PR_1_2013-cz_1.vp:CorelVentura 7.0

Pharmacological Reports
Copyright © 2013
2013, 65, 173–178
by Institute of Pharmacology
ISSN 1734-1140
Polish Academy of Sciences
Effects of acetylsalicylic acid on the levels of
sulfane sulfur and non-protein sulfhydryl groups
in mouse tissues
Anna Bilska-Wilkosz1, Magdalena Ochenduszka1, Ma³gorzata Iciek1,
Maria Soko³owska-Je¿ewicz1, Bogdan Wiliñski2, Marta Góralska2,
Zbigniew Srebro2, Lidia W³odek1
1
2
Chair of Medical Biochemistry, Department of Human Developmental Biology, Jagiellonian University,
Medical College, Kopernika 7, PL 31-034 Kraków, Poland
Correspondence: Anna Bilska-Wilkosz, e-mail: [email protected]
Abstract:
Background: The study is focused on searching for the link between acetylsalicylic acid (ASA) and sulfur metabolism. The present
work aimed to investigate the effect of ASA on the level of the sulfane sulfur and non-protein thiol compounds (NPSH) in the liver
and kidneys of mice.
Methods: The study was conducted on female albino Swiss mice weighing approximately 20 g. The experimental group was treated
intraperitoneally (ip) with aspirin, lysine salt, at a dose of 10 mg (on pure aspirin basis)/kg for 5 days. The control group was treated
ip with 0.9% NaCl in a volume of 0.2 ml for 5 days. The experimental and control mice were sacrificed by cervical dislocation on 5th
day of the experiment, 3 h after the last injection. The homogenates of livers and kidneys were next used for assaying the levels of
NPSH and sulfane sulfur-containing compounds.
Results: The results indicate that in the liver of ASA-treated mice, the level of the cyanolysable sulfane sulfur decreased compared
to the control group, whereas in the kidney, its level significantly increased. The opposite results in the liver and kidney were observed also for NPSH, namely ASA elicited a drop in NPSH level in the liver, and elevated it in the kidney.
Conclusion: The results of the present study suggest that ASA affects sulfur metabolism, in particular, renal and hepatic production
of sulfane sulfur and NPSH in mice.
Key words:
acetylsalicylic acid (aspirin), sulfane sulfur, non-protein sulfhydryl groups
Introduction
The history of aspirin (ASA) begun in 1897 and since
then its staggering career has been unceasingly flourishing worldwide. “The most important therapeutic of all
time”, “symbol of the 20th century”, “vitamin S” these
are only some of phrases currently used to describe aspirin [5, 20, 21]. This enthusiasm is justified by the fact
that more specific molecular mechanisms underpinning aspirin’s action have come to light and new medicinal indications for this drug have been reported.
We focused our attention on the link between ASA and
sulfur metabolism. In mammals, methionine, an essential
amino acid for protein synthesis and the source of sulfur,
is of dietary origin. Methionine is demethylated to homocysteine, an intermediate in cysteine synthesis. Cysteine is
incorporated into proteins and glutathione (GSH). Homo-
Pharmacological Reports, 2013, 65, 173–178
173
cysteine, cysteine and GSH are a natural reservoir of
reductive capacities of the cells. The remaining low
molecular weight thiol compounds are derivatives of
cysteine (coenzyme A, lipoic acid) or GSH (cysteinylglycine). The sum of low molecular weight thiols
(in reduced form), such as homocysteine, cysteine,
GSH and cysteinylglycine is referred to as nonprotein thiol compounds (NPSH). GSH constitutes
about 95% of the cellular NPSH pool.
Apart from incorporation into protein and GSH,
cysteine can be also catabolized via two main routes.
One of them involves oxidation of -SH group to cysteine sulfinate, which may be decarboxylated to hypotaurine or converted to pyruvate and sulfite. Sulfates
and taurine are end products of cysteine catabolism
via aerobic pathway. The second metabolic route, anaerobic path referred to as “desulfhydration”, results
in sulfane sulfur-containing compounds.
