I-OXICOLOGY
AND
Effect
APPLIED
PHARMACOLOGY
of Spironolactone
33,366-375
on the
(1975)
Distribution
of Mercury’
CURTIS D. KLAASSEN~ Clinical Pharmacology and Toxicology Center, Department of Pharmacology, University of Kansas Medical Center, Kansas City, Kansas 66103 Received January 7,1975; accepted March 26,1975
Effect of Spironolactone on the Distribution of Mercury. KLAASSEN, 33,366-375. The distribution and biliary excretion of 203HgC12 (0.3 mg Hg/kg) iv was measuredin rats treated with spironolactone(SP, 75 mg/kg, ip) for various time intervals. SPhad its greatesteffect whenadministeredasa singledose15min before HgC12.SPdecreasedthe concentration of 203Hgin the plasmafrom 1.5 to 0.05 fig/ml, while it increasedthe blood concentration from 1.5 to 5 pg/ml. This treatment increasedthe content of Hg in the lung 12,heart 6, spleen3, brain 3, muscle2, stomach1.7, and liver 1.5 timescontrol, had no effect on the concentrationof 203Hgin the intestine,bone, and testes,and markedly decreasedthe amount in the kidney to 10% of controls. Biliary excretion of Hg wasnot increased.When SPwasadministered90 min or 3 hr before administration of the 203HgC12,qualitatively similar but lessdramatic effects on the distribution of Hg were obtained. SP administered15min after HgC12administration had a similar effect on the distribution of Hg as when administered30 min before Hg&, with the exception that the concentrationof Hg in the kidney wasnot decreased.The two major metabolic products of SP, canrenoneand thioacetic acid, were also given to determinetheir effect on Hg distribution. Canrenonehad no effect while thioacetic acidproducedan effect similarto that producedby SP.It appears that the alteration in the distribution of Hg after SPtreatment is dueto the sulfur portion of the molecule.It seemslikely that the sulfur moiety complexesthe Hg; this complex distributesin the body similar to organic mercurial compounds,which in comparisonto inorganic mercurials,reach a lower concentration in the plasmaand kidney and a higher concentration in the blood and other tissues.The decreasein the concentration of Hg in the kidney producedby SPisprobably responsiblefor the decreased toxicity of Hg after SPtreatment. C. D. (1975).Toxicol. Appl. Pharmacol.
Pretreatment with spironolactone
has been shown to protect rats against a variety of chemicals including digitoxin (Selye et al., 1969; Buck and Lage, 1971; Klaassen, 1974), indomethacin (Selye, 1969; Solymoss et al., 1971), and mercury (Selye, 1970). Spironolactone increases the microsomal drug metabolizing enzymes in the liver (Feller and Gerald, 1971; Hamreck et al., 1973), and this property is thought to be responsible for decreasing digitoxin (Solymoss et al., 1971; Castle and Lage, 1972, 1973a, b, c) and indomethacin toxicity (Solymoss et al., 1971); however, this effect on the microsomal enzymes is probably not responsible for decreasing the toxicity of mercury. 1The studywassupportedby fundsfrom PublicHealthServiceGrant GM 15956. z The authoris a recipientof a PublicHealthServiceCareerDevelopment Award GM 30996.
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any from reserved. Printed in Great Britain
366
SPIRONOLACTONE
AND
Hg
367
Selye (1970) demonstrated that spironolactone pretreatment for 4 days, twice daily. protects rats from renal damage and death which would otherwise result from iv administration of HgCl,, but was of no value when given after HgCl,. Garg et al. (1971) demonstrated that spironolactone when given 1 hr before HgCl, was also effective in preventing the kidney lesions. This was accompanied by a lower concentration of Hg in tissues of the rat and a higher amount in the feces. Haddow and Marshall (1972) also observed an increased excretion of Hg into the stool after spironolactone treatment. In a further study Haddow et al. (1972) reported an increase in the RBC content of Hg and an increase in the biliary excretion of Hg when spironolactone was given before the HgCl, and suggested that the latter was the mechanism by which spironolactone protects against mercury poisoning. However, Garg et a/. (1971) did not observe an increase in biliary excretion of Hg after spironolactone pretreatment. Therefore, the purpose of the present investigation is to examine the biliary excretion and distribution of Hg after spironolactone pretreatment in an attempt to determine the mechanism by which spironolactone protects against mercury toxicity. METHODS
Animals and Drugs
Thorp Sprague-Dawley male rats (150-250 g) were used throughout. Mercuric chloride and methyl mercuric chloride were obtained from Fisher Scientific Co. (Pittsburgh, Penn.) and K and K Laboratories (Plainview, NY), respectively. *03Mercurie chloride and [203Hg] methyl mercuric chloride were obtained from New England Nuclear (Boston, Mass.). Spironolactone, canrenone, and [3H]spironolactone were graciously supplied by Searle and Company (Chicago, Ill.). Thioacetic acid was purchased from Sigma Chemical Co. (St. Louis, MO.). Spironolactone (75 mg/kg) was suspended in propylene glycol and administered ip in a volume of 5 ml/kg. Equimolar doses of canrenone (61 mg/kg) and thioacetic acid (13.7 mg/kg) were prepared similarly. Distribution of Mercury
