Chem. Res. Toxicol. 1991,4, 669-677 (40) Sahar, E., and Latt, S. A. (1980)Energy transfer and binding competition between dyes used to enhance staining differentiation in metaphase chromosomes. Chromosoma 79,1-28. (41) Portugal, J., and Waring, M. J. (1988)Assignment of DNA binding sites for DAPI (4',6'-diamidine-2-phenylindone)and Hoechst 33258 (bis-benzimide). A comparative footprinting study.
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Biochim. Biophys. Acta 949,158-168. (42) Jorgenson, K.F., Varshney, V., and van de Sande, J. H. (1988) Interaction of Hoechst 33258 with repeating synthetic DNA polymers and natural DNA. J. Biomol. Struct. Dyn.5,1005-1023. (43) Beerman, T.A., Sigmund, R., McHugh, M., L o w , J. W., Rao, K. E., and Bathini, Y. (1991)Biochim. Biophys. Acta (in press).
Formation of Glutathionyl-Spironolactone Disulfide by Rat Liver Cytochromes P450 or Hog Liver Flavin-Containing Monooxygenases: A Functional Probe of Two-Electron Oxidations of the Thiosteroid? Caroline J. Decker,+ John R. Cashman,t Katsumi Sugiyama,? David Maltby,t and Maria Almira Correia*pf Departments of Pharmacology and Pharmaceutical Chemistry and Liver Center, University of California, Sun Francisco, California 94143 Received July 15, 1991 We have previously reported that the diuretic thiosteroid spironolactone (SPL) inactivates rat liver microsomal cytochromes P450 [P450 (P450 3A and P450 2C11)] in a mechanism-based fashion, and we have identified two polar SPL metabolites (SPL-sulfinic acid and -sulfonic acid), formed in a partition ratio of =201 in such rat liver microsomal'incubations [Decker et al. (1989) Biochemistry 28,5128-51361, We proposed a t the time that these metabolites were most likely derived from further enzymatic (or nonenzymatic) oxidations of the one-electron oxidation product [SPL-thiyl radical (SPL-S')] and/or the two-electron-oxidized species [SPL-sulfenic acid (SPL-SOH)]. In those studies, glutathione (GSH) was found to attenuate both SPL-mediated P450 loss as well as polar metabolite formation by ~ 4 0 % .We have now reexamined this in greater detail and report that it is due to GSH trapping of an electrophilic oxidized SPL species to form an adduct that we have isolated and unambiguously characterized by mass spectral analyses as the glutathionyl-SPL adduct (SPL-SSG). Moreover, we have found not only that rat liver microsomal formation of this adduct is enhanced a t pH 9.0, the pH optimum for flavin-containing monooxygenase (FMO), but also that such adduct formation was indeed efficiently catalyzed by purified hog liver FMO. Because FMO oxidations of thiols are thought to entail a two-electron process to form the corresponding sulfenic acids, we infer that such a SPL-SSG adduct most likely reflects FMO-catalyzed oxidation of SPL to SPL-SOH, which on leaving the FMO active site is then trapped by GSH. Moreover, if not intercepted by external nucleophiles, FMO-generated SPL-SOH may attack rat liver microsomal P45Os, thus accounting for the GSH-inhibitable component of SPL-mediated P450 loss. This possibility is strengthened by the finding that GSH-mediated attenuation of such a SPL-mediated P450 loss was diminished by thermal inactivation of rat liver microsomal FMO. The isolation and characterization of the SPL-SSG adduct, we believe, not only rationalize the previously reported GSH-mediated attenuation of P450 loss but also provide the first direct evidence for the intermediacy of GSH conjugates in reduction of FMO-generated sulfenic acids. Furthermore, because we detected no polar oxidized SPL metabolites in purified FMO-catalyzed systems even in the absence of GSH, it appears that these metabolites must be derived from the one-electron-oxidized SPL-S' species.
Introduction The antiminer~ocorticoid spironolactone (spL)lor its deacetylated (SPL-SH) is known to inactivate the adrenal and testicular cytochromes P450 (P450s),in various species (1-5). It is now well established that SPL deacetylation is critical to its P450 inactivation, since tissues deficient in microsomal deacetylases or blockade
* T~whom correspondence should be addressed at the Department of Pharmacology, Box 0450, University of California, San Francisco, San Francisco, CA 94143. 'Department of Pharmacology and Liver Center. t Department of Pharmaceutical Chemistry and Liver Center. 0893-228x/91/2704-0669$02.50/0
of deacetylation by specific inhibitors of microsomal esterases result in little or no SPL-mediated P450 inactivation (1, 3, 4). We have shown that in vivo administration of Abbreviations: CID, collision-induced dissociation; MS/MS, mass spectrometry; P450, cytochrome P450; SPL-SH, deacetylated spironolactone; DEX, dexamethasone; DETAPAC, diethylenetriaminepentaacetic acid; DTT, dithiothreitol; ESR, electron spin resonance; FMO, flavin-containing monooxygenase; GSH, glutathione; GSSG,oxidized glutathione; GS', glutathionyl radical; HPLC, high-performance liquid chromatography; +LSIMS, liquid secondary ion mass spectral analyses in the positive mode; NADPH, nicotinamide adenine dinucleotide phosphate, reduced form; RSOH, organic sulfenic acid; RSSC, organic (aryl alkyl) glutathionyl disulfide; SPL, spironolactone; SPLSSG, S P L GSdadduct; SPL-S', SPL-thiyl radical; SPL-SOH, SPL-sulfenic acid.
