Selenoxides inhibit δ-aminolevulinic acid dehydratase

Article abstract of DOI:10.1016/S0378-4274(00)00296-4

The effect of two selenides and their selenoxides on δ-aminolevulinic acid dehydratase (δ-ALA-D) from liver of adult rats was investigated. In vivo, selenides can be oxidized to selenoxides by flavin-containing monooxygenases (FMO) and selenoxides can regenerate selenides by thiol oxidation. Phenyl methyl selenide (PhSeCH₃) and 1-hexynyl methyl selenide (C₄H₉C≡CSeCH₃) were converted to selenoxides by reaction with H₂O₂. PhSeCH₃ and C₄H₉C≡CSeCH₃ had no effect on δ-ALA-D up to 400 μM. Conversely, their selenoxides inhibited δ-ALA-D, and the IC₅₀ for enzyme inhibition was about 100 and 70 μM, respectively. Partially purified δ-ALA-D (P₅₅) from swine liver was also inhibited by these selenoxides. The inhibitory action of selenoxides was antagonized by dithiotreitol (DTT). Moreover, δ-ALA-D from a plant source was inhibited by the selenoxides, suggesting a possible involvement of -SH groups in a distinct site of the homologous region implicated in Zn²⁺ binding in mammalian δ-ALA-D. After exposure to PhSeCH₃ (500 μmol/kg/day) for 45 or 30 days, the activity of δ-ALA-D from liver of mice decreased to about 50% of the control group. The in vivo inhibitory action of this compound was not antagonized by DTT. PhSeCH₃ and C₄H₉C≡CSeCH₃ had no effect on the rate of DTT oxidation, but their selenoxides oxidized DTT. The results of the present study suggest that hepatic δ-ALA-D of rodents is a potential molecular target for selenides as a consequence of their metabolism to selenoxides by FMO.

Full text of DOI:10.1016/S0378-4274(00)00296-4

Toxicology Letters 119 (2001) 27–37
www.elsevier.com/locate/toxlet
Selenoxides inhibit d-aminolevulinic acid dehydratase
a,b
a
a
Marcelo Farina , Vanderlei Folmer , Rodrigo C. Bolzan ,
a
a
a
a,
Leandro H. Andrade , Gilson Zeni , Ant oˆ nio L. Braga , Jo a˜ o B.T. Rocha *
a
Departamento de Qu ´ı mica, Centro de Ci eˆ ncias Naturais e Exatas, Uni6ersidade Federal de Santa Maria, 97105-900,
Santa Maria, RS, Brazil
b
Curso de Farm a´ cia, Centro de Ci eˆ ncias da Sa u´ de, Uni6ersidade Regional Integrada do Alto Uruguai e das Miss o˜ es, Erechim,
RS, Brazil
Received 17 July 2000; received in revised form 20 October 2000; accepted 23 October 2000
Abstract
The effect of two selenides and their selenoxides on d-aminolevulinic acid dehydratase (d-ALA-D) from liver of
adult rats was investigated. In vivo, selenides can be oxidized to selenoxides by flavin-containing monooxygenases
(
FMO) and selenoxides can regenerate selenides by thiol oxidation. Phenyl methyl selenide (PhSeCH ) and 1-hexynyl
3
methyl selenide (C H C/CSeCH ) were converted to selenoxides by reaction with H O . PhSeCH and
4
9
3
2
2
3
C H C/CSeCH had no effect on d-ALA-D up to 400 mM. Conversely, their selenoxides inhibited d-ALA-D, and
4
9
3
the IC50 for enzyme inhibition was about 100 and 70 mM, respectively. Partially purified d-ALA-D (P ) from swine
55
liver was also inhibited by these selenoxides. The inhibitory action of selenoxides was antagonized by dithiotreitol
(
DTT). Moreover, d-ALA-D from a plant source was inhibited by the selenoxides, suggesting a possible involvement
2+
of ꢀSH groups in a distinct site of the homologous region implicated in Zn
binding in mammalian d-ALA-D. After
exposure to PhSeCH (500 mmol/kg/day) for 45 or 30 days, the activity of d-ALA-D from liver of mice decreased to
3
about 50% of the control group. The in vivo inhibitory action of this compound was not antagonized by DTT.
