Comparison of various aryl-dithiolethiones and aryl-dithiolones as hydrogen sulfide donors in the presence of rat liver microsomes

Article abstract of DOI:10.1124/DMD.119.090274

It has been reported that microsomal metabolism of ADT (5-(p-methoxyphenyl)-3H-1,2-dithiole-3-thione, anetholedithiolethione, Sulfarlem) and ADO (5-(p-methoxyphenyl)-3H-1,2-dithiole-3-one, anetholedithiolone) led to formation of H₂S mainly deri

Full text of DOI:10.1124/DMD.119.090274

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Comparison of various aryl-dithiolethiones and aryl-dithiolones as hydrogen sulfide,
H2S, donors in the presence of rat liver microsomes
Madou-Marilyn Dali, Patrick M Dansette, Daniel Mansuy, and Jean-Luc Boucher
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS UMR 8601,
University Paris Descartes, 45 rue des Saints-Pères., 75270 Paris. France (M.-M.D., P.D.,
D.M., J.-L.B.)
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Running title: Dithiolethiones and dithiolones as H2S donors
Correspondence to :
Jean-Luc Boucher, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques,
UMR 8601 CNRS, University Paris Descartes, 45 rue des Saints Pères, 75006 Paris, France.
Telephone: 33 (0)1 42 86 21 91
Fax: 33 (0)1 42 86 83 87
Email: jean-luc.boucher@parisdescartes.fr
Text pages:
Tables:
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Figures:
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References:
Abstract:
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227 words
336 words
Introduction:
Results and Discussion:
1788 words
Abbreviations
ADT, 5-(p-methoxyphenyl)-3H-1,2-dithiole-3-thione; ADO, 5-(p-methoxyphenyl)-3H-1,2-
dithiole-3-one; ADOSO, 5-(p-methoxyphenyl)-3H-1,2-dithiole-3-one-1-sulfoxide; ADTSO, 5-
(p-methoxyphenyl)-3H-1,2-dithiole-3-thione-sulfoxide; β-ME, β-mercapto-ethanol; Bz-ImH,
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N-benzyl-imidazole; CYP, cytochrome P450; Cys, L-cysteine; dmADT, 5-(p-hydroxyphenyl)-
3H-1,2-dithiole-3-thione; DPD, N,N-dimethylamino-para-phenylene-diamine; DO, dithiolone;
DT, dithiolethione; DTT, dithio-threitol; GSH, glutathione; NAC, N-acetyl-L-cysteine; PB,
Phenobarbital; pMA, para-methoxy-acetophenone.
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Abstract
It has been reported that microsomal metabolism of ADT (anetholedithiolethione, Sulfarlem)
and ADO (anetholedithiolone) led to formation of H2S mainly derived from oxidations
catalyzed by cytochrome P450 (CYP)-dependent monooxygenases, and that ADO was a better
H2S-donor than ADT under these conditions. This article compares the H2S-donor abilities of
18 dithiolethione and dithiolone analogs of ADT and ADO upon incubation with rat liver
microsomes. It shows that, for all the studied compounds, maximal H2S formation was obtained
after incubation with microsomes and NADPH, and that this formation greatly decreased in the
presence of N-benzyl imidazole (Bz-ImH), a known inhibitor of CYP. This indicates that H2S
formation from all the studied compounds requires, as previously observed in the case of ADT
and ADO, oxidations catalyzed by CYP-dependent monooxygenases. Under these conditions,
the studied dithiolones were almost always better H2S-donors than the corresponding
dithiolethiones. Interestingly, the best H2S yields (up to 75%) were observed in microsomal
oxidation of ADO and its close analogs, pCl-Ph-DO and Ph-DO, in the presence of glutathione
(GSH), whereas only small amounts of H2S were formed in microsomal incubations of those
compounds with GSH but in the absence of NADPH. A possible mechanism for this effect of
GSH is proposed on the basis of results obtained from reactions of GSH with ADOSO (ADO
sulfoxide), the ADO metabolite involved in H2S formation in microsomal oxidation of ADO.