0 1991 American Chemical Society
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670 Chem. Res. Toxicol., Vol. 4, No. 6, 1991
SPL also causes loss of hepatic P450 in untreated rats and that this loss is magnified after pretreatment of rats with dexamethasone (DEX), an inducer of P450 3A isozymes (6, 7). Selective functional markers have allowed us to identify rat hepatic microsomal P45Os 3A and 2 C l l as the targets of SPL inactivation, a finding directly confirmed in the case of P450 2 C l l with the purified functionally reconstituted enzyme (7). In vitro studies with liver microsomes from untreated or DEX-pretreated rats incubated with SPL with or without NADPH have permitted us not only to characterize such P450 inactivation as a mechanism-based suicide process with SPL-SH as the "active" inactivating intermediate but also to isolate and unequivocally identify the ultimate polar SPL-SH metabolites of P450-catalyzed oxidations as the sulfinic and sulfonic acid species (7). On the basis of these findings, we have proposed that these relatively polar metabolites were derived either from the one-electron oxidation product [SPL-thiyl radical (SPLS')] or from the two-electron-oxidized species [ SPLsulfenic acid (SPL-SOH)] generated by P450-dependent metabolism of SPL-SH (7). The well-recognized high reactivity of either SPL intermediate has preempted its individual characterization, although we have, albeit unsuccessfully, attempted to spin trap the thiyl radical with ESR probes.2 However, as previously reported (7), the relatively polar nucleophile GSH was capable of partially attenuating both in vitro SPL-mediated P450 loss and polar metabolite formation by ~ 4 0 % This . intrigued us, because GSH is considered too hydrophilic to gain access into the P450 active site, leading us to postulate that such GSH-induced attenuation most likely was due to its trapping of diffusible SPL-S' or SPL-SOH species which escaped the P450 active site after generation. Alternatively, it was also conceivable that as in the case of methimazole-induced P450 loss (8,9), the attenuation of SPL-induced P450 loss by GSH was a reflection of its protective trapping of a destructive metabolite generated by another rat liver microsomal enzyme, the flavin-containing monooxygenase (FMO). Indeed, hog liver FMOs are known to sulfoxidize thioamides and other thio compounds (10-15), including 7a-thiomethyl-SPL (16),to the corresponding sulfoxides. On the other hand, FMO-catalyzed oxygenations of thiocarbamides and organic thiols have been proposed to proceed via a two-electron oxidation process to afford the corresponding sulfenic acids (11-15). Although to our knowledge, the latter products have never directly been identified as FMO-catalyzed products of SPL-SH, nevertheless, such SPL-sulfenic acid generation by FMO in liver microsomes could contribute to SPLmediated microsomal P450 loss. Because sulfenic acids are proposed to react with thiols (11-15,17), it was conceivable that inclusion of GSH in the incubation system would protect P450 simply by trapping the FMO-generated SPL-SOH. Although GSH is also precluded from the FMO active site (15),it can efficiently trap electrophilic metabolites generated by FMO. Thus, irrespective of the catalyst (FMO or P450), a SPL-GSH adduct (SPL-SSG), if formed, should be isolable from the microsomal incubation mixtures. To determine if formation of such an adduct could indeed account for the protective effect of GSH on SPL-mediated P450 loss, the relative formation of SPL-SSG in NADPH-supplemented incubations of liver microsomes from DEX-pretreated rats or purified hepatic FMO from untreated hogs was examined under conditions optimized for either P450- or FMO-dependent SPL-SH
* C. Decker, C. Kennedy, R. P. Mason, and M. A. Correia, unpublished observations.
oxidation. Our findings of these studies and the isolation and unequivocal characterization of the SPL-SSG adduct from such reaction mixtures are described below.
Experimental Procedures Materials. DEX, GSH, dilauroylphosphatidylcholine, ~ - a lecithin, and SPL were purchased from Sigma Chemical Co, St Louis, MO, and Calbiochem Inc., San Diego, CA, respectively. SPL-SH was a gift from G. D. Searle & Co, Skokie, IL. All other chemicals were of analytical reagent grade and were obtained from either Fisher Chemical Co., San Jose, CA, or Mallinckrodt Chemical Works, St Louis, MO. Animal Treatment. Male Sprague-Dawleyrats (200-220g) obtained from Bantin & Kingman, San Jose, CA, were fed and given water ad libitum and then injected intraperitoneallywith DEX (100 mg/ kg, dissolved in corn oil) daily, for 3-4 days. In some experiments, 24 h later, they were given a single intraperitoneal injection of SPL (200 mg/kg) suspended in water with Tween-80 (3 drops/lO mL) and killed 2 h later. Enzyme Preparations. Liver microsomes were prepared as described before (18),from livers that were perfused in situ with ice-cold 0.15 M KCl solution, removed, and homogenized. Protein concentration and P450 content were determined by the method of Lowry et al. (19) and Omura and Sato (20),respectively. Liver FMO was isolated and purified from liver microsomes (a generous gift from Dr. D. M. Ziegler, University of Texas, Austin) obtained from hogs (that to our knowledge had not been treated with any pharmacological agent) by a modification [inclusionof L-a-lecithin (2 mg/mL) throughout the isolation] of the procedure previously described (21). The molar content of the purified FMO preparation was assessed from its absorbance at 450 nm (t = 11.8mM-' cm-') after reduction with NADPH. Incubation Systems. A typical incubation mixture (final volume 3 or 6 mL) contained hepatic microsomes from DEXpretreated rats (3 pM P450), NADPH (1mM), DETAPAC (1.5 mM), SPL (0.5 mM), dissolved in "PC" buffer containing phosphatidylcholine (25 mg/100 mL sonicated in 0.1 M phosphate buffer, pH 7.4), and 0.1 M phosphate buffer, pH 7.4. Appropriate control incubations which excluded SPL or NADPH were always included in parallel. Reactions were started by addition of NADPH and terminated at designated times by gassing with CO and chilling in ice. In some experiments, GSH (5 mM) was also included in the reaction mixtures. In others, the final pH of the incubation was adjusted to 9.0 with 0.25 M sodium borate buffer. Mixtures (finalvolume 0.5 mL) consisting