PhSeCH and C H C/CSeCH had no effect on the rate of DTT oxidation, but their selenoxides oxidized DTT. The
3
4
9
3
results of the present study suggest that hepatic d-ALA-D of rodents is a potential molecular target for selenides as
a consequence of their metabolism to selenoxides by FMO. © 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: d-Aminolevulinate dehydratase; Selenoxides; Flavin-containing monooxygenase
(
Forstrom et al. 1978; Landestein et al., 1979;
1
. Introduction
Wingler and Brigelius-Floh e´ , 1999). Moreover,
this element is a component of other se-
lenoproteins like 5%-deioidinase (Behne and Kyri-
akopoulos, 1990) and selenoprotein P (Linder,
Selenium is an essential trace element that con-
stitutes the active center of glutathione peroxidase
1
990). In view of the fact that selenium was found
*
Corresponding author. Tel.: +55-55-2208140; Fax: +55-
to be an essential dietary micronutrient for many
mammalian animal species (Oldfield, 1987; Lin-
5
5-2208031.
E-mail address: jbtrocha@base.ufsm.br (J.B.T. Rocha).
0
378-4274/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0378-4274(00)00296-4

2
8
M. Farina et al. / Toxicology Letters 119 (2001) 27–37
der, 1990), dietary selenium supplementation is
accepted by the scientific community. On the
other hand, it is well known that selenium is
highly toxic to several species of mammals like
cattle (Hartley and Grant, 1961), sheep (Blodgett
and Bevill, 1987), pigs (Penrith, 1995), rats
al., 1995; Sassa, 1998). d-ALA-D is a sulfhydryl-
containing enzyme and its activity is highly sensi-
tive to the presence of elements such as mercury
(Rocha et al., 1993, 1995; Emanuelli et al., 1996,
1998), lead (Rodrigues et al., 1989, 1996; Burns
and Godwin, 1991; Flora et al., 1991; Goering,
1993), copper (Nelson et al., 1981), and tin (Chiba
and Kikuchi, 1984), which possess high affinity
for ꢀSH groups. The enzyme is also inhibited by
elements such as aluminum (Schetinger et al.,
1999; Vieira et al. 2000), gallium arsenide (Kondo
et al., 1996; Flora et al., 1998), and organic sele-
nium and tellurium compounds (Barbosa et al.,
1998; Maciel et al., 2000) that oxidize ꢀSH groups
or tentatively compete with Zn . d-ALA-D inhi-
bition may impair heme biosynthesis and can
result in the accumulation of ALA, which has
some pro-oxidant activity (Bechara et al., 1993).
The selenoxides generated by oxidation of se-
lenides via FMO are potent thiol oxidants and,
consequently, can oxidize ꢀSH groups of d-ALA-
D, causing enzyme inactivation. In the present
study, we reported the in vitro effects of one
dialkyl- and one alkylaryl-selenide on the
sulfhydryl-containing enzyme d-ALA-D from a
mammalian source. The selenides were reacted
with hydroperoxide in order to form their respec-
tive selenoxides and the products of these reac-
tions were studied as in vitro inhibitors of
d-ALA-D. The effect of exposure to phenyl
methyl selenide on d-ALA-D activity was also
studied in order to identify possible toxic effects
of organoseleno compounds towards the
sulfhydryl containing enzyme d-ALA-D.
(
Anundi et al., 1984) and humans (Cheftel and
Truffert, 1972; Frost, 1972; Louria et al., 1972).
