Biochemical
and Molecular Roles of Nutrients
JOAN C. FISCHER, RANDALL L TACKETT,* E. W. HOWERTH* AND MARY ANN JOHNSON3 Department of Foods and Nutrition, *Department of Pharmacology and Toxicology, and Department of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602 Because antioxidant nutrients can prevent doxoru bicin-induced lipid peroxidation and cardiomyopathy, the purpose of the present study was to determine how Cu and Se status affect the toxicity of doxoru bicin. Copper and Se are recognized as antioxidants because Se is necessary for full activity of glutathione peroxidase (GSH-Px, EC 1.11.1.9)4 (Arthur et al. 1987) and Cu is a component of Cu,Zn Superoxide dis mutase (Cu,Zn SOD, EC 1.15.1.1) (as cited by Johnson et al. 1992). Moreover, the antioxidants dimethyl sulfoxide and t-butylhydroquinone protect against the cardiac damage found in Cu-deficient rats (Johnson and Saari 1989, Saari and Medeiros 1991). However, little is known about the practical significance of the antioxidant functions of Cu and Se, such as their influence on drug-induced free radical production. A limited amount of information is available on the influence of dietary Cu (Fischer et al. 1991, Zidenberg-Cherr et al. 1989) and Se (Chen et al. 1986, Doroshow et al. 1980, Nakano et al. 1989) defi ciencies on doxorubicin toxicity, but few of these
ABSTRACT This study tests the hypothesis that Cu and Se deficiencies enhance doxorubicin-induced cardiotoxicity and anemia. Male Sprague-Dawley rats (n = 48) were fed Cu and Se-adequate (+Cu+Se), Cu-deficient (-Cu), Se-deficient (-Se) or Cu and Se-deficient (-Cu-Se) diets for 5.5 wk. Doxorubicin (4 mg/kg body wt) or saline was administered once weekly for the last 4 wk of the study. Copper deficiency was confirmed by 79% lower liver Cu, 67% lower liver Cu.Zn Superoxide dismutase (Cu.Zn SOD) activity and 76% lower erythrocyte Cu.Zn SOD activity. Selenium deficiency was confirmed by 90% lower liver glutathione peroxidase activity. Rats fed the -Cu diet had greater reductions in hematocrit than did those fed the +Cu diet after adminis tration of doxorubicin. Doxorubicin, Cu deficiency and Se deficiency all produced electrocardiographic abnormal ities and ultrastructural anatomical lesions. However, the dietary deficiencies did not enhance doxorubicininduced cardiotoxicity. Doxorubicin, but not Cu or Se deficiency, raised lipid peroxidation 16% in liver (P < 0.01) and 18% ¡nheart (not significant). These data suggest that the cardiomyopathies caused by doxoru bicin and Cu and Se deficiencies have some similarities, but cardiac changes may be related to mechanisms other than lipid peroxidation. J. Nutr. 122: 2128-2137, 1992. INDEXING KEY WORDS: •doxorubicin
•selenium
•copper
Presented in part at the Annual Meeting of the Federation of American Societies for Experimental Biology, April 1991, Atlanta GA (Fischer, ]., Tackett, R. & Johnson, M. A. (1991) Interactions between copper deficiency, selenium deficiency and adriamycin toxicity. FASEB I. 4: A6281 (abs.)]. 2Funded in part by International Life Sciences InstituteNutrition Foundation, Future Leaders Award (MAJ|; University of Georgia Experiment Station, Hatch Project #H630 (MAJ); and The University of Georgia Graduate School (JF). 3To whom correspondence should be addressed. 4Abbreviations used: Cu,Zn SOD, Cu,Zn Superoxide dismutase (EC 1.15.1.1); CK, creatine kinase (EC 2.7.3.2); EKG, electrocardi ogram,- GSH-Px, glutathione peroxidase (EC 1.11.1.9); CP, ceruloplasmin (EC 1.16.3.1). Diet abbreviations: +Cu+Se, copper and selenium-adequate diet; -Cu, copper-deficient diet; -Se, selenium-deficient diet; -Cu-Se, copper and selenium-deficient diets.
rats
•heart
The nutrients a-tocopherol, ß-carotene, ascorbic acid, zinc, manganese, copper (Cu) and selenium (Se) are critical components of the antioxidant system in all tissues (Machlin and Bendich 1987). The chemotherapeutic drug doxorubicin enhances lipid peroxidation and causes severe cardiac damage (Singal et al. 1987) that can be reduced or prevented by nutritional antioxidants such as a-tocopherol (Myers et al. 1977) and ascorbic acid (Shimpo et al. 1991). 0022-3166/92
$3.00 ©1992 American
Institute
of Nutrition.