of purified hog liver FMO (2.5 nmol), SPL-SH (0.5 mM), *GSH (5.0 mM), and NADPH (1 mM) were incubated either at pH 7.4 (0.1 M phosphate buffer) or at pH 9.0 (0.25 M sodium borate buffer), at 33 O C for 15 min. Reactions were initiated by the addition of SPL-SH, after a 2-min incubation period, and were terminated with perchloric acid (5% final concentration). Assays. Cytochrome P450 destruction assays were conducted exactly as detailed previously (7), and P450 content was monitored in the incubates as described (22). In some experiments,microsomal FMO was inactivated by preincubating rat liver microsomes at 37 "C for 30 min, before assessment of SPL-induced P450 loss. Control microsomes incubated in parallel at 0 "C for 30 min served as the corresponding controls. Regio- and stereoselective testosterone hydroxylase activitieswere monitored by HPLC by the method reported previously (23). Liver microsomes from DEX-pretreated rats or purified hog liver FMO had characteristically high (p-methoxyphenyl)-l,3dithiolane S-oxygenase activity [3.20 0.58 at pH 7.4 or 548 nmol/(mg of proteimmin) at pH 9.0, respectively], assayed as described (24). FMO-dependent cimetidine S-oxidation was assayed in rat liver microsomes preincubated at 0 or 37 "C for 30 min in an incubation system containing liver microsomes (2 mg of protein/mL), cimetidine (0.2 mM), and NADPH (1 mM) in a final volume of 0.5 mL, by a modification of a procedure previously described (24). The reactions were terminated by the addition of 2 volumes of cold CH,C12/2-propanol(1:l v/v), and after thorough mixing, the insoluble material was removed by a brief centrifugation. After filtration through a 4-rm nylon filter and evaporation, the extract was taken up in methanol for separation and quantificationby HPLC as described previously (25).
Generation of Glutathionyl-Spironolactone Adduct Liver GSH content was determined as described (26). The extent of "redox cycling" of SPL-SH was assessed by assaying the GSSG formed in incubations containing liver microsomes from DEX-pretreated rats (2 mg of protein/mL), SPL (1.0 mM), DETAPAC (1.5 mM), GSH (5.0 mM) with and without NADPH (1.0 mM), and 0.1 M phosphate buffer, pH 7.4, carried out at 37 "C for 30 min. The incubates were sedimented to pellet out the microsomes, and the GSSG in the supernatants was assayed by the method of Hill and Burk (27). In parallel, the FMO substrate ethylenethiourea (1.0 mM) that is well established to undergo redox cycling (12) was included as a positive control. In Vitro Generation and Isolation of SPL Metabolites. Polar metabolites (SPL-sulfinicacid and -sulfonic acid derivatives) were isolated from reaction mixtures identical to that described above, incubated a t 37 "C for 15 min, terminated by gassing with CO and chilling on ice, and then centrifuged a t lOOOOOg for 30 min. The supernatanb were collected, filtered through a 0.45-pm Rainin Nylon-66 filter, and subjected to HPLC on a Rainin Dynamax C18 column (5 pm, 4.6 mm X 20 cm) interfaced with a Hewlett-Packard diode array detector set a t 240 nm, exactly as described (7). Polar metabolites were quantified by integration of the area under the absorption peaks. In some incubations (final volume 6 mL), when SPL-SSG adduct formation was monitored, [3H]GSH (7 pCi) and/or GSH (5 mM) was included and incubated at 37 OC for 15 or 30 min, and reactions were terminated either with perchloric acid (final concentration 5 % ) or by chilling in ice. The mixtures were sedimented at 5000g to remove the precipitated proteins, or at 105000g to remove microsomes, before addition of S-(p-nitrobenzyl)-GSH to the supernatants as an internal standard. The supernatants were then subjected to HPLC on a Rainin Dynamax C18 column (5 pm, 4.6 mm x 20 cm), fitted with a C18 guard column (4.6 mm X 5 cm) interfaced with a Hewlett-Packard diode array (UV) detector set a t 240 nm. The columns were preequilibrated with 38% CH30H/62% 0.02 M CH3COONa (pH 4.0) a t a flow rate of 1 mL/min. Immediately on sample injection, a linear gradient (0-100% over 25 min) of CH30H was developed. The SPL-SSG adduct had a retention time of 16.8min under these conditions3and was quantified either by its [3H]GSHradioactivity or by integration of the area under the corresponding 240-nm absorbance peak. The SPL-SSG adduct was found to be recovered almost quantitatively (~98%)from acid-precipitated incubation mixtures. Similar values were obtained using either method of adduct quantification. For mass spectral analyses of the SPL-SSG adduct, the HPLC conditions were identical to those described above except that the mobile phase consisted of 0.02 M CH3COONH4 (pH 4.0)/ CH30H (5050 v/v). The peak material eluting a t 16.8 min, and characterized by its [3H]GSH radioactivity and 240-nm absorbance, was collected, purified by rechromatography using C H 3 0 H / H 2 0(5050 v/v) as the mobile phase, evaporated under a stream of N2, and lyophilized. The lyophilized material was then esterified (hexylated) with acetyl chloride (0.2 N; 5 pL) in dry hexanol, a t 45 "C for 1 h (28). Aliquots (1 pL) were then subjected to mass spectrometric analyses as described below. Chemical Synthesis of Authentic SPL-SSG Adduct. SPL-SH (2.5 mg) was added to a 50% solution of 0.25 M sodium borate buffer (pH 9.0) in acetonitrile, containing a 2-fold molar excess of oxidized GSH (GSSG). The mixture was stirred at room temperature for 2 h and then subjected to isolation and purification by the HPLC procedures exactly as described above. Material (yield = 0.25 mg) with the characteristic 240-nm absorbance and eluting with the same retention time as the putative enzymatically generated SPL-SSG adduct was isolated from these reactions, derivatized, and subjected to mass spectral analyses as described below. Mass Spectrometric Analyses of SPL-SSG Adduct. Liquid secondary ion mass spectral analyses in the positive mode (+LSIMS) were performed on the derivatized samples (see above) as described (28). A MS-50s (Kratos Analytical Instruments, The differences in retention times (16.8 and 14.5 min) exhibited by the SPL-SSG adduct from rat liver microsomal and hog liver FMO catalyzed incubations, we believe, largely reflect slight differences in the HPLC conditions (columns,temperature) used in each study. In all cases the adduct eluted -1.5 min after the two polar metabolites.