While the mechanism of selenium toxicity has not
been clearly elucidated, Painter (1941) emphasized
that the toxicity of inorganic selenium could be
related to the oxidation of thiols with the con-
comitant formation of derivatives of the RSSeSR
type (selenotrisulfides). After the theoretical
Painter proposal, interaction of thiols with inor-
ganic selenium was confirmed experimentally by
various investigators ( Tsen and Tappel, 1958;
Ganther, 1968). In fact, inorganic selenium com-
pounds (selenite) catalytically increase the oxida-
tion of glutathione and this reaction produces
2
+
ꢀ
−
superoxide (O2 ) (Seko et al., 1989).
Flavin-containing monooxygenases (FMO) are
flavoproteins of some mammalian tissues that cat-
alyze NADPH- and oxygen-dependent oxidation
of a wide variety of xenobiotics (Ziegler, 1988;
Cashman, 1995). The catalytic mechanism of the
FMO is known in some detail only for the hog
liver enzyme (Poulsen and Ziegler, 1979; Beaty
and Ballou, 1980), but it is likely that the major
steps are essentially the same in other mammals.
Kinetic studies have indicated that, in the cell, the
enzyme is present largely in the hydroperoxyflavin
form and, in principle, compounds that can be
oxidized by an organic hydroperoxide are poten-
tial substrates for this enzyme (Beaty and Ballou,
1981). In addition, several studies have demon-
strated the monooxygenation of dialkyl- and alky-
laryl-selenides to their selenoxides by FMO
2. Materials and methods
(
Bruice, 1982; Goeger and Ganther, 1994). Se-
2.1. Compounds
lenoxides are potent thiol oxidants and can ini-
tiate a futile cycle that catalyzes the oxidation of
thiols at the expense of NADPH and oxygen
Dithiothreitol
(DTT),
5,5%-dithio-bis(2-ni-
trobenzoic acid), magnesium chloride, zinc chlo-
ride, 5-aminolevulinic acid, Coomassie brilliant
blue G, p-dimethylaminobenzaldehyde and
purified catalase from bovine liver were obtained
from Sigma (St. Louis, MO, USA). Hydrogen
peroxide (30%), dimethylsulfoxide (DMSO), ethy-
lacetate, potassium permanganate, mono- and
(
Ziegler et al., 1991).
d-Aminolevulinate dehydratase (d-ALA-D) is
an essential enzyme in most organisms, catalyzing
the condensation of two molecules of 5-
aminolevulinic acid (ALA) to form the monopy-
rrole porphobilinogen (Sassa et al., 1989; Jaffe et

M. Farina et al. / Toxicology Letters 119 (2001) 27–37
29
dibasic potassium phosphate, acetic acid, ortho-
2.4. Preparation of partially-purified l-ALA-D
Partially-purified d-ALA-D (P ) was obtained
as previously described (Emanuelli et al., 1998).
2.5. In 6i6o exposure to phenyl methyl selenide
Mice were injected with phenyl methyl selenide
phosphoric acid, trichloroacetic acid and sodium
chloride were obtained from Merck (Darmstadt,
Germany). The selenocompounds phenyl methyl
selenide and 1-hexynyl methyl selenide were pre-
pared according to literature methods (Braga et
al., 1996). Their structures are presented in
Scheme 1.
55
(
500 mmol/kg/day) once a day, subcutaneously,
five times per week for 30 or 45 days. Subcuta-
neous route of exposure was selected because
phenyl methyl selenide is highly lipophilic. The
phenyl methyl selenide was diluted in DMSO and
injected at a proportion of 2.5 ml/kg. Control
animals were injected with DMSO at a proportion
of 2.5ml/kg. The animals were killed 24 h after the
last injection and the liver was removed for tissue
preparation.
2
.2. Animals
Adult male Wistar rats (aged 2–3 months) and
male mice (aged 2–3 months) from our own
breeding colony were maintained in a conditioned
room (20–25°C) under natural lighting conditions
with water and food (Guabi-RS, Brasil) ad libi-
tum. The hogs used for d-ALA-D purification
were obtained from the Sector of Swine Culture
of our University.
