Water-soluble organotellurium compounds: Catalytic protection against peroxynitrite and release of zinc from metallothionein

Article abstract of DOI:10.1021/tx990156g

The antioxidant properties of a number of water-soluble diorganyl tellurides have been investigated. These organotellurium compounds efficiently protect against peroxynitrite-mediated oxidation of dihydrorhodamine 123, hydroxylation of benzoate, and nitra

Full text of DOI:10.1021/tx990156g

Chem. Res. Toxicol. 2000, 13, 3-9
3
Articles
Wa ter -Solu ble Or ga n otellu r iu m Com p ou n d s: Ca ta lytic
P r otection a ga in st P er oxyn itr ite a n d Relea se of Zin c
fr om Meta lloth ion ein
Claus J acob,†,‡,§ Gavin E. Arteel,†,‡ Takahiro Kanda, Lars Engman, and
|
|
,
†
Helmut Sies*
Institut f u¨ r Physiologische Chemie I, Heinrich-Heine-Universit a¨ t D u¨ sseldorf,
D-40001 D u¨ sseldorf, Germany, and Institute of Chemistry, Department of Organic Chemistry,
Uppsala University, S-751 21 Uppsala, Sweden
Received August 31, 1999
The antioxidant properties of a number of water-soluble diorganyl tellurides have been
investigated. These organotellurium compounds efficiently protect against peroxynitrite-
mediated oxidation of dihydrorhodamine 123, hydroxylation of benzoate, and nitration of
4
-hydroxyphenyl acetate. The peroxidation of the zinc storage protein, metallothionein, by tert-
butyl hydroperoxide is also catalyzed by the water-soluble organotellurium compounds. As
compared to selenium-containing compounds (e.g., ebselen and selenocystamine), some of the
tellurides that were tested {e.g., 3-[4-(N,N-dimethylamino)benzenetellurenyl]propanesulfonic
acid, sodium salt} exhibit a significantly higher reactivity in these assays, making them some
of the most effective compounds tested thus far. The catalysis of destruction of zinc-sulfur
clusters by water-soluble organotellurium compounds could have implications for the bioavail-
ability of zinc in vivo. These compounds might be lead compounds for the development of a
new class of water-soluble, tellurium-based antioxidant and zinc-releasing drugs.
In tr od u ction
peroxynitrite reductase (7), and recent work showed that
selenoprotein P in human plasma and thioredoxin re-
ductase may also act to protect against peroxynitrite (8,
1
Peroxynitrite, a potent oxidizing and nitrating species,
leads to tissue damage in a number of pathological
conditions in humans and in experimental animals (1,
9
). The specific redox chemistry of selenium as exhibited
in glutathione peroxidase has led to the synthesis of
organoselenium compounds that exhibit glutathione per-
oxidase-like activity. In general, selenium-containing
amino acids and low-molecular weight organoselenium
compounds (e.g., ebselen) rapidly react with peroxynitrite
2
). At the level of the whole organism, the reactivity of
peroxynitrite can be considered beneficial, because of its
cytotoxicity to bacteria (3) and other invading organisms.
However, excessive production of peroxynitrite can dam-
age normal tissue. Indeed, the formation of protein
(10, 11) and protect against DNA damage caused by
3
-nitrotyrosine in vivo has been seen in a number of
peroxynitrite (12, 13) better than their sulfur analogues.
inflammatory conditions in humans and experimental
animals (4). Therefore, defense against peroxynitrite is
of biological importance and is of interest for drug
development (5).
We proposed that cellular proteins containing seleno-
amino acids may selectively protect against damage due
to peroxynitrite (6). Glutathione peroxidase can act as a
Synthetic organoselenium compounds (e.g., ebselen)
protect against neuronal tissue injury clinically (14-16).
Organotellurium compounds have also exhibited potent
antioxidative properties in model systems (17-19) and
in increasingly complex biological systems (20-22). In-
deed, the synthetic compound bis(4-aminophenyl) tel-
luride has a higher glutathione peroxidase-like activity
and protects against peroxynitrite-mediated oxidation of
dihydrorhodamine 123 more efficiently than its selenium
analogue or ebselen (23). However, the compounds previ-
ously tested were highly water-insoluble, possibly de-
creasing their therapeutic potential.
