts have

Recibido: 27-1-09. Aceptado: 15-9-09. Arch. Zootec. 60 (230): 275-282. 2011. AJUWON, IDOWU, AFOLABI, KEHINDE, OGUNTOLA AND OLATUNBOSUN been used for the supplementation of ani- acid composition have been observed in the mal feeds (Guo et al., 2001). Copper is erythrocytes and hepatocytes of copper routinely supplemented to swine and exposed animals (Zhang et al., 2000) and poultry diets at concentrations above erythrocytes of human (Hochstein et requirement of the animals, because al.,1980). Erythrocytes are particularly pharmacological concentrations of copper sensitive to oxidative stress and like other act as a growth stimulant in these species, cells of aerobic organisms, they are supplied thereby improving growth performance, with protective antioxidant mechanisms in including increase in body weight gain and order to counteract the toxic action of feed intake (Cromwell et al., 1989; Bakalli et oxygen radicals. Defense is provided by al., 1995). Lien et al. (2004), found enzyme activities like superoxide dismutase, supplemental copper to reduce VLDL- catalase and glutathione peroxidase. cholesterol and enhance HDL-cholesterol Superoxide dismutase is the enzyme that in egg yolk of laying hens. Also CuSO4 catalyses the dismutation of the highly supplementation has been reported to redu- reactive superoxide anion to molecular ce the saturated fatty acid proportion in oxygen (O2) and to the less reactive species, abdominal fat and increase PUFA:SFA ratio, hydrogen peroxide (H2O2). Glutathione as well as reduces the cholesterol content in (GSH), GSH peroxidase and catalase, under meat of broiler chickens (Skrivan et al., most circumstances eliminate the resulting

2000, Xia et al., 2004). Research evidences H2O2 (Fridovich, 1995, Teixeira et al., 1998). have shown that copper regulates choles- Although reports of studies on the use terol biosynthesis by reducing hepatic of copper as a feed supplement in poultry glutathione concentration (Kim et al., 1992). has been widely published, however to the A large decrease in the reducing capacity of best of our knowledge, information regarding the cellular redox couples, such as gluta- the toxicity of copper in broiler chickens is thione has been used as an indication of still limited. Therefore, prompted by this oxidative stress in rats and other animal paucity of data, we evaluated the copper models (Schafer and Buettner, 2001). burden, status of lipid peroxidation and Although copper is a major essential antioxidant systems in the erythrocyte and element, serious toxic effects of this metal liver of broiler chickens whose diet has been has been reported when it is over loaded supplemented with copper. (Toplan et al., 2005, Zhang et al., 2000, Bremner, 1998). Copper ions are powerful MATERIALS AND METHODS catalysts of free radical damage. Its ability to induce oxidative damage is generally ANIMALS AND EXPERIMENTAL DESIGN attributed to the formation of highly reactive This study protocol was approved by hydroxyl radical (OH-) from hydrogen the University of Agriculture, Abeokuta peroxide (H2O2) via the Haber-Weiss (Nigeria), animal use and welfare committee reaction (Bremner, 1998; Kadiiska et al., and all investigation adhered strictly to the 1993). The OH- radical generated during statement for the use of animals in research. copper-induced oxidative stress may lead Forty unsexed, three-week old broiler to lipid peroxidation and formation of chickens (Anak 2000) were purchased from reactive products which may be involved in the UNAAB-LEVENTIS Agro-Allied Indus- severe damage of cell molecules and try, Abeokuta, Nigeria. The birds were kept structures (Videla et al., 2003). Lipid in standard battery cages with automatic peroxidation as evidenced by increased nipple drinkers and standard feeding trough. malondialdehyde content, as well as Feeds and water were given ad libitum. The alteration in membrane integrity and fatty birds were divided into two groups of twenty

