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Abstract
The drug anetholedithiolethione (ADT) and its analogs have been extensively used as H2S donors. However, the mechanism of H2S formation from ADT under biologic conditions remains almost completely unknown. This article shows that only small amounts of H2S are formed during incubation of ADT and of its metabolite anetholedithiolone (ADO) with rat liver cytosol or with rat liver microsomes (RLM) in the absence of NADPH, indicating that H2S formation under these conditions is of hydrolytic origin only to a minor extent. By contrast, much greater amounts of H2S are formed upon incubation of ADT and ADO with RLM in the presence of NADPH and dioxygen, with a concomitant formation of H2S and para-methoxy-acetophenone (pMA). Moreover, H2S and pMA formation under those conditions are greatly inhibited in the presence of N-benzyl-imidazole indicating the involvement of cytochrome P450-dependent monooxygenases. Mechanistic studies show the intermediate formation of the ADT-derived 1,2-dithiolium cation and of the ADO sulfoxide during microsomal metabolism of ADT and ADO, respectively. This article proposes the first detailed mechanisms for the formation of H2S from microsomal metabolism of ADT and ADO in agreement with those data and with previously published data on the metabolism of compounds involving a C=S bond. Finally, this article shows for the first time that ADO is a better H2S donor than ADT under those conditions.
SIGNIFICANCE STATEMENT Incubation of anetholedithiolethione (ADT) or its metabolite anetholedithiolone (ADO) in the presence of rat liver microsomes, NADPH, and O2 leads to H2S. This article shows for the first time that this H2S formation involves several steps catalyzed by microsomal monooxygenases and that ADO is a better H2S donor than ADT. We propose the first detailed mechanisms for the formation of H2S from the microsomal metabolism of ADT and ADO.
Introduction
Anetholedithiolethione (ADT, formula in Fig. 1) is a drug that has been used for many years for its choleretic and sialogogic properties (Christen, 1995; Hamada et al., 1999; Nagano and Takeyama, 2001). ADT and several dithiolethiones are also tested as cancer chemoprevention agents whose mechanisms of action involve activation of Nrf2 signaling and induction of phase 2 enzymes (Zhang and Munday, 2008; Ansari et al., 2018). ADT and its O-demethylated derivative dmADT were also extensively used as H2S donors, and the coupling of dmADT with numerous anti-inflammatory drugs has led to a variety of compounds described for their H2S-donor properties and therapeutic effects (Li et al., 2007; Chen et al., 2010; Lee et al., 2010; Sparatore et al., 2011; Kashfi and Olson, 2013; Couto et al., 2015; Szabo and Papapetropoulos, 2017; Ansari et al., 2018; Powell et al., 2018). However, the mechanism of H2S formation from ADT, dmADT, and their derivatives under biologic conditions remains almost completely unknown (Szabo and Papapetropoulos, 2017; Ansari et al., 2018; Powell et al., 2018).
Formula of the main metabolites formed during microsomal metabolism of ADT [from Dulac et al., 2018].
We have recently reported that the oxidative metabolism of ADT by liver microsomes not only leads to dmADT but also to several products deriving from an S-oxidation of ADT, such as ADTSO, ADO, and para-methoxy-acetophenone (pMA) (Fig. 1) (Dulac et al., 2018). Under these conditions, the corresponding products deriving from dmADT, such as dmADTSO, dmADO, and para-hydroxy-acetophenone (pHA), are also formed (Fig. 1) (Dulac et al., 2018). Metabolism of ADO by the same microsomes leads to pMA, in which reaction the two sulfur atoms and one carbon atom of ADO are lost. Is there formation of H2S during the cleavage of the heterocycle of ADT and ADO? In the study described in this article, we followed the formation of H2S during the metabolism of ADT and ADO by rat liver microsomes (RLM) and found that ADO is a much better H2S donor than ADT under these conditions. H2S formation mainly occurs in the presence of liver microsomes and NADPH, the necessary cofactor of microsomal monooxygenases. Possible mechanisms for H2S and pMA formation from ADT and ADO are proposed on the basis of these results.
Materials and Methods
General Reagents and Synthesis of Authentic Samples.
