Toxicology 184 (2003) 179 /187

www.elsevier.com/locate/toxicol

Profile of nonprotein thiols, lipid peroxidation and daminolevulinate dehydratase activity in mouse kidney and liver
in response to acute exposure to mercuric chloride and sodium
selenite
Marcelo Farina a,c,
, Ricardo Brandao a, Fabiana S. de Lara a,
Letcia B. Pagliosa a, Felix A. Soares c, Diogo O. Souza c, Joao B.T. Rocha b
a

Curso de Farmacia, Centro de Ciencias da Saude, Universidade Regional Integrada do Alto Uruguai e Missoes, C
ampus de Erechim,
99700-000 Erechim, RS, Brazil
b
Departamento de Qumica, Centro de Ciencias Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS,
Brazil
c
Departamento de Bioqumica, ICBS, Instituto de Ciencias Basicas da Saude, Universidade Federal do Rio Grande do Sul, Rua Ramiro
Barcelos, 2600 Anexo, Bairro Santana, 90035-003 Porto Alegre, RS, Brazil
Received 9 May 2002; received in revised form 11 September 2002; accepted 15 September 2002

Abstract
The effects of mercury (Hg2) and selenite (Se4 ) on d-aminolevulinic acid dehydratase (d-ALA-D) activity, 2thiobarbituric acid reactive substances (TBARS) and nonprotein sulfhydryl content (NPSH) in mouse kidney and liver
were investigated. Male mice were given a single i.p. injection of Hg2 and/or Se4 (25 mmol/kg) and were killed at 6,
12, 24 and 48 h after treatment. Hg2 inhibited renal d-ALA-D at 6 and 12 h after treatment. Se4 abolished the
inhibitory effect of mercury on renal d-ALA-D at 12 h after treatment. Renal and hepatic NPSH content decreased
after Hg2 exposure and selenite inhibited, at least in part, the Hg-induced oxidation of renal and hepatic NPSH. Se4
and Hg2 , when injected alone, did not alter hepatic or renal TBARS levels; however, simultaneous exposure to these
compounds increased hepatic and renal TBARS levels at 12 and 48 h after treatment, respectively. Present results
suggest that selenium abolishes the interaction of Hg2 with sulfhydryl groups of protein and nonprotein sources.
# 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Thiol groups; Lipid peroxidation; d-Aminolevulinate dehydratase; Selenium; Mercury; Interaction

1. Introduction

Corresponding author. Tel.: /51-33-16-5559; fax: /51-3316-5540

E-mail address: marcelofarina@zipmail.com.br (M. Farina).

It has been well documented that mercury and

selenium interact in the body of mammals, and the
co-administration of both reduces the toxicity of
each other (Magos and Webb, 1980; Cuvin-Aralar

0300-483X/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 3 0 0 - 4 8 3 X ( 0 2 ) 0 0 5 7 6 - 0

