0013-7227/90/1273-1495$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 3 Printed in U.S.A.
Adriamycin-Induced Increase in Serum Aldosterone Levels: Effects in Riboflavin-Sufficient and Riboflavin-Deficient Rats* JOHN T. PINTO, BRADLEY N. DELMAN, PURABI DUTTA, AND JEROME NISSELBAUM Memorial Sloan-Kettering Cancer Center, Departments of Medicine and Clinical Chemistry, New York, New York 10021
ABSTRACT. Previous studies in rats have demonstrated that 1) aldosterone enhances biosynthesis of renal flavin coenzymes; 2) riboflavin analogs inhibit the synthesis of aldosterone; and 3) adriamycin inhibits flavin coenzyme biosynthesis. In their entirety, these findings suggest that both diminished flavin coenzyme biosynthesis induced by adriamycin and a dietary riboflavin deficiency would result in decreased formation of aldosterone. The present study examined the effects of adriamycin treatment on serum aldosterone in rats consuming either a diet adequate in riboflavin or a riboflavin-deficient diet. Groups of rats fed specially prepared diets were injected for 3 days with adriamycin (cumulative dose range, 6-24 mg/kg BW). Pair-fed controls were given saline. After death, adrenal glands were excised, and blood samples were analyzed for aldosterone levels. No changes in adrenal weights or protein and potassium concentrations were observed after adriamycin treatment. In contrast
I
NCREASING knowledge has accumulated concerning modulation of riboflavin metabolism by hormones (1-4) and drugs (5-8). To be physiologically active, riboflavin must first be phosphorylated to riboflavin-5'phosphate (FMN) by flavokinase (ATP:riboflavin-5'phosphotransferase; EC 2.7.1.26) and then be pyrophosphorylated to flavin adenine dinucleotide (FAD) by FAD synthetase (ATP:FMN adenylyltransferase; EC 2.7.7.2). Both FAD and FMN bind to a variety of flavoenzymes that participate in drugs (5, 6), steroid (9), fatty acid (10, 11), and overall energy metabolism (12). Previous investigations have recognized that pituitary and/or adrenal function may control activation of riboReceived January 29, 1990. Address requests for reprints to: John Thomas Pinto, Ph.D., Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 140, New York, New York 10021. * Preliminary findings were published in an abstract form in Clin Res 37:333, 1989 (Abstract). This work was supported by the Clinical Nutrition Research Unit, Grants CA-29502 and CA-08748 from the NIH, and grants from the NIA (5-K01-AG-00399), Hoffmann-LaRoche, Inc., and the Stella and Charles Guttman Foundation. The research was performed in the Nutrition Research Laboratory of SloanKettering Institute.
to initial predictions, in riboflavin-sufficient rats, serum aldosterone levels were markedly enhanced by adriamycin in a doserelated manner. Riboflavin-deficient animals had lower basal aldosterone levels and markedly attenuated responses to adriamycin than did riboflavin-sufficient rats. In separate groups of adriamycin-treated rats fed a normal chow diet, serum aldosterone levels increased, and serum corticosterone levels showed a small but significant decline. In addition, adriamycin treatment caused an increase in urinary potassium excretion and a decrease in sodium excretion. These results suggest that flavins play a decisive role in regulating the levels of aldosterone and raise the possibility that the adriamycin-induced increase in serum aldosterone may be part of the pathogenetic mechanisms of cardiovascular toxicity and overall muscular weakness. (Endocrinology 127: 1495-1501, 1990)
flavin and its subsequent involvement with the mitochondrial cytochrome P-450 reductase system and corticosteroidogenesis. Studies by Fazekas and Sandor (3, 9) have indicated that ACTH-treated rats exhibit enhanced formation of renal flavin nucleotides and that tissue-specific induction of flavokinase may explain this increase. Subsequent studies by Trachewsky et al. (4, 13), using enzyme-linked immunosorbent assay, revealed that aldosterone stimulates de novo renal biosynthesis of flavokinase and that spironolactone (mineralocorticoid type I receptor antagonist), actinomycin-D (RNA synthesis inhibitor), or cycloheximide (protein synthesis inhibitor) can abolish this stimulatory effect. These data support the concept that mineralocorticoids mediate renal flavin biosynthesis and that enhanced formation of flavin coenzymes by aldosterone may be associated with positive sodium balance. To extend the relationship between the formation of flavin coenzymes and aldosterone levels, Trachewsky and Kem (14-17) demonstrated that analogs of riboflavin substituted in the N-10 position of the isoalloxazine ring possess antihypertensive activity. Thus, evidence to date suggests that aldosterone 1495
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 06 July 2015. at 09:10 For personal use only. No other uses without permission. . All rights reserved.
