Journal of Molecular andCellular Cardiology (1978) 10,945-95 1
Glutathione Peroxidase Activity and Selenium Concentration in the Hearts of Doxorubicin-treated Rabbits* N. W. REVIS University
AND NIVES
MARUSIC
of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, and Biology Division, Oak Ridge .hfational Laboratory, Oak Ridge, Tennessee 37830, U.S.A. (Received 15 December
1977, accepted in revised form 13 February 1978)
N. W. R~vrs AND N. Gum. Glutathione Peroxidase Activity and Selenium Concentration in the Hearts of Doxorubicin-treated Rabbits. 3ouwd of Molecular and Cellular Cardiolop (1978) 10,945-951. Doxorubicin-treated cardiotoxicity was studied in the rabbit. Rabbits given intravenous injections of 1.5 mg doxorubicin/kg body weight (3 times per week for 3 weeks) developed morphological and histological alterations in their hearts. In addition to these changes, both glutathione peroxidase activity and selenium concentration were significantly reduced in the hearts of the doxorubicin-treated rabbits. The decrease in glutathione peroxidase activity is probably related to the decrease in the concentration of selenium, since this enzyme requires selenium for activity and, furthermore, since in vitro studies failed to show any effect of doxorubicin on glutathione peroxidase. Although the mechanism responsible for the observed decrease in selenium is not known, an alteration in the selenium flux in the myocardial cell may account for the observation. The results of the present studies suggest that the observed increase in lipid hydroperoxides in the hearts of doxorubicin-treated animals may be the result of a decrease in glutathione peroxidase activity. KEY WORDS: Doxorubicin;
Cardiotoxicity;
Glutathione
peroxidase; Selenium.
1. Introduction Doxorubicin is an antibiotic of the anthracycline class which is used in the treatment of various types of cancer [q. Its use, however, has been limited due to its cardiotoxic effects, which include myocardial necrosis and fibrosis [16]. The mechanism(s) responsible for these effects are not known. However, in several recent reports it has been demonstrated that doxorubicin induces lipid peroxidation in the hearts of mice [9, 101. Based on this observation Meyers et al. [9] have suggested that the cardiotoxic effects of doxorubicin may be the result of membrane lipid peroxidation induced by the doxorubicin. Several investigators have suggested that glutathione peroxidase (glutathione: * Research supported by the Department of Energy under contract with Union Carbide Corporation. By acceptance of this article, the publisher or recipient acknowledges the right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.
946
N. W. REVIS
AND
N. MARUSIC
hydrogen-peroxide oxidoreductase, EC 1.11.1.9) prevents membrane lipid peroxidation by catabolizing hydrogen peroxide and lipid hydroperoxides [4, 14. The present studies were therefore performed to determine if doxorubicin treatment affects the activity of this enzyme. Since glutathione peroxidase requires selenium for activity fI5], selenium concentrations were also measured. The results show that doxorubicin treatment affects both the activity of the enzyme and the concentrations of selenium.
2. Materials
and Methods
Male New Zealand rabbits with body weights of 2.4 to 3.2 kg (obtained from Jackson Laboratory) were observed for at least 14 days prior to the experiment for signs of respiratory or intestinal infection. After that period 1.5 mg doxorubicin/kg was injected intravenously (3 times per week for 3 weeks). Following the 3 weeks of treatment, rabbits were killed by cervical dislocation, and the hearts, livers, and kidneys were excised, blotted, and placed in ice-cold solution containing 0.25 M sucrose and 5 mM EDTA buffered at pH 7.4 with 5 mM Tris-HCl and homogenized. Homogenates were centrifuged at 750 g for 20 min, and the supernatant was recentrifuged at 100 000 g for 60 min. The pellet fraction (which contained plasma membrane fragments, mitochondria, and microsomes) was discarded, and the soluble fraction obtained was used to determine the activity of glutathione peroxidase. Glutathione peroxidase was measured by a modification of the coupled-assay procedure of Paglia and Valentine [12]. The reaction mixture consisted of (final concentration) 40 mM potassium phosphate buffer (pH 7.0), 1 nm EDTA, 1 mu NaNs, 0.5 mM NADPH, 1 i.u. GSSG-reductaselml (Sigma Chem.), 1.5 mM GSH, and 0.88 mM Hs 02 in a total volume of 1 ml. All ingredients except enzyme source and peroxide solution were combined at the beginning of each day. Enzyme source (0.1 ml) was added to 0.8 ml of the above mixture and allowed to incubate 5 min at room temperature before initiation of the reaction by the addition of 0.1 ml peroxide solution. Absorbance at 340 nm was recorded for 5 min, and the activity was calculated from the slope of these lines as pmol NADPH oxidized per min. The glutathione peroxidase reaction was always checked for linearity with time and the amount of enzyme added from the tissue isolated from the controls and doxorubicin-treated rabbits. No difference was observed in the linearity of this reaction with time or the amount of enzyme added. Blank reactions with the enzyme source replaced by distiiled water were subtracted from each assay. Protein was measured by the method of Lowry et al. [5]. For electron microscopic studies, sections of heart were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate containing 3% sucrose for 6 h at 4°C. Sections were washed in 0.1 M cacodylate (pH 7.2) containing 6% sucrose and postfixed for 1 h