180

M. Farina et al. / Toxicology 184 (2003) 179 /187

and Furness, 1991). Although the mechanism

underlying the protective effects of selenite against
mercury toxicity is still unsolved, there is evidence
that selenide (produced from selenite in the presence of glutathione) forms a (metal /Se/S) complex, which then binds to selenoprotein P to form a
ternary complex, (metal /Se/S) /Sel P (Sasakura
and Suzuki, 1998).
Mercuric chloride (Hg2) and sodium selenide
(Se4) may react with and deplete sulfhydryl (/
SH) groups of biomolecules. Hg2 reacts with
sulfhydryl groups on proteins forming stable
mercaptides (Strycks and Kolthoff, 1953) or with
nonprotein thiols, such as reduced glutathione
(GSH) and cysteine (Cys), forming large amounts
of complexes of the Hg /GSH and Cys /Hg types
(Rabenstein, 1989; Lash et al., 1998). In contrast,
Se4 reacts with thiols such as GSH, generating
relatively unstable derivatives of the RSSeSR
(selenotrisulfides) type (Tsen and Tappel, 1958;
Ganther, 1968) which can generate reactive oxygen
species in subsequent steps (Spallholz, 1997; Nogueira et al., 2001).
Lipid peroxidation serves as a marker of cellular
oxidative stress and has been observed after Hg2
(Fukino et al., 1984; Huang et al., 1996) and Se4
(Shen et al., 1999; Moak and Christensen, 2001)
exposure. Hg2 can stimulate lipid peroxidation
by enhancing H2O2 formation in mitochondria
(Lund et al., 1991), while selenite-induced lipid
peroxidation seems to be related to generation of
superoxide (Spallholz, 1997). However, some lines
of evidence indicate that lipid peroxidation is not
the cause of mercury- (Ichikawa et al., 1987) or
selenite- (Lashneva et al., 1996) induced cell death.
Moreover, selenite has been considered to be a
radical scavenger (Rodemann et al., 1999).
d-Aminolevulinate dehydratase (d-ALA-D) is
an enzyme sensitive to sulfhydryl-blocking agents
such as mercury (Rocha et al., 1995, 2001;
Nogueira et al., 2002), lead (Rodrigues et al.,
1989) and copper (Nelson et al., 1981). d-ALA-D
is also inhibited by elements such as aluminum
(Vieira et al., 2000), selenium and tellurium
compounds (Barbosa et al., 1998; Farina et al.,
2001, 2002; Maciel et al., 2000) that oxidize /SH
groups or compete with Zn2 at the active center
of the enzyme. This enzyme participates in the

second step of heme biosynthesis catalyzing the

condensation of two molecules of 5-aminolevulinic
acid to porphobilinogen (Jaffe, 1995) and consequently plays a fundamental role in the aerobic
metabolism of animals.
In view of the controversy in the literature about
the effects of mercury and selenite on lipid
peroxidation and the lack of studies concerning
the inhibition or even the antagonism between the
effects of Hg2 and Se4 on d-ALA-D activity,
our direct aim was to investigate the effects of
acute exposure to mercuric chloride and/or selenite
on lipid peroxidation and d-ALA-D activity in
mouse liver and kidney and their relationship with
nonprotein thiols. Considering the interaction of
selenide (produced from selenite in the presence of
glutathione) with Hg2 in the body (Sasakura and
Suzuki, 1998), we hypothesized that simultaneous
exposure to selenite and inorganic mercury can
reduce or cancel out the toxic effects observed
after poisoning with individual compounds.

2. Methods
2.1. Compounds
Reduced glutathione, 5,5?-dithio-bis(2-nitrobenzoic acid) (DTNB), 5-aminolevulinic acid, brilliant
blue G, 2-thiobarbituric acid, acetic acid and p dimethylaminobenzaldehyde were obtained from
Sigma Chemical Co. (St. Louis, MO, USA).
Bovine serum albumin, mono- and dibasic potassium phosphate, acetic acid, ortho -phosphoric
acid, trichloroacetic acid, sodium chloride, sodium
selenide and mercuric chloride were obtained from
Merck (Darmstadt, Germany).
2.2. Animals and treatment
Adult male mice (Swiss albino) from our own
breeding colony were maintained under controlled
temperature (22 /25 8C) and natural lighting conditions, with free access to water and food
(Nuvital-PR, Brazil). A total of 80 mice were
used. Mercuric chloride and sodium selenite were
dissolved in physiological saline solution to allow
for single intraperitoneal administrations of vo-

M. Farina et al. / Toxicology 184 (2003) 179 /187

181

lume doses of 10 ml/kg. The doses used were the

same (25 mmol/kg) for both compounds. Mercury
dose was based on the study of Fukino et al.
(1984), where a similar acute exposure to mercuric
chloride increased lipid peroxidation in renal
tissue. The selenite dose was identical to that of
mercuric chloride considering that the molar ratio
of Hg2 to Se is 1:1 in the ternary complex [(Hg /
Se/S) /Sel P] (Sasakura and Suzuki, 1998). Hg /Se
mice received 25 mmol/kg of mercuric chloride plus
25 mmol/kg of sodium selenite. Control mice
received two intraperitoneal injections of physiological saline solution (10 ml/kg). Mice receiving
the same treatment were divided into four subgroups and killed at 6, 12, 24 and 48 h after
administration. A total of 16 subgroups of five
animals each were used.

and free /SH groups were determined in the clear

supernatant (which was neutralized with 0.1 M
NaOH) by the method of Ellman (1959).