The rats were anesthetized with urethane (800 mg/kg, ip). The femoral vein and artery were cannulated with polyethylene tubing (PE-50) for administering the mercury compounds and sampling the blood, respectively. The bile duct was exposed by a midline abdominal incision and cannulated with PE-IO tubing. Alteration in biliary flow and excretion due to hypothermia (Roberts et al., 1967) was prevented by maintaining the rectal temperature at 37°C with a heat lamp-temperature regulator device. The rats were injected with spironolactone once either 15 min, 90 min, or 3 hr before or 15 min after the iv administration of 203HgC1, (0.3 mg Hg/kg). Bile was collected during timed intervals thereafter and measured gravimetrically. Blood was collected into tubes containing heparin. 203Hg contents was determined in the entire bile sample while aliquots of blood and plasma were used. The concentration of ‘03Hg in the various samples was determined in a Packard Auto-Gamma spectrometer (La Grange, Ill.). The concentration of 203Hg was measured in the kidney, liver, heart, muscle, lung, spleen, bone, testes, and brain of the rat. For most of these tissues the entire organ was 13
368
CURTIS
D. KLAASSEN
used. The tibia was used as a representative bone and the soleus and gastrocnemius as muscle. The wet tissue weight of each sample was used in the calculation of Hg content. These tissues were removed from the rats 90 min after administration of zo3Hg. The experimental protocol described above was used throughout, but minor modifications were made in some of the experiments. Canrenone (61 mg/kg) or thioacetic acid (13.7 mg/kg) was substituted for spironolactone, [ 203Hg]methyl mercuric chloride was substituted for 203mercuric chloride, and rats were killed at other time intervals. Plasma Concentration of [3H]Spironolactone {20-3H}Spironolactone was administered ip in propylene glycol(5 ml/kg) and blood samples were collected into tubes containing heparin at timed intervals thereafter. The amount of 3H in the plasma was determined by the addition of 50-100 ~1 of plasma to 10 ml of Aquasol (New England Nuclear, Boston, Mass.) scintillation medium. Radioactivity was estimated with a liquid scintillation spectrometer (model 3320, Packard Industries, La Grange, Ill.). The amount of quenching was determined by automatic external standardization. Statistics The means of the treated groups were compared to the control mean by Students’ t test (Steele and Torrie, 1960). RESULTS
The effect of spironolactone (75 mg/kg), when administered ip at various times before and after the administration of 203HgC12, on the plasma and blood concentration of 203Hg is shown in Fig. 1. When administered 15 min before the HgCl,, the concentration of Hg in the blood was five times higher than that in the control animals. When given 90 min or 3 hr before HgC12, qualitatively similar but quantitatively less pronounced results were observed as when spironolactone was administered 15 min before HgCl,. When spironolactone was given 15 min after the HgCl,, only a small increase in the blood concentration of Hg was observed. Spironolactone produced an even greater effect on the concentration of Hg in the plasma than in the blood. When spironolactone was administered 15 min before the HgCl,, the plasma concentration was decreased to less than 5 % of that seen in control rats. Spironolactone was less effective when administered earlier. When spironolactone was given 15 min after the HgCl,, a marked decrease in the plasma Hg concentration was observed, similar to that observed when the spironolactone was given 15 min before H&l,. The effect of spironolactone on the biliary excretion of Hg is shown in Table 1. While the mean excretion of Hg was increased at all time intervals after spironolactone administration, this increase was significant only when spironolactone was administered 15 min after HgCl,. The effect of spironolactone, administered at various time intervals, on the concentration of Hg in the various tissues is shown in Fig. 2. The administration of spironolactone 15 min before HgCl, produced a decrease in the concentration of Hg in the plasma and kidney whereas it produced an increased concentration of Hg in most of the other tissues, especially in the blood, brain, heart, lung, and spleen. Qualitatively similar
SPIRONOLACTONE
AND
369
Hg
II-HIN
SP PRE-TREATMENT
SO-HIN
SP PRE-TREATMENT
3-M SP PRE-TREATMENT IS-MIN SP POST-TREATMENT CONTROL
I,
5
,
I
I5
30
I
I
45
60
90
MINUTES
FIG. I. Concentration of ‘03Hg in the blood and plasma at various times after the iv administration of 203HgC12 (0.3 mg Hg/kg). Rats were given spironolactone ip (75 mg/kg) 3 hr, 90 min, or 15 min before the HgCl, or 15 min after the HgCl,. Each value represents the mean +SE of five to six rats. TABLE EFFECT
1
OF SPIRONOLACTONE ON THE BILIARY OF MERCURY IN RATS”
Spironolactone
treatment
Control Pre-15 min Pre-90 min Pre-3 hr Post-15 min
EXCRETION
Biliary excretion 1.26 2.98 5.01 3.09 5.08
& -t * 5 +
(,ug,/kg)
0.31” 1.40 2.20 1.14 1.48”
’ 203HgCl, (0.3 mg Hg/kg) was administered iv and bile collected for 90 min. b Each value represents the mean +SE of five rats. ’ Significantly different from controls (p < 0.05).
results were obtained when spironolactone was given 90 min or 3 hr before the HgC12, but quantitatively the changes were less pronounced. When spironolactone was administered 15 min after HgCI,, it produced an increased concentration of Hg in the blood, brain, heart, lung, and spleen and decreasedthe concentration of Hg in the plasma similarly as when the spironolactone was administered 15 min before the HgCI, : however, it did not produce a decreasein the amount of Hg in the kidney.
370
CURTISD. KLAASSEN
CONTROL 3-M SP PRE-TREATMENT 90-MIN SP PRE-TREATMENT I5-MIN SP PRE-TREATMENT 15-MIN SP POST-TREATMENT
eTm U C?B B e
FIG 2. Concentration of ‘03Hg in various tissues 90 min after the iv administration of 203HgC13 (0.3 ig Hg/kg). Rats were given spironolactone ip (75 mg/kg) 3 hr, 90 min, or 15 min before the HgC12. Each value represents the mean *SE of five to six rats.
CONTROL
9-
IS-MN I 5
1 15
I 30 MINUTES6
1
SP PRE-TREATMENT
1 90
FIG 3 Concentration of 203Hg in the kidneys of control rats and rats pretreated 15 min before the 203Hg&; (iv, 0.3 mg Hg/kg) with spironolactone (ip, 75 mg/kg). Each value represents the mean _+SE of five to seven rats.