*
To whom correspondence should be addressed: Institut f u¨ r
Physiologische Chemie I, Heinrich-Heine-Universit a¨ t D u¨ sseldorf, Post-
fach 101007, D-40001 D u¨ sseldorf, Germany. Telephone: +49-211-811-
2
707. Fax: +49-211-811-3029. E-mail: helmut.sies@uni-duesseldorf.de.
†
Heinrich-Heine-Universit a¨ t D u¨ sseldorf.
‡
§
These authors contributed equally to this work.
Current address: Department of Chemistry, University of Exeter,
Here, the abilities of several water-soluble organotel-
lurium compounds (1-9, Figure 1) to protect against
oxidation and nitration reactions caused by peroxynitrite
were assessed. The catalytic activity of these compounds
toward the peroxidation of the zinc storage protein
Stocker Road, Exeter EX4 4QD, United Kingdom.
|
Uppsala University.
1
The term peroxynitrite is used sometimes to refer to both the
-
peroxynitrite anion (ONOO ) and peroxynitrous acid (ONOOH); the
IUPAC names are oxoperoxonitrate(1-) and hydrogen oxoperoxonitrate,
respectively.
1
0.1021/tx990156g CCC: $19.00 © 2000 American Chemical Society
Published on Web 12/11/1999

4
Chem. Res. Toxicol., Vol. 13, No. 1, 2000
J acob et al.
To a stirred suspension of elemental tellurium (0.127 g, 1.0
mmol) in tetrahydrofuran (5 mL) in a round-bottomed two-
necked flask was added superhydride (lithium triethylborohy-
dride; 3 mL, 1.0 M) in tetrahydrofuran (3.0 mmol) dropwise at
2
0 °C, and stirring was continued for 0.5 h under the same
conditions. To this mixture was directly added 1,3-propanesul-
tone (3-hydroxy-1-propanesulfonic acid γ-sultone; 0.249 g, 2.10
mmol). After the mixture had been stirred for an additional 6 h
at ambient temperature, the crystalline precipitate was filtered
and washed with diethyl ether (3 × 5 mL) to afford 0.180 g (47%)
1
of the title compound as colorless crystals: mp >250 °C;
H
NMR (CD
3
OD, 300 MHz) δ 2.09 (quint, J ) 7.5 Hz, 2 H), 2.68
t, J ) 7.5 Hz, 2 H), 2.80 (t, J ) 7.5 Hz, J Te-H ) 22 Hz, 2 H);
2
(
1
3
1
C NMR (CD
3
OD, 75 MHz) δ 1.3 ( J Te-C ) 136 Hz), 28.8, 54.4.
Te: C, 18.68; H, 3.14. Found: C,
Anal. Calcd for C
8.76; H, 3.31.
Peroxynitrite was synthesized from sodium nitrite and H
using a quenched-flow reactor (29), and H was eliminated
by passage of the peroxynitrite solution over MnO powder. The
final peroxynitrite concentration was determined spectropho-
6 2 6 2
H12Li O S
1
2
O
2
2
O
2
2
-
1
-1
tometrically at 302 nm (ꢀ ) 1700 M cm ). Double-distilled
water was used throughout the experiments. Millipore water
and Chelex-treated and nitrogen-purged buffer solutions were
used for assays involving MT. Stock solutions of tested com-
pounds (10 or 20 mM) were prepared in H
4-aminophenyl) telluride were dissolved in methanol.
Oxid a tion of Dih yd r or h od a m in e 123 by P er oxyn itr ite.
2
O. Ebselen and bis-
(
Oxidation of DHR-123 in the presence of peroxynitrite was
followed fluorometrically (7, 23). DHR-123 (500 nM) was added
to 100 mM potassium phosphate buffer (pH 7.3) containing 0.1
mM DTPA. Peroxynitrite (100 nM) was added by bolus addition
under vigorous stirring, and changes in fluorescence (λex ) 500
nm, λem ) 536) were measured 20 s after peroxynitrite addition
with a Perkin-Elmer LS-5 luminescence spectrometer. Under
these conditions, the effects of tested compounds were deter-
mined relative to the effect of peroxynitrite alone (“no addition”).