ADRIAMYCIN-INDUCED INCREASE IN ALDOSTERONE
1496
enhances renal biosynthesis of FAD and that flavin coenzymes, directly or mediated through flavoenzymes, govern the formation of aldosterone. Investigations in this laboratory have focused upon the physiological consequences to riboflavin metabolism associated with the administration of tri- and tetracyclic drugs (7,18-20). Each of these compounds when used in clinically relevant doses in laboratory animals diminishes the formation of FAD. Recent investigations (21-23) with the antineoplastic agent adriamycin have demonstrated a dose-related and organ-selective decrease in FAD formation, particularly in cardiac and skeletal muscle. In addition, studies performed in vitro by Fisher et al. (24) showed that adriamycin can bind competitively to sites similar to those that bind flavin coenzymes. These drugs, which are recognized to have diverse pharmacological action, may derive a common cytotoxic efficacy by inhibiting FAD biosynthesis. These findings raise the possibility that clinically significant riboflavin deficiency may result from the use of these drugs despite a diet adequate in the vitamin. The present study was initially conducted to determine whether alterations in riboflavin metabolism produced by diet and/or drugs affect the level of serum aldosterone, a hormone that stimulates renal flavokinase activity. Our investigations examined the effects of graded doses of adriamycin on aldosterone levels in serum of riboflavin-sufficient and riboflavin-deficient rats. Contrary to our initial prediction, administration of adriamycin to riboflavin-sufficient animals resulted in a marked doserelated increase in serum aldosterone levels. In animals that are already deficient in riboflavin due to diminished dietary intake, adriamycin was considerably less effective in elevating serum aldosterone. Further experiments investigated whether adriamycin administered to normal chow-fed animals induced aldosterone formation through alteration of its precursor, corticosterone, and whether aldosterone-responsive excretion of electrolytes was affected by adriamycin. Materials and Methods Animals, diets, and drugs Three-week-old male Sprague-Dawley rats were purchased from Charles River Breeding Laboratories, Inc. (Wilmington, MA). All animals were randomly distributed and housed individually in wire-bottom metabolic cages in our animal facility with controlled temperature and 12-h lighting cycle. In all experiments, tap water was allowed ad libitum, and animals were handled at least twice daily during cage acclimation periods. For the first experiment, riboflavin deficiency was produced in groups of animals by feeding a specially prepared riboflavin-deficient diet (25). At intervals during a 7-week feeding and cage acclimation period, rats were bled from the suborbital sinus, and the erythrocyte glutathione reductase
Endo • 1990 Vol 127 • No 3
activity coefficient (EGRAC) was determined periodically to assess the degree of deficiency. Under these conditions, biochemical evidence of riboflavin deprivation is evident after 24 weeks on the deficient diet, i.e. significant elevation of EGRAC, while overt clinical signs may appear after 7-8 weeks. Riboflavin-sufficient animals were age matched and fed a diet identical in composition to the riboflavin-deficient diet, but with supplemental riboflavin (8.5 /ug/g diet) (25). This figure represents approximately 3 times the recommended dietary allowance for riboflavin for the rat. Both diets were prepared by Dyets, Inc. (Bethlehem, PA). In later experiments, animals were maintained on standard pelleted Purina rat chow (Ralston-Purina Co., St. Louis, MO), which had been determined in our laboratory to contain approximately 8.5 pig riboflavin/g diet. Adriamycin was purchased from Adria Laboratories (Columbus, OH) as a crystalline powder and reconstituted with saline before injection into animals. Glutathione reductase activity and activity coefficient The activities of glutathione reductase measured first in the absence of exogenous FAD (basal activity) and then again in the presence of FAD were