Chem. Res. Toxicol. 1992,5,163-166
163
NADPH-Dependent Oxidation of Reduced Ebselen, 2-Selenylbenzanilide, and of 2-( Methylseleno)benzanilide Catalyzed by Pig Liver Flavin-Containing Monooxygenase Daniel M. Ziegler,tJ Peter Graf,t Lawrence L. Poulsen,s Wilhelm Stahl,? and Helmut Sies*vt Institut fur Physiologische Chemie I, Moorenstrasse 5, Universitat Diisseldorf, W-4000Diisseldorf, Germany, and Clayton F o u n d a t i o n Biochemical Institute, University of T e x a s ut Austin, Austin, Texas 78712 Received S e p t e m b e r 17, 1991
The selenazole ring-opened metabolites of ebselen, 2-selenylbenzanilide and 2-(methylseleno)benzanilide, are substrates for flavin-containing monooxygenase from pig liver. The K,,, values were 25 and 3 p M ,respectively, measured at 37 "C,pH 7.4, in the presence of 1mM GSH. The V,, values were 390 mU/mg of protein, similar to those obtained with methimazole or other substrates for FMO1. Although ebselen also appears to be a substrate in the absence of GSH, -it progressively inactivates the enzyme, apparently by binding covalently to essential enzyme thiols. The oxidation products of the selenol and methylseleno derivatives are rapidly reduced by GSH, regenerating the parent substrates. Rapid reduction of the selenide oxide by GSH was unexpected and suggests that, unlike S-oxidation of sulfides, Se-oxidation of selenides may be a route for bioactivation. The data show that in the presence of FMOl micromolar amounts of either of these ringopened metabolites establish a futile cycle catalyzing the oxidation of GSH to GSSG by NADPH and oxygen.
Introduction Ebselen [2-phenyl-1,2-benzisoselenazol-3(2If)-one], a selenoorganic compound, mimics the catalytic activity of glutathione peroxidase in vitro (1,2)and exhibits antiinflammatory activity (3-6). Both these properties are apparently due to the ability of ebselen to catalyze the reduction of hydroperoxides by glutathione or other thiols. In the presence of thiols ebselen is reduced to 2-selenylbenzanilide, which rapidly reduces hydrogen peroxide and organic hydroperoxides to water and an alcohol, respectively, regenerating ebselen (7,8). Metabolic studies (9-12) showed that in liver ebselen is reduced to 2-selenylbenzanilide, which is then either glucuronidated to 2-(glucuronylseleno)benzanilide or methylated to 2-(methylse1eno)benzanilide. The latter metabolite is further para-hydroxylated to N-(4-hydroxyphenyl)-2-(methylseleno)benzamide,which is excreted as the glucuronide (11,12).In addition to metabolic reactions leading to disposition, a recent study (13)suggested that rat liver microsomes catalyze the oxidation of 2-(methylse1eno)benzanilide to 2-(methylseleninyl)benzanilide. Both 2-selenylbenzanilide and 2-(methylseleno)benzanilide are potential substrates for microsomal monooxygenases, especially the flavin-containing monooxygenases which preferentially catalyze the oxidation of xenobiotic soft nucleophiles (14,15). In this report we describe kinetic studies on the oxidation of the selenol and methylseleno metabolites of ebselen catalyzed by the purified flavin-containing monooxygenase (FMO1)' from pig liver and examine the reactivity of the enzyme-generated products with glutathione. Materlals and Methods Reagents. Ebselen and 2 - ( m e t h y l s e l e n o ) b e ~ d ewere kind gifts from Drs. N. Dereu and E. Graf, Rh6nePoulenc-Nattermannann,
* Address correspondence to this author. +
Universitiit Diisseldorf.