In reverse-addition control experiments, the compounds used
in this study did not interfere with the fluorescence of the
rhodamine 123 that formed.
F igu r e 1. Structures of new water-soluble organotellurium
compounds.
2
metallothionein (MT) with subsequent zinc release was
Nitr a tion of 4-Hyd r oxyp h en yl Aceta te by P er oxyn i-
tr ite. Transition metal-catalyzed nitration of HPA by perox-
ynitrite was performed as described previously (23). Complexed
Fe(III)-EDTA was made by mixing equimolar (10 mM) amounts
also investigated (24, 25). Several compounds not only
were efficient scavengers of peroxynitrite but also cata-
lyzed peroxidation of MT in the presence of tert-butyl
hydroperoxide. The effect on peroxide-induced MT oxida-
tion is a measure of how well the compounds can perform
as antioxidants. Taken together, these data suggest
potential therapeutic properties of these organotellurium
compounds as antioxidants as well as regulators of zinc
metabolism.
3
of aqueous solutions of FeCl and EDTA. HPA (10 mM) was
dissolved in 100 mM sodium phosphate buffer (pH 7.3) contain-
ing 0.5 mM Fe(III)-EDTA. Peroxynitrite (100 µM) was added
to the assay solution by bolus addition under constant vortexing.
After 30 min, the pH was adjusted to 10-11 with 1 M NaOH
and the concentration of 4-hydroxy-3-nitrophenyl acetate de-
-
1
-1
termined spectrophotometrically (ꢀ430 ) 4400 M cm ) (30).
The concentration of nitrated HPA formed in the presence of
tested compounds (20 µM) was compared with that of nitrated
HPA formed with peroxynitrite alone (no addition).
Exp er im en ta l P r oced u r es
Rea gen ts. Benzoic acid, 4-hydroxyphenyl acetate (HPA),
Chelex, glutathione (GSH), N-(1-naphthyl)ethylenediamine,
sulfanilamide, selenocystamine, and metallothionein-2 (rabbit
liver, cadmium form) were purchased from Sigma (Deisenhofen,
Hyd r oxyla tion of Ben zoa te Ca u sed by Stea d y-Sta te
In fu sion of P er oxyn itr ite. While protection against DHR-123
oxidation and HPA nitration caused by bolus addition of
peroxynitrite was used as a relative measure of the efficiency
with which a compound reacts directly with peroxynitrite,
protection against hydroxylation of benzoate was used as a
measure of the efficiency with which the reaction can be
catalytically maintained in the presence of reducing equivalents,
in this case, GSH (23, 31). Peroxynitrite was infused with a
FRG). The zinc form, Zn
literature procedures (26) and exclusively used in experiments.
-(2-Pyridylazo)resorcinol (PAR) and MnO were from Fluka
Buchs, Switzerland). DHR-123 was purchased from Molecular
Probes (Eugene, OR). 2-Phenyl-1,2-benzioselenazol-3(2H)-one
ebselen) was kindly provided by Rh oˆ ne-Poulenc-Rorer (Cologne,
7
MT-2, was reconstituted according to
4
2
(
(
-1
micropump at a rate of 35 µL min from a 450 µM stock solution
under constant mixing with a magnetic stirrer at room tem-
perature into a mixture (0.4 mL) containing benzoic acid (1 mM)
and DTPA (0.1 mM) in 0.5 M potassium phosphate buffer (pH
Germany).
Bis(4-aminophenyl) telluride (27) and compounds 1-7 and 9
28) were prepared according to literature methods. Bis(sulfo-
(
propyl) telluride dilithium salt (8) was synthesized as follows.
6
.8); the final volume was 470 µL. The steady-state input
concentration of peroxynitrite (0.8 µM) was calculated by using
2
Abbreviations: DHR-123, dihydrorhodamine 123; DTPA, diethyl-
the infusion rate of peroxynitrite and its decay rate in phosphate
enetriaminepentaacetic acid; ebselen, 2-phenyl-1,2-benzisoselenazol-
(2H)-one; GSH, glutathione (reduced form); Hepes, 4-(2-hydroxyethyl)-
piperazine-1-ethanesulfonic acid; HPA, 4-hydroxyphenyl acetate; MT,
zinc metallothionein-2 (rabbit liver); PAR, 4-(2-pyridylazo)resorcinol.