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Significance Statement
A series of 18 dithiolethiones and dithiolones was compared for their ability to form hydrogen
sulphide (H2S) in oxidations catalyzed by microsomal monooxygenases. The studied
dithiolones were better H2S-donors than the corresponding dithiolethiones and the addition of
glutathione to the incubations strongly increased H2S formation. A possible mechanism for this
effect of GSH is proposed on the basis of results obtained from reactions of GSH with 5-(p-
methoxyphenyl)-3H-1,2-dithiole-3-one-1-sulfoxide, a metabolite of the choleretic and
sialologic drug Sulfarlem.
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Introduction
Anetholedithiolethione (ADT, Sulfarlem, Figure 1) is a well known choleretic and sialologic
drug (Christen. 1995; Nagano et al., 2001). Its O-demethylated derivative dmADT, and many
compounds resulting from the coupling of dmADT with various anti-inflammatory drugs have
been extensively described for their H2S-donor properties and therapeutic effects (Chen et al.,
2010; Couto et al., 2015; Sparatore et al., 2009; Sparatore et al., 2011; Kashfi and Olson, 2013;
Ansari et al., 2018; Powell et al., 2018). Only very small amounts of H2S are formed upon
incubation of ADT with rat or human liver microsomes in the absence of NADPH, whereas
much greater amounts of H2S are formed upon incubation with liver microsomes containing
NADPH and O2, the two cofactors required for microsomal monooxygenases (Dulac et al.,
2019). Microsomal oxidation of ADT leads to the concomitant formation of H2S and para-
methoxy-acetophenone (pMA) (Dulac et al., 2019), and to other metabolites such as dmADT
and products resulting from an ADT S-oxidation such as anetholedithiolone, ADO and ADTSO
(Fig. 1) (Dulac et al., 2018). Interestingly, ADO was found as a much better H2S donor than
ADT under these conditions of microsomal oxidative metabolism (Dulac et al., 2019).
Moreover, a detailed mechanism for H2S formation upon microsomal oxidation of ADT and
ADO, implying the intermediate formation of ADOSO (Fig. 1) that results from an S-oxidation
of ADO, was proposed (Dulac et al., 2019).
In this article, one compares the abilities of several dithiolethione (DT) and dithiolone (DO)
analogs of ADT and ADO to act as H2S donors in the presence of NADPH-containing liver
microsomes, and shows that, in a general manner, dithiolones are better H2S donors than
dithiolethiones under these conditions. The greatest amounts of H2S were obtained upon
microsomal oxidation of close analogs of ADO, and the presence of glutathione (GSH) led to a
great increase of H2S formation (yields based on starting dithiolone up to 75%). A possible
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mechanism for this GSH effects is proposed on the basis of experiments performed between
ADOSO, the S-oxide of ADO, with GSH.
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Materials and Methods
General reagents, authentic samples and proteins
The commercial origins of ADT, pMA, NADPH, Bz-ImH and the solvents used were indicated
previously (Dulac et al., 2018). Naproxene, Aspirine (sodium salts), N.N-dimethyl-para-
phenylenediamine sulfate (DPD) and Oltipraz were from Sigma-Aldrich (Saint-Quentin
Fallavier, France). Authentic metabolites of ADT, ADO, dmADT and dmADO, were prepared
as described previously (Dulac et al., 2018). ADOSO was prepared from ADO following
modifications of a previously reported protocol (Tardif and Harp, 2000; Dulac et al., 2019).
NMR data were obtained from a Bruker 500 AV2 spectrometer. Mass spectra were recorded on
an Exactive HRMS-instrument (Thermo, Les Ulis, France). Preparation of liver microsomes
from phenobarbital (PB)-pretreated rats, measurements of protein concentrations and CYP
contents were made as reported previously (Dulac et al., 2019).
Chemistry
Dithiolethiones were obtained by treatment of the corresponding β-keto-esters with Lawesson’s
reagent and elemental sulfur in anhydrous toluene following a previously described method
(Pedersen and Lawesson, 1979; Biard et al., 1992).
1
5-(Phenyl)-3H-1,2-dithiole-3-thione (Ph-DT). H NMR (d6-acetone) 7.90 (d, 2H, J =
8.0), 7.65 (t, 1H, J = 8.0), 7.61 (s, 1H), 7.58 (m, 2H); HRMS (ESI+): m/z value for C9H6S3 + H+
210.9711, found 210.9704.