determined using the method of Beutler (26). Activity coefficients were calculated by dividing the activity of glutathione reductase in the presence of FAD by the basal activity. We defined as riboflavin deficient those animals whose EGRAC was greater than 1.7. Treatment of animals In the first study animals were maintained on specially prepared diets for 7 weeks before treatment with adriamycin. To determine the effect of adriamycin on aldosterone formation in animals consuming either a diet adequate in riboflavin or a riboflavin-deficient diet, we performed the following experiment. Beginning on day 1, two groups each of 10-week-old riboflavin-sufficient and riboflavin-deficient rats were injected ip for 3 days with adriamycin, representing cumulative doses of 6, 16, and 24 mg/kg BW. A dose of 10 mg/kg in rats is equivalent to 60 mg/m2 in humans. Pair-fed controls for each drug-treated group of riboflavin-sufficient and riboflavin-deficient animals were given comparable volumes of saline. The number of rats used in each group is indicated in the figure legends. On day 4 of the study, 18 h after the last injection, animals were killed by exposing them to carbon dioxide, and blood samples were drawn via cardiac puncture for aldosterone determinations. Adrenal glands were excised, weighed, and analyzed for protein and potassium levels. In a second experiment to corroborate our findings in rats fed the specially prepared diet adequate in riboflavin, groups of animals were acclimated to metabolic cages for 10 days and allowed to feed ad libitum on a standard pelleted Purina rat chow diet. This study was performed 1) to determine whether the observed elevation in aldosterone was not a stress-induced manifestation of the specially prepared diet coupled with diminished food intake and 2) to investigate more closely the dose-related rise in aldosterone by including among the previous cumulative dose regimen of 6, 16, and 24 mg/kg intermediate doses of adriamycin at 8 and 18 mg/kg BW. All
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 06 July 2015. at 09:10 For personal use only. No other uses without permission. . All rights reserved.
ADRIAMYCIN-INDUCED INCREASE IN ALDOSTERONE injections were administered ip in divided doses over a 3-day injection period. On day 4 and 18 h after the last injection, animals were killed, and blood samples were analyzed for aldosterone. In a third study to determine the effects of adriamycin on corticosterone levels and aldosterone-responsive parameters, 10 Sprague-Dawley rats consuming a Purina rat chow diet were placed in metabolic cages and acclimated for 1 week. At the end of this period and before drug administration, all animals had blood samples taken from the suborbital sinus and analyzed for aldosterone and corticosterone levels; 24-h urine samples were collected and measured for pretreatment levels of sodium, potassium, and creatinine. The 24-h collection period for urine samples was from 1000 h to 1000 h the next day. After pretreatment collection of blood and urine specimens, on day 1 of study, five animals received saline, and five corresponding pairweighed rats received adriamycin (3 mg/kg twice daily for 3 days; cumulative dose, 18 mg/kg). Saline-treated rats received a similar volume of isotonic saline. On day 3 of the adriamycin injection period, a second 24-h urine collection was begun. On day 4, after urine samples were collected, animals were killed, and blood samples were drawn via cardiac puncture for determinations of aldosterone and corticosterone. Aldosterone RIA Serum aldosterone concentrations were measured using a standard RIA kit (Coat-A-Count no extraction RIA) purchased from Diagnostic Products Corp. (Los Angeles, CA). The average interassay coefficient of variance was 10%. Aliquots of serum were transferred to incubation tubes that were coated with antiserum, and 1.0 ml [125I]aldosterone was added. After 3 h at 37 C the tubes were decanted, and bound [125I]aldosterone was measured by counting for 1 min in a 7-counter. A logit/log graphic analysis was used to linearize the standard curve over a range of 25-1200 