SPIRONOLACTONE
AND
Hg
371
The time course of the concentration of Hg in the kidney following the administration of HgCI, to control rats and rats pretreated with spironolactone 1.5min before the HgClz is shown in Fig. 3. The concentration of Hgin the kidneys of the rats that were pretreated with spironolactone 15 min earlier was lower at all time intervals after Hg administration, and at the 90-min time interval was about lo:!;; of controls. When the rats were
25
I
I 15
1 30
I 60
I 90 MINUTES
I 120
, 150
I I 160
FIG. 4. Concentration of spironolactone and its metabolites in the plasma at various times after the ip administration ofi3H} spironolactone(75 mg/kg). Each valuerepresents the mean k SE of four rats,
given the spironolactone 15 min after HgCl, administration, the concentration of Hg in the kidney was not significantly different from controls and thus are not depicted in the graph. The concentration of Hg in the liver of rats pretreated with spironolactone was 1.9 times higher than in controls at 5 min, 3.8 at I5 min, 2.1 at 30 min, 2.2 at 60 min. and 1.4 at 90 min. The amount of radioactivity in plasma following intraperitoneal administration of {3H)spironolactone is shown in Fig. 4. The concentration of 3H in the plasma increased rapidly during the first 15 min and remained relatively constant for the next 3 hr.
SPIRONOLACTONE 0 0? ---
o’/a!?
CANRENONE FIG.
5. Chemical structure of spironolactone and canrenone.
372
CURTIS
D. KLAASSEN
Since spironolactone is known to be rapidly metabolized to canrenone and thioacetic acid (Sadee et al., 1972), the effect of these two biotransformation products (Fig. 5)
CANRENONE CONTROL THIOACETATE
0.01
I 5
15
r
30
45 60 MINUTES
1 so
FIG. 6. Effect of ip administration of spironolactone (75 mg/kg), canrenone (61 mg/kg), or thioacetic acid (13.7 mg/kg) given 15 min before 203HgC12 (0.3 mg Hg/kg) on the concentration of ‘03Hg in the blood and plasma. Each value represents the mean kSE of three to seven rats.
zo- -S-I‘b---
1
i---i----I
C BLOOD
101 3
4 b
-------I
SP BLOOD
50-
C PLASMA SP PLASMA 7
5
I
15
30
45
60
90
MINUTES FIG . 7. Concentration of ‘03Hg in the plasma and blood in control rats and rats given spironolactone 15 min before (75 mg/kg) *‘%H3Hg (1 .Omg Hg/kg). Each value represents the mean *SE of six rats.
on the distribution of Hg was examined (Fig. 6). Equimolar doses of spironolactone, canrenone, or thioacetic acid were given to the rats and 1.5min later the 203HgCl, was administered. Pretreatment with the canrenone had no effect on the distribution of Hg.
SPIRONOLACTONE
AND
Hg
373
However, both spironolactone and thioacetic acid increased the concentration of Hg in blood and decreased the concentration in plasma. The changes in the amounts of Hg in the various tissues were qualitatively similar for thioacetic acid and spironolactone, although the thioacetic acid had a less pronounced effect. The effect of spironolactone pretreatment on the concentration of Hg in the blood and plasma following the administration of methyl mercury is shown in Fig. 7. Pretreatment with spironolactone 15 min before methyl mercury produced a slight decrease in the blood and plasma Hg values. This is very different from the effect of spironolactone on inorganic mercury which was increased in the blood and markedly decreased in the plasma. When spironolactone was given 15 min following methyl mercury, the effect in decreasing the blood and plasma concentrations was greater than when given before the methyl mercury. DISCUSSION The nephrotoxicity of mercuric chloride is well known, although prior treatment with spironolactone will reduce the ability of Hg to produce renal tubular necrosis and death (Selye, 1970). The mechanism by which spironolactone protects against mercuric chloride injury does not appear to be related to its ability to induce microsomal enzymes. Spironolactone is effective when it is given as a single dose 1 or 2 hr before mercuric chloride administration (Garg et al., 1971, Haddow et al., 1972), a time which is insufficient for the microsomal enzymes to become induced. Spironolactone appears to protect against the renal injury produced by mercuric chloride by decreasing the amount of Hg in the kidney. In the present study it was shown that pretreatment with spironolactone prevented the accumulation of Hg in the kidney, being more effective when given 15 min rather than 90 or 180 min before the Hg When given 15 min after the mercuric chloride, spironolactone was ineffective in lowering the concentration of Hg in the kidney. Spironolactone also does not protect against the renal injury and death when given after mercuric chloride (Selye, 1970). Spironolactone alters the distribution of Hg not only in the kidney but in other tissues as well. It markedly decreases the concentration of Hg in the plasma and kidney and increases it in many of the other tissues such as the blood, heart, liver, spleen, and brain. Spironolactone again was more effective in altering the distribution of Hg in these tissues when administered shortly before the Hg. Whereas spironolactone did not alter the concentration of Hg in the kidney when it was given 15 min after the mercuric chloride, it did alter the distribution of Hg in other tissues. Therefore, it appears that once the Hg is bound to the kidney, it cannot be removed by spironolactone. It has been suggested earlier that spironolactone protects against the toxic actions of Hg by increasing its excretion into bile (Haddow and Marshall, 1972; Haddow et al., 1972). In another study spironolactone did not increase the biliary excretion of Hg (Garg et al., 1971). In the present study spironolactone increased biliary excretion of Hg, but this increase was statistically significant only when the spironolactone was given after the Hg. Spironolactone administration at this time does not decrease the concentration of Hg in the kidney, alter the extent of kidney injury, or the incidence of death. Also, in the pretreated rats, a marked decrease in the content of Hg was found in the kidneys 5 min after mercuric chloride administration, a time by which very little
