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Sulfonamide inhibition of rat hepatic uroporphyrinogen I synthetase activity and the biosynthesis of heme
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 201, No. 1, April 15, pp. 88-94, 1980
Sulfonamide
Inhibition Activity
of Rat Hepatic Uroporphyrinogen and the Biosynthesis of Hemel
I Synthetase
PETER G. PETERS, MANOHAR L. SHARMA, DAVID M. HARDWICKE, AND WALTER N. PIPER’ Department
of Phamacology, School of Medicine, lkiversity 91143 San Francisco, California Received May 8, 1979; revised
November
of California,
17, 1979
Sulfonamides are known to provoke attacks of acute intermittent porphyria, which is characterized by a deficiency of uroporphyrinogen I synthetase (EC 4.3.1.8) activity. Various sulfonamides were examined for their potential as inhibitors of purified rat hepatic uroporphyrinogen I synthetase activity in vitro, and 10 were found to be inhibitory. Inhibition of uroporphyrinogen I synthetase activity by sulfonamides is reversible by dialysis and exhibits noncompetitive kinetics. Inhibition constants (KJ ranged from 100 PM for sulfaguanidine to 270 /.LM for sulfadiazine. These observations were extended to irr viva studies using sulfamerazine (K, = 250 PM). Twenty-four hours after administration of sulfamerazine (orally; 1 g/kg) to male rats, hepatic microsomal heme and cytochrome P-450 contents were decreased approximately 33 and 42’70, respectively. This was reflected by the inhibition of both aniline-p-hydroxylase and aminopyrine-N-tlemethylase activities, which are P-450 mediated. Heme oxygenase activity was not altered by sulfamerazine treatment. However, the relative rate of heme biosynthesis, as measured by the incorporation of 6-[4-‘?C]aminolevulinic acid into heme, was 28% lower in sulfamerazine-treated rats. Thus, these results indicate that sulfamerazine acts to inhibit heme biosynthesis. Hepatic &aminolevulinic acid synthetase (EC 2.3.1.37) activity was increased 2.2.fold in the sulfamerazine-treated group, which is probably a compensatory increase caused by depression of the heme content. Consequently, it is suggested that sulfamerazine inhibits the synthesis of heme at the uroporphyrinogen I synthetase step to impair hemoprotein formation and cytochrome P-450-mediat.ed hepatic drug metabolism.
Uroporphyrinogen I synthetase (UROS),3 the third enzymatic step for heme synthesis, is decreasedto 50%of the normal level in acute intermittent porphyria (AIP) (1,2). Sulfonamides, antibiotics that are frequently used for treatment of both topical and systemic bacterial infections, are known to provoke attacks of AIP (3, 4) and to produce toxic disorders of the hemopoietic system.
These phenomena prompted us to investigate the effects of various sulfonamides on URO-S activity, and to study the consequences of this interaction on hepatic heme formation and cytochrome P-450mediated drug metabolism. These observations are presented in this report. EXPERIMENTAL
Materials
’ Supported by Grant ES-01343 from the National Institutes of Health. This is publication 79-4 from the Department of Pharmacology, University of California, San Francisco. 2 To whom reprint requests should be sent. R Abbreviations used: AIP, acute intermittent porphyria; ALA, &aminolevulinic acid; ALAS, &aminolevulinie acid synthetase; SMZ, sulfamerazine; URO-S, uroporphyrinogen I synthetase; PPO, 2,5diphenyloxazole; POPOP, 1,4-bis[2-(5.phenyloxazoyl)]benzene. 0003-9861/80/050088-07$02.00/0 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
PROCEDURES
Sulfonamides, bovine serum albumin Type V, 2,5diphenyloxazole (PPO), 1,4-bis[2-(5.phenyloxazoyl)]benzene (POPOP), NADP, hemin, and glucose 6phosphate were obtained from Sigma Chemical Company, St. Louis, Missouri. 6-[4-‘“C]Aminolevulinic acid (ALA), 25.4 mCi/mol, was purchased from New England Nuclear, Boston, Massachusetts, and porphobilinogen from Porphyrin Products, Logan, Utah. Pyridine, sodium dithionite, and all other laboratory 88
SULFONAMIDE
INHIBITION
OF UROPORPHYRINOGEN
reagents were of analytical grade and purchased from J. T. Baker, Phillipsburg, New Jersey or the Mallinckrodt Chemical Works, St. Louis, Missouri.
Methods Animals. Male Sprague-Dawley rats were obtained from Simonsen Laboratories, Gilroy, California, and weighed 160-200 g. They were allowed water ad lib&m and fasted for 16 h. The experimental group was administered sulfamerazine in water (orally; 1 g/kg; 4 ml/kg), and the controls received an equal volume of water. Uroporphyrinogen I synthetase pur$cation. The enzyme was purified from rat liver as described previously (5). This method employed heat treatment of hepatic cytosol (55°C for 5 min), ammonium sulfate fractionation, DEAE-cellulose chromatography with a O-O.4 M KC1 gradient, and Sephadex G-100 gel chromatography. The enzyme preparations used for this study represented 300. to 400-fold purification from hepatic cytosol and were stable for several weeks when stored at -20°C. Uroporphyrinogen I synthetase assay. The activity of URO-S was assayed by the method of Strand et al. (1). All incubations were carried out for 60 min at 37°C. Uroporphyrin I was measured fluorometrically with an Aminco-Bowman spectrophotofluorometer with excitation and emission wavelengths of 405 and 595 nm, respectively. Reversibility of inhibition of enzyme activity by sulfonamides. URO-S was incubated with each sulfonamide (1 mM) for 1 h at 37°C. Control and sulfonamide preparations were then dialyzed sepapH 8.0 rately against 2 liters of 50 mM Tris-HCl, (with one buffer change after 6 h) for 24 h. At the end of this time period, control and experimental preparations were tested for URO-S activity. Kinetics of inhibition of enzyme activity by sulfonamides. Sulfonamide concentrations of 0, 0.05, and 1 mM and substrate concentrations of 0.5, 1.0, 2.0, and 5.0 pM were employed, and the Lineweaver-Burk method (6) was used for plotting the kinetic data of the enzyme. Inhibition constants were calculated by the method of Dixon (7). Incorporation of &[4-‘4C]aminolevulinic acid into heme. The relative rates of hepatic heme biosynthesis were determined in rats 24 h after a single, oral dose of either water or sulfamerazine in water (1 g/kg; 4 ml/kg). [4-‘?C]ALA (2 pCi) was injected intraperitoneally, and 1 h later rats were sacrificed, livers perfused with 0.9% saline and homogenized in 3 vol of 1.15% KCl-50 mM Tris, pH 7.4. The microsomal fraction was isolated and the heme was extracted into ethyl acetate-acetic acid 4:l (v/v), and washed with water and 1.5 M HCl as described by Bonkowsky et al. (8). A loo-p,1 aliquot of the labeled heme extract was added to 12 ml of scintillation fluid prepared by the method described by Minaga et al.