EFFECT
OF DOXORUBICIN
ON GLUTATHIONE
947
PEROXIDASE
at room temperature in 1y0 0504-5% sucrose in cacodylate buffer (pH 7.2). These sections were dehydrated in ethanol and embedded in Epon. Observations were made in a Hitachi HU-12 electron microscope following uranyl acetate and alkaline lead staining. For determination of selenium, sections of heart and liver were frozen and lyophilized to constant weight. Tissues were reweighed and analyzed for selenium by neutron activation analysis as described by Lyon and Emory [Cl. The dry weight was determined from the lyophilized samples. All changes in the experimental conditions are described in the text or indicated in the Tables and Plates. The results were analyzed by the Student’s t-test, and the differences between values of the group mean were considered significant only if the analysis of variance indicated a probability of less than 0.05%. Doxorubicin hydrochloride was a gift from Adria Laboratories, Inc. 3. Results
Results in Table 1 and Plates 1(a) and (b) and 2(a) and (b) are included in this report to show that doxorubicin-induced cardiotoxicity occurs when the protocol outlined here is followed. Table 1 demonstrates the development of cardiomegaly after doxorubicin treatment. The increase in the ratio of heart weight to body weight was 40%, which was statistically significant (P < 0.01). Although heart weight increased following this treatment, chronic heart failure was not grossly evident at necropsy. The morphology of the control rabbit heart is illustrated in Plate 1(a) and (b) ; the morphology of rabbit heart following doxorubicin treatment is illustrated in Plate 2(a) and (b). The difference between the control and treated hearts is quite obvious. The following were frequently observed after doxorubicin treatment : distension of the sarcoplasmic reticulum, granulation of sarcoplasm, fragmentation and lysis of the myofibrils, mitochondria swelling and degeneration, condensed chromatin, and mild fibrosis (not shown in this study). The effects of doxorubicin treatment on glutathione peroxidase are illustrated in Table 2. Treatment reduced by 40% (2’ < 0.01) the activity of this enzyme in the TABLE
1.
Body weight* Treatment
No. of animals
None
6
Doxorubicin
9
Initial
Final
2240 f 51 2310 & 47 2350 f 35 2260 f 60
Heart wet weight
Heart weight/ body weight*
k)
x 100
4.7 -& 0.98 6.3 f 0.74t
0.20 -J= 0.08 0.28 f 0.013f
* Mean f S.E.M. t P x 0.01. The method used to determine dry weight is shown in the Methods section.
% Dry weight* 21 f 20 f
1.0 1.6
948
N. W.
TABLE
2. Effects oftreatment*
Tissues
Heart Liver Kidney
REVIS
AND
on glutatbione
N. MARUSIC
peroxidase
NADPH Oxidized/proteint Doxorubicin $ treated
N mol min/mg ControlS 320 f 18 1245 f 38 230 & 21
from rabbit
NADPH Oxidized dry weight? Doxorubicin treated
N mol min/g ControlS
190 f 238 1840 f 25 11 180 * 14
tissues
90 f 5.6 341 * 11.2 61 & 7.6
48 & 4.17 471 f 16.311 44 & 5.5
* Rabbits were injected intravenously with 1.5 mg doxorubicin/kg body weight 3 times per week for 3 weeks. The hearts, liver, and kidneys were removed and processed as described in Methods. Control rabbits were treated identically except that saline was used in place of doxorubicin. t Mean f S.E.M. $ Six rabbits were used; &even rabbits were used. 11P< 0.05; TP< 0.01.
heart. The enzyme activity in the liver was significantly increased (P < 0.05), while the activity in the kidney was insignificantly increased (by 20%). It is noted that the activity of this enzyme in the 100 000 g pellet was less than 10% of total activity. Furthermore, the activity of this enzyme in the 100 000 g pellet from the three tissues studied did not change during the period of treatment. In an attempt to expiain the observed changes in glutathione peroxidase, selenium concentrations were measured, since this enzyme requires selenium for activity. Table 3 shows the levels of selenium found in the heart and liver following doxorubicin treatment. A significant decrease (P < 0.01) was observed in hearts from doxorubicin-treated rabbits. Livers from the treated rabbits showed an insignificant increase. Of the other elements measured, significant decreases were observed in copper (heart) and potassium (liver).