The commercial origins of ADT, pHA, pMA, NADPH, N-benzyl-imidazole (Bz-ImH), and the solvents used were indicated previously (Dulac et al., 2018). All authentic metabolites of ADT were prepared as described previously (Dulac et al., 2018) and displayed 1H and 13C NMR, UV-visible, and mass spectra in accordance with their structures. Nuclear magnetic resonance spectra were recorded on a Bruker 500 AV2 spectrometer. The mass spectra were recorded on an Exactive HRMS-instrument (Thermo, Les Ulis, France).
Synthesis of Dithiolium Hydrogensulfate.
Dithiolium hydrogensulfate (DT) was prepared from ADT following a previously described protocol (Klingsberg, 1961). Five millimoles of ADT (1.2 g) were dissolved in 50 ml of acetone, and 2.8 g of 40% peracetic acid was added at 0°C. After 10 minutes, the precipitate was filtered, washed with acetone, and dried (1.4 g, 92%). 1H NMR (D2O): 10.04 (d, 1H, J = 5.0), 8.71 (d, 1H, J = 5.0), 8.06 (d, 2H, J = 9.0), 7.17 (d, 2H, J = 9.0), 3.97 (s, 3H). 13C NMR (D2O): 191.3, 171.2, 165.5, 134.8, 131.9, 123.4, 115.9, 56.0. HRMS: m/z value for C10H9OS2: calc.: 209.0089, found: 209.0084. UV (MeOH) λmax: 413 and 260 nm; UV (0.1 M HCl) λmax: 404 and 290 nm.
Synthesis of ADOSO.
ADOSO was prepared from ADO by oxidation with dimethyldioxirane (see formula in Fig. 3) following modifications of a previously described protocol (Tardif and Harp, 2000). A solution of ADO (11.2 mg, 0.05 mmol) in 1 ml acetone was cooled to – 78°C and 1.4 ml of a solution of 0.04 M dimethyldioxirane in acetone [prepared as indicated in Adam et al. (1987)] was added. The temperature was allowed to rise to room temperature in 1 hour and the solvent was evaporated under vacuum. The crude product was found to be pure ADOSO. 1H NMR (CDCl3): 7.71 (d, 2H, J = 9.0), 7.02 (d, 2H, J = 9.0), 6.81 (s, 1H), 3.88 (s, 3H). 13C NMR (CDCl3): 190.2, 171.6, 163.6, 130.7, 121.8, 121.6, 115.0, 55.9. HRMS: m/z value for C10H8O3S2: calc.: 239.9915, found: 239.9908. UV (MeOH) λmax: 306 nm.
Origin of Rat Liver Cytosols and Microsomes, and of Human Liver Microsomes.
Rat liver microsomes and cytosols were prepared as previously reported (Kremers et al., 1981) from male Sprague-Dawley rats (Charles River, L’Arbresle, France) untreated (UT-RLM) or treated either with phenobarbital (PB) (50 mg.kg−1, in 0.9% NaCl, i.p. for 4 days) or β-naphthoflavone (β-NF) (50 mg.kg−1, in corn oil, i.p. for 4 days), two inducers of cytochromes P450 (P450) (Williams et al., 2005). Human liver microsomes were from Corning Inc. (Corning, Amsterdam, The Netherlands). Protein concentrations and P450 contents were determined as described previously (Bradford, 1976, and Omura and Sato, 1964, respectively).
Typical Microsomal Incubation Procedures with Measurement of H2S Formation.