I SYNTHETASE
89
(9) and counted for radioactivity in a Beckman LS 8000 liquid scintillation counter (97% efficiency). Incorporation of [4-‘“C]ALA into hepatic microsomal heme was known to be linear for the l-h pulse. Cytochrome P-&Xi assay. Hepatic microsomal cytochrome P-450 was determined from the carbon monoxide difference spectrum (450-500 nm) of dithionite-reduced microsomes using an extinction coefficient of 91 mM-’ cm-’ (10). Heme assay. Heme was estimated by the difference spectrum of the oxidized/reduced pyridine hemochromogen between 541 and 557 nm using an extinction coefficient of 20.7 mM-’ cm-’ as described bg Falk (11). GAminolevulinic acid synthetase assay. The activity of ALA synthetase was determined by the method described by Marver et nl. (12). Microsomal heme oxyge,lase assay. Microsomal heme oxygenase activity was determined by the method of Tenhunen et al. (13). Assay of hepatic microsomal drug ~netabolism activity. Aminopyrine-N-demethylase and aniline-phydroxylase activities were determined in incubation media containing 5 mM substrate and 3 mg microsomal protein as described by Schenkman ef al. (14). except that semicarbazide (4.1 mM) was added to trap the formaldehyde produced during the demethylation of aminopyrine. Formaldehyde was estimated by the method of Nash (15) and p-aminophenol, the metabolic product of aniline-p-hydroxylation, was measured by the method of Imai ef al. (16). Samples were incubated with shaking for 20 min at 37°C. Protein. Protein was assayed by the method described by Lowry et al. (17) using bovine serum albumin as the standard. Statistics. For determination of the significance of differences between means, data were analyzed by Student’s t test. RESULTS
Sulfonamide
Inhibition oj Uroporphyrinogen Z Synthetase
Activity The ability of sulfonamides to provoke attacks of AIP raised the question of whether this group of drugs could inhibit the activity of uroporphyrinogen I synthetase. This was examined by assessing the effect of various sulfonamides on the purified enzyme. When present in a final concentration of 1 mM, all the sulfonamides tested were inhibitory except sulfathiazole (Table I). Sulfanilic acid was also tested and failed to inhibit enzyme activity. Of the drugs which were inhibitory, sulfisomidine produced the greatest inhibition (65%)
90
PETERS TABLE
ET AL.
I
INHIBITION OF RAT HEPATIC UROPORPHYRINOGEN I SYNTHETASEACTIVITY BY VARIOUS SULFONAMIDES SULFONAMIDEO
N’(6-indozolylj~ Sulfanilomida
0 ‘““:?I”“”
.&
65
170
60
270
50
250
50
170
50
220
40-50
190
Sultogwnidina
35
100
Sulfamoxole
33
160
Sulfopyridinr
25 20 0
while sulfanilamide
-
produced the least
(20%).
In order to determine whether inhibition was reversible, enzyme aliquots were dialyzed in the presence of each sulfonamide at 1 lllM concentrations. Activity of each inhibited enzyme preparation returned to approximately 90% of control activity. Incubation of sulfonamide inhibitors with control enzyme preparations that had been dialyzed resulted in inhibition of enzymatic activity, demonstrating that dialysis did not render the enzyme insensitive to inhibition by sulfonamides. These findings indicate that inhibition of URO-S activity by sulfonamides is reversible. From Lineweaver-Burk plots, it was determined that each sulfonamide derivative is a noncompetitive inhibitor. A representative plot is shown for sulfamerazine (Fig. l), the sulfonamide drug that was used for the in viwo studies. The inhibition constants for the sulfonamide drugs, illustrated in Table I, range from 100 /.G! for sulfaguanidine to 270 PM for sulfadiazine. The Effect of Sulfameruzine on Hepatic Heme and Cytochrome P-450 Content and Drug Metabolism
The observation that several sulfonamides inhibit uroporphyrinogen I synthetase ac-
FIG. 1. Kinetics of inhibition of rat hepatic uroporphyrinogen I synthetase activity by sulfamerazine. The inset exhibits a Dixon plot for determining the inhibition constant. (0) Sulfamerazine (SMZ); (0) control. Each point represents the mean value of three determinations.
tivity in vitro suggests that they may alter the hepatic heme content in vivo. Alteration of hepatic heme levels would be expected to produce changes in the concentration of certain cytochromes in the hepatocyte, especially those with a short half-life, such as cytochrome P-450 [7-9 h fast phase, 46-48 h slow phase, Levin and Kuntzman (IS)]. In order to test this m mvrRoL 0 SMZ
”
Heme content
Cytahrane P-450 content
Cytcchmme b, caltent
FIG. 2. Hepatic microsomal heme, cytochrome P-450, and cytochrome b, content after treatment of rats with sulfamerazine. Sulfamerazine was administered orally (1 g/kg), and animals were sacrificed at 24 h and the livers assayed as described under Methods. Each bar represents the mean f SEM for three rats. An asterisk denotes significant difference (P < 0.05) between sulfamerazine-treated and control rats.