Chem. Res. Toricol., Vol. 4,No. 6,1991 671 Table I. Relative Effects of GSH on P450 Inactivation and Polar Metabolite and SPL-SSG Adduct Formation in Incubations of SPL with Hepatic Microromes from DEX-Pretreated Rats SPL-SSG system" P450 inactivationb polar metabolites* adductb SPL -NADPH 0 NDc ND' 21.6 f 5.6 ND' +NADPH 1.66 f 0.33 SPL + GSH -NADPH 0 NDc 1.78 f 0.89 +NADPH 0.97 f 0.28d (41.6)a 12.3 2.8' (43.l)r 3.87 f 1.58'
*
The complete incubation system contained microsomal P450 (3 nmol/mL). Other details and assay of P450 inactivation, polar metabolite formation, and GSH adduct are described under Experimental Procedures. Incubations were carried out at pH = 7.4 and 37 O C , for 15 min. All values are mean f SD of 7 independent observations. * nmol/(mL incubation.15 min). ND, not detectable. d-fStatistically significant at d p < 0.002, ' p < 0.005, or f p < 0.02, from corresponding SPL + NADPH or SPL + GSH - NADPH values by Student's t test. GSH-mediated attenuation is listed in parentheses, as percent (%) lowering of corresponding SPL + NADPH values. Manchester, U.K.) equipped with a 23-kG magnet and 10-kV postacceleration detector was used for analyses. The +LSIMS ion source (primary Cs+ ion gun) and coolable sample probe were developed a t the UCSF Mass Spectrometry Facility. Trifluoroacetic acid (1%) in thioglycerol was used as the matrix, and the probe temperatures were maintained a t 5-15 "C.
Results Isolation and Structural Characterization of a SPL-SSG Adduct. Aerobic incubation of liver microsomes from DEX-pretreated rats with SPL ( p H 7.4) at 37 "C for 15 m i n resulted in P450 inactivation and in parallel formation of t h e previously characterized polar SPLsulfinic acid and -sulfonic acid metabolites (7), in an N A D P H - d e p e n d e n t process (Table I). Inclusion of GSH in this incubation system attenuated both P450 inactivation and polar metabolite formation (Table 11), in confirmation of our previous report (7).T o specifically search for a n y SPL-SSG adducts, [3H]GSH was also included in the incubation mixtures, and on termination of t h e reaction, t h e s u p e r n a t a n t s were subjected to HPLC using conditions identical t o those previously employed for separation of t h e polar metabolites, except that both 3H radioactivity and absorbance (Azdonm)of t h e a,@-unsaturated ketone function of SPL were monitored. A peak with retention time of ~ 1 6 . min, 8 and thus considerably more hydrophobic than either of the polar metabolites (retention times 15.3 and 15.7 min, respectively), was observed in chromatograms of microsomal incubations containing SPL, GSH, and NADPH (Figure 1). Omission of NADPH resulted in significant lowering of this peak, but not in its abolition, thereby revealing appreciable nonenzymatic autoxidative formation of this putative SPL-SSG a d d u c t (Figure 1). In contrast, exclusion of either SPL or GSH completely prevented a d d u c t formation, thus indicating i t s dependence on b o t h of these cosubstrates. T h e putative SPL-SSG a d d u c t was further purified b y HPLC using M e O H / H 2 0 as t h e mobile phase, lyophilized, and subjected t o +LSIMS mass spectral analyses after hexyl ester derivatization. The mass spectral data ( m / z = 848, dihexyl ester; m/z = 764, monohexyl ester) of this material were consistent with that of a disulfide a d d u c t of SPL and GSH. F u r t h e r confirmation was obtained b y t a n d e m MS/MS collision-induced dissociation (CID) spectrum of t h e p a r e n t MH+ ion (mlz = 848) which was found t o fragment to m/z = 507 a n d m / z = 341, corresponding t o t h e dihexylated GSS a n d steroid moieties, respectively. As discussed previously (7), the base peak in t h e spectrum at m/z 341 m a y have resulted from can-
672 Chem. Res. Toxicol., Vol. 4, No. 6, 1991
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Table 11. Relative Effects of pH on P450 Loss, Polar Metabolite Formation, and Adduct Formation in Incubations of SPL with Liver Microsomes from DEX-Pretreated Rats' 70 P450 lossb polar metabolitesc SPL-SSG adductd DH addition expt 1 expt 2 expt 1 expt 2 expt 1 expt 2 7.4 NADPH 36 (1.08)e 49 (1.47)' 16.6 16.5 NDf 0 GSH 0 0 NDI NDf 2.3 1.2 14.3 8.8 4.5 2.8 26 (0.78)' 16 (0.48)' NADPH + GSH 9.0 NADPH 26 (O.78le 37 ( 1 . 1 l ) e 10.7 13.0 NDf 0 0 NDi NDf 3.2 1.4 0 GSH 7.9 4.3 16 (0.48)' 6.0 6.3 NADPH + GSH 0
'Incubations with liver microsomes (3 nmol/mL incubation) from DEX-pretreated rats were carried out at pH 7.4 or 9.0 and 37 "C, for 15 min as detailed (Experimental Procedures). % P450 loss, cpolar metabolite [nmol/(mL incubation.15 min)], dSPL-SSG adduct production [nmol/(mL incubation.15 min)], and %mol of P450 inactivated/(mL incubation.15 min) were assayed as described under Experimental Procedures. Each value represents the mean of two individual determinations per experiment. 'ND, not detectable. Table 111. Relative Hepatic Microsomal Testosterone Hydroxylase Activities' in DEX-Pretreated Rats at pH 7.4 and 9.0 testosterone hydroxylase activity, nmol of hydroxstestosterone / (me of protein-min) 4-6Ac 160 60 28 ANDb 1 5 ~ 15a 7a 16a 2a PH 6a 7.4 0.18 2.08 0.34 0.28 20.3 2.34 1.33 2.36 4.60 0.43 5.19 0.15 8.52 0.80 9.0 0.04 0.73 0.39 0.63 0.90 1.59 0.12 2.92 Testosterone hydroxylase activities were determined as described (Experimental Procedures). All values represent means of 2 individual determinations. AND, androstenedione. 4,6 diene product.