pg/ml. Past experience with adriamycintreated animals indicates a need to dilute serum from these rats 1:10 or 1:20 before analysis for aldosterone. Corticosterone [l,2- 3 H]Corticosterone (Amersham Corp., Arlington Heights, IL) was purified by TLC on silica gel. The band that migrated with authentic corticosterone was scraped from the plate, eluted with acetone, concentrated to dryness, and redissolved in a small volume of ethanol. Stock standard was dissolved in absolute ethanol and diluted in 0.05 M Tris-HCl, pH 8.0, buffer containing 0.1 M NaCl and 0.1% BSA before use. A competitive binding assay was performed using 100 n\ monkey serum as the binder. Standards (2-50 Mg/dl) were prepared in 0.05 M Tris-HCl, pH 8.0, buffer containing 0.1 M NaCl and 0.1% BSA. The following protocol was developed for use in our laboratory. One hundred microliters of unknown sample were mixed with 200 n\ buffer and heated in a boiling water bath for 10 min in order to denature endogenous corticosterone-binding proteins. After cooling the samples to room temperature, 100 fA monkey serum were added, and the samples were allowed to stand for 30 min at room temperature. One hundred microliters of [3H]corticosterone (6500 cpm) were added, and incubation
1497
was continued for 90 min at 37 C. Dextran-coated charcoal (0.5 ml) was added, and the mixture was shaken in an ice-water bath for 10 min. The tubes were centrifuged, and the supernatant fraction was decanted into scintillation fluid and counted for 5 min. Maximum binding was approximately 33% of the total counts. Twenty percent displacement occurred at 7 ng/&\, 50% displacement occurred at 15 /ig/dl, and 80% displacement occurred at 27 Urinary electrolyte determinations Urine samples were centrifuged before assay. Sodium and potassium concentrations were measured on an ASTRA-8T (Beckman Instruments, Brea, CA) equipped with ion-selective electrodes. Statistical analysis Statistical analyses were made with Student's t test for paired and unpaired data to compare results in samples from drugand saline-treated, riboflavin-sufficient and riboflavin-deficient animals. P < 0.05 was considered significant. All results are expressed as the mean ± SD.
Results Figure 1 illustrates the effects of graded cumulative doses of adriamycin in riboflavin-sufficient and riboflavin-deficient animals. Since aldosterone levels from saline-treated pair-fed rats within each dietary group did not differ, values were combined and treated statistically as one group. The numerical values of aldosterone in saline-treated animals (SAL) shown in Fig. 1 are 296 ± 49 and 191 ± 33 pg/ml for the riboflavin-sufficient and riboflavin-deficient groups, respectively. The basal levels of serum aldosterone were measured in blood drawn via cardiac puncture and represent values that are 2-3 times higher than those determined by others in nonstressed Sprague-Dawley rats (27, 28). A dose of 6 mg/kg more than doubled serum aldosterone levels in riboflavin-sufficient rats, but was ineffective in rats fed a riboflavindeficient diet. In riboflavin-sufficient animals given 16 and 24 mg/kg adriamycin, serum aldosterone levels increased 8- and 17-fold, respectively, over those in salinetreated controls, while similar doses administered to riboflavin-deficient animals elevated serum aldosterone 3- and 9-fold over values in saline-treated controls. In riboflavin-deficient animals, significant elevations in aldosterone were not observed until a cumulative dose of 16 mg/kg BW was administered. As indicated in Table 1, treatment with adriamycin over the 3-day injection period did not significantly affect adrenal wet weights, total protein content, or intracellular potassium concentrations in either the riboflavinsufficient or riboflavin-deficient group. The results in Table 1 represent values obtained from the group of animals that had received 16 mg/kg adriamycin.
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RIBOFLAVIN SUFFICIENT DIET
2
RIBOFLAVIN DEFICIENT DIET
6 p
"
X
I O* 4-1-
o LU
Endo• 1990 Voll27-No3
ADRIAMYCIN-INDUCED INCREASE IN ALDOSTERONE
1498
2--
Vol. 127, No. 3 Printed in U.S.A.