Sulfane sulfur-containing compounds are characterized by the presence of exceptionally reactive sulfur atom, occurring in the 0 or –1 oxidation state,
which is covalently bound to another sulfur atom (RS-S*-H). Sulfur with such properties easily leaves
structure of the compound, and can be transferred to
different acceptors, like sulfates(IV) (SO32–) or cyanide (CN-) (Fig. 1).
The problem of ASA effect on the sulfur metabolism
has already been approached by other scientific centers.
In 1972, Dowdy and Nielsen demonstrated that ASA
was effective in promoting increased sulfate incorporation into bone-forming regions of the chick tibia, regardless of the zinc status of the bird [6]. Studies of recent
years suggest that ASA and other non-steroidal antiinflammatory drugs (NSAIDs) suppress progression of
different cancers, including breast ovarian, colorectal
cancer and also lung, esophagus and stomach cancers [5,
24, 33]. It has been reported that ibuprofen, indomethacin and ASA have comparable potential anticancer efficacy against chemically induced mammary gland cancer
as thiol compounds N-acetylcysteine and lipoic acid
[17]. Srebro et al. demonstrated that ASA augmented the
concentration of endogenous hydrogen sulfide in the
mouse brain and liver [26].
Histopathological studies of the mammalian brain
have demonstrated that glial cells contain Gomoripositive cytoplasmic granulation (GPCG), rich in reduced sulfur, whose biological role is unknown [27].
In 2008, Srebro et al. discovered that the glial
Gomori-positive material contained sulfane sulfur
[25]. The studies of Sura et al. indicated that ASA affected Gomori-positive glia in the mouse brain [29]. Results of in vivo studies of Bilska et al. revealed that ASA
caused a drop in GPCG number in the mouse brain [3].
In this study, we decided to determine whether ASA
administration can influence the level of sulfane sulfur
compounds and NPSH in the liver and kidney of mice.
We hope that our study is interesting, the more so that
searching for new mechanisms of action of ASA is important both from theoretical and practical perspective.
Materials and Methods
Fig. 1. Anaerobic cysteine catabolism is the source of sulfane sulfurcontaining compounds. Sulfane sulfur plays a significant role in the
transformation of cyanides intro less toxic thiocyanates (rhodanates,
SCN-). Also sulfate (IV) can be a sulfane sulfur acceptor which leads
to the formation of thiosulfate excreted with urine. Both cyanide detoxification to thiocyanates and thiosulfate production are catalyzed
by sulfurtransferases: rhodanese (EC.2.8.1.1; TST) and 3-mercaptopyruvate sulfurtransferase (EC.2.8.1.2; MST)
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Pharmacological Reports, 2013, 65, 173–178
Animals
Female albino Swiss mice weighing approximately
20 g were used. Animals were housed in plastic cages
Aspirin and sulfur compounds in mouse tissue
Anna Bilska-Wilkosz et al.
in a room at a constant temperature of 20 ± 2°C with
natural light-dark cycle. They had free access to standard pellet diet and water. All experiments were conducted according to guidelines of the Animal Use and
Care Committee of the Jagiellonian University (Kraków,
Poland).
Chemicals
Laspal (DL-lysine acetylsalicylate) was obtained from
Sanofi-Synthelabo (Le Plessis Robinson, France). 5,5Dithiobis-(2-nitrobenzoic) acid (DTNB), Folin-Ciocalteau reagent, potassium cyanide (KCN), reduced
glutathione (GSH) and trichloroacetic acid (TCA)
were provided by Sigma Chemical Co. (St. Louis,
MO, USA). All the other reagents were of analytical
grade and were obtained from Polish Chemical Reagent Company (POCh, Gliwice, Poland).
The level of NPSH was estimated with DTNB
according to the Sedlak method [23]. In this assay
DTNB is reduced by non protein sulfhydryl groups
present in TCA extract to 2-nitro-5-mercaptobenzoic
acid having a characteristic yellow color. Absorbance
of the colored product is measured at 412 nm.
Protein content was assayed by the method of
Lowry et al. [16].
Statistical analysis
All statistical calculations were carried out with the
STATISTICA 8.0 computer program. The mean and
standard deviation of the mean were calculated for
each group. Normal distribution was verified with the
Kolmogorow-Smirnow test. Data were analyzed by
Student’s t-test. Values of p < 0.05 were considered
statistically significant.