SULFONAMIDE
INHIRITION
OF UROPORPHYRIKOGEN
FIG. 3. Hepatic microsomal drug metabolism after treatment of rats with sulfamerazine. Experimental conditions were identical to those described for Fig. 2. Each bar represents the mean -t SEM for four rats. An asterisk denotes significant difference (P < 0.05) between sulfamerazine-treated and control rats.
91
I SYNTHETASE a
CONTROL
0
SMZ
Micmsomal Heme Oxygenase
FIG. 4. tivity after perimental for Fig. 2. four rats.
Hepatic microsomal heme oxygenase actreatment of rats with sulfamerazine. Exconditions were identical to those described Each bar represents the mean 2 SEM for
hypothesis, sulfamerazine was used as a representative of the sulfonamide group of drugs and the heme, cytochrome P-450, and cytochrome b, contents of rat hepatic microsomes were measured in control and icant difference in the activity of microsomal treated animals. The data indicate that. heme oxygenase between sulfamerazinesulfamerazine caused a 33% decrease in the treated and control rats. Therefore, it is microsomal heme content and a 41% de- unlikely that sulfamerazine depressed hecrease in the cytochrome P-450 content patic heme and cytochrome P-450 content (Fig. 2). The hepatic microsomal content of cytochrome 6, was not altered by sulfam CONTROL merazine treatment. 4oor n=4 asMz Cytochrome P-450-mediated drug metabolism was determined by measuring the N-demethylation of aminopyrine (a Type I 2E 300 . . . . n=3 .. . substrate) and the p-hydroxylation of ani.::: 22 .::: .::: line (a Type II substrate). Sulfamerazine .::: 2 a::: administration to rats produced decreases .::: = 200 ‘::: -::: of aminopyrine-N-demethylase and aniline.;:: h .::: .::: p-hydroxylase activities of 42 and 28%#, 8 .::: .::: 100 .::: respectively (Fig. 3). 1::: The E.ffect of Sulfamerazine on Microsomal Heme Oxygenase Activity
The observed loss of heme and cytochrome P-450 produced by sulfamerazine could have been the result of an increase in the rate of heme catabolism. Microsomal heme oxygenase, the first and rate-limiting enzymatic step in the breakdown uf heme, was assayed in sulfamerazine-treated rats to see whether its activity had increased relative to the controls. The results of this investigation are presented in Fig. 4 and show that there was no signif-
J-
....* .::: !I .:.*: .::: .::: .::: *::: . . . .. .
-
Incorporation of ALA% into Hepatic Microsomal Heme
FIG. 5. Incorporation of [‘“CIALA into hepatic microsomal heme after treatment of rats with sulfamerazine. [%]ALA (2 &i) was injected intraperitoneally 24 h after administration of sulfamerazine (orally; 1 g/kg). After a l-h pulse, the rats were sacrificed and microsomal heme was extracted and measured as described under Methods. Numbers above the bars denote the number of rats. An asterisk denotes significant difference (P < 0.05) between sulfamerazinetreated and control rats.
92
PETERS
ET AL.
by accelerating the rate of heme catabolism by stimulation of heme oxygenase activity. The Effect of Sulfameraxine on the Incorporation of &[4-14Cylminolevulinic Acid into Hepatic Microsomal Heme in Vivo
The observations that URO-S activity is inhibited by sulfonamides in vitro and that sulfamerazine administration did not appear to enhance the rate of heme breakdown suggested that the decrease in the microsomal heme content was the result of inhibition of the rate of heme biosynthesis. This hypothesis was tested by comparing the rate of incorporation of [4J4C]ALA into hepatic microsomal heme in both sulfamerazine-treated and control rats. The results (Fig. 5) show that the sulfamerazinetreated rats have a rate which is 28% lower than that for control animals. The Effect of Sulfameraxine on GAminolevulinic Acid Synthetase Activity
A deficiency or partial inhibition of URO-S activity could produce a drop in the level of heme, resulting in a compensatory increase of ALAS activity for restoration of the synthesis of heme (19-21). Therefore, the activity of hepatic ALAS was measured following pretreatment of rats with sulfamerazine. The activity of hepatic ALAS was found to be approximately 2.2-fold greater than controls in the sulfamerazine-treated animals (Fig. 6). DISCUSSION
Several sulfonamides have been found to be reversible, noncompetitive inhibitors of rat hepatic uroporphyrinogen I synthetase activity in vitro. Administration of suifamerazine to rats decreased hepatic microsomal heme, cytochrome P-450 and drug metabolism, and depressed the rate of incorporation of [14C]ALA into microsomal heme. Sulfamerazine administration failed to alter hepatic microsomal heme oxygenase activity. These results indicate that sulfamerazine depresses the synthesis of heme by inhibiting the activity of the
20
r
a
CONTROL
0
SMZ
*
6 -aminolevulinic acid synthetase activity FIG. 6. Hepatic S-aminolevulinic acid synthetase activity after treatment of rats with sulfamerazine. Experimental conditions were identical to those described for Fig. 2. Each bar represents the mean 2 SEM for three rats. An asterisk denotes significant difference between sulfamerazine-treated and control rats.