Received 13 April 1992. Accepted 7 fuly 1992.
2128
Downloaded from https://academic.oup.com/jn/article-abstract/122/11/2128/4754707 by Tulane University Medical Library user on 03 February 2019
Copper and Selenium Deficiencies Do Not Enhance the Cardiotoxicity in Rats Due to Chronic Doxorubicin Treatment1'2
Cu AND Se DEFICIENCIES
AND DOXORUBICIN
studies have thoroughly examined the role of changes in both antioxidant enzymes and lipid peroxidation as mediators of doxorubicin-induced cardiotoxicity. Thus, this study examined the interaction of Cu, Se and doxorubicin using biochemical, physiological and anatomical indices.
Animals, diet and experimental design. Male Sprague-Dawley rats [n = 48, initial weight 46-52 g; HarÃ-an Sprague Dawley, Indianapolis, IN) were housed individually in stainless steel wire-bottom cages in a room with controlled temperature (21 ± 1°C),humidity and light (12-h light:dark cycle, lights on 0600-1800 h). All animal procedures were ap proved by the University of Georgia Institutional Animal Care and Use Committee. Rats were assigned to four dietary groups (12 rats per group) and fed a Cu and Se-adequate diet (+Cu+Se diet), a Cu-deficient and Se-adequate diet (-Cu), a Cu-adequate and Sedeficient diet (-Se) or a Cu and Se-deficient diet (-Cu-Se).5 Rats were fed the diets with ad libitum access for 5.5 wk. Copper-adequate diets were supplemented with copper sulfate and Se-adequate diets with sodium selenite. The -Cu and -Cu-Se diets contained 0.52 mg Cu/kg and the +Cu+Se and -Se diets contained 8.43 mg Cu/kg, as determined by atomic absorption spectrophotometry. The -Se and -Cu-Se diets con tained 0.05 mg Se/kg and the +Cu+Se and -Cu diets contained 0.48 mg Se/kg, as determined by a fluori metrie method (Watkinson 1966). Diets contained 46 mg Fe/kg and 51 mg Zn/kg, as determined by atomic absorption spectrophotometry. Body weight and food intake were recorded daily. Doxorubicin hydrochloride (Adria Labs, Columbus, OH) (4 mg/kg body wt) or an equal volume of saline (9 g/L) was injected intraperitoneally, once a week for 4 wk, beginning at wk 2 of the study, resulting in a cumulative dose of 16 mg doxorubicin/kg body wt. Four days after the final injection, rats were anesthetized with CÛ2, placed in a supine position and a lead II electrocardiogram (EKG) was recorded. All EKGs were recorded on a Grass Polygraph (Quincy, MA) at a voltage setting of l mV/cm and at a paper speed of 25 mm/s. Blood was collected and separated as previously described (Johnson and Murphy 1988). The liver, heart and spleen were removed, rinsed thoroughly with ice-cold saline, blotted dry and weighed. The heart was cut sagittally. After removing a 1-mm3 section from the left ventricular wall for transmission electron microscopy, one half of the heart was fixed in 10% buffered formalin for light microscopy. Sections cut for electron microscopy were immediately immersed in a mixture of 2%
2129
paraformaldehyde, 2% gluteraldehyde and 0.2% picric acid in 0.1 mol/L cacodylate buffer (pH 7.2-7.4). The formalin-fixed heart was sectioned transversely halfway between the interventricular groove and apex so that histopathologic sections included right and left ventricular free walls and interventricular septum. Trimmed samples were then embedded in paraffin, sectioned at 4 urn, and stained with hematoxylin and eosin. Tissues for electron microscopy were postfixed in 1% osmium tetraoxide, dehydrated and embedded in Spurr low viscosity resin. Toluidine blue thick sections were examined, and thin sections were taken from areas where myofibers were in longitudinal orientation. Thin sections were stained with uranyl acetate and lead citrate. Sections were coded so that their identity was unknown during histological evaluation. The re maining portion of each heart and 1-g pieces of liver were homogenized separately in four volumes of icecold buffer (pH 7.0, 0.05 mol/L phosphate) with a Teflon pestle in a glass tube. Analysis of samples. Plasma was kept at 4°Cfor 24 h until creatine kinase (CK, EC 2.7.3.2) activity could be assessed (#520, Sigma Chemical). Other enzyme activities measured were Cu,Zn SOD, using 4 mmol/ L pyrogallol as the substrate (Johnson and Murphy 1988), GSH-Px with 0.3 mmol/L t-butyl hydroperoxide (Paglia and Valentine 1979) and ceruloplasmin (CP, EC 1.16.3.1) with 7.88 mmol/L odiansidine dihydrochloride (Schosinsky et al. 1974). Lipid peroxidation in fresh whole tissue homogenates was assessed indirectly by measurement of thiobarbituric acid-reactive substances using the method of Buege and Aust (1978). All enzyme activities and lipid peroxidation were expressed per milligram of protein (Lowry et al. 1951). Dietary Cu, Fe and Zn, and Cu and Fe in the liver and spleen, were determined with flame atomic ab sorption spectrophotometry (model 5000, PerkinElmer, Norwalk, CT) as described by Johnson (1986). Accuracy of the analysis was verified by determi nation of the Cu and Fe content of a bovine liver standard obtained from the National Institute of Stan dards and Technology (Gaithersburg, MD) (recovery: 90% Cu, n = 3; 98% Fe, n = 6). Diet samples were digested with perchloric-nitric acid, reduced with hydrochloric acid and analyzed for Se by fluorometric methods (Watkinson 1966).