Archivos de zootecnia vol. 60, núm. 230, p. 276. DIETARY COPPER AND OXIDATIVE AND ANTIOXIDANT SYSTEMS IN BROILERS birds each. Group 1 served as the control the erythrocytes washed thrice using 0.9% and was fed on a basal diet purchased from NaCl for lipid peroxidation, catalase, UNAAB-LEVENTIS Agro-Allied Industry. glutathione and superoxide dismutase Group 2, the treatment group, were fed on activity determination. The birds were the same basal diet supplemented with 250 sacrificed and liver excised, perfused with mg CuSO4/kg diet. This feeding protocol ice-cold 0.9% NaCl solution to remove resi- continued for a total of 42 days. The basal dual blood. Half of the liver was homoge- diet was formulated following the procedure nized in ice-cold 50 mM sodium phosphate of Idowu et al. (2003), and its composition buffer and 0.1 mM Na2EDTA (pH 7.8). The is shown in table I. soluble fraction was prepared by centrifugation at 1000 x g for 20 minutes. The SAMPLE PREPARATION remaining half was stored frozen for copper At the end of the feeding period, birds in content determination. the two groups were starved overnight for 12 hours. Exactly 10 ml of blood was drawn COPPER CONTENT DETERMINATION from the brachial vein of each bird into Exactly 1ml of blood was digested with heparinized tubes and 2 ml aliquot of blood 10 ml concentrated nitric acid and digests sample was transferred into another set of brought to 25 ml with deionised water tubes for copper content determination. The (Ademuyiwa, 1995). For liver, 1g was dried remaining blood was centrifuged at 750 x g to a constant weight at 85°C. Dried samples for 5 minutes, supernatants discarded and were cold digested in 2 ml of nitric acid overnight. They were then hot digested on a block digester at 120°C until all the organic Table I. Composition of the basal diet. (Com- matter was dissolved. Two milliliters of 30% posición de la dieta basal). (w/v) hydrogen peroxide were added during Ingredients digestion to enhance oxidization. The Maize % 44.00 digests were allowed to cool, and then Soya bean full fat % 36.00 diluted to 25 ml with deionised water (Alonso Rice husk % 12.33 et al., 2000). Copper concentrations in the Palm oil % 4.00 digests were determined by atomic absorp- Bone meal % 2.50 tion spectrometry (Buck Scientific, Model Oyster shell % 0.50 210, Connecticut, USA). Values were Vitamin/mineral premix % 0.25 expressed as µg/ml of blood or µg/g of Salt (NaCl) % 0.25 tissue. Methionine % 0.17 MEASUREMENT OF INDICES OF OXIDATIVE Determined analysis Crude protein % 19.90 STRESS Fat % 11.00 Lipid peroxidation: The extent of lipid Ash % 14.31 peroxidation was estimated in terms of Fibre % 4.78 thiobarbituric acid reactive substances (TBARS), using malondialdehyde (MDA) Calculated analysis as standard by method of Beuge and Aust Energy (ME) kcal/kg 3203.76 (1978). Briefly, 1 ml of erythrocyte or liver Energy/protein ratio 160.20 homogenate was added with 2 ml of the Methionine % 0.45 TCA-TBA-HCl reagent [15% (w/v) TCA, Calcium % 1.33 0.375% (w/v) TBA and 0.25N HCl]. The Phosphorus % 0.69 Copper mg/kg 5.90 contents were boiled for 15 minutes, cooled and centrifuged at 1000 x g to remove the

Archivos de zootecnia vol. 60, núm. 230, p. 277. AJUWON, IDOWU, AFOLABI, KEHINDE, OGUNTOLA AND OLATUNBOSUN precipitate. The absorbance was read at 535 dissolved in 100 ml of distilled water). After nm and the MDA concentration of the sample centrifugation, 2 ml of the supernatant was calculated using extinction coefficient of mixed with 0.2 ml of 0.4 M Na2HPO4 and 1 ml 1.56 x 105M-1Cm-1 and expressed as µmol of DTNB (5, 5' -Dithio-bis-(2-nitrobenzoic MDA/g Hb for erythrocyte and µmol MDA/ acid) reagent (40 mg DTNB in 100 ml of g tissue for liver. aqueous 1% trisodium citrate). Absorbance Catalase determination was carried out was read at 412 nm within 2 minutes. GSH following the method described by Clair- concentration was expressed as mmol/g Hb borne (1986), in which the disappearance of or mmol/g tissue.