enzyme uroporphyrinogen I synthetase. Hepatic microsomal cytochrome b, content was not significantly altered 24 h after treatment of rats with sulfamerazine. This observation could be anticipated, since the half-life of this cytochrome is reported to range from 2.3-5 days (22-24). Sulfonamide inhibition of the synthesis of heme is a plausible explanation for the potentiation of the anticoagulant effect of warfarin by concomitant drug therapy with sulfisoxazole (25), sulfamethizole (26), and Co-trimoxazole (27-29). The decreased hepatic content of heme and the microsomal hemoprotein cytochrome P-450, produced by inhibition of the enzyme URO-S, would be expected to result in increased circulating levels of warfarin and a potentiation of the anticoagulant effect. Sulfonamides have been thought to potentiate the effect of warfarin by displacement from plasma protein (30). However, the results reported herein indicate that inhibition of hepatic microsomal drug metabolism may be an important mechanism
SULFONAMIDE
INHIBITION
OF UROPORPHYRINOGEN
whereby various sulfonamides potentiate the anticoagulant effect of warfarin. Assuming a half-life of 6 h, a volume of distribution of 11.2 liters in a 70-kg man (31), and the usual therapeutic dosage regimen of 4 g initially followed by 1 g every 6 h thereafter (32), we have calculated that the apparent total body concentration of sulfisoxazole will be approximately 600 j..&M at 24 h. This value exceeds the value of the inhibition constant reported in Table I for sulfisoxazole (170 PM) anal suggests that in vivo concentrations greater than the Ki can be attained. Thus, it is probable that in vivo inhibition of uroporphyrinogen I synthetase activity occurs during sulfonamide therapy. The reciprocal relationship between heme content and ALAS activity (19-21) provides an explanation for the elevation of ALAS activity following the loss of heme after sulfonamide administration. The depression of heme content produced by sulfonamide inhibition of URO-S would be the feedback signal for induction of ALAS as a compensatory mechanism in an attempt to synthesize more heme (20). ALAS is regarded as the rate-limiting step in heme biosynthesis, based partly upon the fact that its activity is normally very low. Meyer (33) and DeMatteis (34) have reported that the V values of ALAS and URO-S are similar and the lowest of the enzymes involved in the biosynthesis of heme. Sulfonamide administration, . which produces elevation of ALAS activity as a probable compensatory response to the decreased heme content following inhibition of URO-S activity, may render URO-S the new rate-limiting step for the biosynthesis of hepatic heme. Such an event may be critical in individuals afflicted with acute intermittent porphyria, who already have a diminished cellular activity of URO-S. REFERENCES 1. STRAND, L. J., MEYER, U. A., FELSHER, B. F., REDEKER, A. G., AND MARVER, H. S. (1972) J. Clin. Invest. 51, 2530-2536. 2. MEYER, U. A., STRAND, L. J., Doss, M., REES, A. C., AND MARVER, H. S. (1972) Nezc Engl. J. Med. 286, 1277-1282. 3. STEIN, J. A., AND TSCHUDY, D. P. (19’70) Medicine 49, l- 16.
I SYNTHETASE
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4. EALES, L. (1971) S. Afr. J. Lab. Clin. Med. 17, 120- 125. 5. PIPER, W. N., AND VAN LIER, R. B. L. (1977) Mol. Phamnacol. 13, 1126-1135. 6. LINEWEAVER, H., AND BURK, D. (1934) J. Amer. Chem. Sot. 56, 658-666. 7. DIXON, M. (1953) Biochern. J. 55, 170-171. 8. BONKOWSKY, H. L., BEMENT, W. J., AND ERNY, R. (1978) Biochim. Biophys. Acta 541, 119123. 9. MINAGA, T., SHARMA, M. L., KUN, E., AND PIPER, W. N. (1978) Biochim. Biophys. Actn 538, 417-425. 10. OMURA, T., AND SATO, R. (1964) J. Riol. Chem. 239, 2370-2378. and Metallo11. FALK, J. E. (1964) Porphyrins porphyrins, Biochim. Biophys. Acta Library Vol. 2, pp. 182, 241, Elsevier, Amsterdam. 12. MARVER, H. S., TSCHUDY, D. P., PERLROTH, M. G., AND COLLINS, A. (1966) J. Biol. Chem. 241, 2803-2809. 13. TENHUNEN, R., MARVER, H. S., AND SCHMID, R. (1969) J. Biol. Chem. 244, 6388-6394. 14. SCHENKMAN, J. B., REMMER, H., AND ESTABROOK, R. W. (1967) ,uol. Pham~acol. 3, 113-123. 15. NASH, T. (1953) Biochem. J. 55, 416-421. 16. IMAI, Y., ITO, A., AND SATO, R. (1966) b. Biochern. 60, 417-428. 17. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chew. 193, 265-275. 18. LEVIN, W., AND KUNTZMAN, R. (1969) Mol. Phamnacol. 5, 499-506. 19. BURNHAM, B. F., AND LASCELLES, J. (1963) Biochern. J. 87, 462-472. 20. GRANICK, S. (1966) J. Biol. Chem. 241, 13591375. 21. DE MATTEIS, F. (1978) in Heme and Hemoproteins (De Matteis, F., and Aldridge, W. N., eds.), Handbook of Experimental Pharmacology Vol. 44, p. 132, Springer-Verlag, New York. 22. OMURA, T., SIEKEVITZ, P., AND PALADE, G. E. (1967) J. Biol. Chem. 242, 2389-2396. 23. KURIYAMA, Y., OMURA, T., SIEKEVITZ, P., AND PALADE, G. E. (1969) J. Biol. Chem. 244, 2017-2026. 24. DRUYAN, R., DEBERNARD, B., AND RABINOWITZ, M. (1969) J. Biol. Chem. 244, 58745878. 25. SELF, T. H., EVANS, W., AND FERGUSON, T. (1975) Circulation 52, 528. 26. LUMHOLTZ, B., SIERSBAEK-NIELSEN, K., SKOVSTED, L., KAMPMANN, J., AND HANSEN, J. M. (1975) Clin. Pharmacol. They. 17, 731-734. 27. BARNETT, D. B., AND HANCOCK, B. W. (1975) h-it. Med. J. 1, 608-609. 28. HASSALL, C., FEETAM, C. L., LEACH, R. H.,
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PETERS AND MEYNELL, 2, 684.
M. J. (1975) &it.
Med. J.
29. ERRICK, J. K., AND KEYS, P. W. (1978) Amer. J. Hosp. Pharm. 35, 1399-1401. 30. KOCH-WESER, J., AND SELLERS, E. M. (1971) New Engl. J. Med. 285, 547-558. 31. RITSCHEL, W. A. (1976) Handbook of Basic Pharmacokinetics, Drug Intelligence Publications, Hamilton, 111.
ET AL. 32. WEINSTEIN, L. (1975) in The Pharmacological Basis of Therapeutics (Goodman, L. S., and Gilman, A., eds.), pp. 1113-1129. Macmillan, New York. 33. MEYER, U. (1978) in Diagnosis and Therapy of Porphyrins and Lead Intoxication (Doss, M., ed.), pp. 3-7, Springer-Verlag, New York. 34. DE MATTEIS, F. (1975) in Enzyme Induction (Parke, D. V., ed.), pp. 185-205, Plenum, New York.