2.3. Tissue preparation

Protein was measured by the method of Bradford (1976) using bovine serum albumin as the
standard.

Mice were killed by decapitation and the livers

and kidneys were quickly removed and homogenized in 7 volumes of 150 mM NaCl. The
homogenates were centrifuged at 4000 /g at
4 8C for 10 min to yield low speed supernatant
fractions (S1) that were used for the d-ALA-D
assay and for measurement of NPSH.
2.4. Enzyme assay
d-ALA-D activity was assayed by the method of
Sassa (1982) by measuring the rate of product
(porphobilinogen) formation except that 84 mM
potassium phosphate buffer, pH 6.4 and 2.5 mM
of aminolevulinic acid (ALA) were used (Barbosa
et al., 1998). Incubations were carried out for 1
(liver) and 2 h (kidney) at 39 8C. The reaction
product was determined using modified Ehrlichs
reagent at 555 nm, with a molar absorption
coefficient of 6.1 /104 per M for the Ehrlich/
porphobilinogen salt.
2.5. Determination of nonprotein thiols (NPSH)
To determine NPSH, 500 ml of 10% trichloroacetic acid were added to 500 ml of low speed
supernatant (S1). After centrifugation (4000 /g at
4 8C for 10 min), the protein pellet was discarded

2.6. Determination of thiobarbituric acid-reactive

substances (TBARS)
TBARS were determined in tissue homogenates
by the method of Ohkawa et al. (1979), in which
malondialdehyde (MDA), an end-product of fatty
acid peroxidation, reacts with thiobarbituric acid
(TBA) to form a colored complex. MDA values
were determined with the absorbance coefficient of
the MDA /TBA complex at 532 nm /1.56 /105
per cm/mmol.
2.7. Protein quantification

2.8. Statistical analysis

Data were analyzed by three-way ANOVA [2
with/without Hg2 /2 with/without Se4 /4
times of sampling (6, 12, 24 or 48 h)]. Differences
between groups were analyzed by one-way ANOVA, followed by Duncans multiple range test
when appropriate. A possible relationship between
thiol depletion, ALA-D inhibition and lipoperoxidation was also evaluated by two-tailed Pearson
Correlation.

3. Results
The renal and hepatic d-ALA-D activities are
reported in Tables 1 and 2, respectively. Hg2,
when administered alone, inhibited renal d-ALAD at 6 and 12 h, but not at 24 and 48 h after
treatment (Table 1), Selenite, when administered
alone, had no effect on renal d-ALA-D at either
timepoint. When both compounds were administered simultaneously, the inhibitory effect of Hg2
was abolished at 12 h after treatment in kidneys.
In liver (Table 2), the enzyme was not inhibited
after acute exposure to Se4 and Hg2. The

M. Farina et al. / Toxicology 184 (2003) 179 /187

182

Table 1
d-ALA-D activity from mouse kidney after acute exposure to mercuric chloride and/or sodium selenite
6h
Control
Na2SeO3
HgCl2
Na2SeO3 plus HgCl2
P value (treatment effect)

12 h

8.019/0.12
7.259/0.71
5.819/0.38a,
6.149/0.36a
0.016

24 h

7.959/0.95
7.189/0.39
6.249/0.50a,1,2
7.459/0.25
0.047

48 h

7.999/0.21
8.079/0.20
7.889/0.802,3
7.579/0.24
0.467

P value (time effect)

8.269/0.29
8.009/0.28
8.369/0.493
7.409/0.58
0.165

0.667
0.171
0.025
0.095

Mice were killed at 6, 12, 24 and 48 h after treatment. Enzyme activity is expressed as nmol of formed porphobilinogen per mg
protein per h. The results are presented as mean9/S.E. (n/5 animals per group). Means not sharing the same superscript letters were
different at the P B/0.05 level (Duncans multiple range test). Comparisons were made within each column (treatment effect). Means
not sharing the same superscript number were different at the P B/0.05 level (Duncans multiple range test). Comparisons were made
within each row (time effect).