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5-(4-Chlorophenyl)-3H-1,2-dithiole-3-thione (p-Cl-Ph-DT). 1H NMR (d6-acetone)
7.93 (d, 2H, J = 8.0), 7.62 (d, 1H, J = 8.0), 7.61 (s, 1H); HRMS (ESI+): m/z value for
C9H5ClS3 + H+ 244.9321, found 244.9662.
5-(2-Chlorophenyl)-3H-1,2-dithiole-3-thione (o-Cl-Ph-DT). 1H NMR (d6-acetone) 7.77
(dd, 1H, J = 8.0 and 1.5), 7.67 (d, 1H, J = 8.0), 7.61 (td, 1H, J = 7.5 and 1.5), 7.54 (t, 1H, J =
8.0), 7.37 (s, 1H); HRMS (ESI+): m/z value for C9H5ClS3 + H+ 244.9321, found 244.9458.
5-(3-Hydroxy-4-methoxy-phenyl)-3H-1,2-dithiole-3-thione (o-OH-ADT). 1H NMR (d6-
acetone) 7.56 (s, 1H), 7.47 (d, 1H, J = 2.0), 7.42 (d, 1H, J = 8.0), 6.98 (d, 1H, J = 8.0), 3.90 (s,
3H); HRMS (ESI+): m/z value for C10H8O2S3 + H+ 256.9765, found 256.9755.
Naproxene-dmADT (ATB345) and aspirine-dmADT (ACS 14) were prepared as previously
described (Wallace et al., 2007) by reaction of naproxene or acetylsalicylic acid with dmADT
in the presence of hydroxybenzotriazole and di-cyclo-hexyl-carbodiimide in dry N,N-
dimethylformamide.
4-(5-Thioxo-5H-[1,2]dithiol-3-yl)phenyl-2-(2-methoxynaphthaleny-6-yl)propanoate
(Naproxene-dmADT). 1H NMR (d6-acetone) 7.88 (d, 2H, J = 8.5), 7.85-7.82 (m, 3H), 7.55 (s,
1H), 7.53 (d, 1H, J = 1.5), 7.31 (d, 1H, J = 3.0), 7.24 (d, 2H, J = 8.5), 7.17 (dd, 1H, J = 9.0 and
2.5), 4.24 (q, 1H, J = 7.0), 3.91 (s, 3H), 1.96 (d, 3H, J = 7.0); HRMS (ESI+): m/z value for
C23H18O3S3 + H+ 439.0497, found 439.0681.
4-(5-Thioxo-5H-1,2-dithiol-3-yl)phenyl-2-acetoxybenzoate (Aspirine-dmADT). 1H
NMR (d6-acetone) 8.24 (dd, 1H, J = 7.5 and 2.0), (8.03 d, 2H, J = 9.0), 7.78 (t, 1H, J = 7.5),
7.64 (s, 1H), 7.49 (m, 3H), 7.32 (d, 1H, J = 8.0), 2.32 (s, 3H); HRMS (ESI-): m/z value for
C18H12O4S3 – H+ 386.9819, found 386.9819.
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Dithiolones were obtained by treatment for 24 h at room temperature of the corresponding
dithiolethiones with mercuric acetate in a dichloromethane and acetic acid mixture following a
previously described method (Klingsberg, 1972).
1
5-Phenyl-3H-1,2-dithiole-3-one (Ph-DO): H NMR (d6-acetone): 7.83 (d, 2H, J = 7.0),
7.61 (m, 1H), 7.56 (m, 2H), 7.10 (s, 1H); HRMS (ESI+): m/z value for C9H6OS2 + H+ 194.9939,
found 194.9964.
1
5-(4-Chlorophenyl)-3H-1,2-dithiole-3-one (p-Cl-Ph-DO): H NMR (d6-acetone) 7.85
(d, 2H, J = 8.5), 7.61 (d, 2H, J = 8.5), 7.13 (s, 1H); HRMS (ESI+): m/z value for C9H6ClOS2
228.9549, found 228.9581.
1
5-(2-Chlorophenyl)-3H-1,2-dithiole-3-one (o-Cl-Ph-DO): H NMR (CDCl3) 7.51 (d,
2H, J = 8.5), 7.43 (t, 1H, J = 8.5), 7.36 (t, 1H, J = 8.0), 6.77 (s, 1H); HRMS (ESI+): m/z value
for C9H5ClOS2 + H+ 228.9549, found 228.9580.