Sulfonamide
Inhibition Activity
of Rat Hepatic Uroporphyrinogen and the Biosynthesis of Hemel
I Synthetase
PETER G. PETERS, MANOHAR L. SHARMA, DAVID M. HARDWICKE, AND WALTER N. PIPER’ Department
of Phamacology, School of Medicine, lkiversity 91143 San Francisco, California Received May 8, 1979; revised
November
of California,
17, 1979
Sulfonamides are known to provoke attacks of acute intermittent porphyria, which is characterized by a deficiency of uroporphyrinogen I synthetase (EC 4.3.1.8) activity. Various sulfonamides were examined for their potential as inhibitors of purified rat hepatic uroporphyrinogen I synthetase activity in vitro, and 10 were found to be inhibitory. Inhibition of uroporphyrinogen I synthetase activity by sulfonamides is reversible by dialysis and exhibits noncompetitive kinetics. Inhibition constants (KJ ranged from 100 PM for sulfaguanidine to 270 /.LM for sulfadiazine. These observations were extended to irr viva studies using sulfamerazine (K, = 250 PM). Twenty-four hours after administration of sulfamerazine (orally; 1 g/kg) to male rats, hepatic microsomal heme and cytochrome P-450 contents were decreased approximately 33 and 42’70, respectively. This was reflected by the inhibition of both aniline-p-hydroxylase and aminopyrine-N-tlemethylase activities, which are P-450 mediated. Heme oxygenase activity was not altered by sulfamerazine treatment. However, the relative rate of heme biosynthesis, as measured by the incorporation of 6-[4-‘?C]aminolevulinic acid into heme, was 28% lower in sulfamerazine-treated rats. Thus, these results indicate that sulfamerazine acts to inhibit heme biosynthesis. Hepatic &aminolevulinic acid synthetase (EC 2.3.1.37) activity was increased 2.2.fold in the sulfamerazine-treated group, which is probably a compensatory increase caused by depression of the heme content. Consequently, it is suggested that sulfamerazine inhibits the synthesis of heme at the uroporphyrinogen I synthetase step to impair hemoprotein formation and cytochrome P-450-mediat.ed hepatic drug metabolism.
Uroporphyrinogen I synthetase (UROS),3 the third enzymatic step for heme synthesis, is decreasedto 50%of the normal level in acute intermittent porphyria (AIP) (1,2). Sulfonamides, antibiotics that are frequently used for treatment of both topical and systemic bacterial infections, are known to provoke attacks of AIP (3, 4) and to produce toxic disorders of the hemopoietic system.
These phenomena prompted us to investigate the effects of various sulfonamides on URO-S activity, and to study the consequences of this interaction on hepatic heme formation and cytochrome P-450mediated drug metabolism. These observations are presented in this report. EXPERIMENTAL
Materials
’ Supported by Grant ES-01343 from the National Institutes of Health. This is publication 79-4 from the Department of Pharmacology, University of California, San Francisco. 2 To whom reprint requests should be sent. R Abbreviations used: AIP, acute intermittent porphyria; ALA, &aminolevulinic acid; ALAS, &aminolevulinie acid synthetase; SMZ, sulfamerazine; URO-S, uroporphyrinogen I synthetase; PPO, 2,5diphenyloxazole; POPOP, 1,4-bis[2-(5.phenyloxazoyl)]benzene. 0003-9861/80/050088-07$02.00/0 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
PROCEDURES
Sulfonamides, bovine serum albumin Type V, 2,5diphenyloxazole (PPO), 1,4-bis[2-(5.phenyloxazoyl)]benzene (POPOP), NADP, hemin, and glucose 6phosphate were obtained from Sigma Chemical Company, St. Louis, Missouri. 6-[4-‘“C]Aminolevulinic acid (ALA), 25.4 mCi/mol, was purchased from New England Nuclear, Boston, Massachusetts, and porphobilinogen from Porphyrin Products, Logan, Utah. Pyridine, sodium dithionite, and all other laboratory 88
SULFONAMIDE
INHIBITION
OF UROPORPHYRINOGEN
reagents were of analytical grade and purchased from J. T. Baker, Phillipsburg, New Jersey or the Mallinckrodt Chemical Works, St. Louis, Missouri.
Methods Animals. Male Sprague-Dawley rats were obtained from Simonsen Laboratories, Gilroy, California, and weighed 160-200 g. They were allowed water ad lib&m and fasted for 16 h. The experimental group was administered sulfamerazine in water (orally; 1 g/kg; 4 ml/kg), and the controls received an equal volume of water. Uroporphyrinogen I synthetase pur$cation. The enzyme was purified from rat liver as described previously (5). This method employed heat treatment of hepatic cytosol (55°C for 5 min), ammonium sulfate fractionation, DEAE-cellulose chromatography with a O-O.4 M KC1 gradient, and Sephadex G-100 gel chromatography. The enzyme preparations used for this study represented 300. to 400-fold purification from hepatic cytosol and were stable for several weeks when stored at -20°C. Uroporphyrinogen I synthetase assay. The activity of URO-S was assayed by the method of Strand et al. (1). All incubations were carried out for 60 min at 37°C. Uroporphyrin I was measured fluorometrically with an Aminco-Bowman spectrophotofluorometer with excitation and emission wavelengths of 405 and 595 nm, respectively. Reversibility of inhibition of enzyme activity by sulfonamides. URO-S was incubated with each sulfonamide (1 mM) for 1 h at 37°C. Control and sulfonamide preparations were then dialyzed sepapH 8.0 rately against 2 liters of 50 mM Tris-HCl, (with one buffer change after 6 h) for 24 h. At the end of this time period, control and experimental preparations were tested for URO-S activity. Kinetics of inhibition of enzyme activity by sulfonamides. Sulfonamide concentrations of 0, 0.05, and 1 mM and substrate concentrations of 0.5, 1.0, 2.0, and 5.0 pM were employed, and the Lineweaver-Burk method (6) was used for plotting the kinetic data of the enzyme. Inhibition constants were calculated by the method of Dixon (7). Incorporation of &[4-‘4C]aminolevulinic acid into heme. The relative rates of hepatic heme biosynthesis were determined in rats 24 h after a single, oral dose of either water or sulfamerazine in water (1 g/kg; 4 ml/kg). [4-‘?C]ALA (2 pCi) was injected intraperitoneally, and 1 h later rats were sacrificed, livers perfused with 0.9% saline and homogenized in 3 vol of 1.15% KCl-50 mM Tris, pH 7.4. The microsomal fraction was isolated and the heme was extracted into ethyl acetate-acetic acid 4:l (v/v), and washed with water and 1.5 M HCl as described by Bonkowsky et al. (8). A loo-p,1 aliquot of the labeled heme extract was added to 12 ml of scintillation fluid prepared by the method described by Minaga et al.