Adriamycin-Induced Increase in Serum Aldosterone Levels: Effects in Riboflavin-Sufficient and Riboflavin-Deficient Rats* JOHN T. PINTO, BRADLEY N. DELMAN, PURABI DUTTA, AND JEROME NISSELBAUM Memorial Sloan-Kettering Cancer Center, Departments of Medicine and Clinical Chemistry, New York, New York 10021
ABSTRACT. Previous studies in rats have demonstrated that 1) aldosterone enhances biosynthesis of renal flavin coenzymes; 2) riboflavin analogs inhibit the synthesis of aldosterone; and 3) adriamycin inhibits flavin coenzyme biosynthesis. In their entirety, these findings suggest that both diminished flavin coenzyme biosynthesis induced by adriamycin and a dietary riboflavin deficiency would result in decreased formation of aldosterone. The present study examined the effects of adriamycin treatment on serum aldosterone in rats consuming either a diet adequate in riboflavin or a riboflavin-deficient diet. Groups of rats fed specially prepared diets were injected for 3 days with adriamycin (cumulative dose range, 6-24 mg/kg BW). Pair-fed controls were given saline. After death, adrenal glands were excised, and blood samples were analyzed for aldosterone levels. No changes in adrenal weights or protein and potassium concentrations were observed after adriamycin treatment. In contrast
I
NCREASING knowledge has accumulated concerning modulation of riboflavin metabolism by hormones (1-4) and drugs (5-8). To be physiologically active, riboflavin must first be phosphorylated to riboflavin-5'phosphate (FMN) by flavokinase (ATP:riboflavin-5'phosphotransferase; EC 2.7.1.26) and then be pyrophosphorylated to flavin adenine dinucleotide (FAD) by FAD synthetase (ATP:FMN adenylyltransferase; EC 2.7.7.2). Both FAD and FMN bind to a variety of flavoenzymes that participate in drugs (5, 6), steroid (9), fatty acid (10, 11), and overall energy metabolism (12). Previous investigations have recognized that pituitary and/or adrenal function may control activation of riboReceived January 29, 1990. Address requests for reprints to: John Thomas Pinto, Ph.D., Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 140, New York, New York 10021. * Preliminary findings were published in an abstract form in Clin Res 37:333, 1989 (Abstract). This work was supported by the Clinical Nutrition Research Unit, Grants CA-29502 and CA-08748 from the NIH, and grants from the NIA (5-K01-AG-00399), Hoffmann-LaRoche, Inc., and the Stella and Charles Guttman Foundation. The research was performed in the Nutrition Research Laboratory of SloanKettering Institute.
to initial predictions, in riboflavin-sufficient rats, serum aldosterone levels were markedly enhanced by adriamycin in a doserelated manner. Riboflavin-deficient animals had lower basal aldosterone levels and markedly attenuated responses to adriamycin than did riboflavin-sufficient rats. In separate groups of adriamycin-treated rats fed a normal chow diet, serum aldosterone levels increased, and serum corticosterone levels showed a small but significant decline. In addition, adriamycin treatment caused an increase in urinary potassium excretion and a decrease in sodium excretion. These results suggest that flavins play a decisive role in regulating the levels of aldosterone and raise the possibility that the adriamycin-induced increase in serum aldosterone may be part of the pathogenetic mechanisms of cardiovascular toxicity and overall muscular weakness. (Endocrinology 127: 1495-1501, 1990)
flavin and its subsequent involvement with the mitochondrial cytochrome P-450 reductase system and corticosteroidogenesis. Studies by Fazekas and Sandor (3, 9) have indicated that ACTH-treated rats exhibit enhanced formation of renal flavin nucleotides and that tissue-specific induction of flavokinase may explain this increase. Subsequent studies by Trachewsky et al. (4, 13), using enzyme-linked immunosorbent assay, revealed that aldosterone stimulates de novo renal biosynthesis of flavokinase and that spironolactone (mineralocorticoid type I receptor antagonist), actinomycin-D (RNA synthesis inhibitor), or cycloheximide (protein synthesis inhibitor) can abolish this stimulatory effect. These data support the concept that mineralocorticoids mediate renal flavin biosynthesis and that enhanced formation of flavin coenzymes by aldosterone may be associated with positive sodium balance. To extend the relationship between the formation of flavin coenzymes and aldosterone levels, Trachewsky and Kem (14-17) demonstrated that analogs of riboflavin substituted in the N-10 position of the isoalloxazine ring possess antihypertensive activity. Thus, evidence to date suggests that aldosterone 1495
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 06 July 2015. at 09:10 For personal use only. No other uses without permission. . All rights reserved.