H2O2 was monitored spectrophotometrically Protein content was determined accor- at 240 nm. Briefly, 20 µl of erythrocyte or ding to the method of Bradford (1976) using liver homogenate was added to 1 ml of bovine serum albumin as standard. All 0.05M phosphate buffer pH 7.0. Then, 0.5 ml chemical used in the enzymatic activity of 30% H2O2 was added to the buffered determination were of analytical purity and sample to initiate the reaction. The change were obtained from Sigma Chemical. in absorbance for 1 minute at 10 seconds interval was recorded. One unit activity is STATISTICAL ANALYSIS defined as the amount of enzyme catalyzing The results of all measurements were the formation of water and oxygen from expressed as mean ± standard deviation. hydrogen peroxide under assay condition. Data collected were subjected to analysis of Superoxide dismutase was assayed variance (ANOVA) and means separated according to a modified procedure of Das et using student's t-test with a probability al. (2000). In this method, 1.4 ml aliquot of factor of p<0.05 considered significant. the reaction mixture (comprising 1.11 ml of 50 mM phosphate buffer, pH 7.4, 0.075 ml of RESULTS 20 mM L-Methionine, 0.04 ml of 1% (v/v) Triton X-100, 0.075 ml of 10 mM hydro- BIOCHEMICAL FINDINGS xylamine hydrochloride and 0.1 ml of 50 mM Copper burden, lipid peroxidation, SOD, EDTA) was added to 100 µl of the erythro- CAT and GSH levels in the erythrocyte and cyte or liver homogenate and incubated at liver for each group are presented in tables 30°C for 5 minutes. 80 µl of riboflavin was II and III respectively. Copper supple- then added and the tubes were exposed to mentation in the diet significantly (p<0.05) 20W-Philips fluorescent lamps for 10 increased the copper burden in the blood minutes. After the exposure time, 1 ml of and liver of treated birds compared with Greiss reagent (mixture of equal volume of control. The mean copper concentration in 1% sulphanilamide in 5% phosphoric acid) the blood and liver of treated group were was added and absorbance of the colour respectively 2.15 and 1.67 times higher than formed measured at 543 nm. One unit of the control. A significant elevation (p<0.05) enzyme activity was measured as the amount was noted in lipid peroxidation in the copper of SOD capable of inhibiting 50% of nitrite exposed group with values being 3.67 and formation under assay condition. 1.25 times higher than control in both Glutathione concentration was deter- erythrocyte and liver respectively. mined in samples according to the method Values of SOD activity showed a of Boyne and Ellman (1972). Briefly, 1 ml of significant reduction (p<0.05) in the erythrocyte or liver homogenate was treated erythrocyte (10.9 ± 1.09 vs. 18.3 ± 1.21) and with 4.0 ml of metaphosphoric acid preci- liver (49.8 ± 14.94 vs. 77.4 ± 13.60) of copper pitating solution (1.67 g of glacial meta- exposed birds compared to the control. phosphoric acid, 0.2 g EDTA and 30 g NaCl Similarly, a significant decrease was noted

Archivos de zootecnia vol. 60, núm. 230, p. 278. DIETARY COPPER AND OXIDATIVE AND ANTIOXIDANT SYSTEMS IN BROILERS

Table II. Blood copper burden, erythrocyte Table III. Liver copper burden, lipid lipid peroxidation, SOD, CAT, and GSH peroxidation, SOD, CAT and GSH concen- concentrations. (Carga de cobre, peroxidación trations. (Carga de cobre, peroxidación lipídica lipídica en los eritrocitos y concentraciones de en los eritrocitos y concentraciones de SOD, CAT SOD, CAT y GSH en la sangre). y GSH en el hígado).

Parameter Control Treatment Parameter Control Treatment (n=20) (n=20) (n=20) (n=20)

Copper burden1 2.0 ± 0.73b 4.3 ± 0.28a Copper burden1 5.1 ± 0.61b 8.5 ± 0.66a Lipid peroxidation2 0.3 ± 0.07b 1.1 ± 0.19a Lipid peroxidation2 0.4 ± 0.02b 0.5 ± 0.08a SOD 18.3 ± 1.21a 10.9 ± 1.09b SOD 77.4 ± 13.60a 49.8 ± 14.94b CAT 3.8 ± 0.65a 1.8 ± 0.35b CAT 16.0 ± 1.26a 9.6 ± 2.44b GSH 0.9 ± 0.17a 0.3 ± 0.19b GSH 3.9 ± 0.17a 2.5 ± 0.43b

