L-arginine, a nitric oxide precursor, reduces dapsone-induced
methemoglobinemia in rats
Natália Valadares de Moraes, Mateus Machado Bergamaschi, Maria de Lourdes Pires Bianchi,
Juliana Bordinassi Bragheto, Wilson Roberto Malfará, Regina Helena Costa Queiroz
Department of Clinical, Toxicological and Food Sciences Analysis, School of Pharmaceutical Sciences of Ribeirão Preto,
University of São Paulo
Dapsone use is frequently associated to hematological side effects such as methemoglobinemia and
hemolytic anemia, which are related to N-hydroxylation mediated by the P450 enzyme system. The aim
of the present study was to evaluate the influence of L-arginine supplementation, a precursor for the
synthesis of nitric oxide, as single or multiple dose regimens on dapsone-induced methemoglobinemia.
Male Wistar rats were treated with L-arginine at 5, 15, 30, 60 and 180 mg/kg doses (p.o., gavage) in
single or multiple dose regimens 2 hours prior to dapsone administration (40 mg/kg, i.p.). The effect of
the nitric oxide synthase inhibitor L-NAME was investigated by treatment with multiple doses of 30
mg/kg (p.o., gavage) 2 hours before dapsone administration. Blood samples were collected 2 hours after
dapsone administration. Erythrocytic methemoglobin levels were assayed by spectrophotometry. The
results showed that multiple dose supplementations with 5 and 15 mg/kg L-arginine reduced dapsoneinduced methemoglobin levels. This effect is mediated by nitric oxide formation, since the reduction in
methemoglobin levels by L-arginine is blocked by simultaneous administration with L-NAME, a nitric
oxide synthase inhibitor.
Uniterms: Dapsone. Methemoglobinemia. L-arginine. Nitric oxide. L-NAME.
O uso da dapsona é frequentemente associado a efeitos adversos hematológicos, como a metemoglobinemia
e anemia hemolítica, ambos relacionados com a N-hidroxilação mediada pelo sistema P450. O objetivo
do estudo foi avaliar a influência da suplementação de L-arginina, um precursor da síntese de óxido
nítrico, administrado em regime de dose única ou múltipla na metemoglobinemia induzida pela dapsona.
Ratos machos Wistar foram tratados com L-arginina (po, gavagem) em dose única ou múltipla de 5, 15,
30, 60 e 180 mg/kg 2 horas antes da administração de dapsona (40 mg/kg, ip). O efeito do L-NAME,
um inibidor de óxido nítrico sintase (NOS), foi avaliado através do tratamento com doses múltiplas de
30 mg/kg. Amostras de sangue foram coletadas duas horas após a administração de dapsona. A
concentração de metemoglobina eritrocitária foi analisada por espectrofotometria. Os resultados
mostraram que a suplementação em dose múltipla de 5 e 15 mg/kg de L-arginina reduziu os níveis de
metemoglobina induzida pela dapsona. Este efeito é mediado pela formação de óxido nítrico, uma vez
que a redução nos níveis de metemoglobina pela L-arginina é bloqueada pela administração simultânea
de L-NAME, um inibidor da óxido nítrico sintase.
Unitermos: Dapsona. Metemoglobinemia. L-arginina. Óxido nítrico. L-NAME.
INTRODUCTION
Dapsone (4,4’-diaminodiphenylsulfone, DDS) is
*Correspondence: R. H. C. Queiroz. Departamento de Análises Clínicas,
Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de
Ribeirão Preto - USP. Av. do Café, s/n, 14040-903 - Ribeirão Preto - SP, Brazil.
E-mail: rqueiroz@fcfrp.usp.br.
a potent antibacterial and anti-inflammatory compound
and has been clinically used in the treatment of leprosy
as a component of a multidrug therapy that includes a
combination of DDS, clofazimine and rifampicin (Katoch
2002; Walker, Lockwood 2007). The drug is also used
for the treatment of malaria and Pneumocystic carinii
pneumonia in patients with acquired immunodeficiency
Article
Brazilian Journal of
Pharmaceutical Sciences
vol. 48, n. 1, jan./mar., 2012
88
N. V. Moraes, M. M. Bergamaschi, M. L. P. Bianchi, J. B. Bragheto, W. R. Malfará, R. H. C. Queiroz
syndrome (Powell et al., 1967; Mills et al., 1988; Castro
1998; Tobin-D’Angelo et al., 2004; Nyunt, Plowe 2007).