374
CURTISD. KLAASSEN
Hg hasbeen excreted into the bile. Thus it appearsthat spironolactone can increasethe biliary excretion of Hg, but this is probably the result of the alteration in the distribution of Hg in the body rather than the mechanism which protects against the toxic effects of Hg. The mechanismby which spironolactone alters the distribution of Hg doesnot appear to be related to the steroid moiety of the molecule. The concentration of 3H in the plasma after administration of [3H]spironolactone wasfound to be relatively constant between 15 min and 3 hr after intraperitoneal administration while spironolactone had a much greater effect on the distribution of Hg when given 15 min before spironolactone than when given 3 hr before. Since spironolactone is metabolized very rapidly to canrenone and thioacetic acid (Sadee et al., 1972), the effect of equimolar amounts of these two portions of the spironolactone molecule on the distribution of Hg were measured.The steroid portion of the molecule (canrenone) had no effect on the distribution of Hg whereas thioacetic acid produced qualitatively similar changes to those produced by spironolactone, but quantitatively thesechangeswere somewhat less.This quantitative difference is probably due to differences in the absorption, distribution and excretion rates of thioacetic acid when given alone compared to when given complexed to the steroid. It seemslikely that the mechanismby which spironolactone protects against Hg toxicity involves the sulfur portion of the molecule. Selye (1970) stated that two other thioacetylated steroids, emdabol and spiroxasone, also inhibit the renal toxicity of Hg. The thioacetate probably forms a ligand with Hg, which thus converts the inorganic mercury into an organic mercury. The Hg then distributes like an organic mercury, which is characterized by a high blood concentration, a very low plasma concentration and relatively similar concentrations in the other tissues(Klaassen, 1975). After the organic complex is formed in the body, a decreasein the amount of Hg in the kidney and the plasma, and an increase in many of the other tissuesoccurs as a result of the change in the form of the Hg. This decreasein the concentration of Hg in the kidney appears to be the primary reason why the kidney lesion is reduced. Similar results would probably be observed with other sulphydryl containing compounds. ACKNOWLEDGMENTS The author would like to thank the very able technical assistanceof Roxanne Barfneckt and CelesteSoutee.The author would also like to thank Searleand Company for spironolactone, [‘Hlspironolactone and canrenone. REFERENCES BUCK, S. H. AND LAGE, 0. L. (1971).Possiblemechanism of theprevention of digitoxin toxicity by spironolactonein the mouse.Arch. Int. Pharmacodyn. 189, 192-197. CASTLE, M. C. AND LAGE, G. L. (1972).Effect of pretreatment with spironolactone,pheno-
barbital or j%diethylaminoethyldiphenylpropyl acetate(SKF 525-A) on tritium levels in blood, heart, and liver of rats at various timesafter administrationof (3H)-digitoxin. Biothem. Pharmacol. 21,1449-1455. CASTLE, M. C. AND LACE, G. L. (1973a).Enhancedbiliary excretion of digitoxin following spironolactoneas it relatesto the prevention of digitoxin toxicity. Res. Commun. Chem. Pathol. Pharmacol. 5.99-108.
SPIRONOLACTONE AND Hg
375
CASTLE, M. C. AND LAGE, G. L. (1973b). Excretion of H3-digitoxin following spironolactone pretreatmentin rats. Drug Metab. Disp. 1, 590-597. CASTLE, M. C. ANDLACE,G. L. (1973~). Biliary excretion and hepaticconcentrationsof H3digitoxin and its metabolitesfollowing spironolactonepretreatment of rats. Res. Commun. Chem. Pathol. Pharmacol. 6, 601-612. FELLER, D. R. ANDGERALD, M. C. (1971).Interactions of spironolactonewith hepatic microsomaldrug metabolizingenzymesystems.Biochem. Pharmacol. 20, 1991-2000. GARG,B. D., S~LYMOSS, B. AND TUCHWEBER, B. (1971).Effect of spironolactoneon the distribution and excretion of ‘03HgC12in the rat. Ar~neimittleforschung 21, 815-816. HADDOW,J. E., FISH,C. A., MARSHALL,P. C. ANDLESTER, R. (1972). Biliary excretion of mercury enhancedby spironolactone.Gastroenterology 63, 1053-1058. HADDOW, J. E. ANDMARSHALL, P. (1972). Increasedstool mercury excretion in the rat: the effect of spironolactone.Proc. Sot. Exp. Biol. Med. 140, 707-709. HAMRECK, M. E., ZAMPAGLIONE, N. C., STRIPP, B. ANDGILLETTE, J. R. (1973)Investigation of the effectsof methyltestosterone,cortisoneand spironolactoneon the hepatic microsomal mixed function oxidasesystemin maleand femalerats. Biochem. Pharmacol. 22, 293-3 10. KLAASSEN, C. D. (1974).Effect of microsomalenzyme inducerson the biliary excretion of cardiacglycosides.J. Pharmaeof. Exp. Ther. 191,201-211. KLAASSEN, C. D. (1975).Biliary excretion of mercury compounds.Toxicol. Appl. Pharmacoi. 33, 352-361.
ROBERTS, R. J., KLAASSEN, C. D. AND PLAA,G. L. (1967).Maximum biliary excretion of bilirubin and sulfobromophthaleinduring anasthesia-induced alteration of rectal temperature. Proc. Sot. Exp. Biol. Med. 125, 313-316.
SADEE,W., RIEGELMAN, S.ANDJONES, S. C. (1972).Plasmalevelsof spironolactonein the dog. J. Pharm. Sci. 61, 1129-l 131.
SELYE,H. (1969).Prevention of indomethacin-inducedintestinal ulcersby spironolactoneand norbolethone.Can. J. Physiol. Pharmacol. 47, 981-983. SELYE,H. (1970).Mercury poisoning:prevention by spironolactoneScience 169, 775-776. SELYE,H., KRAJNEY,M. AND SAVOIE, L. (1969). Digitoxin poisoning: prevention by spironolactone.Science 164, 842-843. SOLYMOSS, B., TOTH,S., VARGA, S. AND SELYE, H. (1971).Protection by spironolactoneand oxandroloneagainstchronic digitoxin or indomethacinintoxication. Tosicol. Appl. Phmmmol. X3,586-592.