5-(3-Hydroxy-4-methoxy-phenyl)-3H-1,2-dithiole-3-one (o-OH-ADO): 1H NMR
(CDCl3) 7.18 (dd, 1H, J = 8.5 and 2.0), 7.05 (d, 1H, J = 2.0), 6.97 (d, 1H, J = 8.5), 6.73 (s, 1H),
6.00 (s, 1H), 3.94 (s, 3H); HRMS (ESI-): m/z value for C10H8O3S2 – H+ 238.9836, found
238.9834.
4-Methyl-5-pyrazinyl-3H-1,2-dithiole-3-one (Oltipraz-DO) (O’Dwyer et al., 1997; Ko et al.,
2006): 1H NMR (CDCl3) 8.98 (s, 1H), 8.69 (d, 2H, J = 7.5), 2.32 (s, 3H); HRMS (ESI+): m/z
value for C8H7ON2S2 211.0001, found 210.9991.
Naproxene-dmADO and Aspirine-dmADO were obtained by reaction of naproxene or
acetylsalicylic acid with dmADO in the presence of hydroxybenzotriazole and di-cyclo-hexyl-
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carbodiimide in dry N,N-dimethylformamide following a described protocol (Wallace et al.,
2007).
4-(5-Oxo-5H-[1,2]dithiol-3-yl)phenyl-2-(2-methoxynaphthaleny-6-yl)propanoate
1
(Naproxene-dmADO). H NMR (d6-acetone); 7.85-7.82 (m, 3H), 7.68 (d, 2H, J = 8.5), 7.54
(dd, 1H, J = 8.5 and 2.0), 7.30 (d, 1H, J = 2.5), 7.22 (d, 2H, J = 8.5), 7.17 (dd, 1H, J = 9.0 and
2.5), 7.04 (s, 1H), 4.23 (q, 1H, J = 7.0), 3.90 (s, 3H), 1.66 (d, 3H, J = 7.0); HRMS (ESI+): m/z
value for C23H18O4S2 + H+ 423.0726, found 423.0786.
1
4-(5-Oxo-5H-1,2-dithiol-3-yl)phenyl-2-acetoxybenzoate (Aspirine-dmADO) H NMR
(d6-acetone) 8.22 (dd, 1H, J = 7.5 and 1.5), 7.93 (d, 2H, J = 8.0), 7.78 (t, 1H, J = 7.5), 7.49 (m,
1H), 7.46 (d, 2H, J = 8.0), 7.30 (d, 1H, J = 7.5), 7.12 (s, 1H), 2.26 (s, 3H); HRMS (ESI-): m/z
value for C18H12O5S2 – H+ 371.0047, found 371.0045.
Typical microsomal incubation procedures with measurement of H2S formation
Hydrogen sulfide formation was measured using the methylene blue method (Fogo and
Popowsky, 1949; Giustarini et al., 2014) using microsomal incubation procedures previously
described (Dulac et al. 2019). HPLC-HRMS analyses of metabolites from incubations of ADO
and ADOSO were performed as previously described (Dulac et al., 2018).
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Results and Discussion
Formation of H2S upon incubation of various dithiolethione analogs of ADT
The formulae of the compounds used in this study are shown in Figure 2. This includes
Oltipraz, a drug available for treatment of schistosomiasis and also tested as a cancer
chemopreventive agent (Bueding et al., 1982; Rao et al., 1993; O’Dwyer et al., 1997; Ko et al.,
2006), close analogs of ADT, such as Ph-DT, p-Cl-Ph-DT, o-Cl-Ph-DT, dmADT, o-OH-ADT,
and compounds resulting from acylation of the OH group of dmADT by anti-inflammatory
drugs, Naproxene-dmADT, and Aspirine-dmADT. The H2S-donor properties of these
compounds were compared to those of the corresponding dithiolone derivatives in which the
C=S function is replaced with a C=O function. They are indicated in Table 2 by abbreviations
similar to those used for the corresponding dithiolethiones (Table 1 and Figure 2) in which DT
was replaced with DO. The origin or synthesis of all these compounds is described in Materials
and Methods.