TABLE
3. Concentrations of the various doxorubicin-treated rabbits
Element Sodium Magnesium Chlorine Potassium Selenium Copper
Concentration Heart Treated
Control 4566 904 5452 1.8 2.5 25
h f f f & &
elements
404 266 690 0.6 0.8 2.3
* Ma f S.E.M.ofsix experiments. t P < 0.05; tP < 0.01.
4506 953 5623 1.4 1.4 16
f 5 f f f f
in hearts
and livers
in tissue ( yg/g dry wt)* Liver Control 528 216 471 0.3 0.4: 1.9t
6237 f 1137 + 100000 & 2.4 & 6.1 f 12 &
617 301 811 0.9 1.3 1.6
from
control
and
Treated 6237 920 7730 1.6 7.5 14
& * f f f f
598 193 555 0.77 1.2 2.4
PLATE 1. Section of heart from rabbit injected with saline (a) ID, intercalated disc; M, mitochondria; MY, myofibril. GV, Golgi vesicle; ER, endoplasmic reticulum; N, nucleus.
3 times per week for 3 weeks. x 14000. (b) F, fat droplet; DV, dense vesicle;
PLATE 2 (a) and (b). Section of heart from rabbit injected with 1.5 mg doxorubicin,‘kg body weight 3 times per week for 3 weeks. Note distension of sarcoplasmic reticulum (SR) vacuolization of the sarcoplasm (V), granulation of sarcoplasm (arrows). nuclrus iN) and degenerating mitochondria (M). x 14000.
EFFECT
OF DOXORUBICIN
ON CLUTATHIONE
PEROXIDASE
949
To explore the mechanism responsible for the decreased glutathione peroxidase activity and selenium concentration, glutathione peroxidase activity from heart and liver was measured in the presence of doxorubicin (1 x 10-4 M). Several different procedures (i.e. addition of doxorubicin to the reaction mixture or preincubation of enzyme with doxorubicin at 37°C for 1 h or at 4% for 24 h) showed that doxorubitin did not affect the glutathione peroxidase activity in heart or liver. In addition to measuring the direct effect of doxorubicin on this enzyme, selenium (10-O. 1 mM) was added to 10 000 g supernatant prior to assaying enzyme activity. Of the three tissues studied from the controls and doxorubicin-treated rabbits no change in the activity of glutathione peroxidase was observed following treatment with selenium.
4. Discussion
The pathological alterations reported here demonstrate the cardiotoxic effects of doxorubicin. The total dose given during the experimental period was 13.5 mg doxorubicin/kg body weight, which is lower than the dose required to induce doxorubicin cardiotoxicity in rats (26 mg/kg body weight) or mice (20 mg/kg body weight) [S, II]. Furthermore, the time required to induce doxorubicin cardiotoxicity in rats is 10 weeks, which is considerably longer than was required in the present studies. This suggests that the rabbit is more sensitive to the cardiotoxic effects of doxorubicin than the rat or mouse. As discussed above, lipid peroxidation has been shown to occur in the hearts of mice treated with doxorubicin. The mechanism responsible for this change is not known. However, Handa and Sato [3] have shown that microsomes exposed to anthracyclines stimulate the formation of a superoxide radical ion. Previous studies have shown that the superoxide radical ion can decompose to yield a hydroxyl radical and hydrogen peroxide [I3]. These, in turn, are known to initiate freeradical mediated chain reactions which result in conversion of the membrane unsaturated fatty acids to lipid peroxides [17]. That glutathione peroxidase and selenium are both depressed in the hearts of doxorubicin-treated rabbits suggests a possible explanation of the reported increase in lipid hydroperoxides. Glutathione peroxidase is thought to maintain low levels of hydrogen peroxide and lipid hydroperoxides. However, McCay et al. [7] in a recent report observed that glutathione peroxidase was unable to metabolize membrane lipid hydroperoxides in vitro. Based on this observation, they have suggested that glutathione peroxidase serves the role of preventing lipid peroxidation by scavenging hydrogen peroxide. Since in vitro studies failed to show any effect of doxorubicin on glutathione peroxidase, the observed decrease in this enzyme is probably related to an effect of doxorubicin on selenium metabolism. The decrease in the selenium concentration in the hearts of doxorubicin-treated rabbits is not understood presently. Changes in
950
N. W. REVIS AND N. MARUSIC
the calcium concentration in the hearts of treated rabbits have been observed in this Iaboratory (unpublished observations). This suggests that doxorubicin treatment affects the flux of calcium into the myocardial cell, an effect which may be related to selenium flux into this cell. Meyers et al. [IO] have shown that vitamin E pretreatment reduces doxorubicin cardiotoxicity. They have suggested that this reduction is related to the stabilization of the myocardial membrane by vitamin E. However, vitamin E is also known to affect the cellular transport properties of selenium [2]. Further studies are in progress to determine if giving supplemental selenium to doxorubicin-treated rabbits will restore the activity of glutathione peroxidase and if this will reduce doxorubicin cardiotoxicity.