I SYNTHETASE
89
(9) and counted for radioactivity in a Beckman LS 8000 liquid scintillation counter (97% efficiency). Incorporation of [4-‘“C]ALA into hepatic microsomal heme was known to be linear for the l-h pulse. Cytochrome P-&Xi assay. Hepatic microsomal cytochrome P-450 was determined from the carbon monoxide difference spectrum (450-500 nm) of dithionite-reduced microsomes using an extinction coefficient of 91 mM-’ cm-’ (10). Heme assay. Heme was estimated by the difference spectrum of the oxidized/reduced pyridine hemochromogen between 541 and 557 nm using an extinction coefficient of 20.7 mM-’ cm-’ as described bg Falk (11). GAminolevulinic acid synthetase assay. The activity of ALA synthetase was determined by the method described by Marver et nl. (12). Microsomal heme oxyge,lase assay. Microsomal heme oxygenase activity was determined by the method of Tenhunen et al. (13). Assay of hepatic microsomal drug ~netabolism activity. Aminopyrine-N-demethylase and aniline-phydroxylase activities were determined in incubation media containing 5 mM substrate and 3 mg microsomal protein as described by Schenkman ef al. (14). except that semicarbazide (4.1 mM) was added to trap the formaldehyde produced during the demethylation of aminopyrine. Formaldehyde was estimated by the method of Nash (15) and p-aminophenol, the metabolic product of aniline-p-hydroxylation, was measured by the method of Imai ef al. (16). Samples were incubated with shaking for 20 min at 37°C. Protein. Protein was assayed by the method described by Lowry et al. (17) using bovine serum albumin as the standard. Statistics. For determination of the significance of differences between means, data were analyzed by Student’s t test. RESULTS
Sulfonamide
Inhibition oj Uroporphyrinogen Z Synthetase
Activity The ability of sulfonamides to provoke attacks of AIP raised the question of whether this group of drugs could inhibit the activity of uroporphyrinogen I synthetase. This was examined by assessing the effect of various sulfonamides on the purified enzyme. When present in a final concentration of 1 mM, all the sulfonamides tested were inhibitory except sulfathiazole (Table I). Sulfanilic acid was also tested and failed to inhibit enzyme activity. Of the drugs which were inhibitory, sulfisomidine produced the greatest inhibition (65%)
90
PETERS TABLE
ET AL.
I
INHIBITION OF RAT HEPATIC UROPORPHYRINOGEN I SYNTHETASEACTIVITY BY VARIOUS SULFONAMIDES SULFONAMIDEO
N’(6-indozolylj~ Sulfanilomida
0 ‘““:?I”“”
.&
65
170
60
270
50
250
50
170
50
220
40-50
190
Sultogwnidina
35
100
Sulfamoxole
33
160
Sulfopyridinr
25 20 0
while sulfanilamide
-
produced the least
(20%).
In order to determine whether inhibition was reversible, enzyme aliquots were dialyzed in the presence of each sulfonamide at 1 lllM concentrations. Activity of each inhibited enzyme preparation returned to approximately 90% of control activity. Incubation of sulfonamide inhibitors with control enzyme preparations that had been dialyzed resulted in inhibition of enzymatic activity, demonstrating that dialysis did not render the enzyme insensitive to inhibition by sulfonamides. These findings indicate that inhibition of URO-S activity by sulfonamides is reversible. From Lineweaver-Burk plots, it was determined that each sulfonamide derivative is a noncompetitive inhibitor. A representative plot is shown for sulfamerazine (Fig. l), the sulfonamide drug that was used for the in viwo studies. The inhibition constants for the sulfonamide drugs, illustrated in Table I, range from 100 /.G! for sulfaguanidine to 270 PM for sulfadiazine. The Effect of Sulfameruzine on Hepatic Heme and Cytochrome P-450 Content and Drug Metabolism
The observation that several sulfonamides inhibit uroporphyrinogen I synthetase ac-
FIG. 1. Kinetics of inhibition of rat hepatic uroporphyrinogen I synthetase activity by sulfamerazine. The inset exhibits a Dixon plot for determining the inhibition constant. (0) Sulfamerazine (SMZ); (0) control. Each point represents the mean value of three determinations.
tivity in vitro suggests that they may alter the hepatic heme content in vivo. Alteration of hepatic heme levels would be expected to produce changes in the concentration of certain cytochromes in the hepatocyte, especially those with a short half-life, such as cytochrome P-450 [7-9 h fast phase, 46-48 h slow phase, Levin and Kuntzman (IS)]. In order to test this m mvrRoL 0 SMZ
”
Heme content
Cytahrane P-450 content
Cytcchmme b, caltent
FIG. 2. Hepatic microsomal heme, cytochrome P-450, and cytochrome b, content after treatment of rats with sulfamerazine. Sulfamerazine was administered orally (1 g/kg), and animals were sacrificed at 24 h and the livers assayed as described under Methods. Each bar represents the mean f SEM for three rats. An asterisk denotes significant difference (P < 0.05) between sulfamerazine-treated and control rats.
SULFONAMIDE
INHIRITION
OF UROPORPHYRIKOGEN
FIG. 3. Hepatic microsomal drug metabolism after treatment of rats with sulfamerazine. Experimental conditions were identical to those described for Fig. 2. Each bar represents the mean -t SEM for four rats. An asterisk denotes significant difference (P < 0.05) between sulfamerazine-treated and control rats.