Hydrogen sulfide formation was measured using the methylene blue method (Fogo and Popowsky, 1949; Li et al., 2007; Giustarini et al., 2014). In this assay, hydrogen sulfide is trapped by ZnSO4 forming ZnS. Under acidic conditions, H2S redissolves and reacts with N,N-dimethyl-para-phenylenediamine in an oxidative coupling reaction catalyzed by ferric chloride and forming methylene blue detected spectrophotometrically (λmax: 670 nm). The usual assay mixture (final volume 300 μl) contained cytosolic (2.5 mg/ml) or microsomal (1.5 mg/ml, about 2.5 μM P450) proteins, 0.1 mM substrate, and 1 mM ZnSO4 in 50 mM phosphate buffer, pH 7.4. The mixtures were preincubated for 2 minutes at 37°C, and the reactions were started by the addition of 1 mM NADPH. Usual incubations were carried out for 0–90 minutes at 37°C. The reactions were quenched by the addition of 100 μl of 20 mM N,N-dimethyl-para-phenylenediamine sulfate (in 7.2 M HCl) and 100 μl of 30 mM FeCl3 (in 1.2 M HCl). After 10 minutes at room temperature, the proteins were precipitated by centrifugation (13,000 rpm, 10 minutes), and absorbance at 690 nm of aliquots (200 μl) was determined using a 96-well microplate reader (PowerWaveXS; BioTek Instrument, Colmar, France). The H2S concentrations were calculated against a calibration curve of Na2S treated under identical conditions as the incubation mixtures. Incubations performed in the absence of O2 were conducted as above, except that all components were prepared in potassium phosphate buffer previously bubbled with Argon for 30 minutes. Data are means ± S.D. from three to six experiments. Square deviation errors were determined by using the Microsoft Office Excel 2007 (STDEVPA function) software.
Analyses of Metabolites.
Incubations (final volumes 200 μl) were performed under conditions identical to those described earlier, except that ZnSO4 was omitted and reactions were quenched by the addition of 100 μl of cold methanol containing 50 μM 5-(p-chlorophenyl)-3H-1,2-dithiole-3-thione (internal standard). Treatment of the mixtures and their study by high-performance liquid chromatography–mass spectrometry (HPLC-MS) were done as described previously (Dulac et al., 2018).
Results and Discussion
Formation of H2S upon Microsomal Metabolism of ADT and ADO.
The formation of H2S was followed by using the methylene blue method (Fogo and Popowsky, 1949; Giustarini et al., 2014) during the metabolism of ADT and ADO by liver cytosol or microsomes from PB-pretreated rats (see Materials and Methods). Table 1 shows that H2S was formed upon incubation of 100 μM ADT with liver microsomes from PB-treated rats in the presence of NADPH in a yield of about 13% after 1 hour. Interestingly, metabolism of ADO led to a much better H2S yield (about 40%) under identical conditions. It is noteworthy that only low amounts of H2S were formed under the same conditions but in the absence of NADPH. Moreover, incubation of ADT or ADO with rat liver cytosol under the same conditions in the presence or in the absence of NADPH led to very low amounts of H2S (Table 1). H2S formation from metabolism of ADT or ADO with liver microsomes in the presence of NADPH greatly decreased in incubations performed under anaerobic conditions (Table1). These data indicated that microsomal formation of H2S from ADT or ADO mainly comes from an oxidation of these substrates catalyzed by microsomal monooxygenases. The inhibition of H2S formation in aerobic microsomal incubations performed in the presence of Bz-ImH, a usual inhibitor of P450 (Testa and Jenner, 1981; Correia and Ortiz de Montellano, 2005) (Table 1), showed that the microsomal oxidations of ADT and ADO responsible for H2S formation are mainly catalyzed by P450-dependent monooxygenases.
Formation of H2S upon metabolism of ADT and ADO by liver cytosol or microsomes from PB-pretreated rats
Similar results were obtained when either liver microsomes from untreated or β-NF-pretreated rats (UT-RLM or β-NF RLM) or human liver microsomes (HLM) were used, in the presence of NADPH, which led to yields of about 6% and 22% (UT-RLM), 12% and 42% (β-NF-RLM), or 9% and 17% (HLM) H2S formation from ADT and ADO, respectively (data not shown).