Table IV. Effects of GSH on SPL-Mediated P450 Loss in 0 and 37 "C Preincubated Rat Liver Microsomes' preincubation cimetidine S-oxidation, incubation P450 content, P450 loss, conditions nmol/(mg of proteinamin) conditions nmol/mg of protein (70basal) nmol/mg of protein 0 "C/30 min 2.47 SPL 1.1 (100) SPL + NADPH 0.57 (52) 0.53 SPL + NADPH + GSH 0.82 (75) 0.28 (53)b 37 OC/30 min 0.35 SPL 1.2 (100) SPL + NADPH 0.52 (44) 0.68 SPL + NADPH + GSH 0.66 (56) 0.54 (21)b
'Liver microsomes from DEX-pretreated rats were preincubated for 30 min at either 0 or 37 "C and then assayed for FMO-dependent cimetidine S-oxidase activity. Aliquots were used to assess SPL-mediated loss in the presence and absence of GSH (5 mM). Values are the mean of two separate determinations. Percent (%) GSH-mediated attenuation of SPL-mediated P450 loss. renone formed on elimination of the dihexylated GSS moiety from the MH+ species. Such structural characterization of SPL-SSG thus confirmed that GSH was capable of trapping an electrophilic SPL species to form a disulfide in an NADPH-dependent fashion. These data however provided no clue as to the nature of this electrophilic species (SPL-S' or SPL-SOH?) or whether it was catalyzed by P450- or FMO-dependent oxidation. Relative Formation of SPL-SSG Adduct under Conditions Optimized for P450- or FMO-Catalyzed S-Oxidations. As an initial approach to identifying the specific S-oxygenase,we exploited the reported difference in the pH optima of the two oxygenases (13,14,29). Thus, the relative SPL-mediated P450 inactivation and formation of the SPL-SSG adduct and the polar metabolites in incubations of SPL/NADPH with DEX-pretreated rat liver microsomes were examined at pH 7.4 and 9.0, the pH optima for P450- and FMO-dependent oxidations, respectively (13,14,29). At pH 7.4, in the absence of GSH, SPL produced a mean P450 loss of 42% and combined polar metabolite formation of the order of 16.5 nmol/(mL incubation.15 min) (loo%), but as expected, no SPL-SSG adduct formation was detected in two separate experiments (Table 11). In contrast, GSH inclusion in the incubations attenuated P450 loss and polar metabolite formation to 21% and 70%, respectively, while doubling the value of SPL-SSG adduct formation over that seen in the absence of NADPH. At pH 9.0, on the other hand, both SPL-mediated P450 loss and polar metabolite formation were considerably reduced (Table 11),consistent with suboptimal P450-dependent catalyses. This was independently confirmed when the relative testosterone hydroxylase
activity of liver microsomes from DEX-pretreated rats was assessed at pH values of 7.4 and 9.0 (Table 111). However, at pH 9.0, GSH further suppressed SPL-elicited P450 loss (Table 11). More importantly, at this pH, although the basal levels of the autoxidatively formed adduct increased appreciably, possibly due to facile nonenzymatic oxidation of thiols a t alkaline pH, GSH inclusion considerably enhanced the NADPH-dependent formation of the SPL-SSG adduct (Table 11). Such relative enhancement of SPL-SSG adduct formation a t a pH that is clearly suboptimal for P450-dependent catalyses but optimal for FMO-dependent oxidations implicated FMO as a key catalyst in the formation of the reactive SPL species, which was capable of destroying microsomal P450 content, if not trapped by GSH. To determine whether FMO was indeed responsible for the formation of a reactive oxidized SPL species, as in the case of methimazole (9), the well-recognized thermal lability of FMO was exploited as follows: Liver microsomes from DEX-pretreated rats were preincubated (in the absence of NADPH) at 0 or 37 "C at pH 7.4 for 30 min, before GSH-induced attenuation of SPL-mediated P450 destruction was examined (Table IV). In parallel, FMOdependent cimetidine S-oxidation of these 0 or 37 "C preincubated microsomes was assayed to determine their FMO activity. Preincubation of liver microsomes at 37 "C (in the presence of DETAPAC to protect P450 3A isozymes) resulted in 86% loss of FMO-dependent activity (Table IV). When these microsomes were further incubated with SPL f NADPH f GSH, although the basal values of SPL-mediated P450 loss were only minimally lowered in 37 "C preincubated microsomes, the GSH-in-
Chem. Res. Toxicol., Vol. 4, No. 6, 1991 673
Generation of Glutathionyl-Spironolactone Adduct
Table V. Formation of the SPL-SSG Adduct in Vitro"with Purified Hog Liver FMO SPL-SSG adduct, nmol/(nmol of FMO-15 min) expt 1 expt 2 system pH = 9.0 pH = 9.0 pH = 7.4 complete 19.2 40.0 5.66 -NADPH 2.12 9.43 2.73 -GSH NDb -FMO 2.77 -FMO, -NADPH 8.76 1.31 -SPL-SH NDb =Complete incubation systems with purified FMO, in 0.25 M sodium borate buffer (pH = 9.0) or 0.1 M potassium phosphate buffer (pH = 7.4), were as detailed (ExperimentalProcedures). All values represent means of 2 individual determinations with separate FMO preparations used in each experiment. bND, not detectable.