REFERENCES 1. DE MARCO, A., GAETONI, M. & SCARPINATO, B. M. Adriamycin (NSC-123, 127): a new antibiotic with antitumor activity. Cancer ChemotherapyReports 53, 33-37 (1969). 2. DIPLOCIC, A. Metabolic aspects of selenium action and toxicity. Critical Reviews in Toxicology 70,27 l-320 (1976). 3. HANDA, K. & SATO, S. Generation of free radicals of quinone-containing anti-cancer chemicals in NADPH-microsome system evidenced by initiation of sulfite oxidation. GANiV66,43-48 (1975). 4. LAWRENCE, R. A. & BURK, R. F. Glutathione peroxidase activity in selenium deficient rat liver. Biochemical and Biophysical ResearchCommunications71,952-958 ( 1976), 5. LOWRY, 0. H., ROSENBROUGH, N. S., FARR, A. L. & RANDALL, T. Protein measurement with folin phenol reagent. Journal of Biologkal Chemistry 193,265-275 (1951). 6. LYON, W. S. & EMORY, J. G. Neutron activation analysis applied to the study of elements entering and leaving a coal-fired steam plant. International 3ournal of Environmental Analytical Chemistry 4, 125-133 (1975). 7. MCCAY, P. B., GIVSON, D. D., FONG, K.-L. & ROGER, K. Effects of glutathione peroxidase activity on lipid peroxidation in biological membranes. Biochimica et biophytica acta 431,359-468 (1976). 8. METTL.ER, F. P., YOUNG, D. M. St WARD, J. M. Adriamycin-induced cardiotoxicity (cardiomyopathy and congestive heart failure) in rats. Cancer Research37, 2705-2713 (1977). 9. MEYERS, C. E., MCGUIRE, W. P., LISS, R., IFRIM, I., FROTZINGER, K. & YOUNG, R. C. Adriamycin: the role of lipid peroxidation in cardiac toxicity and tumor response. Science197,165-167 (1976). 10. MEYERS, C. E., MCGUIRE, W. P. & YOUNG, R. C. Adriamycin: amelioration of toxicity of a-tocopherol. CancerChemotherapyReports 60,96 l-962 ( 1976). 11. OMAYE, S. T. & TAPPEL, A. L. Glutathione peroxidase, glutathione reductase and thiobarbituric acid-reactive products in muscle of chickens and mice with genetic muscular dystrophy. I;3 Sciences15, 137-145 (1974). 12. PAGLIA, D. E. & VALENTINE, W. N. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. 3ournal of Laboratory and Clinizal Medicine 70, 158-169 ( 1967). 13. ROSEN, H. & KLEBANOFF, F. Chemilumincscence and superoxide production by myeloperoxidase-deficient leukocytes. Journal of Clinical Investigation 58,50-&O (1976).
EFFECT
OF DOXORUBICIN
ON GLUTATHIONE
PEROXIDASE
951
14. ROSENOFF, S. H., BROOKS, E., BOSTICK, F. & YOUNG, R. C. Adriamycin-induced cardiac damage in the mouse: a small-animal model of cardiotoxicity. Journal of t/u .hbtionuECuncer Institute 55, 191-194 (1975). 15. ROTRUCK, J. T., HOEKSTFU, W. G. & POPE, A. L. Glucose-dependent protection by dietary selenium against haemolysis of rat erythrocytes in vitro. Nuture New Biology 231, 223-225 (1971). 16. YOUNG, D. M. Pathologic effects of adriamycin (NSC-123, 127) in experimental systems. CancerChemothru~y Reports 6, 159-175 (1975). 17. ZIMMERMANN, R., FLOHE, L., WESER, U. & HAJIRTMANN, H. J. Inhibition of lipid peroxidation in isolated inner membrane of rat liver mitochondria by superoxide dismutase. FEBS Letters 29, 117-20 (1973).