Experimental design
The animals were divided randomly into two groups
of 6 animals each. The first group was treated ip with
0.9% NaCl in a volume of 0.2 ml for 5 days and was
considered to be the control. The second group was
treated ip with aspirin, lysine salt, at a dose of 10 mg
(on pure aspirin basis) per kg of body weight for
5 days. The experimental and control mice were sacrificed by cervical dislocation on the 5th day of the experiment, 3 h after the last injection. The livers and
kidneys were removed, washed in 0.9% NaCl, placed
in liquid nitrogen and stored at -80°C until biochemical tests were performed.
Preparation of tissue homogenates
The frozen livers and kidneys were weighed and homogenates were prepared by homogenization of 1 g
of the tissue in 4 ml of 0.1 M phosphate buffer, pH
7.4, using the IKA-ULTRA-TURRAX T8 homogenizer. The homogenates were next used for assaying the levels of NPSH and sulfane sulfur-containing
compounds.
Biochemical investigations
The level of the compounds containing sulfane sulfur
was determined by the method of Wood based on cold
cyanolysis and colorimetric detection of the ferric
thiocyanate complex ion at 460 nm [38].
Results
Figures 2 and 3 show the mean values of NPSH and
sulfane sulfur levels in the liver of the ASA-treated
and control animals. ASA significantly lowered the
level of sulfane sulfur compounds (2.98 ± 0.74 nmol/
mg of protein) and NPSH (28.5 ± 2.2 nmol/mg of protein) compared to control (3.94 ± 0.79 and 37.31
± 8.27 nmol/mg of protein, respectively). However, in
the kidney, as it can be seen in Figures 4 and 5, ASA
significantly increased the concentration of both sulfane sulfur (2.91. ± 0.27 nmol/mg of protein) and
NPSH (8.33 ± 1.72 nmol/mg of protein) compared to
control (2.48 ± 0.21 and 6.86 ± 1.57 nmol/mg of protein, respectively).
Discussion
The results of the present study suggest that ASA affects sulfur metabolism, in particular, renal and hepatic production of sulfane sulfur and NPSH in mice.
We recently reported that ASA significantly lowered
the level of bound sulfane sulfur in the liver of mice
[3]. Our present study further evidenced that ASA
lowered the level of sulfane sulfur in the liver of mice.
Pharmacological Reports, 2013, 65, 173–178
175
**
***
3.0
2.5
2.0
1.5
1.0
0.5
0.0
control
control
aspirin
aspirin
Fig. 2. The effect of ASA treatment on the sulfane sulfur level in the
mouse liver. Data are shown as the means ± SD. * Significant vs. control group; ** p < 0.01. The sulfane sulfur content was expressed in
nmol per 1 mg of protein
nmol/mg of protein
nmol/mg of protein
3.5
50
45
40
35
30
25
20
15
10
5
0
Fig. 4. The effect of ASA treatment on the sulfane sulfur level in the
mouse kidney. Data are shown as the means ± SD. * Significant vs.
control group; *** p < 0.001. The sulfane sulfur content was expressed in nmol per 1 mg of protein
12
***
nmol/mg of protein
nmol/mg of protein
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
**
10
8
6
4
2
0
control
aspirin
control
aspirin
Fig. 3. The effect of ASA treatment on the non-protein sulfhydryl
groups (NPSH) level in the mouse liver. Data are shown as the means
± SD. * Significant vs. control group; ** p < 0.01. The NPSH content
was expressed in nmol per 1 mg of protein
Fig. 5. The effect of ASA treatment on the non-protein sulfhydryl
groups (NPSH) level in the mouse kidney. Data are shown as the
means ± SD. * Significant vs. control group; ** p < 0.01. The NPSH
content was expressed in nmol per 1 mg of protein
The term “bound sulfane sulfur” was introduced by
Ogasawara, to describe sulfur released by dithiothreitol reduction [22]. According to some authors, “bound
sulfur” can constitute a sulfane sulfur fraction [10,
22]. Further, we demonstrated that ASA increased sulfane sulfur concentrations in the kidney. Thus, ASA
produced opposing effects in these organs. Therefore,
our findings suggest that despite a similar metabolic
profile, the roles of the kidney and the liver in sulfur
metabolism are disparate. Literature data lead to the
same conclusion. Aebi et al. observed that cysteine
concentration in the kidney was 8 times higher than in
the liver [2]. Cysteine is a toxic amino acid and its
physiological concentration is very low compared
with other amino acids. Thus, it is an interesting and
unresolved problem why cysteine does not show toxic
effects in the kidney and what role it plays in this or-
gan. Moreover, a crucial role of the kidney in sulfur
metabolism is corroborated by the fact that plasma
levels of sulfur-containing amino acids are changed
the most in terminal renal failure compared to other
amino acids [4, 36, 37].