The major metabolic pathway of DDS is acetylation,
producing monoacetyldapsone (MADDS). DDS is also
metabolized by N-hydroxylation mediated by cytochrome
P450 isozymes CYP2C19, CYP2C9, CYP3A4 and
CYP2E1 in man, and isozymes CYP2C6/11 and CYP3A1 in
rats, producing DDS hydroxylamine (DDS-NOH) (Fleming
et al., 1992; Vage, Svensson, 1994; Mitra et al., 1995; Gill
et al., 1995; Ganesan et al., 2010). Glucuronidation of
DDS and DDS-NOH is catalyzed by the enzyme UDPglucuronosyltransferase (UGT) allowing its excretion in
urine and bile (Coleman et al., 1996; Tingle et al., 1997).
The co-oxidation of DDS-NOH and hemoglobin
produces nitroso derivatives and methemoglobin, thus
causing methemoglobinemia and hemolysis (Tingle et
al., 1990; Coleman, 1995), which are the major dosedependent side effects of DDS chronic users (Kaluarachchi
et al., 2001). MADDS-NOH has been shown to be a more
potent methemoglobin (metHb) former than DDS-NOH
in human erythrocytes in vitro (Coleman, Holden, 2004),
whilst both metabolites present the same potency in rats
and humans (Vage et al., 1994).
The reduction of xenobiotic-induced metHb formation and the mechanisms underlying this effect have been
widely investigated in the last few years. The various
attempts to reduce methemoglobinemia have included:
preventing CYP-mediated oxidative metabolism of xenobiotics to hydroxylamines (Coleman et al., 1990; Malfará
et al., 2002); biochemical attenuation of metHb formation
with antioxidants (Prussick et al., 1992; Wright et al.,
1996; Dötsch et al., 1998; Wright et al., 1998; Dötsch et
al., 2000; Tanen et al., 2000; Matteuci et al., 2003; De
Moraes et al., 2008; Jo et al., 2008); and reduction of
metHb to hemoglobin by stimulating NADH diaphorase
or NADPH diaphorase (Dötsch et al., 2000).
L-arginine (ARG), a semi-essential amino acid, is
the nitrogenous precursor for the synthesis of nitric oxide
(NO) by a NADPH-dependent NO synthase (NOS) and
regulates vital metabolic pathways. NO is sufficiently nonpolar to cross membranes without a carrier and is known to
modulate vasorelaxation and exhibit antioxidant properties
due to superoxide scavenger and heme oxygenase inductor
activities (Wood et al., 2008).
In spite of all the evidence pointing to the importance
of ARG in vital pathways, the role of ARG supplementation in DDS-induced methemoglobinemia has yet to be
described in the literature. The aim of the present study
was to evaluate the role of ARG in single and multiple
dose regimens in DDS-induced methemoglobinemia. We
also evaluated whether the effect of ARG on DDS-induced
methemoglobinemia can be modulated by pretreatment
with N-nitro-L-arginine methyl ester (L-NAME), a nonspecific NOS inhibitor.
MATERIAL AND METHODS
Dapsone was supplied by FURP (Fundação para o
Remédio Popular; Guarulhos, Brazil) and L-(+)-arginine
was supplied by Acros organics (Morris Plains, NJ, USA).
L-NAME was purchased from Sigma-Aldrich (St. Louis,
MO, USA). KCN was supplied by Merck (Darmstadt,
Germany) and K3Fe(CN)6 was supplied by Merck (Rio de
Janeiro, Brazil). Water was purified with the Milli-Q Plus
system (Millipore, Bedford, MA, USA).
Experimental study
The experimental study was approved by the Ethics Committee for the Use of Animals of Ribeirão Preto
Campus, University of São Paulo, Brazil, in accordance
with the US National Institutes of Health Guide for the
Care and Use of Laboratory Animals (Protocol number
06.1.461.53.6). Male Wistar rats (200 ± 20 g) were kept for
48 hours before the experiment in a room under controlled
temperature (21-23 °C) and humidity (40-60%) and on a
12 h light:12 h dark cycle. The animals had free access to
chow and water throughout the experiment. The animals
(n = 8 per group) were treated in single or multiple dose
regimens. ARG was administered orally (p.o. gavage,
200 μL), dissolved in sterile physiologic saline, whereas
DDS was dissolved in dimethylsulphoxide (DMSO) and
administered intraperitoneally (i.p., 200 μL). L-NAME at
30 mg/kg was administered using a multiple dose regimen
in the same solution as ARG.