-
1
buffer at 25 °C and pH 6.8 (0.65 s ). The amount of hydroxy-
lation of benzoate was determined fluorometrically (λex ) 300
nm, λem ) 410 nm) 0, 0.5, 1, and 2 min after peroxynitrite
3

Antioxidant Protection by Organotellurides
Chem. Res. Toxicol., Vol. 13, No. 1, 2000
5
infusion with a fluorescence microcuvette. Under these condi-
tions, the individual and combined effects of organotellurium
compound (2.5 µM) and GSH (60 µM) were determined relative
to the effect of peroxynitrite alone (no addition). In control,
reverse-order experiments, the compounds used in this study
did not significantly interfere with the fluorescence determina-
tion of benzoate hydroxylation.
Nitr ite F or m a tion d u r in g P er oxyn itr ite In fu sion . To
determine the effect of organotellurium compounds on nitrite
formation by peroxynitrite, peroxynitrite was infused under
conditions identical to those described above for hydroxylation
of benzoate, with the exception that the phosphate buffer
contained no benzoic acid and GSH concentrations were 120 µM.
Nitrite concentrations in samples were determined with the
Griess reaction. Briefly, Griess reagents [50 µL of 0.1% N-(1-
naphthyl)ethylenediamine and 200 µL of 2.5% sulfanilamide in
1
5% phosphoric acid] were added to 750 µL of diluted sample;
the resultant product was detected colorimetrically by its
absorbance at 540 nm, and nitrite standards (0-20 µM) were
used to calculate standard curves.
Zin c Relea se fr om Meta lloth ion ein . The rate of zinc
release was measured as described previously (32). MT (0.5 µM)
was dissolved in 20 mM Hepes (pH 7.4) containing 100 µM 4-(2-
pyridylazo)resorcinol (PAR). Reactions were initiated by adding
tert-butyl hydroperoxide (500 µM), and zinc release was moni-
-1
-1
tored spectrophotometrically (ꢀ500 ) 65 000 M cm ) for 30 min.
Tested compounds (100 nM) were added before tert-butyl
hydroperoxide addition. The extent of total zinc transfer was
measured by addition of ebselen (50 µM) in the absence of tert-
butyl hydroperoxide and set to 100%. Initial zero-order rates
for zinc release were estimated for the time interval from 2 to
1
2 min. Tested compounds in the presence or absence of tert-
butyl hydroperoxide did not react with PAR. The influence of
tested compounds on basal zinc release in the absence of tert-
butyl hydroperoxide was assessed.
F igu r e 2. Protection of ebselen and organotellurium com-
pounds against dihydrorhodamine 123 oxidation caused by
peroxynitrite. Oxidation of dihydrorhodamine 123 was per-
formed as described previously (1). Peroxynitrite (100 nM) was
added to 0.5 µM dihydrorhodamine 123, and different concen-
trations of ebselen (b), bis(4-aminophenyl) telluride (9), or
compound 3 (2) in 0.1 M phosphate buffer and 0.1 mM DTPA
Resu lts
P r ot ect ion b y Or ga n ot ellu r iu m Com p ou n d s
a ga in st th e Oxid a tion of Dih yd r or h od a m in e d u r in g
Bolu s Ad d ition of P er oxyn itr ite. Figure 2 shows the
protection by selected organotellurium compounds against
peroxynitrite-mediated oxidation of DHR-123. Table 1
summarizes the half-maximal inhibitory concentration
of the compounds that were tested. As a comparison, the
results obtained with ebselen are also shown. Compound
(pH 7.3) under intense stirring at room temperature. Data are
means ( SD (n ) 3-6). The concentration required to inhibit
oxidation of dihydrorhodamine 123 by 50% was determined from
these curves (see Table 1).