2.6. Enzyme assay
2
2
.3. Tissue preparation
Mammalian 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 ALA were used. Moreover, for the
partially purified d-ALA-D, the medium con-
tained 100 mM D,L-dithiothreitol. For the plant
enzyme, the medium contained 50 mM Tris–HCl
buffer (pH 9.0) and 2.5 mM ALA. All experi-
ments were carried out after 10 min of pre-incuba-
tion. The reaction was started 10 min after the
addition of the enzyme preparation by adding the
substrate. Incubations were carried out for 1 h at
.3.1. Animals
Rats and mice were killed by decapitation. The
liver was quickly removed, placed on ice and
homogenized in 7 volumes 150 mM NaCl. The
homogenate was centrifuged at 4000×g at 4°C
for 10 min to yield a low-speed supernatant frac-
tion (S ) that was used for enzyme assay.
1
2
.3.2. Plants
Cucumber (Pepino oadai ) seeds were germi-
nated for 5–7 days at 25°C. Leaves were homoge-
nized in a medium containing 5 volumes 10 mM
Tris–HCl buffer (pH 9.0). The homogenate was
then centrifuged as already described for animal
tissue and a low-speed supernatant fraction (S1)
obtained was used for enzyme assay.
3
9°C for the mammalian enzyme and for 90 min
at 35°C for the plant enzyme. The reaction
product was determined using modified Ehrlich’s
reagent at 555 nm, with a molar absorption coeffi-
4
−1
−1
cient of 6.1×10 cm
M
for the Ehrlich-por-
phobilinogen salt. The reaction rates were linear
with respect to time of incubation and added
protein for all experimental conditions.
2.7. Reaction of selenides with hydroperoxide
The selenides (28.5 mM–2.85 mM) dissolved in
0 ml ethanol were mixed with 25 ml H O to give
1
2
2
Scheme 1. Structures of selenides.
a final concentration of 30 mM. The reaction was

3
0
M. Farina et al. / Toxicology Letters 119 (2001) 27–37
Table 1
carried out for 1 h at 39°C and stopped by adding
catalase (200 U). The same tubes were used for
the enzyme assay and the final concentrations of
the chalcogens ranged from 4 to 400 mM. The
absence of H O was assessed in parallel tubes by
IC50 (mM) values for d-ALA-D inhibition by PhSeCH3 and
a
C H C/CSeCH and their reaction products with H O
4
9
3
2
2
Compound
Rat (S1)
Hog (P55
)
Plant (S1)
2
2
PhSeCH3
\400
104
\400
61
\400
97
tritiating with potassium permanganate (KMnO4).
PhSeCH +H O
2
3
2
C H C/CSeCH
\400
72
\400
123
\400
210
4
9
3
2.8. Determination of the rate of DTT oxidation
C H C/CSeCH
4
9
3
+
H O
2 2
Reaction of selenides with H O were carried
2
2
a
Reaction of selenides with H O was carried out for 1 h
out as described in Section 2.7, except that H O
2
2
2
2
and the remaining H O was eliminated by adding catalase
200 U). Enzyme pre-incubation with selenides or products
2
2
(
125 mM) and selenides (0.7–14 mM) were used,
(
and the final concentrations of chalcogens ranged
from 0.1 to 2 mM. After catalase addition, 84
mM potassium phosphate buffer (pH 6.4) was
added. DTT (2 mM) oxidation was evaluated by
measuring the disappearance of ꢀSH groups by
the method of Ellman (1959). Incubation at 39°C
was initiated by adding DTT in a total volume of
from the reaction of selenides with H O was started by
2
2
adding the supernatant from rat (S ) or partially purified hog
1
liver d-ALA-D (P55) to a medium containing 84 mM potas-
sium phosphate buffer (pH 6.4). For plant d-ALA-D, superna-
tants from cucumber leaves (S ) was added to a medium
1
containing 50 mM Tris–HCl buffer (pH 9.0). The reaction was
started 10 min later by adding ALA (2.5 mM). Data are
expressed as the mean of five to seven independent experi-
ments. S.E.M. was less than 15% of respective means.