Formation of H2S was measured by using the methylene blue method (Giustarini et al., 2014)
during incubation of the DT and DO compounds with liver microsomes from PB-pretreated
rats. As previously reported (Dulac et al., 2019), incubation of 100 µM ADT with those liver
microsomes in the presence of 1mM NADPH led to H2S in a yield of about 12% after 1h
(Table 1). Without NADPH or in the presence of 1mM Bz-ImH, a known inhibitor of CYP-
dependent monooxygenases (Testa and Jenner, 1981; Correia and Ortiz de Montellano, 2005),
only low amounts of H2S were formed (Table 1, Dulac et al., 2019). Table 1 also shows that the
eight ADT analogs used in this study are generally less efficient H2S-donors than ADT after
microsomal metabolism, if one excepts o-OH-ADT that gave similar H2S yields. Moreover, for
most of these compounds one observed, as in the case of ADT, a decrease of H2S formation
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after incubation without NADPH or in the presence of Bz-ImH. These results indicate that
microsomal H2S formation from the studied dithiolethiones would mainly derive, as in the case
of ADT (Dulac et al., 2019), from an oxidation catalyzed by CYP-dependent monooxygenases.
When incubation of ADT with NADPH supplemented microsomes was performed under
identical conditions but in the presence of 1 mM glutathione, one observed a decrease of H2S
yield (Table 1). Microsomal incubations in the presence of GSH of the other DT indicated in
Table 1 led to H2S yields either slightly higher or lower than that found in the case of ADT
(Table 1).
The above data show that the eight ADT analogs used in this study are generally less efficient
H2S-donors than ADT after microsomal metabolism, if one excepts o-OH-ADT that gave
similar H2S yields (Table 1). The first step of the microsomal metabolism of ADT leading to
H2S formation seems to be the S-oxidation of the ADT C=S group (Dulac et al., 2018). This S-
oxidation is in competition with the CYP-dependent oxidative demethylation of ADT leading
to dmADT (Dulac et al., 2018). The lower yields of H2S formation observed with most other
DT could be due either to competitive microsomal oxidations more efficient than the ADT
oxidative demethylation, or to less efficient C=S S-oxidation compared to ADT. In that context,
we have performed preliminary HPLC-MS studies of microsomal oxidations of two other DTs
mentioned in Table 1: dmADT, and o-OH-ADT. In the case of dmADT, a metabolite
characterized by a molecular ion at m/z = 242.9596, that should result from the hydroxylation
of the phenyl ring of dmADT, was detected (m/z calculated for C9H7O2S3 (dmADT + O) + H+)
= 242.9609). Finally, in microsomal oxidation of o-OH-ADT, a metabolite characterized by a
molecular ion at m/z 242.9595 was found and should result from an oxidative demethylation of
o-OH-ADT (m/z calculated for C9H7O2S3 (o-OH-ADT - CH2) + H+ = 242.9609). Other kinds of
reactions are occurring in competition with the S-oxidation of the C=S function of Aspirine-
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ADT, Naproxene-ADT and Oltipraz. In the particular case of Aspirine-ADT and Naproxene-
ADT, we found that incubation of these compounds with liver microsomes led to dmADT that
should result from the hydrolysis of their ester function by microsomal esterases. In the case of
Oltipraz, literature data reported that several metabolites not resulting from the S-oxidation of
its C=S group are formed in high amounts (Bieder et al., 1983).
Formation of H2S upon incubation of various dithiolone analogs of ADO
As reported previously, incubation of ADO with NADPH-supplemented liver microsomes
under the above described conditions led to H2S yields higher than those observed with ADT
(Table 2 and Dulac et al., 2019). Without NADPH or in the presence of 1mM Bz-ImH, the H2S
yields were very low which confirmed that H2S formation is mainly catalyzed by CYP-
dependent monooxygenases (Table 2 and Dulac et al., 2019). Similar or even higher H2S yields
(22 to 36%) were observed after microsomal metabolism of Ph-DO, p-Cl-Ph-DO and o-Cl-Ph-
DO (Table 2). On the contrary, o-OH-ADO, Oltipraz-DO, Naproxene-dmADO and Aspirine-
dmADO led to lower H2S yields (about 10%). Moreover, all studied DO (except o-OH-ADO)
led to higher H2S yields than the corresponding DT (compare Tables 1 and 2). The H2S yields
are 2.5 to 4.8 fold increased in the case of ADO, Ph-DO, p-Cl-Ph-DO and o-Cl-Ph-DO, but
only 1.5 to 2.0 fold increased in the case of Oltipraz-DO, Naproxene-dmADO and Aspirine-
dmADO). In a general manner, in incubations performed without NADPH or in the presence of
Bz-ImH, all studied DO led to low H2S yields. This indicates that H2S formation from these
DO mainly depends on CYP-dependent monooxygenases. This would be in agreement with the
mechanism previously proposed for H2S formation from microsomal oxidation of ADO that
would involve an S-oxidation leading to ADOSO (Figure 1) as the first step (Dulac et al.,
2019).