* University of Texas at Austin. 0893-228x/92/2705-0163$03.00/0
Cologne, FRG. Biochemicals and chemicals were obtained from Boehringer, Mannheim, and Merck, Darmstadt, FRG. 2-(Methylseleninyl)benzanilide, the oxide of 2-(methylse1eno)benzanilide, was synthesized as described (13). The flavin-containing monooxygenase, isolated from pig liver in Austin, TX, by the published method (16),was lyophilized and sealed in vessels for transport. The dried powder, resuspended in 0.25 M sucrose containing 50 mM potassium phosphate, pH 7.4, retained 7 6 8 0 % of the original activity @,(FAD) llO/mm, at pH 8.4, with saturating substrates and positive effector noctylamine). The resuspended enzyme, diluted to an activity of 3 U/mL with methimazole as substrate a t 37 "C, pH 7.4, was stored in 30-WrL aliquota at -20 O C . Activity of aliquots thawed as needed remained practically constant over 4 months. Activity Measurements. Reactions catalyzed by FMOl or microsomes were carried out at 37 OC in 100 mM potassium phosphate, pH 7.4, containing 1 mM EDTA and either 0.2 mM NADPH or an NADPH-generating system consisting of 0.2 mM NADP+, 5 mM glucose 6-phosphate, and glucose-6-phosphate dehydrogenase. The reaction was generally initiated by adding no more than 10 rL/mL of the selenium compounds dissolved in ethanol. Other additions were as indicated in specific figures or tables. Velocities of reactions catalyzed by FMOl were measured spectrophotometrically by following substrate-dependent oxidation of NADPH or by measuring the concentration of oxidation products in aliquota of the reaction medium withdrawn a t regular intervals, usually 0, 3, 6, 9, and 12 min. The concentration of GSSG in 0.1-mL aliquota of the reaction medium transferred to tubes containing 0.05 mL of phosphoric acid (5%) was determined as described (18). The concentration of 2-(methylseleniny1)benzanilide as a function of reaction time was calculated by the following method. Aliquots (0.5 mL) of the reaction medium were transferred into 1.0 mL of ethyl acetate, mixed with a vortex mixer, and centrifuged. An aliquot of the ethyl acetate extract (0.8 mL) was ~~
Abbreviations: FMO1, FM02, flavin-containing monooxygenase forms similar in substrate specificity and structure to the major pig liver and rabbit lung enzymes, respectively (19); EDTA, ethylenediaminetetraacetate; GSSG, glutathione disulfide; GSH, glutathione; NADPH and NADP+, reduced and oxidized nicotinamide adenine dinucleotide, respectively; methimazole, 2-mercapto-l-methylimidazole; U and mu, units of enzyme activity catalyzing the conversion of 1pmol and 1nmol of substrate/min, respectively. 0 1992 American Chemical Society
Ziegler e t al.
164 Chem. Res. Toxicol., Vol. 5, No. 2, 1992 Table I. Kinetic Constants for the Oxidation of Selenium Compounds Catalyzed by Flavin-Containing Monooxygenase (FMO1) from Pig Liver" Vmm,
mU/mg substrate additions K,, UM of protein ebselen 120-150b GSH (1mM) 25 370 5' 390 2-(methylseleno)benzanilide GSH (1mM) 3 390 10 380 methimazole Constants were calculated from substrate-dependent oxidation of NADPH at 37 O C in potassium phosphate (0.1 M), pH 7.4, containing EDTA (1 mM) with saturating NADPH and 200 pM oxygen at substrate concentrations above and below K,. *Rate for the first minute after addition of ebselen (50 pM); inhibition of enzyme by ebselen in the absence of GSH precludes further kinetic analysis. K, calculated by the procedure of Poulsen et al. (17) by the change in velocity with limiting substrate. a
removed, dried under a stream of nitrogen, and redissolved in 0.4 mL of ethanol/water (1/1 v/v). Twenty microliters was injected onto a reversed-phase LiChrospher R P 18e end-capped 5-rm 4- x 125-mm column. The selenium compounds were resolved with 50% ethanol in water (v/v) on a Merck-Hitachi L6210 HPLC unit at a flow rate of 1 mL/min. The effluent was monitored with an L4250 UV/vis detector. The oxide was fully resolved from its parent substrate and from potential rearrangement products; retention times, in min, were as follows: ebselen, 6; 2-(methylseleno)benzanilide, 8; and 2-(methylseleninyl)benzanilide, 4. The concentration of the oxide was calculated from peak heights of known amounts of the chemically synthesized product carried through the extraction procedure and HPLC analysis. The oxide extracted quantitatively (97%) into ethyl acetate and was quite stable in neutral solution free from low-molecular-weight thiols or other reductants. Components present in purified FMOl did not interfere with the extraction or analysis of the oxide by this method.