2
50 ml. Aliquots of 50 ml were sampled at 0, 30
and 60 min to determine the amount of ꢀSH
groups at 412 nm.
by one-way analysis of variance (ANOVA) fol-
lowed by Duncan’s Multiple Range Test when
appropriate. The data of experiments with a fixed
DTT concentration were analyzed by three-way
analysis of variance. All other results were ana-
lyzed by two-way ANOVA, followed by Duncan’s
Multiple Range Test when appropriate. Differ-
ences between groups were considered to be sig-
nificant when P50.05.
2
.9. Protein quantification
Protein was measured by the method of Brad-
ford (1976) using bovine serum albumin as
standard.
2.10. IC50 determination
The IC50 for in vitro inhibition of d-ALA-D
was calculated by the method of Dixon and Webb
(
1964).
3. Results
1
2
.11. H-NMR studies
3.1. Selenocompounds×H O ×l-ALA-D
2
2
acti6ity
The selenides were incubated with H O at a
2
2
ratio 1:10 (mol/mol) at 39°C for 24 h. The organic
phase was extracted with ethylacetate and ana-
The compounds phenyl methyl selenide
(PhSeCH3) and 1-hexynyl methyl selenide
(C H C/CSeCH ) did not inhibit hepatic d-
lyzed with a Brucker DPX-200 (200 MH ) spec-
Z
4
9
3
trometer
for
solution
in
CDCl3
with
ALA-D. However, the products of reaction of
phenyl methyl selenide or 1-hexynyl selenide with
H O inhibited d-ALA-D from rat liver with IC
tetramethylsilane as internal standard.
2
2
50
2.12. Statistical analysis
values in the micromolar range (Table 1). Statisti-
cal analysis indicated a significant (P50.01) in-
teraction between selenide and H O , indicating
In vivo experiments and the enzymatic assays
with varying DTT concentrations were analyzed
2
2
that the reactions of the selenides with H O2
2

M. Farina et al. / Toxicology Letters 119 (2001) 27–37
31
generated more potent products that inhibited
d-ALA-D from rat liver.
dized during tissue preparation. Similar protec-
tion by DTT against the inhibitory effects of
products of reaction of selenides with H O was
observed when plant d-ALA-D was used (data
not shown).
d-ALA-D was partially purified to assess if
2
2
endogenous liver substances existing in S would
1
contribute to the inactivation of d-ALA-D by
these selenocompounds. The inhibitory effects of
phenyl methyl selenide and 1-hexynyl methyl sele-
nide on d-ALA-D activity from hog (P ) were
1
3
.2. H-NMR studies for chemical
5
5
characterization of the selenoxides
similar to those obtained using S (Table 1). As
1
observed with S , the selenides did not inhibit the
1
1
H-NMR studies were carried out in order to
partially purified enzyme and the products of the
reactions of these compounds with H O inhibited
characterize the chemical structure of products of
2
2
ALA-D with IC50 values in the micromolar range
(
Table 1).
d-ALA-D from mammals possesses at least two
sites that contain cysteinyl residues. One of these
sites binds zinc with relatively low affinity (B site),
while the other binds zinc with relatively high
affinity (A site) (Dent et al. 1990). In plants, the
site homologous to mammalian B site is charac-
terized by the substitution of cysteinyl residues by
acidic amino acids, which apparently renders the
enzyme less susceptible to oxidation (Jaffe et al.,
1995). Similarly to that obtained with mammalian
enzyme, the products of the reaction of the se-
lenides with H O inhibited d-ALA-D from cu-
2
2
cumber leaves. The IC50 values for d-ALA-D
inhibition by the products of reaction between
H O and phenyl methyl selenide and 1-hexynyl
2
2
methyl selenide were in the micromolar range
(
Table 1).