D
676
I
I
31 1
I
130
IS7
315
I 3 (
I*
MSO
Figure 1. Formation of polar metabolites (PM) and SPL-SSG adduct from SPL in rat liver microsomal incubations: HPLC profiles and mass spectral characterization of the SPL-SSG adduct. Top: Formation of P M and/or SPL-SSG adduct in incubations of liver microsomes from DEX-pretreated rats, supplemented with (A) SPL + NADPH; (B) SPL +NADPH + GSH; and (C) SPL + GSH. For details see Experimental Procedures. Note: Aliquots of incubation supernatants subjected to HPLC were 0.5,0.85,and 0.85 mL, respectively, in panels A, B, and C. The two polar metabolites (same order of elution) have been previously identified as the sulfonic and sulfinic acid derivatives of SPGSH (7).Bottom: (D) +LSIMS data, (E) tandem MS/MS CID of the parent dihexylated MH' ion (m/z = 848). For details see Experimental Procedures.
duced attenuation of P450 loss was considerably reduced from 53% in fresh (0 "C) microsomes to 21% in 37 "C preincubated microsomes (Table IV). These findings, as in the case of methimazole (9),revealed that FMO might significantly contribute to GSH-preventable P450 loss. Unfortunately, the relative thermal lability of P450 3A isozymes in DEX-pretreated rat liver microsomes precluded the longer preincubation time required for full inactivation of FMO. Role of FMO in SPL-SSGFormation. To directly examine the role of this enzyme in metabolizing SPL to a GSH-trappable electrophilic species, the relative formation of the SPL-SSG adduct by purified hog liver FMO was examined in vitro at pH 7.4 and 9.0 (Table V). Deacetylated SPL (SPL-SH) was used as the substrate, and incubations were carried out at 33 "C for 15 min, followed by HPLC under conditions similar to those described above. The adduct was identified by its characteristic retention time3 and UV-absorption characteristics (Figure 2). Although detectable nonenzymatic SPL-SSG formation was observed a t pH 9.0 when FMO or NADPH was excluded, inclusion of both stimulated SPL-SSG formation 4-6-fold in two separate experiments, whereas a t pH 7.4, only a 2-fold increase was observed (Table V). These findings thus directly show that, in the presence of GSH, FMO efficiently catalyzes the formation of a SPL-derived species that has the spectral and chromatographic characteristics of the corresponding adduct isolated from P450-catalyzed reactions (see above). To unequivocally characterize this species as the SPLSSG adduct, the material was separated by HPLC, collected, and purified by rechromatography. The purified adduct was derivatized (hexylation) and subjected to +LSIMS, along with the chemically synthesized counterpart, which was subjected to identical separation, purification, and derivatization procedures. The mass spectral data for both the FMO-catalyzed product and the synthetic SPL-SSG adduct ( m / z = 848 and m/z = 932, corresponding to the dihexyl and trihexyl esters of SPL-SSG adducts, respectively) are consistent with the presence of the adduct in this peak material. Collision-induced dissociation (CID) MS/MS, of the MH' "parent ion" 848, afforded the corresponding daughter ions a t m / z = 507 for the dihexyl GSSH fragment and m / z = 341 for the steroid moiety. These findings thus unambiguously establish the structural identity of the FMO-catalyzed adduct as SPL-SSG. GSSG Formation Associated with GSH-Mediated Attenuation of Oxidized SPL Metabolites. FMOmediated oxidation of some thiol compounds (meth-
Decker et al.
674 Chem. Res. ToxicoZ., VoZ. 4, No. 6,1991
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Figure 2. Purified hog liver FMO-catalyzed SPL-SSG adduct formation: HPLC profiles and mass spectral characterization. Panels A-C: Formation of SPL-SSG adduct in incubations of purified hog liver FMO, supplemented with (A) SPL-SH + NADPH + GSH and (B) SPL-SH + GSH. (C) SPL-SH + GSH incubated under the same conditions without FMO or NADPH. For details see Experimental Procedures. Equivalent aliquots (0.25 mL) were subjected to HPLC in all cases. Panels D-G: (D) +LSIMS data; (E) tandem MS/MS CID of the parent MH+ ion ( m / z = 848) of FMO-generated SPL-SSG adduct. (F and G) Corresponding data for the chemically synthesized SPL-SSG adduct. These particular spectra were obtained with a VG-70 mass spectrometer (Manchester, England). For other details see Experimental Procedures. Note: We believe the exhaustive hexylation conditions produced a mixture of di- and trihexylated SPL-SSG adducts, as indicated by the presence of both m / z = 848 and m / z = 932 in the +LSIMS spectra. The latter was most likely derived from hexylation of the lactone and elimination of an H atom to yield a double bond. Consistent with this assignment, addition of sodium to this species in the gas phase resulted in the observed m / z = 954.