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Table 2 also shows that the presence of 1 mM GSH in microsomal incubation of ADO and the
other DO led, in many cases, to an increase in the H2S yields. This increase is particularly
important in the case of ADO, p-Cl-Ph-DO, o-Cl-Ph-DO and Ph-DO, as incubation of Ph-DO
with NADPH- and GSH-supplemented microsomes led to H2S in a 75% yield. It is noteworthy
that such high H2S yields were not observed upon incubation of the DO with GSH alone or
with GSH and liver microsomes without NADPH (yields observed with ADO under those
conditions were 2.0 + 0.8 and 3.0 + 1.0 %. respectively).
The mechanism previously proposed for H2S formation upon microsomal oxidation of ADO
(Dulac et al., 2019) involves an S-oxidation of ADO with formation of ADOSO, as a first step
(Figure 3). The next steps would be the opening of ADOSO by H2O leading to intermediate 1,
the hydrolysis of the HSC=O function of 1 leading to H2S and 2, and the decarboxylation of 2
leading to the disulfoxide of para-methoxy-thioacetophenone 3. Finally, monooxygenations of
-
3 would lead to pMA and HSO4 , 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 Kramer, 2007). The same mechanism could be at the origin of H2S
formation upon microsomal metabolism of the other studied DO. In the presence of GSH, a
nucleophilic attack of GSH on the S=O group of ADOSO with opening of the SO-S bond
leading to intermediate 4 could occur. Hydrolysis of the HSC=O function of 4 should lead to
H2S (Figure 4). One could expect that the opening of the O=S-S bond of ADOSO by GSH
would be faster than the opening of this bond by H2O, a much weaker nucleophile.
H2S formation in reaction of ADOSO with GSH
In order to better understand the effects of GSH on H2S formation upon ADO microsomal
oxidation, we have studied the reaction of ADOSO with GSH and found that incubation of 100
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µM ADOSO with 250 µM GSH in phosphate buffer pH 7.4, 1h at 37°C, led to H2S in a yield of
about 95% based on starting ADOSO (Table 3), whereas incubations of ADOSO alone in the
same buffer or incubations of ADO with GSH under identical conditions only led to H2S yields
lower than 3% (Table 3). Figure 5 shows that the H2S yields observed after reaction of ADOSO
with increasing concentrations of GSH (from 100 µM to 1 mM) increased from 30 to about
200%. Other thiols (all at 250 µM), such as β-mercapto-ethanol (β-ME), dithiothreitol (DTT),
L-cysteine (Cys), or N-acetyl-L-cysteine (NAC) reacted with ADOSO with formation of H2S in
yields between 70 and 130% (Table 3). Those very high H2S yields, often higher than 100%,
obtained upon reaction of ADOSO with thiols indicated that more than one sulfur atom of
ADOSO was converted to H2S. A possible explanation for those results is shown in Figure 4 in
which intermediate 5, resulting from the opening of ADOSO by GSH and hydrolysis of the
HSC=O function of 4, is reduced by GSH with formation of 6 and intermediate 7.
Decarboxylation of 7 should lead to para-methoxy-thioacetophenone 8. Finally, reaction of
GSH with 8 would lead to H2S and pMA (Figure 4). In agreement with this hypothesis, an
HPLC-MS study of the reaction of 100 µM ADOSO with 250 µM GSH in phosphate buffer pH
7.4 (1 h at 37°C) showed the formation of pMA in a 84% yield (data not shown).