The interaction between selenocompounds and
ꢀ
SH groups has been frequently reported (Tsen
and Tappel, 1958; Ganther, 1968; Barbosa et al.,
998; Maciel et al., 2000). Moreover, selenoxides
1
are rapidly reduced by glutathione (GSH), yield-
ing oxidized glutathione and the corresponding
selenide (Chen and Ziegler, 1994; Goeger and
Ganther, 1994). Involvement of cysteinyl groups
in d-ALA-D inhibition by the products of the
reaction between the selenides and H O were
Fig. 1. Effect of PhSeCH (A) and C H C/CSeCH (B), and
3
4
9
3
of their products of reaction with H O on d-ALA-D from
2
2
liver. Reaction of selenides with H O was carried out for 1 h
2
2
2
2
and the remaining H O was eliminated by adding catalase
2
2
examined by testing the effect of DTT on d-ALA-
D. Addition of DTT (2 mM) increased d-ALA-D
activity by 28–35% and protected d-ALA-D from
the inhibition caused by the products of reaction
between the selenides and H O (Fig. 1A,B). The
(
200 U). Enzyme pre-incubation with selenides or products
from the reaction of selenides with H O was started by
2
2
adding the liver supernatant (S1) to a medium containing 84
mM potassium phosphate buffer (pH 6.4) in the absence or in
the presence of 2 mM DTT. The d-ALA-D reaction was
started 10 min later by adding ALA (to give a final concentra-
tion of 2.5 mM and a volume of 0.25 ml). Data are expressed
as mean9S.E.M. for three independent experiments.
2
2
increase in d-ALA-D activity caused by DTT is
due to reactivation of enzyme molecules that oxi-

3
2
M. Farina et al. / Toxicology Letters 119 (2001) 27–37
compound that corresponds to methyl phenyl se-
the reaction between phenyl methyl selenide and
H O . Analysis revealed the presence of a single
lenoxide (d=2.63 ppm (3H, s, ꢀCH )) (Chen and
3
2
2
1
Ziegler, 1994). The H-NMR spectrum of the
1
-hexynyl methyl selenide yielded a sign of meth-
ylene hydrogens linked to the selenium atom at
d=2.26 ppm (3H, s, SeCH ) (Braga et al.
3
1
993a,b). After reaction between 1-hexynyl methyl
1
selenide and hydroperoxide, the H-NMR spec-
trum revealed a sign of methylene hydrogens
linked to the selenoxide group at d=2.55 ppm
(
3H, s, ꢀCH ), indicating the formation of 1-
3
hexynyl methyl selenoxide.
3
.3. Selenocompounds×H O ×DTT oxidation
2
2
Although the selenides did not affect DTT oxi-
dation (Figs. 2 and 3), the products of reaction
between phenyl methyl selenide and 1-hexynyl
methyl selenide with H O2 decreased the total
2
amount of free ꢀSH groups from DTT. Phenyl
methyl selenoxide oxidized DTT immediately (at
zero time) and stoichometrically, since 2 mM
DTT was totally oxidized by 2 mM of this se-
lenoxide (Fig. 2). The products of reaction be-
tween 1-hexynyl methyl selenide (2 mM) and
H O also oxidized practically all DTT. However,
2
2
this was seen only after 30 min reaction (Fig. 3).
Thus, the reactivity of these selenoxides toward
ꢀ
SH groups of DTT were quite different.