It is noteworthy that although there exist three alternative cell need-dependent homocysteine metabolic paths, the transulfuration pathway is the predominant pathway for homocysteine metabolism in
the kidney, which suggests that the kidney can produce significant amounts of sulfane sulfur and hydrogen sulfide [9]. It is worth to add that according to recent literature, endogenously produced hydrogen sulfide (H2S) may be stored in the form of sulfane sulfur
[11, 13, 14, 32].
In mammals, sulfane sulfur is primarily synthesized in the liver and kidneys, and then transported in
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Aspirin and sulfur compounds in mouse tissue
Anna Bilska-Wilkosz et al.
plasma to other organs in the form of albumin persulfides [30, 31]. Two key enzymes participate in the
formation and transport of sulfane sulfur compounds,
namely cystathionase (EC.4.4.1.1; CSE) and 3-mercaptopyruvate sulfurtransferase (EC.2.8.1.2; MST); in
addition, rhodanese (EC.2.8.1.1; TST) is involved in
transport of sulfane sulfur compounds [28, 30, 31, 34,
35, 38]. Our previous experiments did not show any
ASA effects on cystathionase and rhodanese activities
in the liver of mice [3].
The present study showed that in the liver ASA significantly lowered the level of NPSH (Fig. 3). Having
in mind that glutathione constitutes about 95% of the
cellular NPSH pool, our result is confirmed by the
study of Kaplowitz et al., which showed depletion of
hepatic glutathione in ASA-treated rats [12]. It is generally accepted that GSH is synthesized in the liver
and this is also the organ containing the highest concentrations of this compound, from where it is transported to plasma and bile. GSH biosynthesis is a part
of the metabolic process discovered by Meister and
called the g-glutamyl cycle [18]. Apart from the enzymes involved in GSH biosynthesis, i.e., g-glutamylcysteine synthetase (EC.6.3.2.2) and glutathione synthetase (EC.6.3.2.3), this cycle includes GSH-degrading
enzymes, namely g-glutamyl transpeptidase (EC.2.3.2.2;
g-GT) and cysteinylglycine dipeptidase (EC.3.4.13.16).
The g-GT is a key step in the g-glutamyl cycle, which
is a series of degrading and synthetic reactions that
participate in cellular GSH metabolism [18, 19].
Hinnchman et al. demonstrated that g-GT activity in
the rat kidney was almost 900 times higher than in
the liver [8]. Several experimental studies indicated
that 90% of GSH present in plasma was synthesized
in the liver, of which 80% was biodegraded in the
kidney [1, 15].
Our results showed that in the kidney ASA significantly increased the level of NPSH (Fig. 5). Similar
results, i.e., elevation of GSH level in the kidney tissue and a drop in the liver were obtained by Hardings
et al. in mice with genetic g-GT deficiency [7]. Based
on this observation, it can be suggested that the
changes in NPSH level in the liver and kidney of mice
observed in the present study can be connected with
ASA effect on the activity of g-GT. Obviously, it is
only a hypothesis because the effect of ASA on the
activity of g-GT has not been investigated so far.
In conclusion, the current study showed that ASA
affected sulfur metabolism, in particular, renal and
hepatic production of sulfane sulfur and non-protein
sulfhydryl groups in mice. The drug increased sulfane
sulfur and NPSH concentrations in the kidney and
lowered them in the liver. So, ASA produced opposing effects in both these organs.
However, confirmation of the hypothesis postulated by us that g-GT is a cellular target for ASA
requires further studies.
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Received: January 25, 2012; in the revised form: August 28, 2012;
accepted: September 14, 2012.