Those reactions between ADOSO and GSH could occur in microsomal incubations of ADO in
the presence of NADPH and GSH, and this could explain the increase of the H2S yields
observed when GSH is added to microsomal incubations. However, in such microsomal
incubations, the formation of ADOSO by monooxygenation of ADO is in competition with
other oxidations of ADO such as its oxidative O-demethylation (Dulac et al., 2018). Moreover,
in such incubations of ADO with NADPH-supplemented microsomes in the presence of GSH,
reactions of intermediates 5, 6 and 8 with GSH leading to H2S (Figure 4) should be in
-
competition with monooxygenation of those intermediates that would eventually lead to HSO4
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as in the case of 3 (Figure 3). The occurrence of those competitive reactions in microsomal
incubations of ADO should explain the much lower H2S yields observed (Table 2) when
compared to those found in reactions of ADOSO with GSH in the absence of microsomes
(Table 3 and Figure 5).
The mechanisms proposed for H2S formation from ADO in Figures 3 and 4 should be operative
for H2S formation from the other studied DO. Accordingly, the results concerning Ph-DO, o-
Cl-Ph-DO and p-Cl-Ph-DO were very similar to those observed with ADO (Table 2). The
much lower H2S yields obtained in microsomal oxidation of dmADO, o-OH-ADO, Oltipraz-
DO, Naproxene-dmADO, and Aspirine-dmADO (Table 2) could be related to
monooxygenations of those molecules occurring at positions different from the S atom
involved in the formation of ADOSO from ADO.
Conclusion
This article compares the H2S-donor abilities of a series of 18 dithiolethione or dithiolone
analogs of ADT or ADO incubated with NADPH-supplemented rat liver microsomes. It shows
that only minor amounts of H2S are formed in incubations performed without NADPH or with
NADPH and Bz-ImH, which indicates that H2S formation requires oxidations catalyzed by
CYP-dependent monooxygenases. With NADPH-supplemented microsomes, the best DT H2S-
donors were ADT itself and o-OH-ADT, and the studied DO were almost always better H2S-
donors than the corresponding DT. The presence of GSH in microsomal incubations had only
minor effects on the H2S yields observed with DT. However, it led to a great increase of the
H2S yields in microsomal incubations of ADO and its close analogs, o-Cl-Ph-DO, p-Cl-Ph-DO
and Ph-DO, the best H2S yield (75%) being obtained in the case of Ph-DO. A possible
mechanism for this effect of GSH is proposed on the basis of a study of reactions of GSH with
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ADOSO, the ADO metabolite involved in H2S formation in microsomal oxidation of ADO.
Since GSH is present in most cells, one should expect that DO related to ADO act as
particularly good H2S-donors in in vivo situations.
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Acknowledgments
The authors thank P. Gerardo for his help in HPLC-MS experiments, and B. Ramassamy for
technical assistance.
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Authorship contributions
Participated in research design: Dansette, Boucher, Mansuy.
Conducted experiments: Dali, Boucher.
Contributed new reagents or analysis tools: Dali, Boucher, Dansette.
Performed data analysis: Dali, Boucher, Dansette, Mansuy.
Wrote or contributing to the writing of the manuscript: Mansuy, Boucher, Dansette.
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Figure Legends
Figure 1: Formulae of ADT and some of its metabolites formed upon microsomal oxidation.
Figure 2: Formulae of the dithiolethiones used in this study.
Figure 3: Mechanism previously proposed for H2S formation upon metabolism of ADO by
NADPH-supplemented liver microsomes (Dulac et al., 2019). Ar = p-methoxyphenyl. +”O” on
some arrows indicates that the corresponding step involves a monooxygenation of the substrate.
Figure 4: Possible mechanism for the formation of H2S upon reaction of ADOSO with GSH.
Figure 5: Representative curve showing the H2S yields obtained after the reaction of 100 µM
ADOSO with increasing GSH concentrations in 50 mM phosphate buffer pH 7.4. ADOSO
(100 µM) in 50 mM phosphate buffer pH 7.4 was incubated for 1 h at 37°C in the presence of
increasing concentrations of GSH and H2S formation was measured as indicated in Materials
and Methods. Data are means + S.D. from 3 experiments.
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Table 1: H2S formation upon metabolism of various dithiolethione analogs of ADT by liver
microsomes from PB-treated rats.