Values in the same row with different superscript Values in the same row with different superscript are significantly different at p<0.05. are significantly different at p<0.05. 1µg/ml; 2µmol MDA/g Hb (MDA= malondialdehyde); 1µg/g tissue; 2µmol MDA/g tissue (MDA= malondial- SOD: superoxide dismutase (unit/mg protein); CAT: dehyde); SOD: superoxide dismutase (unit/mg catalase (unit/mg protein); GSH: glutathione (mmol/ protein); CAT: catalase (unit/mg protein); GSH: g Hb). glutathione (mmol/g tissue). in catalase activity after copper exposure in oxidative stress in tissue. Copper produce - both the erythrocyte and liver (tables II and OH radical from H2O2 and this is capable of III). The level of glutathione indicated a reacting with practically every biological significant depletion (p<0.05) in both molecule, initiating oxidative damage by erythrocyte (67% lower) and liver (36% abstracting the hydrogen from an amino lower) of birds exposed to copper compared bearing carbon to form a carbon centered to those of the control. protein radical and from an unsaturated fatty acid to form a lipid radical (Powell, 2000). DISCUSSION Studies by Toplan et al. (2005) and Zhang et al. (2000) also corroborated our present Numerous studies have reported toxic findings. effects induced when animals and humans Lipid peroxidation is a free radical- are exposed to certain metals (Valko et al., mediated chain reaction, since it is self 2005, Stohs and Bagchi, 1995). Several perpetuating. The length of the propagation mechanisms have been proposed to explain depends upon chain breaking antioxidants, copper-induced cellular toxicity. Most often such as the enzymes SOD, catalase and the basis for these theories is the ability of glutathione peroxidase (Diniz et al., 2003). free Cu ions to participate in the formation In this study, supplementation of CuSO4 of reactive oxygen species (ROS) and other (250 mg/kg diet) in the diet of broilers free radicals (Gaetke and Chow, 2003). The resulted in a reduction in the activities of most important consequences of free radi- SOD and catalase compared with birds in cal production are lipid peroxidation increase the control group. The results of research and change in permeability of cell membrane. on copper influence on SOD activity are The significant increase in lipid peroxidation divergent. Previous studies by Toplan et al. in the experimental group observed in this (2005), in which rats were given 250 mg/l study could be explained by damage due to copper for 9 weeks and Zhang et al. (2000)

Archivos de zootecnia vol. 60, núm. 230, p. 279. AJUWON, IDOWU, AFOLABI, KEHINDE, OGUNTOLA AND OLATUNBOSUN when rats were overloaded with 500 mg Cu/ oxygen species (Ishikawa and Sies, 1989; kg/bw for eight weeks showed that SOD Seidegard and Ekstrom, 1997). In this study, activity was significantly reduced, as also GSH levels were lower in the copper treated observed in this study. However Ozcelik et group compared with the control group. al. (2003), administered water containing The reduction in the GSH levels observed in 100 µg/ml copper for 4 weeks to rats and this study might be due to the reaction of observed an increase in the activity of SOD GSH with copper or with free radicals in experimental animals compared to those promoted by copper supplementation of the control. Sansinanea et al. (1998), in a (Ishikawa and Sies, 1989; Deleve and study to explain the cytotoxicity of Kaplowitz, 1991). Also an impairment of excessive free radical production in the liver GSH synthesis or regenerating enzyme of rats, gave water containing 0.2% solution could be responsible for the observed of CuSO4 and observed SOD activity to be reduction of GSH (Hultberg et al., 1997). higher in experimental group animals than that of control group animals. SOD catalyses CONCLUSION the dismutation of superoxide radical leading to formation of hydrogen peroxide which in In conclusion, this study has de- turn is detoxified by the enzyme catalase monstrated that copper intake at high

(Fridovich, 1995). Therefore, the decreased concentration (250 mg CuSO4/kg diet) SOD and CAT activity observed in this induced adverse effects on the oxidative study may be due to the depletion or and antioxidant systems in broiler chickens inactivation of the enzymes by production as shown by the increase in lipid peroxidation of free radicals (Kono and Fridovich, 1982) and reduction in the activities of SOD and catalase, as well as the concentration of such as superoxide and H2O2 which in turn generate hydroxyl radical resulting in glutathione in both the erythrocyte and the initiation and propagation of lipid pero- liver. This in turn may be detrimental to the xidation. health of the birds since oxidative stress One of the mechanisms by which heavy can lead to increased prevalence of metals produce effect is through their infectious diseases via impairment of immune interaction with cellular sulphydryl groups cell functions. Nevertheless, the beneficial in proteins. Sulphydryl groups thus serves effects of Cu supplementation in poultry as a source of electrons for reduction and may be utilized by reducing the copper supplementation level in the diet. Further- also mediate the methylation process. When more, dose-response relationship experi- the availability of free thiol group is low, ments are suggested in order to determine a enhanced expression of toxicity in the form threshold dose below which there would be of oxidative stress could occur. Since most no toxicity. metals have high affinity for the sulphydryl (SH) groups, endogenous SH compounds ACKNOWLEDGEMENTS such as metallothionein (MT) and GSH are known to be involved in metal toxicity. GSH The authors are grateful to Mrs. O. performs a pivotal role in maintaining the Sorinola for her technical assistance. We metabolic and transport function of cells. It are also indebted to Professors O. may function as an intracellular chelater and Ademuyiwa and T.A. Arowolo for going a metal detoxifying agent. The importance through the manuscript, and also to Dr. of GSH in metal detoxification is also O.M. Sogunle for helping out with the supported by its role in removal of toxic statistical analysis.