ADRIAMYCIN-INDUCED INCREASE IN ALDOSTERONE
1496
enhances renal biosynthesis of FAD and that flavin coenzymes, directly or mediated through flavoenzymes, govern the formation of aldosterone. Investigations in this laboratory have focused upon the physiological consequences to riboflavin metabolism associated with the administration of tri- and tetracyclic drugs (7,18-20). Each of these compounds when used in clinically relevant doses in laboratory animals diminishes the formation of FAD. Recent investigations (21-23) with the antineoplastic agent adriamycin have demonstrated a dose-related and organ-selective decrease in FAD formation, particularly in cardiac and skeletal muscle. In addition, studies performed in vitro by Fisher et al. (24) showed that adriamycin can bind competitively to sites similar to those that bind flavin coenzymes. These drugs, which are recognized to have diverse pharmacological action, may derive a common cytotoxic efficacy by inhibiting FAD biosynthesis. These findings raise the possibility that clinically significant riboflavin deficiency may result from the use of these drugs despite a diet adequate in the vitamin. The present study was initially conducted to determine whether alterations in riboflavin metabolism produced by diet and/or drugs affect the level of serum aldosterone, a hormone that stimulates renal flavokinase activity. Our investigations examined the effects of graded doses of adriamycin on aldosterone levels in serum of riboflavin-sufficient and riboflavin-deficient rats. Contrary to our initial prediction, administration of adriamycin to riboflavin-sufficient animals resulted in a marked doserelated increase in serum aldosterone levels. In animals that are already deficient in riboflavin due to diminished dietary intake, adriamycin was considerably less effective in elevating serum aldosterone. Further experiments investigated whether adriamycin administered to normal chow-fed animals induced aldosterone formation through alteration of its precursor, corticosterone, and whether aldosterone-responsive excretion of electrolytes was affected by adriamycin. Materials and Methods Animals, diets, and drugs Three-week-old male Sprague-Dawley rats were purchased from Charles River Breeding Laboratories, Inc. (Wilmington, MA). All animals were randomly distributed and housed individually in wire-bottom metabolic cages in our animal facility with controlled temperature and 12-h lighting cycle. In all experiments, tap water was allowed ad libitum, and animals were handled at least twice daily during cage acclimation periods. For the first experiment, riboflavin deficiency was produced in groups of animals by feeding a specially prepared riboflavin-deficient diet (25). At intervals during a 7-week feeding and cage acclimation period, rats were bled from the suborbital sinus, and the erythrocyte glutathione reductase
Endo • 1990 Vol 127 • No 3
activity coefficient (EGRAC) was determined periodically to assess the degree of deficiency. Under these conditions, biochemical evidence of riboflavin deprivation is evident after 24 weeks on the deficient diet, i.e. significant elevation of EGRAC, while overt clinical signs may appear after 7-8 weeks. Riboflavin-sufficient animals were age matched and fed a diet identical in composition to the riboflavin-deficient diet, but with supplemental riboflavin (8.5 /ug/g diet) (25). This figure represents approximately 3 times the recommended dietary allowance for riboflavin for the rat. Both diets were prepared by Dyets, Inc. (Bethlehem, PA). In later experiments, animals were maintained on standard pelleted Purina rat chow (Ralston-Purina Co., St. Louis, MO), which had been determined in our laboratory to contain approximately 8.5 pig riboflavin/g diet. Adriamycin was purchased from Adria Laboratories (Columbus, OH) as a crystalline powder and reconstituted with saline before injection into animals. Glutathione reductase activity and activity coefficient The activities of glutathione reductase measured first in the absence of exogenous FAD (basal activity) and then again in the presence of FAD