When one simultaneously followed the formation of H2S and the organic products derived from ADT or ADO in the presence of NADPH-supplemented microsomes, one always observed a concomitant formation of H2S and pMA as a function of time. This is illustrated in Fig. 2, which shows the formation of H2S and pMA during the metabolism of ADO by liver microsomes from PB-pretreated rats (35% yield on the basis of starting ADO, corresponding to about 14 total P450 turnovers). This is also illustrated in Table 1, which shows that similar amounts of H2S and pMA are formed in all the conditions used to metabolize ADT and ADO. Altogether these data indicate that H2S is formed during the metabolism of ADT and ADO to pMA. This is in agreement with the much lower yield of H2S observed upon microsomal metabolism of ADT that was found to lead to dmADT as a major product (about 65% yield) and only to lower amounts of pMA under similar conditions (Dulac et al., 2018). The concomitant formation of H2S and pMA from microsomal oxidation of ADT and ADO is in agreement with the fact that both H2S (this work) and pMA (Dulac et al., 2018) are mainly derived from reactions catalyzed by microsomal monooxygenases.
Representative kinetics of formation of H2S and pMA upon oxidation of 100 μM ADO by liver microsomes from PB-pretreated rats (2.5 μM P450) in the presence of 1 mM NADPH. Incubations (500 μl) were performed and stopped at the times indicated in Materials and Methods. H2S amounts (●) were measured by the methylene blue assay from 300 μl aliquots, whereas the pMA levels (❍) were obtained from 200 μl aliquots analyzed by HPLC with UV-visible detection.
Possible Mechanism of H2S Formation during Microsomal Metabolism of ADO.
The results just described indicate that H2S formation from microsomal metabolism of ADO is catalyzed by monooxygenases. A possible mechanism for this reaction is shown in Fig. 3. It would involve an S-oxidation of ADO followed by the opening of the S–S bond of the intermediate (ADOSO) by H2O to give intermediate 1. The hydrolysis of the HSC=O function of 1 should lead to H2S and compound 2. Decarboxylation of 2 would lead to 3, the disulfoxide of para-methoxy-thioacetophenone. Further monooxygenation of 3 should lead to pMA, as previously found in microsomal oxidation of compounds involving a C=S bond (Cashman and Hanzlik, 1982; Hanzlik and Cashman, 1983; Vannelli et al., 2002; Testa and Krämer, 2007).
Possible mechanism for the microsomal oxidation of ADO with formation of H2S and pMA (Ar = CH3O-C6H4-). + “O” on some arrows indicates that the corresponding step involves a monooxygenation of the substrate.
The sulfoxide intermediate proposed in Fig. 3, ADOSO, was synthesized following a previously described method (Tardif and Harp, 2000) (see Materials and Methods) and found to be stable in the buffer used for microsomal incubations (Table 2). In the presence of RLM without NADPH, it led to H2S formation in a 33% yield after a 1-hour incubation. This result is in agreement with the mechanism shown in Fig. 3, in which a simple hydrolysis of ADOSO leads to the formation of H2S. However, hydrolysis of the O=S–S bond of ADOSO appears to be catalyzed by liver microsomal proteins as it does not occur in buffer alone (Table 2). In the presence of liver microsomes and NADPH, the H2S yield increased up to about 61% (Table 2). This could be a result of an S-oxidation of the S=O group of ADOSO catalyzed by microsomal monooxygenases followed by the hydrolytic opening of the resulting O2S–S bond and hydrolysis of the HSC=O bond.
Formation of H2S upon incubation of ADOSO and DT in the presence of RLM
Possible Mechanism of H2S Formation during Microsomal Metabolism of ADT.
The possible formation of intermediates in the metabolism of ADT to H2S and pMA was evaluated from a high-performance liquid chromatography–high-resolution mass spectrometry (HRMS) study of incubates of ADT with liver microsomes from PB-pretreated rats in the presence of NADPH. During the metabolism of ADT under these conditions, one could detect, in addition to the previously reported metabolites dmADT, ADTSO, ADO, dmADO, and pMA (Dulac et al., 2018), small amounts of the 1,2-dithiolium cation DT (Fig. 4), derived from a loss of the sulfur atom of the ADT C=S group. DT was characterized by its high-resolution mass spectrometry molecular peak at m/z = 209.0084 (theoretical m/z value for C10H9OS2 = 209.0089). The formation of the hydrogensulfate of such a 1,2-dithiolium ion has been reported in the oxidation of 4-phenyl-1,2-dithiol-3-thione, an ADT analog, by three equivalents of peracetic acid (Klingsberg, 1961). Oxidation of ADT by peracetic acid under such conditions led us to DT hydrogensulfate, the cation characterized by 1H NMR and HRMS (see Materials and Methods). The HRMS spectrum of this authentic compound was identical to that of DT detected during ADT microsomal oxidation. Incubation of 100 μM DT hydrogensulfate with liver microsomes from PB-pretreated rats in the presence of NADPH led to H2S (38% yield, Table 2) and pMA (30% yield, data not shown), respectively, after 1 hour.