STEELE, R. G. ANDTORRIE,J. H. (1960.)Principles andProcedures New York.
of Stati,sFtics. McGraw-Hill,
AND
Effect
APPLIED
PHARMACOLOGY
of Spironolactone
33,366-375
on the
(1975)
Distribution
of Mercury’
CURTIS D. KLAASSEN~ Clinical Pharmacology and Toxicology Center, Department of Pharmacology, University of Kansas Medical Center, Kansas City, Kansas 66103 Received January 7,1975; accepted March 26,1975
Effect of Spironolactone on the Distribution of Mercury. KLAASSEN, 33,366-375. The distribution and biliary excretion of 203HgC12 (0.3 mg Hg/kg) iv was measuredin rats treated with spironolactone(SP, 75 mg/kg, ip) for various time intervals. SPhad its greatesteffect whenadministeredasa singledose15min before HgC12.SPdecreasedthe concentration of 203Hgin the plasmafrom 1.5 to 0.05 fig/ml, while it increasedthe blood concentration from 1.5 to 5 pg/ml. This treatment increasedthe content of Hg in the lung 12,heart 6, spleen3, brain 3, muscle2, stomach1.7, and liver 1.5 timescontrol, had no effect on the concentrationof 203Hgin the intestine,bone, and testes,and markedly decreasedthe amount in the kidney to 10% of controls. Biliary excretion of Hg wasnot increased.When SPwasadministered90 min or 3 hr before administration of the 203HgC12,qualitatively similar but lessdramatic effects on the distribution of Hg were obtained. SP administered15min after HgC12administration had a similar effect on the distribution of Hg as when administered30 min before Hg&, with the exception that the concentrationof Hg in the kidney wasnot decreased.The two major metabolic products of SP, canrenoneand thioacetic acid, were also given to determinetheir effect on Hg distribution. Canrenonehad no effect while thioacetic acidproducedan effect similarto that producedby SP.It appears that the alteration in the distribution of Hg after SPtreatment is dueto the sulfur portion of the molecule.It seemslikely that the sulfur moiety complexesthe Hg; this complex distributesin the body similar to organic mercurial compounds,which in comparisonto inorganic mercurials,reach a lower concentration in the plasmaand kidney and a higher concentration in the blood and other tissues.The decreasein the concentration of Hg in the kidney producedby SPisprobably responsiblefor the decreased toxicity of Hg after SPtreatment. C. D. (1975).Toxicol. Appl. Pharmacol.
Pretreatment with spironolactone
has been shown to protect rats against a variety of chemicals including digitoxin (Selye et al., 1969; Buck and Lage, 1971; Klaassen, 1974), indomethacin (Selye, 1969; Solymoss et al., 1971), and mercury (Selye, 1970). Spironolactone increases the microsomal drug metabolizing enzymes in the liver (Feller and Gerald, 1971; Hamreck et al., 1973), and this property is thought to be responsible for decreasing digitoxin (Solymoss et al., 1971; Castle and Lage, 1972, 1973a, b, c) and indomethacin toxicity (Solymoss et al., 1971); however, this effect on the microsomal enzymes is probably not responsible for decreasing the toxicity of mercury. 1The studywassupportedby fundsfrom PublicHealthServiceGrant GM 15956. z The authoris a recipientof a PublicHealthServiceCareerDevelopment Award GM 30996.
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any from reserved. Printed in Great Britain
366
SPIRONOLACTONE
AND
Hg
367
Selye (1970) demonstrated that spironolactone pretreatment for 4 days, twice daily. protects rats from renal damage and death which would otherwise result from iv administration of HgCl,, but was of no value when given after HgCl,. Garg et al. (1971) demonstrated that spironolactone when given 1 hr before HgCl, was also effective in preventing the kidney lesions. This was accompanied by a lower concentration of Hg in tissues of the rat and a higher amount in the feces. Haddow and Marshall (1972) also observed an increased excretion of Hg into the stool after spironolactone treatment. In a further study Haddow et al. (1972) reported an increase in the RBC content of Hg and an increase in the biliary excretion of Hg when spironolactone was given before the HgCl, and suggested that the latter was the mechanism by which spironolactone protects against mercury poisoning. However, Garg et a/. (1971) did not observe an increase in biliary excretion of Hg after spironolactone pretreatment. Therefore, the purpose of the present investigation is to examine the biliary excretion and distribution of Hg after spironolactone pretreatment in an attempt to determine the mechanism by which spironolactone protects against mercury toxicity. METHODS
Animals and Drugs
Thorp Sprague-Dawley male rats (150-250 g) were used throughout. Mercuric chloride and methyl mercuric chloride were obtained from Fisher Scientific Co. (Pittsburgh, Penn.) and K and K Laboratories (Plainview, NY), respectively. *03Mercurie chloride and [203Hg] methyl mercuric chloride were obtained from New England Nuclear (Boston, Mass.). Spironolactone, canrenone, and [3H]spironolactone were graciously supplied by Searle and Company (Chicago, Ill.). Thioacetic acid was purchased from Sigma Chemical Co. (St. Louis, MO.). Spironolactone (75 mg/kg) was suspended in propylene glycol and administered ip in a volume of 5 ml/kg. Equimolar doses of canrenone (61 mg/kg) and thioacetic acid (13.7 mg/kg) were prepared similarly. Distribution of Mercury