simultaneous injection of both compounds also

did not alter hepatic d-ALA-D activity at either
timepoint.
Renal and hepatic nonprotein thiols (NPSH) are
reported in Tables 3 and 4, respectively. Hg2
decreased renal NPSH at 6 and 12 h after
treatment. In liver, the Hg2-induced NPSH
depletion was more persistent and was observed
6, 12 and 24 h after treatment. In fact, the effect of
Hg2 varied with sampling time, as indicated by a
significant Hg /time interaction for renal (P B/
0.001) and hepatic (P /0.010) NPSH. Se4 alone
decreased the total amount of hepatic NPSH at 24
and 48 h after treatment. When Se4 and Hg2
were administered simultaneously, selenite partially reversed the Hg-induced oxidation of renal
NPSH at 12 h but not at 6 h after treatment in
both kidneys (Table 3) and livers (Table 4). Twotailed Pearson Correlation between NPSH and dALA-D activity was significant at the 0.01 level for
both kidneys and livers (data not shown).

Renal TBARS levels were not changed by either

Hg2 or Se4 injections. However, combined
injection of the two compounds caused a significant increase in renal TBARS contents that was
apparent only 48 h after treatment (Table 5).
Changes in hepatic TBARS levels after Hg2
and Se4 administration displayed a similar
profile to that of renal tissue, but a significant
increase in hepatic TBARS levels in mice treated
simultaneously with Hg2 and Se4 appeared at
12 and 48 h after injection (Table 6).

4. Discussion
The inhibitory effect of Hg2 on renal d-ALAD after administration of mercury alone was
different from that after simultaneous administration of mercury and selenium at 12 h after
administration (Table 1). In fact, selenite abolished
the inhibitory effect of Hg2 on renal enzyme after

Table 2
d-ALA-D activity from mouse liver after acute exposure to mercuric chloride and/or sodium selenite
6h
Control
Na2SeO3
HgCl2
Na2SeO3 plus HgCl2
P value (treatment effect)

23.09/1.66
25.99/1.77
21.99/3.14
20.99/0.65
0.434

12 h
23.09/2.71
25.69/1.08
20.69/1.05
19.99/0.28
0.109

24 h
23.29/0.68
23.09/2.25
22.69/1.78
22.29/0.81
0.951

48 h
23.99/1.32
23.39/0.97
20.79/1.73
20.49/1.91
0.283

P value (time effect)

0.971
0.241
0.879
0.575

Mice were killed at 6, 12, 24 and 48 h after treatment. Enzyme activity is expressed as nmol of formed porphobilinogen per mg
protein per h. The results are presented as mean9/S.E. (n /5 animals per group). Statistical procedures are the same as described in
Table 1.

M. Farina et al. / Toxicology 184 (2003) 179 /187

183

Table 3
NPSH from mouse kidney after acute exposure to mercuric chloride and/or sodium selenite
6h
Control
Na2SeO3
HgCl2
Na2SeO3 plus HgCl2
P value (treatment effect)

4.759/0.09
5.099/0.10
3.809/0.44a
3.489/0.44a
0.011

12 h
5.099/0.10
5.129/0.10
2.579/0.15a,1
3.409/0.20b
B/0.001

24 h

48 h

4.459/0.23
4.939/0.21
5.059/0.192
5.159/0.251
0.609

P value (time effect)

4.629/0.16a
5.199/0.09a,b
5.519/0.20b,2
5.269/0.32a,b,1
0.048

0.301
0.597
B/0.001
0.001

Mice were killed at 6, 12, 24 and 48 h after treatment. NPSH are expressed as nmol/mg protein. The results are presented as mean9/
S.E. (n /5 animals per group). Statistical procedures are the same as described in Table 1.