Possible mechanisms for the microsomal oxidation of ADT with formation of H2S and pMA involving DT or ADO as intermediates. Possible mechanism for the microsomal oxidation of ADO with formation of pMA and H2S are shown in Fig. 3. The meaning of Ar and + “O” are given in the legend of Fig. 3.
Thus, ADO and DT are formed during microsomal metabolism of ADT and lead to H2S and pMA when incubated with RLM in the presence of NADPH. Possible mechanisms for their formation upon microsomal oxidation of ADT are shown in Fig. 4. They would involve a common intermediate, previously found in microsomal S-oxidation of compounds involving a C=S function (Cashman and Hanzlik, 1982; Hanzlik and Cashman, 1983; Vannelli et al., 2002; Testa and Krämer, 2007), that results from two successive S-oxidations of the C=S function (Cashman and Hanzlik, 1982; Hanzlik and Cashman, 1983; Vannelli et al., 2002; Testa and Krämer, 2007), intermediate 4 in the case of ADT. Monooxygenation of 4 leading to 5 could be followed by an attack of H2O either on the carbon atom of 5 leading to 6 (Fig. 4, Pathway A) or on the highly oxidized sulfur atom of 5 leading to 8 (Fig. 4, Pathway B). In pathway A, further S-oxidation of intermediate 6 would lead to intermediate 7. The loss of a hydrogensulfate moiety from 7 would then lead to ADO (Fig. 4). Possible microsomal mechanisms for the formation of H2S from ADO have been discussed in the previous paragraph (Fig. 3). In pathway B, the loss of a hydrogensulfate moiety of intermediate 8 would lead to the DT cation. According to this mechanism, oxidation of ADT to DT should require three successive monooxygenations catalyzed by microsomal monooxygenases. This is in agreement with the oxidation of 1,2-dithiolthiones to dithiolium hydrogensulfates by peracetic acid that was reported to consume 3 mol of peracetic acid (Klingsberg, 1961).
H2S formation from microsomal metabolism of DT could then involve an addition of H2O to DT leading to 9, followed by a S–S bond cleavage resulting in the formation of intermediate 10. Hydrolysis of the HOC=S function of 10 should lead to H2S and 11, the decarboxylation of which should give para-methoxy-thioacetophenone 12 (Fig. 4). This mechanism proposed in Fig. 4 for the formation of H2S by simple hydrolysis of DT is in agreement with the results of Table 2 showing that incubation of DT in buffer alone or with microsomes without NADPH did lead to H2S (with yields of 6% and 26%, respectively). The higher yield observed in the presence of microsomes suggest that the hydrolytic opening of DT would be catalyzed by microsomal proteins. The further increase of the H2S formation yield (38%, Table 2) in the presence of microsomes and NADPH could be the result of an oxidation of DT to ADO, followed by the oxidation of ADO with formation of H2S as described in Fig. 3.
The final steps of the formation of pMA from microsomal metabolism of DT would involve an oxidation of 12 to pMA that should occur as expected in microsomal oxidation of compounds involving a C=S bond (Cashman and Hanzlik, 1982; Hanzlik and Cashman, 1983; Vannelli et al., 2002; Testa and Krämer, 2007). Such an oxidation of 12 to pMA and HSO4− should require four monooxygenations, in agreement with previous data showing that the oxidation by H2O2 of compounds involving a C=S bond into corresponding products involving a C=O bond requires the consumption of four equivalents of H2O2 (Böttcher and Lüttringhaus, 1945). According to the mechanisms proposed in Fig. 4, the oxidation of ADT to pMA and hydrogensulfate would require seven monooxygenation steps. This would be in agreement with a previous article reporting that titration of ADT with H2O2 reach a plateau after consumption of 7 H2O2 equivalents (Böttcher and Lüttringhaus, 1945). It is ironical that the metabolic activation of ADT to release a single molecule of H2S requires seven monoxygenation steps.