Results FMO1-CatalyzedOxidations. The kinetic data s u m marized in Table I indicate that both ebselen and its reduced metabolite, 2-selenylbenzanilide,stimulate the oxidation of NADPH catalyzed by purified FMOl from pig liver. Although these results were not explored in detail, ebselen inactivates FMOl in the absence of GSH. At ebselen concentrations above 30-50 pM the loss of activity was usually 50-709'0 within less than 3 min. The reduction of ebselen with thiols is a very fast reaction that takes place immediately even at -70 "C (7), and in the absence of GSH ebselen may react with essential enzyme thiols. However, stepwise reduction of ebselen by GSH through the intermediate selenyl sulfide as described by Fischer and Dereu (7) generates 2-selenylbenzanilide,which does not inhibit FMO1. Double-reciprocal plots of rate vs ebselen in the presence of GSH were linear. The concentration of 2selenylbenzanilide required to half-saturate the enzyme was 25 pM, with a V,, essentially identical to that of methimazole (Table I), which at saturation measures the maximum turnover rate of FMOl (14). In the presence of 1 mM GSH, ebselen-dependent NADPH oxidation exceeded the concentration of added ebselen, suggesting that the substrate was regenerated by the thiol, perhaps by reduction. This possibility was tested by measuring ebselen-dependent oxidation of GSH in reactions catalyzed by FMO1, and as shown in Figure 1, FMOl can catalyze ebselen-dependent oxidation of GSH to GSSG. The oxidation of GSH was not affected by addition of catalase, and catalase was routinely added to limit the nonenzymic oxidation of the selenol and/or GSH by free peroxide. The 1:l ratio of NADPH oxidized to
0
5 10 Reaction Time (minl
15
Figure 1. FMO1-dependent oxidation of GSH to GSSG in the presence of ebselen. The rate of GSSG formation from GSH (1.0 mM) by FMOl (15 mU/mL) was measured in the presence (A) and absence (A)of ebselen (100 pM) in basal medium containing the NADPH-generating system and catalase (loo0 mU/mL). The was measured rate of ebselen-dependent NADPH oxidation (0) spectrophotometrically under the same conditions, except that 0.2 mM NADPH was substituted for the NADPH-regenerating system.
GSSG formed (Figure 1)also suggests that the enzymegenerated thiol oxidant reacts rapidly and quantitatively with GSH. Like the reduction metabolite of ebselen, the methyl derivative 2-(methylseleno)benzanilide is an excellent substrate for FMOl (Table I). Kinetic constants measured in the presence and absence of GSH are essentially the same. In the absence of GSH the ratio of NADPH oxidized/2-(methylseleno)benzanilide added is 1:l (Figure 2A), but as also shown in Figure 2A, GSH regenerates the substrate from the enzyme-catalyzed oxidation product. From the known mechanism and nature of the enzyme-bound oxidant (14) it was anticipated that FMOl would catalyze the oxidation of 2-(methylse1eno)benzanilide to the selenium oxide, 2-(methylseleniny1)benzanilide. This appears to be the case, as shown by the data summarized in Figure 3. FMOl catalyzes the quantitative conversion of the selenide to a product with the same chromatographic properties as the chemically synthesized oxide. The enzymically generated and chemically synthesized products are also quite stable in neutral solutions for several hours at room temperature. However, unlike analogous sulfoxides, the selenium oxide of 2-(methylse1eno)benzanilide was rapidly reduced by GSH. Analysis of the reaction medium by HPLC indicates that the oxide is reduced quantitatively to 2-(methylse1eno)benzanilide by GSH. The rate of reduction is surprisingly fast, as indicated by the tracings in Figure 2, panels A and B. There was no detectable lag in NADPH oxidation when the enzymaticallygenerated (Figure 2A) or the chemically synthesized oxide (Figure 2B) was reduced by GSH in the presence of FMO1. In the absence of substrates GSH has no effect on NADPH oxidation or O2 uptake by FMOl (17).