Single dose regimen
The control group received the vehicle of ARG
(sterile physiological saline) p.o. by gavage two hours
before the administration of the vehicle used to dissolve
DDS (DMSO) i.p. The DDS group received 40 mg/kg
DDS (i.p.) 2 hours after the administration of saline p.o.
The groups DDS + 5 mg/kg ARG, DDS + 15 mg/kg
ARG, DDS + 30 mg/kg ARG, DDS + 60 mg/kg ARG,
DDS + 180 mg/kg ARG received ARG at 5, 15, 30, 60
and 180 mg/kg doses, respectively, 2 hours before the
administration of 40 mg/kg DDS.
Multiple dose regimen
The control group received saline for five days p.o.
(gavage). On the fifth day, the animals received DMSO
i.p. 2 hours after saline administration. The DDS group
L-arginine, a nitric oxide precursor, reduces dapsone-induced methemoglobinemia in rats
received saline for five days p.o. and 40 mg/kg DDS on
the fifth day, 2 hours after saline administration. Groups
DDS + 5 mg/kg ARG, DDS + 15 mg/kg ARG, DDS + 30
mg/kg ARG, DDS + 60 mg/kg ARG, DDS + 180 mg/kg
ARG received ARG at 5, 15, 30, 60 and 180 mg/kg doses,
respectively, for five days. On the fifth day, 2 hours after
ARG administration, the animals received 40 mg/kg DDS.
Heparinized blood samples were collected two hours
after DDS or DMSO administration, in both dose regimens
(Liquemine 5000 IU, Roche, Rio de Janeiro, Brazil). Methemoglobin levels were determined immediately.
89
The initial blood measurement (A1) is referent to
MetHb and possible interferences. When blood is added
to KCN (A 2), MetHb is converted to cyanomethemoglobin (CNMetHb) and then possible interferences are
eliminated because CNMetHb does not absorb at 635
nm. When blood is added to K3Fe(CN)6 all hemoglobin is
converted to MetHb, and this measurement refers to total
MetHb (A3). Finally, blood is added to K3Fe(CN)6 and
KCN, with all hemoglobin converted to MetHb and then
to CNMetHb (A4).
Statistical analysis
Methemoglobin assay
Methemoglobin levels relative to hemoglobin levels
were determined according to the method described by
Evelyn and Malloy (1938) (modified by Harrison and
Jollow, 1986). Briefly, an aliquot (200 μL) of heparinized
blood was added to 10 mL of 0.02 M phosphate buffer pH
7.8 with 0.05% triton X-100 and then shaken in a mixer
for 30 seconds. The hemolysate was then fractionated into
four tubes. Tube 1 (A1) remained with hemolyzed blood.
An aliquot (50 μL) of 20% K3Fe(CN)6 was added to tubes
3 (A3) and 4 (A4). An aliquot (50 μL) of 10% KCN was
then added to tubes 2 (A2) and 4. The absorbance of each
tube was measured at 635 nm. Methemoglobin levels
relative to hemoglobin levels were then calculated by the
following equation:
GraphPad InStat® software (version 3.01) was used
for the calculation of means ± standard deviation. ANOVA
and the Tukey-Kramer post test for multiple comparisons
(p<0.05) were used to compare groups.
RESULTS
As there is no reference value for methemoglobin
levels in rats, some preliminary studies were conducted in
order to evaluate whether ARG or the vehicles could produce methemoglobinemia. A single dose regimen control
group was evaluated by administrating sterile physiological saline (p.o., 200 μL) and DMSO (i.p., 200 μL). The
administration of these vehicles resulted in 3.77 ± 0.43%
methemoglobin formation. In a pilot study, rats were
treated with 5, 15, 30, 60 and 180 mg/kg of ARG (p.o.,
n = 8) by gavage. Methemoglobin levels were assayed in
these groups, resulting in 1.70 ± 0.09%, 1.81 ± 0.08%,
FIGURE 1 - Effect of single dose ARG on methemoglobin levels (%). Animals were treated with ARG at 5, 15, 30, 60 and
180 mg/kg doses (p.o., gavage) 2 hours prior to the administration of 40 mg/kg of DDS (i.p.). Data expressed as means ± standard
deviation. *p < 0.05 compared to control group.