Glutathione Peroxidase Activity and Selenium Concentration in the Hearts of Doxorubicin-treated Rabbits* N. W. REVIS University
AND NIVES
MARUSIC
of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, and Biology Division, Oak Ridge .hfational Laboratory, Oak Ridge, Tennessee 37830, U.S.A. (Received 15 December
1977, accepted in revised form 13 February 1978)
N. W. R~vrs AND N. Gum. Glutathione Peroxidase Activity and Selenium Concentration in the Hearts of Doxorubicin-treated Rabbits. 3ouwd of Molecular and Cellular Cardiolop (1978) 10,945-951. Doxorubicin-treated cardiotoxicity was studied in the rabbit. Rabbits given intravenous injections of 1.5 mg doxorubicin/kg body weight (3 times per week for 3 weeks) developed morphological and histological alterations in their hearts. In addition to these changes, both glutathione peroxidase activity and selenium concentration were significantly reduced in the hearts of the doxorubicin-treated rabbits. The decrease in glutathione peroxidase activity is probably related to the decrease in the concentration of selenium, since this enzyme requires selenium for activity and, furthermore, since in vitro studies failed to show any effect of doxorubicin on glutathione peroxidase. Although the mechanism responsible for the observed decrease in selenium is not known, an alteration in the selenium flux in the myocardial cell may account for the observation. The results of the present studies suggest that the observed increase in lipid hydroperoxides in the hearts of doxorubicin-treated animals may be the result of a decrease in glutathione peroxidase activity. KEY WORDS: Doxorubicin;
Cardiotoxicity;
Glutathione
peroxidase; Selenium.
1. Introduction Doxorubicin is an antibiotic of the anthracycline class which is used in the treatment of various types of cancer [q. Its use, however, has been limited due to its cardiotoxic effects, which include myocardial necrosis and fibrosis [16]. The mechanism(s) responsible for these effects are not known. However, in several recent reports it has been demonstrated that doxorubicin induces lipid peroxidation in the hearts of mice [9, 101. Based on this observation Meyers et al. [9] have suggested that the cardiotoxic effects of doxorubicin may be the result of membrane lipid peroxidation induced by the doxorubicin. Several investigators have suggested that glutathione peroxidase (glutathione: * Research supported by the Department of Energy under contract with Union Carbide Corporation. By acceptance of this article, the publisher or recipient acknowledges the right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.
946
N. W. REVIS
AND
N. MARUSIC
hydrogen-peroxide oxidoreductase, EC 1.11.1.9) prevents membrane lipid peroxidation by catabolizing hydrogen peroxide and lipid hydroperoxides [4, 14. The present studies were therefore performed to determine if doxorubicin treatment affects the activity of this enzyme. Since glutathione peroxidase requires selenium for activity fI5], selenium concentrations were also measured. The results show that doxorubicin treatment affects both the activity of the enzyme and the concentrations of selenium.
2. Materials
and Methods
Male New Zealand rabbits with body weights of 2.4 to 3.2 kg (obtained from Jackson Laboratory) were observed for at least 14 days prior to the experiment for signs of respiratory or intestinal infection. After that period 1.5 mg doxorubicin/kg was injected intravenously (3 times per week for 3 weeks). Following the 3 weeks of treatment, rabbits were killed by cervical dislocation, and the hearts, livers, and kidneys were excised, blotted, and placed in ice-cold solution containing 0.25 M sucrose and 5 mM EDTA buffered at pH 7.4 with 5 mM Tris-HCl and homogenized. Homogenates were centrifuged at 750 g for 20 min, and the supernatant was recentrifuged at 100 000 g for 60 min. The pellet fraction (which contained plasma membrane fragments, mitochondria, and microsomes) was discarded, and the soluble fraction obtained was used to determine the activity of glutathione peroxidase. Glutathione peroxidase was measured by a modification of the coupled-assay procedure of Paglia and Valentine [12]. The reaction mixture consisted of (final concentration) 40 mM potassium phosphate buffer (pH 7.0), 1 nm EDTA, 1 mu NaNs, 0.5 mM NADPH, 1 i.u. GSSG-reductaselml (Sigma Chem.), 1.5 mM GSH, and 0.88 mM Hs 02 in a total volume of 1 ml. All ingredients except enzyme source and peroxide solution were combined at the beginning of each day. Enzyme source (0.1 ml) was added to 0.8 ml of the above mixture and allowed to incubate 5 min at room temperature before initiation of the reaction by the addition of 0.1 ml peroxide solution. Absorbance at 340 nm was recorded for 5 min, and the activity was calculated from the slope of these lines as pmol NADPH oxidized per min. The glutathione peroxidase reaction was always checked for linearity with time and the amount of enzyme added from the tissue isolated from the controls and doxorubicin-treated rabbits. No difference was observed in the linearity of this reaction with time or the amount of enzyme added. Blank reactions with the enzyme source replaced by distiiled water were subtracted from each assay. Protein was measured by the method of Lowry et al. [5]. For electron microscopic studies, sections of heart were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate containing 3% sucrose for 6 h at 4°C. Sections were washed in 0.1 M cacodylate (pH 7.2) containing 6% sucrose and postfixed for 1 h