Discussion The experiments reported here demonstrate that two of the in vivo metabolites of ebselen, 2-selenylbenzanilide and 2-(methylseleno)benzanilide,are excellent substrates for the microsomal flavin-containing monooxygenase (Table I). The oxidation of these selenium compounds catalyzed by FMOl is fully enzymic and does not involve free peroxides or oxy radicals. Although the oxidation product of only the latter of the two metabolites was positively identified (Figure 3), the similarity in structure suggests that the monooxygenase catalyzes oxygenation of the selenium atom in both of the investigated compounds.
Chem. Res. Toxicol., Vol. 5, No.2, 1992 165
FMOl-Catalyzed Ebselen Oxidation
Scheme I. Sequence of Reactions for the Ebselen- (A) and 2-(Methylseleno)benzanilide- (B) Dependent Oxidation of GSH Catalyzed by FMOl
0 6-
A
E
an
OL-
V S e - H
E -
I
g
n 4:
0 2-
I
0
5
10
Timelmin)
B
2-(Methylseleniryll- benzanilide
pq
06
GSSG +
0.24 I
i
0
10
Timelmin)
Figure 2. FMO1-catalyzed oxidation of NADPH in the presence of 2-(methylse1eno)benzanilide (A) or 2-(methylseleninyl)benzanilide (B). NADPH oxidation was followed at 366 nm. Conditions: potassium phosphate buffer (0.1 M), pH 7.4, EDTA (1 mM), NADPH (0.2 mM), and FMO1,23 mU/mL. After establishing the rate of substrate-independent NADPH oxidation, 5 p L of a 10 mM solution of substrate was added as indicated. After completion of the reaction in (A), the addition of 50 rL of GSH (20 mM) stimulates NADPH oxidation; note that there was no detectable lag. In (B),addition of 10 p L of 2-(methylseleniny1)benzanilide(3.8 mM) had no detectable effect, but the rate increased to the maximum FMO1-dependent rate upon addition of GSH (1 mM).
0
0
5 10 15 Reaction Time(min1
Figure 3. Time course of FMO1-catalyzed oxidation of 2-(methylselen0)- (0) to 2 - ( m e t h y l s e l e n i n y l ) b d e (0).Conditions as in Figure 2, with 12 mU/mL FMO1. For details, see Materials and Methods.