The rats were anesthetized with urethane (800 mg/kg, ip). The femoral vein and artery were cannulated with polyethylene tubing (PE-50) for administering the mercury compounds and sampling the blood, respectively. The bile duct was exposed by a midline abdominal incision and cannulated with PE-IO tubing. Alteration in biliary flow and excretion due to hypothermia (Roberts et al., 1967) was prevented by maintaining the rectal temperature at 37°C with a heat lamp-temperature regulator device. The rats were injected with spironolactone once either 15 min, 90 min, or 3 hr before or 15 min after the iv administration of 203HgC1, (0.3 mg Hg/kg). Bile was collected during timed intervals thereafter and measured gravimetrically. Blood was collected into tubes containing heparin. 203Hg contents was determined in the entire bile sample while aliquots of blood and plasma were used. The concentration of ‘03Hg in the various samples was determined in a Packard Auto-Gamma spectrometer (La Grange, Ill.). The concentration of 203Hg was measured in the kidney, liver, heart, muscle, lung, spleen, bone, testes, and brain of the rat. For most of these tissues the entire organ was 13
368
CURTIS
D. KLAASSEN
used. The tibia was used as a representative bone and the soleus and gastrocnemius as muscle. The wet tissue weight of each sample was used in the calculation of Hg content. These tissues were removed from the rats 90 min after administration of zo3Hg. The experimental protocol described above was used throughout, but minor modifications were made in some of the experiments. Canrenone (61 mg/kg) or thioacetic acid (13.7 mg/kg) was substituted for spironolactone, [ 203Hg]methyl mercuric chloride was substituted for 203mercuric chloride, and rats were killed at other time intervals. Plasma Concentration of [3H]Spironolactone {20-3H}Spironolactone was administered ip in propylene glycol(5 ml/kg) and blood samples were collected into tubes containing heparin at timed intervals thereafter. The amount of 3H in the plasma was determined by the addition of 50-100 ~1 of plasma to 10 ml of Aquasol (New England Nuclear, Boston, Mass.) scintillation medium. Radioactivity was estimated with a liquid scintillation spectrometer (model 3320, Packard Industries, La Grange, Ill.). The amount of quenching was determined by automatic external standardization. Statistics The means of the treated groups were compared to the control mean by Students’ t test (Steele and Torrie, 1960). RESULTS
The effect of spironolactone (75 mg/kg), when administered ip at various times before and after the administration of 203HgC12, on the plasma and blood concentration of 203Hg is shown in Fig. 1. When administered 15 min before the HgCl,, the concentration of Hg in the blood was five times higher than that in the control animals. When given 90 min or 3 hr before HgC12, qualitatively similar but quantitatively less pronounced results were observed as when spironolactone was administered 15 min before HgCl,. When spironolactone was given 15 min after the HgCl,, only a small increase in the blood concentration of Hg was observed. Spironolactone produced an even greater effect on the concentration of Hg in the plasma than in the blood. When spironolactone was administered 15 min before the HgCl,, the plasma concentration was decreased to less than 5 % of that seen in control rats. Spironolactone was less effective when administered earlier. When spironolactone was given 15 min after the HgCl,, a marked decrease in the plasma Hg concentration was observed, similar to that observed when the spironolactone was given 15 min before H&l,. The effect of spironolactone on the biliary excretion of Hg is shown in Table 1. While the mean excretion of Hg was increased at all time intervals after spironolactone administration, this increase was significant only when spironolactone was administered 15 min after HgCl,. The effect of spironolactone, administered at various time intervals, on the concentration of Hg in the various tissues is shown in Fig. 2. The administration of spironolactone 15 min before HgCl, produced a decrease in the concentration of Hg in the plasma and kidney whereas it produced an increased concentration of Hg in most of the other tissues, especially in the blood, brain, heart, lung, and spleen. Qualitatively similar
SPIRONOLACTONE
AND
369
Hg
II-HIN
SP PRE-TREATMENT
SO-HIN
SP PRE-TREATMENT
3-M SP PRE-TREATMENT IS-MIN SP POST-TREATMENT CONTROL
I,
5
,
I
I5
30
I
I
45
60
90
MINUTES
FIG. I. Concentration of ‘03Hg in the blood and plasma at various times after the iv administration of 203HgC12 (0.3 mg Hg/kg). Rats were given spironolactone ip (75 mg/kg) 3 hr, 90 min, or 15 min before the HgCl, or 15 min after the HgCl,. Each value represents the mean +SE of five to six rats. TABLE EFFECT
1
OF SPIRONOLACTONE ON THE BILIARY OF MERCURY IN RATS”
Spironolactone
treatment
Control Pre-15 min Pre-90 min Pre-3 hr Post-15 min
EXCRETION
Biliary excretion 1.26 2.98 5.01 3.09 5.08
& -t * 5 +
(,ug,/kg)
0.31” 1.40 2.20 1.14 1.48”
’ 203HgCl, (0.3 mg Hg/kg) was administered iv and bile collected for 90 min. b Each value represents the mean +SE of five rats. ’ Significantly different from controls (p < 0.05).
results were obtained when spironolactone was given 90 min or 3 hr before the HgC12, but quantitatively the changes were less pronounced. When spironolactone was administered 15 min after HgCI,, it produced an increased concentration of Hg in the blood, brain, heart, lung, and spleen and decreasedthe concentration of Hg in the plasma similarly as when the spironolactone was administered 15 min before the HgCI, : however, it did not produce a decreasein the amount of Hg in the kidney.
370
CURTISD. KLAASSEN
CONTROL 3-M SP PRE-TREATMENT 90-MIN SP PRE-TREATMENT I5-MIN SP PRE-TREATMENT 15-MIN SP POST-TREATMENT
eTm U C?B B e
FIG 2. Concentration of ‘03Hg in various tissues 90 min after the iv administration of 203HgC13 (0.3 ig Hg/kg). Rats were given spironolactone ip (75 mg/kg) 3 hr, 90 min, or 15 min before the HgC12. Each value represents the mean *SE of five to six rats.