EFFECT
OF DOXORUBICIN
ON GLUTATHIONE
947
PEROXIDASE
at room temperature in 1y0 0504-5% sucrose in cacodylate buffer (pH 7.2). These sections were dehydrated in ethanol and embedded in Epon. Observations were made in a Hitachi HU-12 electron microscope following uranyl acetate and alkaline lead staining. For determination of selenium, sections of heart and liver were frozen and lyophilized to constant weight. Tissues were reweighed and analyzed for selenium by neutron activation analysis as described by Lyon and Emory [Cl. The dry weight was determined from the lyophilized samples. All changes in the experimental conditions are described in the text or indicated in the Tables and Plates. The results were analyzed by the Student’s t-test, and the differences between values of the group mean were considered significant only if the analysis of variance indicated a probability of less than 0.05%. Doxorubicin hydrochloride was a gift from Adria Laboratories, Inc. 3. Results
Results in Table 1 and Plates 1(a) and (b) and 2(a) and (b) are included in this report to show that doxorubicin-induced cardiotoxicity occurs when the protocol outlined here is followed. Table 1 demonstrates the development of cardiomegaly after doxorubicin treatment. The increase in the ratio of heart weight to body weight was 40%, which was statistically significant (P < 0.01). Although heart weight increased following this treatment, chronic heart failure was not grossly evident at necropsy. The morphology of the control rabbit heart is illustrated in Plate 1(a) and (b) ; the morphology of rabbit heart following doxorubicin treatment is illustrated in Plate 2(a) and (b). The difference between the control and treated hearts is quite obvious. The following were frequently observed after doxorubicin treatment : distension of the sarcoplasmic reticulum, granulation of sarcoplasm, fragmentation and lysis of the myofibrils, mitochondria swelling and degeneration, condensed chromatin, and mild fibrosis (not shown in this study). The effects of doxorubicin treatment on glutathione peroxidase are illustrated in Table 2. Treatment reduced by 40% (2’ < 0.01) the activity of this enzyme in the TABLE
1.
Body weight* Treatment
No. of animals
None
6
Doxorubicin
9
Initial
Final
2240 f 51 2310 & 47 2350 f 35 2260 f 60
Heart wet weight
Heart weight/ body weight*
k)
x 100
4.7 -& 0.98 6.3 f 0.74t
0.20 -J= 0.08 0.28 f 0.013f
* Mean f S.E.M. t P x 0.01. The method used to determine dry weight is shown in the Methods section.
% Dry weight* 21 f 20 f
1.0 1.6
948
N. W.
TABLE
2. Effects oftreatment*
Tissues
Heart Liver Kidney
REVIS
AND
on glutatbione
N. MARUSIC
peroxidase
NADPH Oxidized/proteint Doxorubicin $ treated
N mol min/mg ControlS 320 f 18 1245 f 38 230 & 21
from rabbit
NADPH Oxidized dry weight? Doxorubicin treated
N mol min/g ControlS
190 f 238 1840 f 25 11 180 * 14
tissues
90 f 5.6 341 * 11.2 61 & 7.6
48 & 4.17 471 f 16.311 44 & 5.5
* Rabbits were injected intravenously with 1.5 mg doxorubicin/kg body weight 3 times per week for 3 weeks. The hearts, liver, and kidneys were removed and processed as described in Methods. Control rabbits were treated identically except that saline was used in place of doxorubicin. t Mean f S.E.M. $ Six rabbits were used; &even rabbits were used. 11P< 0.05; TP< 0.01.
heart. The enzyme activity in the liver was significantly increased (P < 0.05), while the activity in the kidney was insignificantly increased (by 20%). It is noted that the activity of this enzyme in the 100 000 g pellet was less than 10% of total activity. Furthermore, the activity of this enzyme in the 100 000 g pellet from the three tissues studied did not change during the period of treatment. In an attempt to expiain the observed changes in glutathione peroxidase, selenium concentrations were measured, since this enzyme requires selenium for activity. Table 3 shows the levels of selenium found in the heart and liver following doxorubicin treatment. A significant decrease (P < 0.01) was observed in hearts from doxorubicin-treated rabbits. Livers from the treated rabbits showed an insignificant increase. Of the other elements measured, significant decreases were observed in copper (heart) and potassium (liver).