12 h of treatment. Yamamoto (1985) demonstrated that Hg2 concentrations in mouse kidney

decreased markedly after simultaneous administration of mercuric chloride and sodium selenite
when compared with the animals that received
mercuric chloride alone. Hg2 showed a different
accumulation in kidney when selenite was administered simultaneously. Selenide (produced from
selenite) forms a Hg /Se/S complex, which then
binds to selenoprotein P to form a ternary complex, (Hg /Se/S) /Sel P (Sasakura and Suzuki,
1998) and the transport of the Se /Hg complex to
the kidney seems to be limited (Yamamoto, 1985).
Although a recent work using analysis of Se and
Hg by extended X-ray absorption fine structure
(EXAFS) spectroscopy demonstrated the presence
of the Se /Hg complex in the plasma of rabbits
treated with mercuric chloride and sodium selenite
(Gailer et al., 2000), the in vivo quantification of
this complex in renal and hepatic tissues have not
yet been performed. In our results, the absence of
inhibitory effect on renal d-ALA-D of Se/Hg mice
after 12 h of treatment indicates the protective
effect of selenite against mercury toxicity. Even

though the Se /Hg complex was not measured in

our work, we realize that the protective effect of
selenite against mercury-induced inhibition on
renal d-ALA-D is related to the formation of a
Se/Hg complex. This conclusion is based on
previous studies demonstrating that concomitant
exposure to Hg2 and Se4 results in the formation of Hg /Se /S which initially involves the
formation of Hg /Se as an intermediary complex
(Yamamoto, 1985; Yoneda and Suzuki, 1997a,b;
Gailer et al., 2000). Recently, this detoxification
product has been elegantly characterized in rabbit
plasma by EXAFS spectroscopy. Furthermore,
these authors also demonstrated that a ternary
complex containing Hg /Se /S can be formed in
vitro by addition of equimolar Hg2 and Se4 to
aqueous, buffered glutathione. The in vitro formation of a complex between Hg /Se and reduced
glutathione raise the possibility of in vivo formation of a similar product, which could help in
explaining the protective effect of Se against Hginduced d-ALA-D inhibition. In addition, selenite
alone did not change the enzyme activity and the
protective effect of selenite against Hg-induced d-

Table 4
NPSH from mouse liver after acute exposure to mercuric chloride and/or sodium selenite
6h
Control
Na2SeO3
HgCl2
Na2SeO3 plus HgCl2
P value (treatment effect)

6.679/0.13
7.209/0.14
5.289/0.70a,1
4.919/0.64a
0.017

12 h
7.359/0.15
7.219/0.15
3.469/0.10a,2
4.759/0.27b
B/0.001

24 h
6.669/0.27
5.879/0.281
4.549/0.39a,1,2
5.339/0.54a,b
0.010

48 h
6.279/0.44
6.379/0.201
5.109/0.631
5.079/0.67
0.173

P value (time effect)

0.149
0.001
0.031
0.903

Mice were killed at 6, 12, 24 and 48 h after treatment. NPSH are expressed as nmol/mg protein. The results are presented as mean9/
S.E. (n /5 animals per group). Statistical procedures are the same as described in Table 1.

M. Farina et al. / Toxicology 184 (2003) 179 /187

184

Table 5
TBARS from mouse kidney after acute exposure to mercuric chloride and/or sodium selenite
6h
Control
Na2SeO3
HgCl2
Na2SeO3 plus HgCl2
P value (treatment effect)

12 h

1.799/0.14
1.869/0.15
1.689/0.10
1.859/0.10
0.737

1.559/0.09
1.539/0.12
1.469/0.15
1.739/0.14
0.533

24 h
1.799/0.07
1.699/0.09
1.549/0.15
1.859/0.21
0.444

48 h
1.499/0.07
1.559/0.07
1.619/0.05
2.129/0.16a
0.001

P value (time effect)

0.088
0.196
0.628
0.401

Mice were killed at 6, 12, 24 and 48 h after treatment, TBARS levels are expressed as nmol of MDA per mg protein. The results are
presented as mean9/S.E. (n/5 animals per group). Statistical procedures are the same as described in Table 1.