91
I SYNTHETASE a
CONTROL
0
SMZ
Micmsomal Heme Oxygenase
FIG. 4. tivity after perimental for Fig. 2. four rats.
Hepatic microsomal heme oxygenase actreatment of rats with sulfamerazine. Exconditions were identical to those described Each bar represents the mean 2 SEM for
hypothesis, sulfamerazine was used as a representative of the sulfonamide group of drugs and the heme, cytochrome P-450, and cytochrome b, contents of rat hepatic microsomes were measured in control and icant difference in the activity of microsomal treated animals. The data indicate that. heme oxygenase between sulfamerazinesulfamerazine caused a 33% decrease in the treated and control rats. Therefore, it is microsomal heme content and a 41% de- unlikely that sulfamerazine depressed hecrease in the cytochrome P-450 content patic heme and cytochrome P-450 content (Fig. 2). The hepatic microsomal content of cytochrome 6, was not altered by sulfam CONTROL merazine treatment. 4oor n=4 asMz Cytochrome P-450-mediated drug metabolism was determined by measuring the N-demethylation of aminopyrine (a Type I 2E 300 . . . . n=3 .. . substrate) and the p-hydroxylation of ani.::: 22 .::: .::: line (a Type II substrate). Sulfamerazine .::: 2 a::: administration to rats produced decreases .::: = 200 ‘::: -::: of aminopyrine-N-demethylase and aniline.;:: h .::: .::: p-hydroxylase activities of 42 and 28%#, 8 .::: .::: 100 .::: respectively (Fig. 3). 1::: The E.ffect of Sulfamerazine on Microsomal Heme Oxygenase Activity
The observed loss of heme and cytochrome P-450 produced by sulfamerazine could have been the result of an increase in the rate of heme catabolism. Microsomal heme oxygenase, the first and rate-limiting enzymatic step in the breakdown uf heme, was assayed in sulfamerazine-treated rats to see whether its activity had increased relative to the controls. The results of this investigation are presented in Fig. 4 and show that there was no signif-
J-
....* .::: !I .:.*: .::: .::: .::: *::: . . . .. .
-
Incorporation of ALA% into Hepatic Microsomal Heme
FIG. 5. Incorporation of [‘“CIALA into hepatic microsomal heme after treatment of rats with sulfamerazine. [%]ALA (2 &i) was injected intraperitoneally 24 h after administration of sulfamerazine (orally; 1 g/kg). After a l-h pulse, the rats were sacrificed and microsomal heme was extracted and measured as described under Methods. Numbers above the bars denote the number of rats. An asterisk denotes significant difference (P < 0.05) between sulfamerazinetreated and control rats.
92
PETERS
ET AL.
by accelerating the rate of heme catabolism by stimulation of heme oxygenase activity. The Effect of Sulfameraxine on the Incorporation of &[4-14Cylminolevulinic Acid into Hepatic Microsomal Heme in Vivo
The observations that URO-S activity is inhibited by sulfonamides in vitro and that sulfamerazine administration did not appear to enhance the rate of heme breakdown suggested that the decrease in the microsomal heme content was the result of inhibition of the rate of heme biosynthesis. This hypothesis was tested by comparing the rate of incorporation of [4J4C]ALA into hepatic microsomal heme in both sulfamerazine-treated and control rats. The results (Fig. 5) show that the sulfamerazinetreated rats have a rate which is 28% lower than that for control animals. The Effect of Sulfameraxine on GAminolevulinic Acid Synthetase Activity
A deficiency or partial inhibition of URO-S activity could produce a drop in the level of heme, resulting in a compensatory increase of ALAS activity for restoration of the synthesis of heme (19-21). Therefore, the activity of hepatic ALAS was measured following pretreatment of rats with sulfamerazine. The activity of hepatic ALAS was found to be approximately 2.2-fold greater than controls in the sulfamerazine-treated animals (Fig. 6). DISCUSSION
Several sulfonamides have been found to be reversible, noncompetitive inhibitors of rat hepatic uroporphyrinogen I synthetase activity in vitro. Administration of suifamerazine to rats decreased hepatic microsomal heme, cytochrome P-450 and drug metabolism, and depressed the rate of incorporation of [14C]ALA into microsomal heme. Sulfamerazine administration failed to alter hepatic microsomal heme oxygenase activity. These results indicate that sulfamerazine depresses the synthesis of heme by inhibiting the activity of the
20
r
a
CONTROL
0
SMZ
*
6 -aminolevulinic acid synthetase activity FIG. 6. Hepatic S-aminolevulinic acid synthetase activity after treatment of rats with sulfamerazine. Experimental conditions were identical to those described for Fig. 2. Each bar represents the mean 2 SEM for three rats. An asterisk denotes significant difference between sulfamerazine-treated and control rats.