TABLE
3. Concentrations of the various doxorubicin-treated rabbits
Element Sodium Magnesium Chlorine Potassium Selenium Copper
Concentration Heart Treated
Control 4566 904 5452 1.8 2.5 25
h f f f & &
elements
404 266 690 0.6 0.8 2.3
* Ma f S.E.M.ofsix experiments. t P < 0.05; tP < 0.01.
4506 953 5623 1.4 1.4 16
f 5 f f f f
in hearts
and livers
in tissue ( yg/g dry wt)* Liver Control 528 216 471 0.3 0.4: 1.9t
6237 f 1137 + 100000 & 2.4 & 6.1 f 12 &
617 301 811 0.9 1.3 1.6
from
control
and
Treated 6237 920 7730 1.6 7.5 14
& * f f f f
598 193 555 0.77 1.2 2.4
PLATE 1. Section of heart from rabbit injected with saline (a) ID, intercalated disc; M, mitochondria; MY, myofibril. GV, Golgi vesicle; ER, endoplasmic reticulum; N, nucleus.
3 times per week for 3 weeks. x 14000. (b) F, fat droplet; DV, dense vesicle;
PLATE 2 (a) and (b). Section of heart from rabbit injected with 1.5 mg doxorubicin,‘kg body weight 3 times per week for 3 weeks. Note distension of sarcoplasmic reticulum (SR) vacuolization of the sarcoplasm (V), granulation of sarcoplasm (arrows). nuclrus iN) and degenerating mitochondria (M). x 14000.
EFFECT
OF DOXORUBICIN
ON CLUTATHIONE
PEROXIDASE
949
To explore the mechanism responsible for the decreased glutathione peroxidase activity and selenium concentration, glutathione peroxidase activity from heart and liver was measured in the presence of doxorubicin (1 x 10-4 M). Several different procedures (i.e. addition of doxorubicin to the reaction mixture or preincubation of enzyme with doxorubicin at 37°C for 1 h or at 4% for 24 h) showed that doxorubitin did not affect the glutathione peroxidase activity in heart or liver. In addition to measuring the direct effect of doxorubicin on this enzyme, selenium (10-O. 1 mM) was added to 10 000 g supernatant prior to assaying enzyme activity. Of the three tissues studied from the controls and doxorubicin-treated rabbits no change in the activity of glutathione peroxidase was observed following treatment with selenium.
4. Discussion
The pathological alterations reported here demonstrate the cardiotoxic effects of doxorubicin. The total dose given during the experimental period was 13.5 mg doxorubicin/kg body weight, which is lower than the dose required to induce doxorubicin cardiotoxicity in rats (26 mg/kg body weight) or mice (20 mg/kg body weight) [S, II]. Furthermore, the time required to induce doxorubicin cardiotoxicity in rats is 10 weeks, which is considerably longer than was required in the present studies. This suggests that the rabbit is more sensitive to the cardiotoxic effects of doxorubicin than the rat or mouse. As discussed above, lipid peroxidation has been shown to occur in the hearts of mice treated with doxorubicin. The mechanism responsible for this change is not known. However, Handa and Sato [3] have shown that microsomes exposed to anthracyclines stimulate the formation of a superoxide radical ion. Previous studies have shown that the superoxide radical ion can decompose to yield a hydroxyl radical and hydrogen peroxide [I3]. These, in turn, are known to initiate freeradical mediated chain reactions which result in conversion of the membrane unsaturated fatty acids to lipid peroxides [17]. That glutathione peroxidase and selenium are both depressed in the hearts of doxorubicin-treated rabbits suggests a possible explanation of the reported increase in lipid hydroperoxides. Glutathione peroxidase is thought to maintain low levels of hydrogen peroxide and lipid hydroperoxides. However, McCay et al. [7] in a recent report observed that glutathione peroxidase was unable to metabolize membrane lipid hydroperoxides in vitro. Based on this observation, they have suggested that glutathione peroxidase serves the role of preventing lipid peroxidation by scavenging hydrogen peroxide. Since in vitro studies failed to show any effect of doxorubicin on glutathione peroxidase, the observed decrease in this enzyme is probably related to an effect of doxorubicin on selenium metabolism. The decrease in the selenium concentration in the hearts of doxorubicin-treated rabbits is not understood presently. Changes in
950
N. W. REVIS AND N. MARUSIC
the calcium concentration in the hearts of treated rabbits have been observed in this Iaboratory (unpublished observations). This suggests that doxorubicin treatment affects the flux of calcium into the myocardial cell, an effect which may be related to selenium flux into this cell. Meyers et al. [IO] have shown that vitamin E pretreatment reduces doxorubicin cardiotoxicity. They have suggested that this reduction is related to the stabilization of the myocardial membrane by vitamin E. However, vitamin E is also known to affect the cellular transport properties of selenium [2]. Further studies are in progress to determine if giving supplemental selenium to doxorubicin-treated rabbits will restore the activity of glutathione peroxidase and if this will reduce doxorubicin cardiotoxicity.