ALA-D inhibition was observed only 12 h after

administration. This suggests that the enzyme was
first inhibited by mercury and selenium restored
enzyme activity.
Mercury inhibits d-ALA-D by reacting with
sulfhydryl groups located at the active center of
the enzyme (Rocha et al., 1995). Renal d-ALA-D
activity decreased in Se/Hg mice at 6 h after
treatment and this inhibitory effect was abolished
at 12 h after treatment. Some studies have
demonstrated that the selenium chemical speciation that complexes with mercury seems to be
selenide (Se2 or HSe) (Sasakura and Suzuki,
1998; Suzuki et al., 1998). Of particular importance for the treatment with Hg2, the result of
renal d-ALA-D strongly suggests that selenium
(selenide) can reactivate thiol-containing enzymes.
In fact, we propose that the translocation of Hg2
from d-ALA-D sulfhydryl groups to selenide
regenerates the enzyme activity at 12 h after
treatment. In agreement with this, selenols and
selenides are more highly reactive toward mercuric
ion than thiols (Clarkson, 1997) and seem to

catalyze the reduction of disulfide to thiols (Singh

and Kats, 1995).
In contrast to renal d-ALA-D, mercury alone
did not inhibit hepatic d-ALA-D (Table 2). Since
the kidney is one of the most sensitive tissues to
mercury poisoning, renal d-ALA-D was preferentially inhibited by mercury when compared with
the hepatic enzyme. Furthermore, the levels of
nonprotein /SH is higher in hepatic tissue than in
kidney (Tables 3 and 4; Maciel et al., 2000; JaquesSilva et al., 2001) which could provide additional
protection for liver d-ALA-D. In the liver, higher
mercury levels were found in rats exposed to
selenite (Potter and Matrone, 1974; Yamamoto,
1985) and the interaction between sodium selenite
and mercuric chloride resulted in a global translocation of mercury from the kidney to the liver
(Komsta-Szumska and Chmielnicka, 1977). The
absence of inhibitory effect on hepatic d-ALA-D
of Se/Hg mice suggests that the Se/Hg complex is
inert toward d-ALA-D sulfhydryl groups.
Hg2 and Se4 may react with and deplete
sulfhydryl groups of protein and nonprotein

Table 6
TBARS from mouse liver after acute exposure to mercuric chloride and/or sodium selenite
6h
Control
Na2SeO3
HgCl2
Na2SeO3 plus HgCl2
P value (treatment effect)

2.489/0.18
2.509/0.14
2.459/0.16
2.259/0.05
0.578

12 h
2.179/0.08a
2.229/0.14a
2.719/0.50a,b
3.229/0.64b
0.042

24 h
2.489/0.09
2.539/0.09
2.409/0.12
2.379/0.08
0.601

48 h
2.279/0.06a
2.459/0.10a,b
2.499/0.11a,b
2.639/0.09b
0.089

P value (time effect)

0.138
0.287
0.837
0.148

Mice were killed at 6, 12, 24 and 48 h after treatment. TBARS levels are expressed as nmol of MDA per mg protein. The results are
presented as mean9/S.E. (n/5 animals per group). Statistical procedures are the same as described in Table 1.

M. Farina et al. / Toxicology 184 (2003) 179 /187

sources. The oxidative effects of Hg2 on renal

and hepatic nonprotein sulfhydryl groups (NPSH)
indicate an apparent high affinity for thiol groups
from nonprotein sources. This is a consequence of
the high concentration of nonprotein thiol groups
(mM) when compared with protein concentrations
in tissues. Consequently, by virtue of the Law of
Mass Action, nonprotein thiol groups more readily react with oxidants, sparing thiol protein
groups from inactivation. This agrees with previous reports indicating the high binding affinity
of Hg for low-molecular weight thiols, such as
GSH and Cys (Rabenstein, 1989; Lash et al.,
1998).
Protein SH groups were not evaluated in this
work for three reasons: first, total amount of
protein thiols does not truly represent d-ALA-D
sulfhydryl content because d-ALA-D represents
only a small proportion of total renal or hepatic
protein. Second, d-ALA-D, in contrast to the bulk
of renal and hepatic proteins possesses thiols in
close proximity (vicinal thiols, Markham et al.,
1993) which, in analogy with low molecular weight
dithiols (Rhead and Scharauzer, 1974; Maciel et
al., 2000; Farina et al., 2002) render this enzyme a
preferential target for oxidants than protein containing non-vicinal thiols. Third, as a consequence
of their high concentrations, nonprotein thiol
groups should react before with oxidants than
the majority of cellular proteins and, in fact, only
those proteins with extreme reactivity (such as dALA-D) would react with oxidants before nonprotein thiols. Furthermore, nonprotein thiols
were preferentially determined in our study because previous work in our laboratory (Barbosa et
al., 1998) showed that glutathione, the main
endogenous nonprotein thiol, presents protective
effect against chalcogen-induced d-ALA-D inhibition. Not surprisingly, in our work, two-tailed
Pearson Correlation was significant for NPSH and
d-ALA-D activity.
The oxidation of GSH by selenite has been well
documented (Ganther, 1968; Seko et al., 1989) and
seems to depend on a variety of factors, including
pH and the GSH:Se ratio (Tsen and Tappel, 1958;
Rhead and Scharauzer, 1974). Our results demonstrated that selenite increased the NPSH oxidation
just in livers (Table 2). Although selenium and