imazole, ethylenethiourea) in the presence of GSH is known to regenerate the parent compound at the expense of two molecules of the reductant, resulting in GSSG in a process termed "redox cycling" (12,17). GSSG would also be generated after GSH-mediated reduction of SPL-S' formed from one-electron oxidation of SPL-SH by either P450 or transition-metal catalysis. Thus, dimerization of two GS' radicals would also afford GSSG. To determine whether either of these possibilities could explain the GSH-mediated attenuation of SPL-elicited P450 loss, the extent of GSSG formation was assessed in incubations of liver microsomes from DEX-pretreated rats, comparable to those described in Table I. Under the same conditions wherein ethylenethiourea, an excellent substrate for hog
liver FMO,produced GSSG (12),no significant GSSG could be detected after inclusion of SPL in GSH- and NADPH-containing systems above that of the corresponding NADPH-devoid controls (Table VI). In Vivo Effects of SPL on Hepatic GSH. GSH is an abundant hepatocellular nucleophile, and its depletion after drug ingestion often serves as an index of the in vivo extent of metabolic activation of drugs to reactive electrophiles (30; ref 31 and references therein). To determine the in vivo extent of FMO- and/or P450-catalyzed reactive SPL-derived species, hepatic GSH content was examined in both untreated and DEX-pretreated rats given SPL (Table VII). Within 2 h of treatment, along with a significant 63% hepatic P450 loss, GSH content was also
Generation of Glutathionyl-Spironolactone Adduct Table VI. Relative Formation of GSSG in NADPH-SupplementedRat Liver Microsomal Incubations of SPL or Other FMO Substratesa drug NADPH GSSG formed, pM 66.5 SPL SPL + 32.3 ethylenethiourea 70.3 ethylenethiourea + 411.0
Chem. Res. Toxicol., Vol. 4, No. 6, 1991 675 Scheme I. P460- and FMO-Dependent Pathways of SPL-SII Oxidation and SPL-SSG Adduct Formation"
"Incubations were carried out at 37 O C for 30 min with liver microsomes from DEX-pretreated rats in the presence of GSH (5 mM) and a drug (1.0 mM), with and without NADPH (1.0 mM) at pH 7.4, as detailed under Experimental Procedures. After incubation, the mixtures were centrifuged and the GSSG was assayed in the supernatants exactly as described by Hill and Burk (27). Values are the means of 2 separate incubations, each assayed in duplicate.
SPL
**
a Rats,
untreated or pretreated with DEX, were administered SPL intraperitoneally as detailed (Experimental Procedures). All values represent the mean &SD of 3 individual animals. P450 in nmol/mg of protein. 'GSH in rmol/g of liver. d*eStatisticalsignificance by Student's t test from corresponding non-SPL-treated controls: dp C 0.01; ' p C 0.05.
significantly lowered in SPL-treated DEX-pretreated rats. Although comparable hepatic GSH loss was also found in SPL-administered untreated rats, it failed to reach statistical significance (Table VII). These findings indicate that SPL results not only in hepatic P450 loss but also in appreciable hepatic GSH depletion in intact rats.
Dlscussion Collectively, the above findings indicate that NADPHsupplemented liver microsomes from DEX-pretreated rats metabolize SPL to an electrophilic species that is trapped by GSH and which we have unambiguously identified as the SPL-SSG adduct. Formation of this adduct is dependent on GSH, NADPH, and SPL, and although significant at pH 7.4, is enhanced at pH 9.0, the pH optimum for FMO- but not P450-dependent reactions. Indeed, replacement of rat liver microsomes with purified hog liver FMO in NADPH- and GSH-supplemented incubations resulted in a highly efficient conversion of SPL to the SPL-SSG adduct. Formation of the SPL-SSG adduct, we believe, accounts for the GSH-mediated protection of rat liver microsomal P450 from SPL-induced destruction. Because such protection was considerably attenuated by thermal inactivation of FMO in these microsomes, it is tempting to speculate that FMO-dependent SPL oxidation may appreciably contribute to the observed GSH-inhibitable component of SPL-mediated P450 destruction. A precedent for a similar contribution of FMO to methimazole-induced P450 destruction has been reported (9). Such events imply that FMO-generated reactive SPL metabolite(s) can diffuse out of the FMO active site and, if not trapped by external nucleophiles such as GSH or accessible microsomal sulfhydryls, attack P4509. Indeed, such a scenario may also partly rationalize our previously reported finding of NADPH-dependent covalent [ 14C]SPL binding to microsomal proteins and its significant attenuation by GSH and/or DTT (7). However, because in vitro levels of SPL-mediated P450 destruction were not substantially
o &
SPL-SOH
Table VII. Effect of SPL on Rat Hepatic P460 and GSH Content in Vivoa treatment DEX pretreated untreated SPL SPL none content none P450b 1.50 0.16 0.59 & 0.13d 0.94 0.13 0.87 0.01 GSH' 4.43 f 0.68 3.16 0.26e 5.60 1.09 4.09 0.70
*
-
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SPL-SH
SPL-S'
dS, __.._._*
0
-
SPL SS0
SPL-SO2H
SPL-SOaH '%
"SPL-S02H and SPL-SOBHare the sulfinic and sulfonic acid derivatives of SPL-SH, respectively.
reduced after FMO inactivation, we infer that P450 destruction can occur directly via its oxidation of SPL in a mechanism-based inactivation p r o c e ~ s as , ~ well as indirectly, by reactive species generated by the neighboring FMO. P450s indeed have been proposed to oxidize other thio compounds (thiopurine, methimazole) to the corresponding sulfenic acids (9,32,33),although in no case has the structure of the metabolite been determined. Thus, although the relative contributions of each enzymatic pathway may vary with the experimental conditions, it remains to be determined whether the SPL-derived destructive species, their site(s) of attack, and thus the mechanism of the P450 destruction are similar or differ in each instance. NADPH-dependent oxidation of SPL-SH by purified FMO in the presence of GSH exclusively yielded the SPL-SSG adduct. No polar SPL metabolites were detected even in the absence of GSH. Similarly, in incubations (devoid of GSH) of liver microsomes from DEXpretreated rats a t pH 9.0, despite the concomitantly increased levels of GSH-trappable SPL species, no corresponding increase in the polar metabolites was detected. These findings together with the observed relative predominance of SPL-SSG adducts versus that of the sulfinic and sulfonic acid species in microsomal incubations optimized for FMO-dependent catalyses, and the reverse situation in those optimized for P450-dependent catalyses, tempt us to speculate that the SPL-SSG adduct might be largely derived from the two-electron-oxidizedsulfenic acid species. Under these circumstances, it is possible that the two polar metabolites might be largely derived from further (n~nenzymatic?)~ oxidations of the one-electron-oxIndeed, our previous finding that n-octylamine (a stimulator of FMO-catalyzed reactions but an inhibitor of P450-dependent oxidations) significant attenuates SPL-mediated P450 loss is consistent with a predominant role for P450 in ita inactivation (7). Although in principle, formation of the SPL-sulfinicacid and -sulfonic acid species by further oxidation of SPL-SOH within the P450 active site is plausible and cannot be entirely excluded, it would require two and three P450 catalytic cycles, respectively.