REFERENCES 1. DE MARCO, A., GAETONI, M. & SCARPINATO, B. M. Adriamycin (NSC-123, 127): a new antibiotic with antitumor activity. Cancer ChemotherapyReports 53, 33-37 (1969). 2. DIPLOCIC, A. Metabolic aspects of selenium action and toxicity. Critical Reviews in Toxicology 70,27 l-320 (1976). 3. HANDA, K. & SATO, S. Generation of free radicals of quinone-containing anti-cancer chemicals in NADPH-microsome system evidenced by initiation of sulfite oxidation. GANiV66,43-48 (1975). 4. LAWRENCE, R. A. & BURK, R. F. Glutathione peroxidase activity in selenium deficient rat liver. Biochemical and Biophysical ResearchCommunications71,952-958 ( 1976), 5. LOWRY, 0. H., ROSENBROUGH, N. S., FARR, A. L. & RANDALL, T. Protein measurement with folin phenol reagent. Journal of Biologkal Chemistry 193,265-275 (1951). 6. LYON, W. S. & EMORY, J. G. Neutron activation analysis applied to the study of elements entering and leaving a coal-fired steam plant. International 3ournal of Environmental Analytical Chemistry 4, 125-133 (1975). 7. MCCAY, P. B., GIVSON, D. D., FONG, K.-L. & ROGER, K. Effects of glutathione peroxidase activity on lipid peroxidation in biological membranes. Biochimica et biophytica acta 431,359-468 (1976). 8. METTL.ER, F. P., YOUNG, D. M. St WARD, J. M. Adriamycin-induced cardiotoxicity (cardiomyopathy and congestive heart failure) in rats. Cancer Research37, 2705-2713 (1977). 9. MEYERS, C. E., MCGUIRE, W. P., LISS, R., IFRIM, I., FROTZINGER, K. & YOUNG, R. C. Adriamycin: the role of lipid peroxidation in cardiac toxicity and tumor response. Science197,165-167 (1976). 10. MEYERS, C. E., MCGUIRE, W. P. & YOUNG, R. C. Adriamycin: amelioration of toxicity of a-tocopherol. CancerChemotherapyReports 60,96 l-962 ( 1976). 11. OMAYE, S. T. & TAPPEL, A. L. Glutathione peroxidase, glutathione reductase and thiobarbituric acid-reactive products in muscle of chickens and mice with genetic muscular dystrophy. I;3 Sciences15, 137-145 (1974). 12. PAGLIA, D. E. & VALENTINE, W. N. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. 3ournal of Laboratory and Clinizal Medicine 70, 158-169 ( 1967). 13. ROSEN, H. & KLEBANOFF, F. Chemilumincscence and superoxide production by myeloperoxidase-deficient leukocytes. Journal of Clinical Investigation 58,50-&O (1976).
EFFECT
OF DOXORUBICIN
ON GLUTATHIONE
PEROXIDASE
951
14. ROSENOFF, S. H., BROOKS, E., BOSTICK, F. & YOUNG, R. C. Adriamycin-induced cardiac damage in the mouse: a small-animal model of cardiotoxicity. Journal of t/u .hbtionuECuncer Institute 55, 191-194 (1975). 15. ROTRUCK, J. T., HOEKSTFU, W. G. & POPE, A. L. Glucose-dependent protection by dietary selenium against haemolysis of rat erythrocytes in vitro. Nuture New Biology 231, 223-225 (1971). 16. YOUNG, D. M. Pathologic effects of adriamycin (NSC-123, 127) in experimental systems. CancerChemothru~y Reports 6, 159-175 (1975). 17. ZIMMERMANN, R., FLOHE, L., WESER, U. & HAJIRTMANN, H. J. Inhibition of lipid peroxidation in isolated inner membrane of rat liver mitochondria by superoxide dismutase. FEBS Letters 29, 117-20 (1973).