.4. In 6i6o exposure to phenyl methyl selenide
Taking into account the fact that phenyl methyl
3
selenide is a substrate for FMO (Chen and
Ziegler, 1994) and the fact that its selenoxide
inhibited d-ALA-D activity, mice were exposed to
Fig. 2. Effect of PhSeCH3 on the rate of DTT oxidation at 0
min (A), 30 min (B), and 60 min (C). Reaction of selenides
with H O was carried out for 1 h and the remaining H O
2
2
2
2
was decomposed by reaction with catalase (200 U) for 45 min
H O +catalase). Parallel tubes with no H O added (con-
(
2
2
2
2
trol), with no catalase added (H O ), or with the addition of
2
2
catalase (catalase) were also run. The reaction was started by
adding DTT to a final concentration of 2 mM in a medium
containing 84 mM potassium phosphate buffer (pH 6.4) and
the selenide (0–2000 mM). Data are expressed as mean9
S.E.M. for three independent experiments: ꢁ, control; ꢂ,
H O ; ꢃ, catalase (200 U); ꢄ, H O +catalase.
Fig. 2. (Continued)
2
2
2
2

M. Farina et al. / Toxicology Letters 119 (2001) 27–37
33
phenyl methyl selenide in order to verify a possi-
ble bioactivation of this xenobiotic. Exposure to
phenyl methyl selenide caused a significant de-
crease in weight gain and an increase in liver as
percentage of body weight when compared with
the control groups (Table 2). ALA-D activity
decreased in the animals exposed to phenyl
methyl selenide. Moreover, the addition of DTT
(
2 mM) to the enzymatic assay did not restore
ALA-D activity in these animals. A parallel group
was also exposed to 1-hexynyl selenide (500 mmol/
kg/day) and the animals died within 3 days of
treatment.
4
. Discussion
The present results demonstrate that the com-
pounds phenyl methyl selenide and 1-hexynyl
methyl selenide did not inhibit d-ALA-D from a
mammalian source, while the products of reaction
of these selenides with H O inhibited the enzyme
2
2
activity with IC50 values in the micromolar range.
Previous studies indicated that the reaction of
some selenides with H O produces their corre-
2
2
sponding oxides (Chen and Ziegler, 1994). In
1
accordance with this, the H-NMR revealed the
presence of the corresponding selenoxides after
reaction between the selenides and H O . Conse-
2
2
quently, the inhibition of d-ALA-D can be as-
cribed to these oxidized forms of selenides.
The inhibition of d-ALA-D by selenium com-
pounds involves the oxidation of essential ꢀSH
groups of the enzyme, since DTT can counteract
this inhibition. Other evidence supporting this
view is the fact that the products of reaction
between the selenides and H O oxidized DTT.
2
2
Previously, Chen and Ziegler (1994) demonstrated
the reduction of selenoxides by low molecular
Fig. 3. Effect of C H C/CSeCH on the rate of DTT oxida-
4
9
3
tion at 0 min (A), 30 min (B) and 60 min (C). Reaction of
selenides with H O was carried out for 1 h and the remaining
2
2
H O was decomposed by reaction with catalase (200 U) for
2
2
4
5 min (H O +catalase). Parallel tubes with no H O added
2
2
2
2
(
control), with no catalase added (H O ), or with the addition
2 2
of catalase (catalase) were also run. The reaction was started
by adding DTT to a final concentration of 2 mM in a medium
containing 84 mM potassium phosphate buffer (pH 6.4) and
the selenide (0–2000 mM). Data are expressed as mean9
S.E.M. for three independent experiments: ꢁ, control; ꢂ,
H O ; ꢃ, catalase (200 U); ꢄ, H O +catalase.
Fig. 3. (Continued)
2
2
2
2

3
4
M. Farina et al. / Toxicology Letters 119 (2001) 27–37
weight thiols such as GSH, thioglycolate and
thiocholine; however, to our knowledge, there are
no data in literature reporting the reduction of
selenoxides by sulfhydryl-containing proteins.