Even though several steps of the mechanisms proposed in Figs. 3 and 4 remain to be established, they are in agreement with the previous mechanisms proposed for the microsomal oxidation of compounds involving a C=S bond (Cashman and Hanzlik, 1982; Hanzlik and Cashman, 1983; Vannelli et al., 2002; Testa and Krämer, 2007). They also are in agreement with the previously described experimental data: 1) the detection of the DT cation during ADT microsomal incubations, 2) the formation of about 0.4 mol of H2S from the microsomal oxidation of 1 mol of ADO that corresponds to the formation of about 0.4 mol of pMA resulting from the cleavage of the ADO-dithiolone ring with loss of the S atoms, and 3) the formation of only about 0.13 mol of H2S from the microsomal oxidation of one mole of ADT that corresponds to the formation of only about 0.2 mol of pMA.
Conclusion
This article shows that only small amounts of H2S are formed during incubation of ADT and ADO with rat liver cytosol or with RLM in the absence of NADPH. This indicates that H2S formation under these conditions is of hydrolytic origin only to a minor extent. By contrast, much greater amounts of H2S are formed upon incubation of ADT and ADO with RLM only in the presence of NADPH and dioxygen, the two necessary reactants of microsomal monooxygenases. Moreover, H2S formation under those conditions is greatly inhibited in the presence of Bz-ImH, indicating that this formation is mainly catalyzed by P450-dependent monooxygenases. This article also shows for the first time that ADO is a much better H2S donor than ADT under these conditions. It also proposes for the first time detailed mechanisms for the formation of H2S from the metabolism of ADT and ADO that are in agreement with the previously described data on the metabolism of these compounds (Dulac et al., 2018) and the mechanisms previously proposed for the metabolic oxidation of compounds involving a C=S bond (Cashman and Hanzlik, 1982; Hanzlik and Cashman, 1983; Vannelli et al., 2002; Testa and Krämer, 2007).
Acknowledgments
The authors thank A. Hessani for her help in HPLC-MS experiments and M. Jaouen and B. Ramassamy for their technical assistance.
Authorship Contributions
Participated in research design: Dansette, Boucher, Mansuy.
Conducted experiments: Dulac, Nagarathinam, Boucher.
Contributed new reagents or analytic tools: Dulac, Boucher, Dansette.
Performed data analysis: Dulac, Boucher, Dansette, Mansuy.
Wrote or contributed to the writing of the manuscript: Mansuy, Boucher, Dansette.
Footnotes
- Received March 18, 2019.
- Accepted June 14, 2019.
Abbreviations
- ADO
- anetholedithiolone, 5-(p-methoxyphenyl)-3H-1,2-dithiole-3-one
- ADOSO
- 5-(p-methoxyphenyl)-3H-1,2-dithiole-3-one sulfoxide
- ADT
- anetholedithiolethione, 5-(p-methoxyphenyl)-3H-1,2-dithiole-3-thione
- ADTSO
- 5-(p-methoxyphenyl)-3H-1,2-dithiole-3-thione sulfoxide
- Bz-ImH
- N-benzyl-imidazole
- dmADT, O-demethylated derivative of ADT
- 5-(p-hydroxyphenyl)-3H-1,2-dithiole-3-thione
- DT
- 5-(p-methoxyphenyl)-1,2-dithiolium hydrogensulfate
- HLM, Human liver microsomes; HPLC-MS
- high-performance liquid chromatography–mass spectrometry
- HRMS
- high-resolution mass spectrometry
- β-NF
- β-naphthoflavone
- P450
- cytochrome P450
- PB
- phenobarbital
- pHA, para-hydroxy-acetophenone; pMA
- para-methoxy-acetophenone
- RLM
- rat liver microsomes
- UT-RLM
- liver microsomes from untreated rats
- Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics
References
In this issue
Mechanism of H2S Formation from Anetholedithiolthione