90
N. V. Moraes, M. M. Bergamaschi, M. L. P. Bianchi, J. B. Bragheto, W. R. Malfará, R. H. C. Queiroz
FIGURE 2 - Effect of multiple dose L-arginine (ARG) on methemoglobin level (%). Animals were treated with saline or ARG at 5,
15, 30, 60 and 180 mg/kg doses (p.o., gavage) or simultaneous gavage of ARG at 5 and 15 mg/kg doses and 30 mg/kg L-NAME for
five days. On the fifth day, rats were treated with 40 mg/kg DDS (i.p.) 2 hours after ARG administration. Data expressed as means
± standard deviation. *p < 0.001 compared to the Control group; #p<0.001 when groups treated with L-NAME are compared to
the respective group without L-NAME.
1.59 ± 0.05%, 1.96 ± 0.08% and 2.03 ± 0.37% of methemoglobin, respectively (data expressed as means ±
standard deviation). These results showed that ARG alone
did not produce methemoglobin.
Administration of 40 mg/kg DDS (i.p.) resulted in
methemoglobin levels of 17.18 ± 1.71% (Figure 1). The
dose of 40 mg/kg (i.p.) of DDS was known to produce
methemoglobinemia in rats based on previous studies by
our group (Malfara et al., 2002; De Moraes et al., 2008;
Bergamaschi et al., 2011). When ARG was administered in
a single dose two hours prior to DDS (40 mg/kg) it failed
to reduce DDS-induced methemoglobinemia (Figure 1).
ARG was also administered at doses 5, 15, 30, 60
and 180 mg/kg (po), in multiple dose regimens. Methemoglobin levels of groups treated with ARG only, in a
multiple drug regimen, did not produce significant levels
of methemoglobin (1.88 ± 0.68%; 2.00 ± 0.13%; 2.56 ±
0.44%; 2.43 ± 0.43% and 1.75 ± 0.76%, respectively),
as observed for a single dose regimen. Animals treated
with 5 or 15 mg/kg ARG for 5 days prior to 40 mg/kg
DDS administration showed a reduction in DDS-induced
methemoglobinemia, with methemoglobin levels similar
to the control group. However, higher doses of ARG (30,
60 and 180 mg/kg) in the multiple dose regimens did not
inhibit methemoglobin formation (Figure 2).
The effect of L-NAME, a NOS inhibitor, was evaluated in order to understand the mechanisms related to the
reduction of DDS-induced methemoglobinemia by ARG.
L-NAME inhibited the reduction in DDS-induced methe-
moglobinemia promoted by ARG, leading to MetHb levels
comparable to DDS administration alone (Figure 2).
DISCUSSION
The concentration of methemoglobin in erythrocytes is regulated by three systems: nicotinamide adenine
dinucleotide (NADH), nicotinamide adenine dinucleotide
phosphate (NADPH) and glutathione systems. Methemoglobin is converted to hemoglobin by the NADH system
when sufficient NADH-methemoglobin reductase is available; it contributes to 95% of methemoglobin reduction to
hemoglobin. The NADPH system reduces methemoglobin
to hemoglobin through the enzyme NADPH-methemoglobin reductase and contributes to 5% of methemoglobin
reduction. Finally, the conversion of reduced glutathione
to glutathione influences methemoglobin levels by reducing oxidizing agents (Evelo et al., 1998; Ward, McCarthy,
1998; Umbreit, 2007).
The standard treatment for methemoglobinemia
includes infusion with methylene blue, whose action
depends on the availability of NADPH within the erythrocytes. This therapy requires glucose-6-phosphate dehydrogenase (G6PD) optimal activity to produce sufficient
amounts of NADPH. In G6PD-deficient subjects, methylene blue therapy has been associated with hemolysis and
methemoglobinemia (Rehman, 2001). Several other substances have been investigated as alternatives to methylene
blue therapy. These have included ascorbic acid (Dötsch
L-arginine, a nitric oxide precursor, reduces dapsone-induced methemoglobinemia in rats
et al., 1998), cimetidine (Coleman et al., 1990; Malfará
et al., 2002), riboflavin (Dötsch et al., 2000), α-lipoic
acid (Coleman, Taylor, 2003), sodium thiosulfate (Matteucci et al., 2003), ethyl pyruvate (Jo et al., 2008) and
N-acetylcysteine (Wright et al., 1996; Wright et al., 1998;
Dötsch et al., 2000; Tanen et al., 2000; De Moraes et al.,
2008). N-acetylcysteine, a precursor of glutathione, used
in combination with DDS in rats, has shown increased
methemoglobin levels in these animals compared to rats
treated with DDS alone. Some authors have suggested
that glutathione can regenerate DDS-NOH from dapsone
nitroso derivatives thus resulting in higher methemoglobin
levels (De Moraes et al., 2008).