Decker et al.
676 Chem. Res. Toxicol., Vol. 4, No. 6,1991
idized SPL-S' species (Scheme I). The observed GSHmediated attenuation of polar metabolites (Table I) thus could be due at least in part to GSH quenching of the SPL-S' species on its migration outside the P450 active site. Although it is generally accepted that FMO catalyses entail two-electron processes (13-15, 17),the role of microsomal P450 in the catalyses of one-electron-oxidized products (34) has recently been challenged, and such radical generation has been attributed largely to transition metals contaminating the microsomal preparations (35). Because of this possibility, we verified polar SPL metabolite formation under conditions reported to largely minimize transition-metal-catalyzed reactions [Chelexed buffers and Chelex-treated rat liver microsomes by the batch method of Buettner (36)l6and found that such polar SPL metabolite formation was only minimally, if at all, attenuated (results not shown). Given these results, we propose that the formation of the SPL-SSG adduct might largely reflect GSH attack on the two-electron-oxidized SPL-SOH species? whereas the polar metabolites might represent further oxidation products of the one-electronoxidized SPL-S' (Scheme I). In addition, we believe the above findings support the reaction scheme for the reduction of FMO-catalyzed organic sulfenic acids (RSOH) by thiols proposed by Ziegler and co-workers (11-15,17). Their scheme envisages nonenzymatic conjugation of the RSOH with a thiol (GSH) to form RSSG, with subsequent reduction of the disulfide by another GSH molecule to regenerate the parent thiol (RSH) and produce GSSG, which is excreted out of the cell. The isolation and identification of the SPL-SSG adduct as a product of FMO-catalyzed oxidations not only are entirely consistent with such a reactino scheme (11-15, 17) but also provide the first tangible evidence of the intermediacy of a sulfenic acid-GSH conjugate in such reactions. However, the formation of the SPL-SSG adduct without any detectable concomitant GSSH accumulation (Table VI) suggests that FMO-catalyzed oxidation of SPL in the presence of GSH appears not to undergo "redox cycling" as observed with other FMO substrates such as ethylenethiourea (Table VI; 12) or methimazole (17). Thus, the SPL-SSG adduct appears to be relatively more chemically stable than the corresponding adducts of the other two drugs. Moreover, lack of GSSG accumulation in NADPH-supplemented incubations of liver microsomes from DEX-pretreated rats with SPL and GSH also argues against significant GSH-mediated reduction of the oneelectron-oxidized SPL-s' radicals to SPL-SH, in this system. Finally, it also appears that the formation of this SPLSSG adduct by either hepatic oxygenase might be physiologically relevant, since SPL administration to rats was found to significantly deplete hepatic GSH. In theory, such depletion could have been due to cooxidation of GSH to GSSG during reduction of SPL-SOH/SPL-SSG, as proposed for other sulfenic acids (17). But the lack of In our hands, in the presence of catalase, this treatment with or without deferoxamine washing of rat liver microsomes did not further attenuate P450-dependent one-electron oxidation of 3,5-dimethylhl-4ethyl-l,4-dihydropyridine to the corresponding 4-deethylatedproduct (K. Su iyama and M. A. Correia, preliminary findings). A t the relative concentrations of SPL-SH and GSH employed,GSH attack on the one-electron-oxidizedSPL-S' species, on the other hand, would principally result in the reduction of the radical to SPL-SH with concomitant formation of GS' and radical recombinationto GSSG rather than SPL-SSG formation. The absence of significant GSSG formation in the NADPH-aupplemented incubations of microsomes with SPL (Table VI) argues against the viability of this SPL-s' detoxification route.
e
substantial GSSG formation at physiologically relevant pHs suggests that direct GSH conjugation with SPL-SOH to form the adduct might primarily account for such hepatic GSH depletion. Although, per se not dramatic, it is conceivable that SPL-induced GSH depletion might compromise the detoxification/ elimination of other coadministered drugs that more heavily rely on this conjugation pathway. Such an impairment may be particularly significant in species (including humans) whose relative hepatic microsomal FMO levels and thus the FMO/P450 ratios are apparently higher than those of hepatic microsomes from Sprague-Dawley rats (37,38),the species employed in this study.
Acknowledgment. These studies were supported in part by NIH Grants DK-26506 (M.A.C.) and GM-36426 (J.R.C.), Training Grant GM-07175, and a Society of Toxicology Hazleton Graduate Student Award (C.J.D.). We gratefully acknowledge the able technical assistance of Ms. E. Soliven in some experiments and Mr. Fred Walls for obtaining the tandem MS/MS data. We also acknowledge the UCSF Liver Center Core Facilities in Spectrophotometry (supported by NIADDK 26743) and Bioorganic Biomedical Mass Spectrometry Resource (A. L. Burlingame, Director; supported by NIADDK 26743 and NIH Division of Research Resources Grant RR016614). Registry No. Spironolactone, 52-01-7; glutathione, 70-18-8; cytochrome P450, 9035-51-2; monooxygenase, 9038-14-6.
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