were determined using the method of Beutler (26). Activity coefficients were calculated by dividing the activity of glutathione reductase in the presence of FAD by the basal activity. We defined as riboflavin deficient those animals whose EGRAC was greater than 1.7. Treatment of animals In the first study animals were maintained on specially prepared diets for 7 weeks before treatment with adriamycin. To determine the effect of adriamycin on aldosterone formation in animals consuming either a diet adequate in riboflavin or a riboflavin-deficient diet, we performed the following experiment. Beginning on day 1, two groups each of 10-week-old riboflavin-sufficient and riboflavin-deficient rats were injected ip for 3 days with adriamycin, representing cumulative doses of 6, 16, and 24 mg/kg BW. A dose of 10 mg/kg in rats is equivalent to 60 mg/m2 in humans. Pair-fed controls for each drug-treated group of riboflavin-sufficient and riboflavin-deficient animals were given comparable volumes of saline. The number of rats used in each group is indicated in the figure legends. On day 4 of the study, 18 h after the last injection, animals were killed by exposing them to carbon dioxide, and blood samples were drawn via cardiac puncture for aldosterone determinations. Adrenal glands were excised, weighed, and analyzed for protein and potassium levels. In a second experiment to corroborate our findings in rats fed the specially prepared diet adequate in riboflavin, groups of animals were acclimated to metabolic cages for 10 days and allowed to feed ad libitum on a standard pelleted Purina rat chow diet. This study was performed 1) to determine whether the observed elevation in aldosterone was not a stress-induced manifestation of the specially prepared diet coupled with diminished food intake and 2) to investigate more closely the dose-related rise in aldosterone by including among the previous cumulative dose regimen of 6, 16, and 24 mg/kg intermediate doses of adriamycin at 8 and 18 mg/kg BW. All
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 06 July 2015. at 09:10 For personal use only. No other uses without permission. . All rights reserved.
ADRIAMYCIN-INDUCED INCREASE IN ALDOSTERONE injections were administered ip in divided doses over a 3-day injection period. On day 4 and 18 h after the last injection, animals were killed, and blood samples were analyzed for aldosterone. In a third study to determine the effects of adriamycin on corticosterone levels and aldosterone-responsive parameters, 10 Sprague-Dawley rats consuming a Purina rat chow diet were placed in metabolic cages and acclimated for 1 week. At the end of this period and before drug administration, all animals had blood samples taken from the suborbital sinus and analyzed for aldosterone and corticosterone levels; 24-h urine samples were collected and measured for pretreatment levels of sodium, potassium, and creatinine. The 24-h collection period for urine samples was from 1000 h to 1000 h the next day. After pretreatment collection of blood and urine specimens, on day 1 of study, five animals received saline, and five corresponding pairweighed rats received adriamycin (3 mg/kg twice daily for 3 days; cumulative dose, 18 mg/kg). Saline-treated rats received a similar volume of isotonic saline. On day 3 of the adriamycin injection period, a second 24-h urine collection was begun. On day 4, after urine samples were collected, animals were killed, and blood samples were drawn via cardiac puncture for determinations of aldosterone and corticosterone. Aldosterone RIA Serum aldosterone concentrations were measured using a standard RIA kit (Coat-A-Count no extraction RIA) purchased from Diagnostic Products Corp. (Los Angeles, CA). The average interassay coefficient of variance was 10%. Aliquots of serum were transferred to incubation tubes that were coated with antiserum, and 1.0 ml [125I]aldosterone was added. After 3 h at 37 C the tubes were decanted, and bound [125I]aldosterone was measured by counting for 1 min in a 7-counter. A logit/log graphic analysis was used to linearize the standard curve over