In addition, the enzyme-generated oxidation products of both compounds oxidize GSH to GSSG (Figures 1and 21, most likely by pathways shown in Scheme I. The major steps in the ebselen-dependent, FMO1-catalyzed oxidation of GSH are quite similar to those proposed for the ebselen-catalyzed oxidation of GSH by peroxides (7,
2 GkH
H20
8). They differ only in the source of the oxidant. In the nonenzymatic system the selenol is oxidized to the selenenic acid by peroxides free in solution, whereas in the FMO1-catalyzed reaction the selenol is oxidized by the enzyme-bound 4a-hydroperoxyflavin which is continuously regenerated by NADPH and oxygen. Reduction of the selenenic acid to the selenol by GSH can occur by either of the pathways shown in Scheme IA. The intramolecular dehydration to ebselen should predominate at low GSH concentrations, but direct reduction of the selenenic acid may become significant at higher concentrations of GSH. However, reduction by either mechanism regenerates the substrate for FMO1, leading to a futile cycle catalyzing the oxidation of GSH to GSSG by NADPH and oxygen. Because 2-selenylbenzanilide is a soft nucleophile structurally similar to xenobiotic thiol substrates for FMOl (14,17), the ability of ebselen to initiate and sustain the cycle shown in Scheme IA was not surprising. On the other hand, the rapid nonenzymatic reduction of 2-(methylseleninyl)benanilide by GSH was unexpected. Unlike sulfoxides which are quite stable in the presence of millimolar concentrations of GSH, the selenoxide of the methyl metabolite of ebselen is reduced extremely fast at concentrations of GSH far less than physiological. As shown in Figure 2, there is no detectable lag in FMO1catalyzed NADPH oxidation after addition of GSH, suggesting that the substrate was regenerated immediately upon mixing. While the rapid reduction of the oxide by GSH would prevent its accumulation in vivo, the methyl metabolite should stimulate biliary efflux of GSSG in intact liver by sustaining the cycle for oxidation of GSH by NADPH and oxygen illustrated in Scheme IB. If the futile cycle described by the studies in this report also occurs in the intact animal, Se-oxidation of selenides, unlike S-oxidation of sulfides, may be a route for metabolic activation rather than detoxication. Potential Relevance to the Human. Whether these selenium compounds are substrates for forms of FMO present in human tissues is an open question. A recent report (19) indicates that a form similar to FMOl is expressed at high amounts only in human fetal liver. FMO
166 Chem. Res. Toxicol., Vol. 5, No. 2, 1992
present in adult human liver is apparently different in ita amino acid sequence. In addition, the studies of Lemoine et al. (20) suggest that FMO present in adult human liver microsomes is catalytically similar to FM02 of rabbit lung rather than FMO1. Like FM02 the human liver enzyme apparently does not N-oxygenate the side-chain nitrogen atom of imipramine or other nucleophilic centers that project less than 6-8 A from a tricyclic ring system (21). Thus, it is possible that ebselen and its metabolites are excluded from the active site of the major adult human liver form of FMO, but are substrates for FMOl present in the human fetal liver. Developmental changes in the oxidative metabolism of ebselen and its metabolites by flavin-containing monooxygenases are a distinct possibility that warrant further study.
Acknowledgment. D.M.Z. is the recipient of a Humboldt Research Award and acknowledges the kind support by the Alexander-von-HumboldtFoundation. In addition, the continued support of his work by the Foundation for Research is deeply appreciated. H.S. gratefully acknowledges support by the National Foundation for Cancer Research, Bethesda, and by the Jug-Stiftung fiir Wissenschaft und Forschung, Hamburg.