Considering the potency of ARG and NO as antioxidant agents, it was proposed that the co-administration of
ARG and DDS might reduce the methemoglobin levels
associated to DDS use. The cationic amino acid ARG is
the precursor for NO biosynthesis mediated by NO synthase. Three isoforms of NO synthase (NOS) occur in a
number of tissues: neuronal NOS (nNOS); inducible NOS
(iNOS) located in glia cells, and endothelial NOS (eNOS)
located in endothelial cells (Palmer et al., 1987; Thomas
et al., 2008). The iNOS can form much larger amounts
of NO compared with other isoforms. In many cells and
pathological conditions the supply of extracellular ARG
is rate-limiting for NO production (Brunini et al., 2007;
Thomas et al., 2008).
In the present study, L-NAME administration
suppressed the reduction in DDS-induced methemoglobinemia mediated by 5 mg/kg and 15 mg/kg ARG in
multiple dose regimens. Considering that L-NAME is
a non-specific NOS inhibitor, our data suggest that the
ARG effect on methemoglobin is mediated by NO. NO is
considered a potent antioxidant agent in vitro and in vivo.
Its antioxidant activity has been proven by suppressing
iron-induced generation of hydroxyl radicals (OH) via the
Fenton reaction, interrupting lipid peroxidation chain reaction, increasing the glutathione antioxidant potency and
inhibiting cysteine proteases (Chiueh, 1999). On the other
hand, increased methemoglobin levels are a known toxic
effect of inhaled NO therapy, commonly used for hypoxic
neonates. NO can combine with hemoglobin to produce
nitrosylhemoglobin and thus form methemoglobin by
oxidation (Weinberger et al., 2001; Hamon et al., 2010).
Based on our observations, we can hypothesize that lower
doses of ARG were beneficial to decrease methemoglobin
levels because of the antioxidant properties of NO. However, higher doses of ARG do not decrease metHb levels
because the antioxidant properties of NO are combined to
its methemoglobinizant effect.
Excess ARG supplementation is also related with the
91
production of NG,NG-dimethyl-L-arginine (ADMA) which
is a NOS inhibitor (Masuda et al. 2002). This metabolite
can convert NO to a superoxide generator (Thomas et al.,
2008). This may explain why NO is beneficial to DDSinduced methemoglobinemia at low ARG concentrations
yet deleterious when excess ARG supplementation is
administered to rats in multiple dose regimens.
NO also produces inhibitory effects in cytochrome
P450 mediated drug metabolism. It is known that NO
forms complexes with the catalytic center of P450 enzymes which results in a decrease in enzymatic activities
of rat microsomes (Khatsenko et al. 1993). The inhibitory effects of NO are rapid, concentration-dependent
and mainly in CYP 2C11 > 2B1/2 > 2E1 = 3A2 > 1A1/2
(Vuppugalla, Mehvar 2004a, 2004b). If NO had inhibited
DDS oxidative metabolism, animals treated with higher
ARG concentration would present lower metHb levels.
However, our results showed that the reduction in MetHb
levels by ARG is observed only when animals are treated
with multiple doses of 5 and 15 mg/kg ARG. Thus, CYP
inhibition by NO does not seem to explain the reduction
in MetHb levels.
In conclusion, ARG reduces DDS-induced methemoglobinemia in rats when low doses (5 and 15 mg/kg)
are administered as a multiple dose regimen. The effect
can be blocked by the simultaneous administration of
L-NAME (a NOS inhibitor). Thus, we can conclude that
ARG supplementation can be an effective reducing agent
for chronic treatment of DDS-induced methemoglobinemia and that its effect is mediated by NO.
ACKNOWLEDGMENTS
This work was supported by CAPES (Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior). The
authors gratefully thank FURP (Fundação para o Remédio Popular) for providing dapsone and Prof. Dr. Lusiane
Maria Bendhack for providing L-NAME.
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Received for publication on 13th April 2011
Accepted for publication on 06th December 2011