CONTROL
9-
IS-MN I 5
1 15
I 30 MINUTES6
1
SP PRE-TREATMENT
1 90
FIG 3 Concentration of 203Hg in the kidneys of control rats and rats pretreated 15 min before the 203Hg&; (iv, 0.3 mg Hg/kg) with spironolactone (ip, 75 mg/kg). Each value represents the mean _+SE of five to seven rats.
SPIRONOLACTONE
AND
Hg
371
The time course of the concentration of Hg in the kidney following the administration of HgCI, to control rats and rats pretreated with spironolactone 1.5min before the HgClz is shown in Fig. 3. The concentration of Hgin the kidneys of the rats that were pretreated with spironolactone 15 min earlier was lower at all time intervals after Hg administration, and at the 90-min time interval was about lo:!;; of controls. When the rats were
25
I
I 15
1 30
I 60
I 90 MINUTES
I 120
, 150
I I 160
FIG. 4. Concentration of spironolactone and its metabolites in the plasma at various times after the ip administration ofi3H} spironolactone(75 mg/kg). Each valuerepresents the mean k SE of four rats,
given the spironolactone 15 min after HgCl, administration, the concentration of Hg in the kidney was not significantly different from controls and thus are not depicted in the graph. The concentration of Hg in the liver of rats pretreated with spironolactone was 1.9 times higher than in controls at 5 min, 3.8 at I5 min, 2.1 at 30 min, 2.2 at 60 min. and 1.4 at 90 min. The amount of radioactivity in plasma following intraperitoneal administration of {3H)spironolactone is shown in Fig. 4. The concentration of 3H in the plasma increased rapidly during the first 15 min and remained relatively constant for the next 3 hr.
SPIRONOLACTONE 0 0? ---
o’/a!?
CANRENONE FIG.
5. Chemical structure of spironolactone and canrenone.
372
CURTIS
D. KLAASSEN
Since spironolactone is known to be rapidly metabolized to canrenone and thioacetic acid (Sadee et al., 1972), the effect of these two biotransformation products (Fig. 5)
CANRENONE CONTROL THIOACETATE
0.01
I 5
15
r
30
45 60 MINUTES
1 so
FIG. 6. Effect of ip administration of spironolactone (75 mg/kg), canrenone (61 mg/kg), or thioacetic acid (13.7 mg/kg) given 15 min before 203HgC12 (0.3 mg Hg/kg) on the concentration of ‘03Hg in the blood and plasma. Each value represents the mean kSE of three to seven rats.
zo- -S-I‘b---
1
i---i----I
C BLOOD
101 3
4 b
-------I
SP BLOOD
50-
C PLASMA SP PLASMA 7
5
I
15
30
45
60
90
MINUTES FIG . 7. Concentration of ‘03Hg in the plasma and blood in control rats and rats given spironolactone 15 min before (75 mg/kg) *‘%H3Hg (1 .Omg Hg/kg). Each value represents the mean *SE of six rats.
on the distribution of Hg was examined (Fig. 6). Equimolar doses of spironolactone, canrenone, or thioacetic acid were given to the rats and 1.5min later the 203HgCl, was administered. Pretreatment with the canrenone had no effect on the distribution of Hg.
SPIRONOLACTONE
AND
Hg
373
However, both spironolactone and thioacetic acid increased the concentration of Hg in blood and decreased the concentration in plasma. The changes in the amounts of Hg in the various tissues were qualitatively similar for thioacetic acid and spironolactone, although the thioacetic acid had a less pronounced effect. The effect of spironolactone pretreatment on the concentration of Hg in the blood and plasma following the administration of methyl mercury is shown in Fig. 7. Pretreatment with spironolactone 15 min before methyl mercury produced a slight decrease in the blood and plasma Hg values. This is very different from the effect of spironolactone on inorganic mercury which was increased in the blood and markedly decreased in the plasma. When spironolactone was given 15 min following methyl mercury, the effect in decreasing the blood and plasma concentrations was greater than when given before the methyl mercury. DISCUSSION The nephrotoxicity of mercuric chloride is well known, although prior treatment with spironolactone will reduce the ability of Hg to produce renal tubular necrosis and death (Selye, 1970). The mechanism by which spironolactone protects against mercuric chloride injury does not appear to be related to its ability to induce microsomal enzymes. Spironolactone is effective when it is given as a single dose 1 or 2 hr before mercuric chloride administration (Garg et al., 1971, Haddow et al., 1972), a time which is insufficient for the microsomal enzymes to become induced. Spironolactone appears to protect against the renal injury produced by mercuric chloride by decreasing the amount of Hg in the kidney. In the present study it was shown that pretreatment with spironolactone prevented the accumulation of Hg in the kidney, being more effective when given 15 min rather than 90 or 180 min before the Hg When given 15 min after the mercuric chloride, spironolactone was ineffective in lowering the concentration of Hg in the kidney. Spironolactone also does not protect against the renal injury and death when given after mercuric chloride (Selye, 1970). Spironolactone alters the distribution of Hg not only in the kidney but in other tissues as well. It markedly decreases the concentration of Hg in the plasma and kidney and increases it in many of the other tissues such as the blood, heart, liver, spleen, and brain. Spironolactone again was more effective in altering the distribution of Hg in these tissues when administered shortly before the Hg. Whereas spironolactone did not alter the concentration of Hg in the kidney when it was given 15 min after the mercuric chloride, it did alter the distribution of Hg in other tissues. Therefore, it appears that once the Hg is bound to the kidney, it cannot be removed by spironolactone. It has been suggested earlier that spironolactone protects against the toxic actions of Hg by increasing its excretion into bile (Haddow and Marshall, 1972; Haddow et al., 1972). In another study spironolactone did not increase the biliary excretion of Hg (Garg et al., 1971). In the present study spironolactone increased biliary excretion of Hg, but this increase was statistically significant only when the spironolactone was given after the Hg. Spironolactone administration at this time does not decrease the concentration of Hg in the kidney, alter the extent of kidney injury, or the incidence of death. Also, in the pretreated rats, a marked decrease in the content of Hg was found in the kidneys 5 min after mercuric chloride administration, a time by which very little