185

mercury concentrations in organs were not determined, the absence of Se4-induced NPSH depletion in kidneys (Table 3) can be related to the
lower deposition of Se in kidney when compared
with liver (Hansen and Kristensen, 1979; Gregus et
al., 2001). In line with this, selenite presents a high
affinity by liver (Wilber, 1980).
Selenium inhibited, at least in part, Hg-induced
oxidation of NPSH at 12 h after treatment. This
phenomenon agrees with that observed for dALA-D activity after simultaneous exposure to
Hg2 and Se4. The translocation of Hg2 from
NPSH groups to selenide (generated from selenite)
may contribute to the decreasing oxidation of
renal and hepatic NPSH after 12 h of treatment
in Se /Hg mice.
Lipid peroxidation has been observed after
Hg2 (Fukino et al., 1984; Huang et al., 1996)
and Se4 (Shen et al., 1999; Moak and Christensen, 2001) exposure. However, mercury- and
selenite-induced lipid peroxidation has been described under different conditions of toxicity,
which refer mainly to different doses, different
time of administration and different strain and age
of the animals. Moak and Christensen (2001)
observed increased hepatic lipid peroxidation in
rats fed a diet containing selenite (2.0 mg selenium
per kg diet) only after 15 weeks of treatment.
Fukino et al. (1984) showed increased TBARS
levels in a study where doses and timepoints were
similar to our experimental protocol, however,
they used rats unless mice. Our results did not
show increased TBARS levels in both livers and
kidneys after individual treatment with selenite or
mercuric chloride at either timepoint. The absence
of increased TBARS levels in animals treated with
selenite or mercuric chloride alone is probably
related to the conditions of intoxication, such as
used doses and animal strain.
The reviewed literature indicates that mercuric
ion binds to selenium to form a biologically inert
complex (Hansen, 1988) and the toxicity of the
complex is very low in comparison with mercuric
chloride or sodium selenite. The formation of such
complex of lower toxicity in animals may play an
important role in the modification of toxicity of
mercuric ion by selenite (Naganuma et al., 1982).
In contrast, our results show increasing hepatic

186

M. Farina et al. / Toxicology 184 (2003) 179 /187

and renal TBARS levels in Se /Hg mice, suggesting, in this case, an apparent toxic effect of the
selenium/mercury complex. The increase in hepatic TBARS levels in Se/Hg mice was significant at
12 and 48 h after treatment. The absence of
increased TBARS levels at 24 h after treatment
could be related to the kinetic of formation of Se/
Hg complex and its transport from blood to liver.
The increase in renal TBARS levels was observed
only at 48 h after treatment. This phenomenon is
probably related to the delayed transport of Se/
Hg complex to the kidneys. In fact, the transport
of the Se /Hg complex to the kidney seems to be
limited (Yamamoto, 1985).
In summary, the results of the present study
indicate that Se4 is effective in restoring Hg2inhibited renal d-ALA-D. Moreover, selenite abolished the interaction of Hg2 with sulfhydryl
groups of nonprotein sources. The major shortcoming of therapeutic interventions against Hg2
intoxication resides in the poor ability of monoand dithiol compounds to remove Hg2 from
thiol-containing proteins. Although selenite (Se4)
can present pro-oxidant properties, the effectiveness of Se4 (possibly mediated by selenide) in
reactivating the thiol content of d-ALA-D indicates that selenide should be carefully considered
for the treatment of Hg2 intoxication.

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