enzyme uroporphyrinogen I synthetase. Hepatic microsomal cytochrome b, content was not significantly altered 24 h after treatment of rats with sulfamerazine. This observation could be anticipated, since the half-life of this cytochrome is reported to range from 2.3-5 days (22-24). Sulfonamide inhibition of the synthesis of heme is a plausible explanation for the potentiation of the anticoagulant effect of warfarin by concomitant drug therapy with sulfisoxazole (25), sulfamethizole (26), and Co-trimoxazole (27-29). The decreased hepatic content of heme and the microsomal hemoprotein cytochrome P-450, produced by inhibition of the enzyme URO-S, would be expected to result in increased circulating levels of warfarin and a potentiation of the anticoagulant effect. Sulfonamides have been thought to potentiate the effect of warfarin by displacement from plasma protein (30). However, the results reported herein indicate that inhibition of hepatic microsomal drug metabolism may be an important mechanism
SULFONAMIDE
INHIBITION
OF UROPORPHYRINOGEN
whereby various sulfonamides potentiate the anticoagulant effect of warfarin. Assuming a half-life of 6 h, a volume of distribution of 11.2 liters in a 70-kg man (31), and the usual therapeutic dosage regimen of 4 g initially followed by 1 g every 6 h thereafter (32), we have calculated that the apparent total body concentration of sulfisoxazole will be approximately 600 j..&M at 24 h. This value exceeds the value of the inhibition constant reported in Table I for sulfisoxazole (170 PM) anal suggests that in vivo concentrations greater than the Ki can be attained. Thus, it is probable that in vivo inhibition of uroporphyrinogen I synthetase activity occurs during sulfonamide therapy. The reciprocal relationship between heme content and ALAS activity (19-21) provides an explanation for the elevation of ALAS activity following the loss of heme after sulfonamide administration. The depression of heme content produced by sulfonamide inhibition of URO-S would be the feedback signal for induction of ALAS as a compensatory mechanism in an attempt to synthesize more heme (20). ALAS is regarded as the rate-limiting step in heme biosynthesis, based partly upon the fact that its activity is normally very low. Meyer (33) and DeMatteis (34) have reported that the V values of ALAS and URO-S are similar and the lowest of the enzymes involved in the biosynthesis of heme. Sulfonamide administration, . which produces elevation of ALAS activity as a probable compensatory response to the decreased heme content following inhibition of URO-S activity, may render URO-S the new rate-limiting step for the biosynthesis of hepatic heme. Such an event may be critical in individuals afflicted with acute intermittent porphyria, who already have a diminished cellular activity of URO-S. REFERENCES 1. STRAND, L. J., MEYER, U. A., FELSHER, B. F., REDEKER, A. G., AND MARVER, H. S. (1972) J. Clin. Invest. 51, 2530-2536. 2. MEYER, U. A., STRAND, L. J., Doss, M., REES, A. C., AND MARVER, H. S. (1972) Nezc Engl. J. Med. 286, 1277-1282. 3. STEIN, J. A., AND TSCHUDY, D. P. (19’70) Medicine 49, l- 16.
I SYNTHETASE
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4. EALES, L. (1971) S. Afr. J. Lab. Clin. Med. 17, 120- 125. 5. PIPER, W. N., AND VAN LIER, R. B. L. (1977) Mol. Phamnacol. 13, 1126-1135. 6. LINEWEAVER, H., AND BURK, D. (1934) J. Amer. Chem. Sot. 56, 658-666. 7. DIXON, M. (1953) Biochern. J. 55, 170-171. 8. BONKOWSKY, H. L., BEMENT, W. J., AND ERNY, R. (1978) Biochim. Biophys. Acta 541, 119123. 9. MINAGA, T., SHARMA, M. L., KUN, E., AND PIPER, W. N. (1978) Biochim. Biophys. Actn 538, 417-425. 10. OMURA, T., AND SATO, R. (1964) J. Riol. Chem. 239, 2370-2378. and Metallo11. FALK, J. E. (1964) Porphyrins porphyrins, Biochim. Biophys. Acta Library Vol. 2, pp. 182, 241, Elsevier, Amsterdam. 12. MARVER, H. S., TSCHUDY, D. P., PERLROTH, M. G., AND COLLINS, A. (1966) J. Biol. Chem. 241, 2803-2809. 13. TENHUNEN, R., MARVER, H. S., AND SCHMID, R. (1969) J. Biol. Chem. 244, 6388-6394. 14. SCHENKMAN, J. B., REMMER, H., AND ESTABROOK, R. W. (1967) ,uol. Pham~acol. 3, 113-123. 15. NASH, T. (1953) Biochem. J. 55, 416-421. 16. IMAI, Y., ITO, A., AND SATO, R. (1966) b. Biochern. 60, 417-428. 17. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chew. 193, 265-275. 18. LEVIN, W., AND KUNTZMAN, R. (1969) Mol. Phamnacol. 5, 499-506. 19. BURNHAM, B. F., AND LASCELLES, J. (1963) Biochern. J. 87, 462-472. 20. GRANICK, S. (1966) J. Biol. Chem. 241, 13591375. 21. DE MATTEIS, F. (1978) in Heme and Hemoproteins (De Matteis, F., and Aldridge, W. N., eds.), Handbook of Experimental Pharmacology Vol. 44, p. 132, Springer-Verlag, New York. 22. OMURA, T., SIEKEVITZ, P., AND PALADE, G. E. (1967) J. Biol. Chem. 242, 2389-2396. 23. KURIYAMA, Y., OMURA, T., SIEKEVITZ, P., AND PALADE, G. E. (1969) J. Biol. Chem. 244, 2017-2026. 24. DRUYAN, R., DEBERNARD, B., AND RABINOWITZ, M. (1969) J. Biol. Chem. 244, 58745878. 25. SELF, T. H., EVANS, W., AND FERGUSON, T. (1975) Circulation 52, 528. 26. LUMHOLTZ, B., SIERSBAEK-NIELSEN, K., SKOVSTED, L., KAMPMANN, J., AND HANSEN, J. M. (1975) Clin. Pharmacol. They. 17, 731-734. 27. BARNETT, D. B., AND HANCOCK, B. W. (1975) h-it. Med. J. 1, 608-609. 28. HASSALL, C., FEETAM, C. L., LEACH, R. H.,
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Med. J.
29. ERRICK, J. K., AND KEYS, P. W. (1978) Amer. J. Hosp. Pharm. 35, 1399-1401. 30. KOCH-WESER, J., AND SELLERS, E. M. (1971) New Engl. J. Med. 285, 547-558. 31. RITSCHEL, W. A. (1976) Handbook of Basic Pharmacokinetics, Drug Intelligence Publications, Hamilton, 111.
ET AL. 32. WEINSTEIN, L. (1975) in The Pharmacological Basis of Therapeutics (Goodman, L. S., and Gilman, A., eds.), pp. 1113-1129. Macmillan, New York. 33. MEYER, U. (1978) in Diagnosis and Therapy of Porphyrins and Lead Intoxication (Doss, M., ed.), pp. 3-7, Springer-Verlag, New York. 34. DE MATTEIS, F. (1975) in Enzyme Induction (Parke, D. V., ed.), pp. 185-205, Plenum, New York.