374
CURTISD. KLAASSEN
Hg hasbeen excreted into the bile. Thus it appearsthat spironolactone can increasethe biliary excretion of Hg, but this is probably the result of the alteration in the distribution of Hg in the body rather than the mechanism which protects against the toxic effects of Hg. The mechanismby which spironolactone alters the distribution of Hg doesnot appear to be related to the steroid moiety of the molecule. The concentration of 3H in the plasma after administration of [3H]spironolactone wasfound to be relatively constant between 15 min and 3 hr after intraperitoneal administration while spironolactone had a much greater effect on the distribution of Hg when given 15 min before spironolactone than when given 3 hr before. Since spironolactone is metabolized very rapidly to canrenone and thioacetic acid (Sadee et al., 1972), the effect of equimolar amounts of these two portions of the spironolactone molecule on the distribution of Hg were measured.The steroid portion of the molecule (canrenone) had no effect on the distribution of Hg whereas thioacetic acid produced qualitatively similar changes to those produced by spironolactone, but quantitatively thesechangeswere somewhat less.This quantitative difference is probably due to differences in the absorption, distribution and excretion rates of thioacetic acid when given alone compared to when given complexed to the steroid. It seemslikely that the mechanismby which spironolactone protects against Hg toxicity involves the sulfur portion of the molecule. Selye (1970) stated that two other thioacetylated steroids, emdabol and spiroxasone, also inhibit the renal toxicity of Hg. The thioacetate probably forms a ligand with Hg, which thus converts the inorganic mercury into an organic mercury. The Hg then distributes like an organic mercury, which is characterized by a high blood concentration, a very low plasma concentration and relatively similar concentrations in the other tissues(Klaassen, 1975). After the organic complex is formed in the body, a decreasein the amount of Hg in the kidney and the plasma, and an increase in many of the other tissuesoccurs as a result of the change in the form of the Hg. This decreasein the concentration of Hg in the kidney appears to be the primary reason why the kidney lesion is reduced. Similar results would probably be observed with other sulphydryl containing compounds. ACKNOWLEDGMENTS The author would like to thank the very able technical assistanceof Roxanne Barfneckt and CelesteSoutee.The author would also like to thank Searleand Company for spironolactone, [‘Hlspironolactone and canrenone. REFERENCES BUCK, S. H. AND LAGE, 0. L. (1971).Possiblemechanism of theprevention of digitoxin toxicity by spironolactonein the mouse.Arch. Int. Pharmacodyn. 189, 192-197. CASTLE, M. C. AND LAGE, G. L. (1972).Effect of pretreatment with spironolactone,pheno-
barbital or j%diethylaminoethyldiphenylpropyl acetate(SKF 525-A) on tritium levels in blood, heart, and liver of rats at various timesafter administrationof (3H)-digitoxin. Biothem. Pharmacol. 21,1449-1455. CASTLE, M. C. AND LACE, G. L. (1973a).Enhancedbiliary excretion of digitoxin following spironolactoneas it relatesto the prevention of digitoxin toxicity. Res. Commun. Chem. Pathol. Pharmacol. 5.99-108.
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CASTLE, M. C. AND LAGE, G. L. (1973b). Excretion of H3-digitoxin following spironolactone pretreatmentin rats. Drug Metab. Disp. 1, 590-597. CASTLE, M. C. ANDLACE,G. L. (1973~). Biliary excretion and hepaticconcentrationsof H3digitoxin and its metabolitesfollowing spironolactonepretreatment of rats. Res. Commun. Chem. Pathol. Pharmacol. 6, 601-612. FELLER, D. R. ANDGERALD, M. C. (1971).Interactions of spironolactonewith hepatic microsomaldrug metabolizingenzymesystems.Biochem. Pharmacol. 20, 1991-2000. GARG,B. D., S~LYMOSS, B. AND TUCHWEBER, B. (1971).Effect of spironolactoneon the distribution and excretion of ‘03HgC12in the rat. Ar~neimittleforschung 21, 815-816. HADDOW,J. E., FISH,C. A., MARSHALL,P. C. ANDLESTER, R. (1972). Biliary excretion of mercury enhancedby spironolactone.Gastroenterology 63, 1053-1058. HADDOW, J. E. ANDMARSHALL, P. (1972). Increasedstool mercury excretion in the rat: the effect of spironolactone.Proc. Sot. Exp. Biol. Med. 140, 707-709. HAMRECK, M. E., ZAMPAGLIONE, N. C., STRIPP, B. ANDGILLETTE, J. R. (1973)Investigation of the effectsof methyltestosterone,cortisoneand spironolactoneon the hepatic microsomal mixed function oxidasesystemin maleand femalerats. Biochem. Pharmacol. 22, 293-3 10. KLAASSEN, C. D. (1974).Effect of microsomalenzyme inducerson the biliary excretion of cardiacglycosides.J. Pharmaeof. Exp. Ther. 191,201-211. KLAASSEN, C. D. (1975).Biliary excretion of mercury compounds.Toxicol. Appl. Pharmacoi. 33, 352-361.
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SELYE,H. (1969).Prevention of indomethacin-inducedintestinal ulcersby spironolactoneand norbolethone.Can. J. Physiol. Pharmacol. 47, 981-983. SELYE,H. (1970).Mercury poisoning:prevention by spironolactoneScience 169, 775-776. SELYE,H., KRAJNEY,M. AND SAVOIE, L. (1969). Digitoxin poisoning: prevention by spironolactone.Science 164, 842-843. SOLYMOSS, B., TOTH,S., VARGA, S. AND SELYE, H. (1971).Protection by spironolactoneand oxandroloneagainstchronic digitoxin or indomethacinintoxication. Tosicol. Appl. Phmmmol. X3,586-592.
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