Archivos de zootecnia vol. 60, núm. 230, p. 280. DIETARY COPPER AND OXIDATIVE AND ANTIOXIDANT SYSTEMS IN BROILERS

REFERENCES

Ademuyiwa, O. 1995. Zur toxikologie der arsen- Fridovich, I. 1995. Superoxide radical and kupfer wechsekwirlung im saugetierorgani superoxide dismutases. Annu. Rev. Biochem., mimus. Untersuchunge an ratten. Ph.D. Thesis. 64: 97-112. University of Munich. Munich. Federal Republic Gaetke, L.M. and Chow, C.K. 2003. Copper toxicity, of Germany. oxidative stress, and antioxidant nutrients. Alonso, M.L., Benedito, J.L., Miranda, M., Castillo, Toxicology, 189: 147-163. C., Hernandez, J. and Shore, R.F. 2000. Arsenic, Guo, R., Henry, P.R., Holwerda, R.A., Cao, J., cadmium, lead, copper and zinc in cattle from Littelli, R.C., Miles, R.D. and Ammerman, C.B. Galicia, NW Spain. Sci. Total Environ., 246: 237- 2001. Chemical characteristics and relative 248. bioavailability of supplemental organic copper Bakalli, R.I., Pesti, G.M., Ragland, W.L. and Konjufca, sources for poultry. J. Anim. Sci., 79: 1132- V. 1995. Dietary copper in excess of nutritional 1141. requirement reduces plasma, and breast muscle Hochstein, P., Kumar, K.S. and Forman, S.J. 1980. cholesterol of chickens. Poultry Sci., 74: 360- Lipid peroxidation and the cytotoxicity of copper. 365. Ann. NY. Acad. Sci., 355: 240-248. Beuge, J.A. and Aust, S.D. 1978. Microsomal lipid Hultberg, B., Anderson, A. and Isaksson, A. 1997. peroxidation. Methods Enzymol., 52: 302-305. Copper ions differ from other thiol reactive metal Boyne, A.F. and Ellman, G.L. 1972. A methodology ions in their effects on the concentration and for analysis of tissue sulphydryl components. redox status of thiols in HeLa cell cultures. Anal. Biochem., 46: 639-653. Toxicology, 117: 89-97. Bradford, M.M. 1976. A rapid and sensitive method Idowu, O.M.O., Eruvbetine, D., Oduguwa, O.O., for the quantification of microgram quantities of Bamgbose, A.M. and Abiola, S.S. 2003. protein utilizing the principle of protein-dye Response of finishing broiler chickens fed three binding. Anal. Biochem., 46: 639-653. energy/protein combinations at fixed E:P ratio. Bremner, I. 1998. Manifestations of copper excess. Nig. J. Anim. Prod., 30: 185-191. Am. J. Clin. Nutr., 67: 1069S-1073S. Ishikawa, T. and Sies, H. 1989. Glutathione as an Clairborne, A. 1986. Catalase activity. In: CRC antioxidant: toxicological aspects. In: Dolphin, Handbook of methods for oxygen radical D., Poulson, R., Avramovic, O. (eds). Glutathione, research. Greenwald RA (ed). CRC Press Inc. chemical, biochemical and medical aspects. Boca Raton, FL. pp. 283-284. Wiley Interscience. New York. pp. 85-111. Cromwell, G.L., Stahly, T.S. and Monegue, H.J. Kadiiska, M.B., Hanna, P.M., Jordan, S.J. and 1989. Effects of source and level of copper on Mason, R.P. 1993. Electron spin resonance performance and liver copper stores in weanling evidence for free radical generation in copper pigs. J. Anim. Sci., 67: 2996-3002. treated vitamin E- and selenium- deficient rats: Das, K., Samanta, L. and Chainy, G.B.N. 2000. A in vivo spin-trapping investigation. Mol. modified spectrophotometric assay of supe- Pharmacol., 44: 222-227. roxide dismutase using nitrite formation by Kim, J.W., Chao, P.Y. and Allen, A. 1992. Inhibition superoxide radicals. Ind. J. Biochem. Biophys., of elevated hepatic glutathione abolishes copper 37: 201-204. deficiency cholesterolemia. FASEB J., 6: 2467- Deleve, L.D. and Kaplowitz, N. 1991. Glutathione 2471. metabolism and its role in hepatotoxicity. Pharm. Kono, Y. and Fridovich, I. 1982. Superoxide radical Ther., 52: 287-305. inhibits catalase. J. Biol. Chem., 257: 5751- Diniz, Y.S., Cicogna, A.C., Padovani, C.R., Silva, 5754. M.D.P., Faine, C.M., Galhardi, C.M., Rodrigues, Lien, T.F., Chen, K.L., Wu, C.P. and Lu, J.J. 2004. H.G. and Novelli, E.L.B. 2003. Dietary restriction Effects of supplemental copper and chromium of fibre supplementation: oxidative stress and on the serum and egg traits of laying hens. Brit. metabolic shifting for cardiac health. Can. J. Poultry Sci., 45: 535-539. Physiol. Pharmacol., 81: 1042-1048. Ozcelik, D., Ozaras, R., Gurel, Z., Uzun, H. and