5The diets contained the following (per kg diet]: 200 g casein (high protein, #160040, Teklad Test Diets, Madison, WI), 400 g cornstarch (Argo, Best Foods, Englewood Cliffs, NJ|, 200 g sucrose (Kroger Co., Cincinnati, OH), 100 g corn oil (Mazóla,Best Foods], 50 g cellulose (#160390, Teklad Test Diets), 35 g Se- and Cu-deficient AIN-76 mineral mix (#TD88243, Teklad Test Diets), 10 g AIN-76A vitamin mix (#40077, Teklad Test Diets), 3 g DL-methionine (#M9500, Sigma Chemical, St. Louis, MO), and 2 g choline dihydrogen citrate (#30220, Teklad Test Diets).
Downloaded from https://academic.oup.com/jn/article-abstract/122/11/2128/4754707 by Tulane University Medical Library user on 03 February 2019
METHODS
TOXICITY
FISCHER ET AL.
2130
RESULTS The development of a Cu-deficient state was verified by a liver Cu of
and Molecular Roles of Nutrients
JOAN C. FISCHER, RANDALL L TACKETT,* E. W. HOWERTH* AND MARY ANN JOHNSON3 Department of Foods and Nutrition, *Department of Pharmacology and Toxicology, and Department of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602 Because antioxidant nutrients can prevent doxoru bicin-induced lipid peroxidation and cardiomyopathy, the purpose of the present study was to determine how Cu and Se status affect the toxicity of doxoru bicin. Copper and Se are recognized as antioxidants because Se is necessary for full activity of glutathione peroxidase (GSH-Px, EC 1.11.1.9)4 (Arthur et al. 1987) and Cu is a component of Cu,Zn Superoxide dis mutase (Cu,Zn SOD, EC 1.15.1.1) (as cited by Johnson et al. 1992). Moreover, the antioxidants dimethyl sulfoxide and t-butylhydroquinone protect against the cardiac damage found in Cu-deficient rats (Johnson and Saari 1989, Saari and Medeiros 1991). However, little is known about the practical significance of the antioxidant functions of Cu and Se, such as their influence on drug-induced free radical production. A limited amount of information is available on the influence of dietary Cu (Fischer et al. 1991, Zidenberg-Cherr et al. 1989) and Se (Chen et al. 1986, Doroshow et al. 1980, Nakano et al. 1989) defi ciencies on doxorubicin toxicity, but few of these
ABSTRACT This study tests the hypothesis that Cu and Se deficiencies enhance doxorubicin-induced cardiotoxicity and anemia. Male Sprague-Dawley rats (n = 48) were fed Cu and Se-adequate (+Cu+Se), Cu-deficient (-Cu), Se-deficient (-Se) or Cu and Se-deficient (-Cu-Se) diets for 5.5 wk. Doxorubicin (4 mg/kg body wt) or saline was administered once weekly for the last 4 wk of the study. Copper deficiency was confirmed by 79% lower liver Cu, 67% lower liver Cu.Zn Superoxide dismutase (Cu.Zn SOD) activity and 76% lower erythrocyte Cu.Zn SOD activity. Selenium deficiency was confirmed by 90% lower liver glutathione peroxidase activity. Rats fed the -Cu diet had greater reductions in hematocrit than did those fed the +Cu diet after adminis tration of doxorubicin. Doxorubicin, Cu deficiency and Se deficiency all produced electrocardiographic abnormal ities and ultrastructural anatomical lesions. However, the dietary deficiencies did not enhance doxorubicininduced cardiotoxicity. Doxorubicin, but not Cu or Se deficiency, raised lipid peroxidation 16% in liver (P < 0.01) and 18% ¡nheart (not significant). These data suggest that the cardiomyopathies caused by doxoru bicin and Cu and Se deficiencies have some similarities, but cardiac changes may be related to mechanisms other than lipid peroxidation. J. Nutr. 122: 2128-2137, 1992. INDEXING KEY WORDS: •doxorubicin
•selenium
•copper