References (1) Miiller, A,, Cadenas, E., Graf, P., and Sies, H. (1984) A novel biologically active seleno-organic compound. Glutathione peroxidase-like activity in vitro and anti-oxidant capacity of PZ 51 (ebselen). Biochem. Pharmacol. 33,3235-3239. (2) Wendel, A., Fausel, M., Safayhi, H., and Otter, R. (1984)A novel biologically active seleno-organic compound. 11. Activity of PZ 51 in relation to glutathione Deroxidase. Biochem. Phuramcol. 33, 3241-3246. (3) Kuhl, P., Borbe, H. O., Fischer, H., RBmer, A., and Safayhi, H. (1986) . . Ebselen reduces the formation of LTB, in human and porcine leukocytes by isomerisation to ita 5S, 12R-6-trans-isomer. Prostaglandins 31,1029-1048. (4) Parnham, M. J., Leyck, S., Dereu, N. Winkelman, J., and Graf, E. (1985) GSH-peroxidase-like organo-selenium compound with antiinflammatory activity. Adu. Inflammation Res. 10,397-401. (5) Wendel, A., and Tiegs, G. (1986) A novel biologically active seleno-organic compound. VI. Protection by ebselen (PZ 51) against galactosamine/endotoxin-inducedhepatitis in mice. Biochem. Pharmacol. 35, 2115-2118. (6) Niederau, C., Ude, K., Niederau, M., Luthen, R., Strohmeyer, G., Ferrell, L. D., and Grendell, J. H. (1991) Effects of the seleno-organic substance ebselen in two different models of acute I
Ziegler et al. pancreatitis. Pancreas 6, 282-290. (7) Fischer, H., and Dereu, N. (1987) Mechanism of the catalytic reduction of hydroperoxides by ebselen: A selenium-77 NMR Chim. Belg. 96, 757-768. study. Bull. SOC. (8) Maiorino, M., Roveri, A., Coassin, M., and Ursini, F. (1988) Kinetic mechanism and substrate specificity of glutathione peroxidase activity of ebselen (PZ 51). Biochem. Pharmacol. 37, 2267-2271. (9) Mtiller, A., Gabriel, H., Sies, H., Terlinden, R., Fischer, H., and Romer, A. (1988) A novel biologically active selenoorganic compound. VII. Biotransformation of ebselen in perfused rat liver. Biochem. Pharmacal. 37, 1103-1109. (10) Fischer, H., Terlinden, R., LBhr, J. P., and Romer, A. (1988) A novel biologically active selenoorganic compound. VIII. Biotransformation of ebselen. Xenobiotica 18, 1347-1359. (11) Sies, H. (1989) Metabolism and disposition of ebselen. In Selenium in Biology and Medicine (Wendel, A., Ed.) pp 153-162, Springer Verlag, Heidelberg. (12) Fischer, H., Hilboll, G., Rbmer, A., and Terlinden, R. (1989) The use of highly enriched "Se in metabolic studies of ebselen in man. In Selenium in Biology and Medicine (Wendel, A., Ed.) pp 163-168, Springer Verlag, Heidelberg. (13) John, N. J., Terlinden, R., Fischer, H., Evers, M., and Sies, H. (1990) Microsomal metabolism of 2-(methylse1eno)benzanilide. Chem. Res. Toxicol. 3, 199-203. (14) Ziegler, D. M. (1988) Flavin-containing monooxygenases: Catalytic mechanism and substrate specificity. Drug Metab. Rev. 19, 1-32. (15) Jakoby, W. B., and Ziegler, D. M. (1990) The enzymes of detoxification. J. Bid. Chem. 265, 20715-20718. (16) Ziegler, D. M., and Poulsen, L. L. (1978) Hepatic microsomal mixed-function amine oxidase. Methods Enxymol. 52,142-151. (17) Poulsen, L. L., Hyslop, R. M., and Ziegler, D. M. (1979) SOxygenation of N-substituted thiourea catalyzed by the liver microsomal FAD-containing monooxygenase. Arch. Biochem. Biophys. 198, 788-789. (18) Akerboom, T. P. M., Bilzer, M., and Sies, H. (1982) The relationship of biliary glutathione efflux and intracellular glutathione disulfide content in perfused rat liver. J. Biol. Chem. 257, 4248-4252. (19) Dolphin, C., Shepard, E. A., Povey, S., Palmer, C. N. A., Ziegler, D. M., Ayesh, R., Smith, R. L., and Phillips, I. R. (1991) Cloning, primary sequence and chromosomal mapping of a human flavincontaining monooxygenase (FMO 1). J. Biol. Chem. 266, 12379-12385. (20) Lemoine, A., Johann, M., and Cresteil, T. (1990) Evidence for the presence of distinct flavin-containing monooxygenases in human tissue. Arch. Biochem. Biophys. 276, 336-342. (21) Nagata, T., Williams, D. E., and Ziegler, D. M. (1990)Substrate specificities of rabbit lung and porcine liver flavin-containing monooxygenases: Differences due to substrate size. Chem. Res. Toxicol. 3, 372-376.