Archivos de zootecnia vol. 60, núm. 230, p. 281. AJUWON, IDOWU, AFOLABI, KEHINDE, OGUNTOLA AND OLATUNBOSUN

Aydin, S. 2003. Copper-mediated oxidative Radical Bio. Med., 18: 321-336. stress in rat liver. Biol. Trace Elem. Res., 96: Teixeira, H.D., Schumacher, R.I. and Meneghini, R. 209-215. 1998. Lower intracellular hydrogen peroxide Powell, S.R. 2000. The antioxidant property of levels in cells over-expressing CuZn-supe- Zinc. J. Nutr., 130: 1447S-1454S. roxide dismutase. Proc. Natl. Acad. Sci., 95: Sansinanea, A.S., Cerone, S.I., Streitenberger, 7872-7875. S.A., Garcia, C. and Auza, N. 1998. Oxidative Toplan, S., Dariyerli, N., Ozcelik, D. and Akyolcu, effect of copper hepatic copper overload. Acta. M.C. 2005. The effects of copper application on Physiol. Pharmacol. Ther. Latinoam., 48: 25-31. oxidative and antioxidant systems in rats. Tra- Schafer, F.Q. and Buettner, G.R. 2001. Redox ce. Elem. Electroly., 22: 178-181. environment of the cell as viewed through the Valko, M., Morris, H. and Cronin, M.T.D. 2005. Metal redox state of the glutathione disulphide/ toxicity and oxidative stress. Curr. Med. Chem., glutathione couple. Free Radical Bio. Med., 30: 12: 1161-1208. 1191-1212. Videla, L.A., Fernandez, V., Tapia, G. and Varela, Seidegard, J. and Ekstrom, G. 1997. The role of P. 2003. Oxidative stress-mediated hepato- human glutathione transferase and epoxide toxicity of iron and copper: role of Kupffer cells. hydrolysis in the metabolism of xenobiotics. Biometals, 16: 103-111. Environ. Health Persp., 105: 791-799. Xia, M.S., Hu, C.H. and Xu, Z.R. 2004. Effects of Skrivan, M., Skrivanova, V., Marounek, M., Tumova, copper bearing montmorillonite on growth per- E. and Wolf, J. 2000. Influence of dietary fat formance, digestive enzyme activities and in- source and copper supplementation on broiler testinal microflora and morphology of male performance, fatty acid profile of meat and broilers. Poultry Sci., 83: 1868-1875. depot fat and on cholesterol content in meat. Zhang, S.S., Noordin, M.M., Rahman, S.O. and Brit. Poultry Sci., 41: 608-614. Haron, J. 2000. Effects of copper overload on Stohs, S.J. and Bagchi, D. 1995. Oxidative hepatic lipid peroxidation and antioxidant defense mechanisms in the toxicity of metal-ions. Free in rats. Vet. Hum. Toxicol., 42: 261-264.

Archivos de zootecnia vol. 60, núm. 230, p. 282.