Presented in part at the Annual Meeting of the Federation of American Societies for Experimental Biology, April 1991, Atlanta GA (Fischer, ]., Tackett, R. & Johnson, M. A. (1991) Interactions between copper deficiency, selenium deficiency and adriamycin toxicity. FASEB I. 4: A6281 (abs.)]. 2Funded in part by International Life Sciences InstituteNutrition Foundation, Future Leaders Award (MAJ|; University of Georgia Experiment Station, Hatch Project #H630 (MAJ); and The University of Georgia Graduate School (JF). 3To whom correspondence should be addressed. 4Abbreviations used: Cu,Zn SOD, Cu,Zn Superoxide dismutase (EC 1.15.1.1); CK, creatine kinase (EC 2.7.3.2); EKG, electrocardi ogram,- GSH-Px, glutathione peroxidase (EC 1.11.1.9); CP, ceruloplasmin (EC 1.16.3.1). Diet abbreviations: +Cu+Se, copper and selenium-adequate diet; -Cu, copper-deficient diet; -Se, selenium-deficient diet; -Cu-Se, copper and selenium-deficient diets.
rats
•heart
The nutrients a-tocopherol, ß-carotene, ascorbic acid, zinc, manganese, copper (Cu) and selenium (Se) are critical components of the antioxidant system in all tissues (Machlin and Bendich 1987). The chemotherapeutic drug doxorubicin enhances lipid peroxidation and causes severe cardiac damage (Singal et al. 1987) that can be reduced or prevented by nutritional antioxidants such as a-tocopherol (Myers et al. 1977) and ascorbic acid (Shimpo et al. 1991). 0022-3166/92
$3.00 ©1992 American
Institute
of Nutrition.
Received 13 April 1992. Accepted 7 fuly 1992.
2128
Downloaded from https://academic.oup.com/jn/article-abstract/122/11/2128/4754707 by Tulane University Medical Library user on 03 February 2019
Copper and Selenium Deficiencies Do Not Enhance the Cardiotoxicity in Rats Due to Chronic Doxorubicin Treatment1'2
Cu AND Se DEFICIENCIES
AND DOXORUBICIN
studies have thoroughly examined the role of changes in both antioxidant enzymes and lipid peroxidation as mediators of doxorubicin-induced cardiotoxicity. Thus, this study examined the interaction of Cu, Se and doxorubicin using biochemical, physiological and anatomical indices.
Animals, diet and experimental design. Male Sprague-Dawley rats [n = 48, initial weight 46-52 g; HarÃ-an Sprague Dawley, Indianapolis, IN) were housed individually in stainless steel wire-bottom cages in a room with controlled temperature (21 ± 1°C),humidity and light (12-h light:dark cycle, lights on 0600-1800 h). All animal procedures were ap proved by the University of Georgia Institutional Animal Care and Use Committee. Rats were assigned to four dietary groups (12 rats per group) and fed a Cu and Se-adequate diet (+Cu+Se diet), a Cu-deficient and Se-adequate diet (-Cu), a Cu-adequate and Sedeficient diet (-Se) or a Cu and Se-deficient diet (-Cu-Se).5 Rats were fed the diets with ad libitum access for 5.5 wk. Copper-adequate diets were supplemented with copper sulfate and Se-adequate diets with sodium selenite. The -Cu and -Cu-Se diets contained 0.52 mg Cu/kg and the +Cu+Se and -Se diets contained 8.43 mg Cu/kg, as determined by atomic absorption spectrophotometry. The -Se and -Cu-Se diets con tained 0.05 mg Se/kg and the +Cu+Se and -Cu diets contained 0.48 mg Se/kg, as determined by a fluori metrie method (Watkinson 1966). Diets contained 46 mg Fe/kg and 51 mg Zn/kg, as determined by atomic absorption spectrophotometry. Body weight and food intake were recorded daily. Doxorubicin hydrochloride (Adria Labs, Columbus, OH) (4 mg/kg body wt) or an equal volume of saline (9 g/L) was injected intraperitoneally, once a week for 4 wk, beginning at wk 2 of the study, resulting in a cumulative dose of 16 mg doxorubicin/kg body wt. Four days after the final injection, rats were anesthetized with CÛ2, placed in a supine position and a lead II electrocardiogram (EKG) was recorded. All EKGs were recorded on a Grass Polygraph (Quincy, MA) at a voltage setting of l mV/cm and at a paper speed of 25 mm/s. Blood was collected and separated as previously described (Johnson and Murphy 1988). The liver, heart and spleen were removed, rinsed thoroughly with ice-cold saline, blotted dry and weighed. The heart was cut sagittally. After removing a 1-mm3 section from the left ventricular wall for transmission electron microscopy, one half of the heart was fixed in 10% buffered formalin for light microscopy. Sections cut for electron microscopy were immediately immersed in a mixture of 2%