C.S. + Bz-ImH c C.S. + GSH d
Dithiolthiones
Ph-DT
C.S. a
C.S. -NADPH b
2.0 0.2
3.1 1.3
2.9 1.7
2.4 2.3
1.2 0.3
3.5 + 1.5
1.5 0.5
2.3 1.8
4.5 0.7
7.5 2.7
12.0 3.5
2.5 1.0
7.0 1.1
6.0 0.3
12.4 2.5
5.7 3.0
6.3 0.7
5.1 1.0
3.6 1.1
2.9 1.0
2.5 1.9
2.0 0.8
1.5 1.0
3.0 0.8
1.9 1.4
4.5 1.0
2.2 1.1
8.5 1.0
6.4 1.6
5.5 1.8
3.3 1.3
1.8 0.8
9.3 1.0
1.5 1.7
4.7 0.7
7.4 3.1
ADT
dmADT
p-Cl-Ph-DT
o-Cl-Ph-DT
o-OH-ADT
Oltipraz
Naproxene-dmADT
Aspirine-dmADT
a
C.S.: Complete system. H2S yields (%) were measured after 1h incubation of 100 µM
dithiolethione in phosphate buffer, pH 7.4 in the presence of liver microsomes (~2.5 µM CYP)
and 1 mM NADPH, as described in Materials and Methods. Data are yields based on starting
b
compound and are means + S.D. from 3 to 8 experiments. Identical conditions but in the
c
absence of NADPH. Identical conditions but 1 mM Bz-ImH was added in the solution.
d
Identical conditions but in the presence of 1 mM GSH.
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Table 2: H2S formation upon metabolism of various dithiolone analogs of ADO by liver
microsomes from PB-treated rats. Abbreviations used for the dithiolones are similar to those
used for the corresponding dithiolethiones (Table 1 and Figure 2) in which DT was replaced
with DO.
Dithiolones
Ph-DO
C.S. a
C.S. – NADPH b C.S. + Bz-ImH c C.S. + GSH d
36.5 1.0
31.1 4.6
11.4 1.2
29.8 3.0
21.7 5.9
8.9 0.1
10.9 3.3
9.9 1.3
9.7 0.1
1.5 0.1
3.1 2.1
3.3 0.5
1.4 0.3
1.2 1.7
3.9 1.6
0.9 1.1
2.6 0.2
2.6 0.8
3.2 2.0
4.1 1.7
4.6 2.0
3.7 0.4
2.9 2.1
1.4 0.2
2.9 3.2
3.6 0.6
4.7 0.4
74.9 0.4
44.2 5.1
21.4 1.6
45.5 3.3
41.2 2.8
4.7 1.8
ADO
dmADO
p-Cl-Ph-DO
o-Cl-Ph-DO
o-OH-ADO
Oltipraz-DO
Naproxene-dmADO
Aspirine-dmADO
9.8 1.1
11.8 0.5
14.7 0.6
a
C.S.: Complete system. H2S yields (%) were measured after 1 h incubation of 100 µM
dithiolone in phosphate buffer, pH 7.4, in the presence of liver microsomes (~2.5 µM CYP) and
1 mM NADPH, as described in Materials and Methods. Data are yields based on starting
b
compound and are means + S.D. from 3 to 8 experiments. Identical conditions but in the
c
absence of NADPH. Identical conditions but 1mM Bz-ImH was added in the solution.
d
Identical conditions but in the presence of 1 mM GSH.
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Table 3. Formation of H2S upon reaction of ADOSO with GSH or other thiols.
Conditions a
H2S yield (%)
2.6 + 0.8
ADOSO + buffer alone
ADOSO + GSH
ADOSO + β-ME
ADOSO + DTT
ADOSO + Cys
93.6 + 10.1
114.2 + 15.8
128.2 + 12.8
97.6 + 12.8
73.8 + 11.0
ADOSO + NAC
a ADOSO (100 µM) was incubated for 1 h at 37°C in 50 mM phosphate buffer pH 7.4, alone or
in the presence of 250 µM of the indicated thiol and H2S yields (%) were measured as indicated
in Materials and Methods. Data are means + S.D. from 4 to 8 experiments.
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Figure 1
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Figure 2
30