In plant d-ALA-D, no cysteinyl residues are
present in the region homologous to that involved
not cause re-activation of inhibited d-ALA-D ac-
tivity in animals exposed to selenium. This may
indicate that these compounds are reducing spe-
cifically the synthesis of this enzyme (the concen-
tration of total protein was not changed by
phenyl methyl selenide; data not shown). Alterna-
tively, the oxidation caused by this organochalco-
gen on d-ALA-D may modify the enzyme
conformation in a such way that DTT did not
reactivate the enzyme. In fact, d-ALA-D has an
octameric structure (Jaffe et al., 1995), and in the
mammalian enzyme an intramonomer disulfide
bond is apparently formed between cysteine 119
and 123 (Markham et al. 1993). However, it is
possible that enzyme oxidation in vivo produces
formation of a disulfide bond between d-ALA-D
monomers that may be not reduced by DTT.
Taking into account the role of FMO in oxidiz-
ing dialkyl- and alkylaryl-selenides (Ziegler et al.,
1991), (Chen and Ziegler, 1994; Goeger and Gan-
ther, 1994) it is plausible that their selenoxides are
generated in vivo during the intoxication of mam-
mals with these compounds. This may contribute
to their toxicity through oxidation of sulfhydryl-
containing enzymes like d-ALA-D. Although the
concentrations of H O used to oxidize the se-
2
+
in Zn
binding in mammalian d-ALA-D and, in
part, this may explain the lower sensitivity of
plant enzyme to some oxidizing agents (Jaffe et
al., 1995). However, plant enzyme is inactivated
by iodoacetamide and N-ethylmaleimide, two
classical sulfhydryl reagents, implying that cys-
teinyl residue(s) located at site(s) other than that
2
+
equivalent to the Zn -binding sites of the mam-
malian enzyme are important for d-ALA-D activ-
ity in plants (Senior et al., 1996). In contrast to
mammalian d-ALA-D, which is inhibited by or-
ganic diselenides, the plant enzyme was not inhib-
ited by diselenides, probably due to the fact that
the inhibition caused by these compounds in-
volves the oxidation of cysteinyl residues located
2
+
at the B Zn -binding site (Barbosa et al., 1998).
The results of the present study suggest that the
selenoxides inhibit plant enzyme, which is in ac-
cordance with the potent thiol oxidant properties
of these compounds (Chen and Ziegler, 1994).
Therefore, differing from diphenyl diselenide, se-
lenoxides like iodoacetamide and N-ethyl-
maleimide oxidize the cysteinyl residue(s) of the
plant enzyme located at site(s) other than that
2
2
lenides to their respective selenoxides were high
and do not approach the hydroperoxide concen-
trations of biological fluids and tissues, the in vivo
oxidation of these selenides is possible because a
sophisticated enzymatic phenomenon exists in
mammalian tissues. In the FMO catalytic mecha-
nism, molecular oxygen oxidizes flavin to yield
2
+
equivalent to the Zn -binding sites of mam-
malian enzyme.
In contrast to that observed in vitro, DTT did
Table 2
a
Effect of in vivo exposure to phenyl methyl selenide on d-ALA-D activity in mice liver
4
5 days of treatment
30 days of treatment
Control
Control
PhSeCH3
PhSeCH3
Body weight (g)
41.892.2
5.790.6
269.8910.2
329.0917.5
33.592.5*
12.690.7*
95.496.9*
114.298.5*
28.592.9
5.990.5
241.3914.5
283.3917.2
20.190.5*
10.790.5*
115.098.2*
128.599.8*
Liver as a percentage of body weight
d-ALA-D activity (nmol/PBG/h)
d-ALA-D activity (DTT, 2mM)
a
Mice were weighed daily and injected with DMSO (control) or PhSeCH (500 mmol/kg) for 45 or 30 days and killed 24 h after
3
the last injection. Tissue preparation and d-ALA-D assay were as described in Section 2. Data are expressed as means9S.E.M.
n=7 for each group). * Significantly different from control.
(

M. Farina et al. / Toxicology Letters 119 (2001) 27–37
35
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Acknowledgements
The financial support by FAPERGS, CAPES
and CNPq is gratefully acknowledged. J.B.T.R.,
R.B. and V.F (PIBIC) are the recipients of CNPq
fellowships. M.F. was the recipient of
FAPERGS fellowship.
a
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