a range of 25-1200 pg/ml. Past experience with adriamycintreated animals indicates a need to dilute serum from these rats 1:10 or 1:20 before analysis for aldosterone. Corticosterone [l,2- 3 H]Corticosterone (Amersham Corp., Arlington Heights, IL) was purified by TLC on silica gel. The band that migrated with authentic corticosterone was scraped from the plate, eluted with acetone, concentrated to dryness, and redissolved in a small volume of ethanol. Stock standard was dissolved in absolute ethanol and diluted in 0.05 M Tris-HCl, pH 8.0, buffer containing 0.1 M NaCl and 0.1% BSA before use. A competitive binding assay was performed using 100 n\ monkey serum as the binder. Standards (2-50 Mg/dl) were prepared in 0.05 M Tris-HCl, pH 8.0, buffer containing 0.1 M NaCl and 0.1% BSA. The following protocol was developed for use in our laboratory. One hundred microliters of unknown sample were mixed with 200 n\ buffer and heated in a boiling water bath for 10 min in order to denature endogenous corticosterone-binding proteins. After cooling the samples to room temperature, 100 fA monkey serum were added, and the samples were allowed to stand for 30 min at room temperature. One hundred microliters of [3H]corticosterone (6500 cpm) were added, and incubation
1497
was continued for 90 min at 37 C. Dextran-coated charcoal (0.5 ml) was added, and the mixture was shaken in an ice-water bath for 10 min. The tubes were centrifuged, and the supernatant fraction was decanted into scintillation fluid and counted for 5 min. Maximum binding was approximately 33% of the total counts. Twenty percent displacement occurred at 7 ng/&\, 50% displacement occurred at 15 /ig/dl, and 80% displacement occurred at 27 Urinary electrolyte determinations Urine samples were centrifuged before assay. Sodium and potassium concentrations were measured on an ASTRA-8T (Beckman Instruments, Brea, CA) equipped with ion-selective electrodes. Statistical analysis Statistical analyses were made with Student's t test for paired and unpaired data to compare results in samples from drugand saline-treated, riboflavin-sufficient and riboflavin-deficient animals. P < 0.05 was considered significant. All results are expressed as the mean ± SD.
Results Figure 1 illustrates the effects of graded cumulative doses of adriamycin in riboflavin-sufficient and riboflavin-deficient animals. Since aldosterone levels from saline-treated pair-fed rats within each dietary group did not differ, values were combined and treated statistically as one group. The numerical values of aldosterone in saline-treated animals (SAL) shown in Fig. 1 are 296 ± 49 and 191 ± 33 pg/ml for the riboflavin-sufficient and riboflavin-deficient groups, respectively. The basal levels of serum aldosterone were measured in blood drawn via cardiac puncture and represent values that are 2-3 times higher than those determined by others in nonstressed Sprague-Dawley rats (27, 28). A dose of 6 mg/kg more than doubled serum aldosterone levels in riboflavin-sufficient rats, but was ineffective in rats fed a riboflavindeficient diet. In riboflavin-sufficient animals given 16 and 24 mg/kg adriamycin, serum aldosterone levels increased 8- and 17-fold, respectively, over those in salinetreated controls, while similar doses administered to riboflavin-deficient animals elevated serum aldosterone 3- and 9-fold over values in saline-treated controls. In riboflavin-deficient animals, significant elevations in aldosterone were not observed until a cumulative dose of 16 mg/kg BW was administered. As indicated in Table 1, treatment with adriamycin over the 3-day injection period did not significantly affect adrenal wet weights, total protein content, or intracellular potassium concentrations in either the riboflavinsufficient or riboflavin-deficient group. The results in Table 1 represent values obtained from the group of animals that had received 16 mg/kg adriamycin.
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RIBOFLAVIN SUFFICIENT DIET
2
RIBOFLAVIN DEFICIENT DIET
6 p
"
X
I O* 4-1-
o LU
Endo• 1990 Voll27-No3
ADRIAMYCIN-INDUCED INCREASE IN ALDOSTERONE
1498
2--