2129
paraformaldehyde, 2% gluteraldehyde and 0.2% picric acid in 0.1 mol/L cacodylate buffer (pH 7.2-7.4). The formalin-fixed heart was sectioned transversely halfway between the interventricular groove and apex so that histopathologic sections included right and left ventricular free walls and interventricular septum. Trimmed samples were then embedded in paraffin, sectioned at 4 urn, and stained with hematoxylin and eosin. Tissues for electron microscopy were postfixed in 1% osmium tetraoxide, dehydrated and embedded in Spurr low viscosity resin. Toluidine blue thick sections were examined, and thin sections were taken from areas where myofibers were in longitudinal orientation. Thin sections were stained with uranyl acetate and lead citrate. Sections were coded so that their identity was unknown during histological evaluation. The re maining portion of each heart and 1-g pieces of liver were homogenized separately in four volumes of icecold buffer (pH 7.0, 0.05 mol/L phosphate) with a Teflon pestle in a glass tube. Analysis of samples. Plasma was kept at 4°Cfor 24 h until creatine kinase (CK, EC 2.7.3.2) activity could be assessed (#520, Sigma Chemical). Other enzyme activities measured were Cu,Zn SOD, using 4 mmol/ L pyrogallol as the substrate (Johnson and Murphy 1988), GSH-Px with 0.3 mmol/L t-butyl hydroperoxide (Paglia and Valentine 1979) and ceruloplasmin (CP, EC 1.16.3.1) with 7.88 mmol/L odiansidine dihydrochloride (Schosinsky et al. 1974). Lipid peroxidation in fresh whole tissue homogenates was assessed indirectly by measurement of thiobarbituric acid-reactive substances using the method of Buege and Aust (1978). All enzyme activities and lipid peroxidation were expressed per milligram of protein (Lowry et al. 1951). Dietary Cu, Fe and Zn, and Cu and Fe in the liver and spleen, were determined with flame atomic ab sorption spectrophotometry (model 5000, PerkinElmer, Norwalk, CT) as described by Johnson (1986). Accuracy of the analysis was verified by determi nation of the Cu and Fe content of a bovine liver standard obtained from the National Institute of Stan dards and Technology (Gaithersburg, MD) (recovery: 90% Cu, n = 3; 98% Fe, n = 6). Diet samples were digested with perchloric-nitric acid, reduced with hydrochloric acid and analyzed for Se by fluorometric methods (Watkinson 1966).
5The diets contained the following (per kg diet]: 200 g casein (high protein, #160040, Teklad Test Diets, Madison, WI), 400 g cornstarch (Argo, Best Foods, Englewood Cliffs, NJ|, 200 g sucrose (Kroger Co., Cincinnati, OH), 100 g corn oil (Mazóla,Best Foods], 50 g cellulose (#160390, Teklad Test Diets), 35 g Se- and Cu-deficient AIN-76 mineral mix (#TD88243, Teklad Test Diets), 10 g AIN-76A vitamin mix (#40077, Teklad Test Diets), 3 g DL-methionine (#M9500, Sigma Chemical, St. Louis, MO), and 2 g choline dihydrogen citrate (#30220, Teklad Test Diets).
Downloaded from https://academic.oup.com/jn/article-abstract/122/11/2128/4754707 by Tulane University Medical Library user on 03 February 2019
METHODS
TOXICITY
FISCHER ET AL.
2130
RESULTS The development of a Cu-deficient state was verified by a liver Cu of