Ta ble 1. Effect of Or ga n otellu r iu m Com p ou n d s a n d
Ebselen on th e Oxid a tion of Dih yd r or h od a m in e 123 a n d
Nitr a tion of 4-Hyd r oxyp h en yl Aceta te by P er oxyn itr ite
3
[Figure 2 (2)] and compound 6 exhibit the smallest half-
half-maximal
nitration of
maximal inhibitory concentration value (80 nM), analo-
gous to the result obtained with bis(4-aminophenyl)
telluride here [Table 1 and Figure 2 (9)] and previously
inhibitory
hydroxyphenyl acetate
concentration (nM)a
b
compound
ebselen
(% of control)
130
80
76.1 ( 7.6
(23). A number of the new tellurium compounds (com-
NDc
bis(4-aminophenyl)
pounds 1, 3, and 6) were as efficient or more efficient
than ebselen [130 nM; Table 1 and Figure 2 (b)]. The
selenium-containing compound 7 had a half-maximal
inhibitory concentration value 6-fold higher than that of
its tellurium-containing analogue (compound 4; Table 1).
While the aliphatic compound 6 was very efficient in
protecting against DHR-123 (Table 1), the other aliphatic
telluride (compound 8) had an intermediate value and
was not as effective as ebselen (Table 1). Compound 2
and compound 3, both of which contain an electron-rich
group in the 4-position on the phenyl ring, have lower
half-maximal inhibitory concentration values (150 and
telluride
1
2
3
4
5
6
7
8
9
130
150
80
270
210
80
1400
250
350
60.5 ( 4.1
40.3 ( 5.0
41.2 ( 7.5
38.6 ( 4.1
45.6 ( 4.1
51.4 ( 2.3
95.4 ( 5.9
52.7 ( 2.8
47.0 ( 1.4
a
Concentration of the compound causing half-maximal inhibi-
tion. See also Figure 2. b The reaction mixture was HPA (10 mM)
in sodium phosphate buffer (pH 7.3) containing 0.5 mM Fe(III)-
EDTA and test compounds (20 µM). Nitration of HPA by bolus
addition of peroxynitrite (100 µM) in the absence of added
compounds yielded a concentration of ∼40 µM; this value was set
to 100%. Data are means ( SD (n ) 3-5). c ND, not determined.
8
0 nM, respectively) than the unsubstituted compound
4
(270 nM). In contrast, the heterocyclic compound 9 (350
nM) had considerably higher half-maximal inhibitory
concentration values than compound 4.
P r ot ect ion b y Or ga n ot ellu r iu m Com p ou n d s
a ga in st Nitr a tion of 4-Hyd r oxyp h en yl Aceta te d u r -
in g Bolu s Ad d ition of P er oxyn itr ite. Table 1 also
summarizes the protection of the tested compounds (20

6
Chem. Res. Toxicol., Vol. 13, No. 1, 2000
J acob et al.
µM) against nitration of HPA by peroxynitrite (100 µM).
The concentration of nitrated HPA in the absence test
compounds was ∼40 µM; this value was set as 100%.
While the organoselenium compounds (ebselen and com-
pound 7) protected against nitration of HPA by 24 and
5
%, respectively, all tellurium compounds protected by
at least 35% (Table 1). The aryl alkyl tellurides (com-
pounds 2-4) were most effective, all protecting by ∼60%
and better than the diaryl telluride (compound 1; Table
1
). The electron-poor alkyl aryl telluride (compound 5)
had an influence similar to that of the alkyl aryl tel-
lurides, while the aliphatic tellurides (compounds 6 and
8
) protected to a lesser degree (∼50%). When peroxyni-
trite was reacted with compounds 3, 4, and 9 in the
absence of HPA, there was an increase in absorbance at
4
30 nm (data not shown), suggesting formation of a
nitration product. This effect was most pronounced with
compound 3. No such product was observed with the
aliphatic compounds (6 and 8).
P r ot ect ion b y Or ga n ot ellu r iu m Com p ou n d s
a ga in st Hyd r oxyla tion of Ben zoa te d u r in g Stea d y-
Sta te In fu sion of P er oxyn itr ite. Figure 3 shows the
influence of catalytic amounts of compound 3 (2.5 µM)
on the hydroxylation of benzoate in the absence and
presence of 60 µM GSH. Benzoate hydroxylation in-
creased linearly with time when, over the course of 2 min,
peroxynitrite was infused with a micropump to maintain
a steady-state concentration of 0.8 µM [Figure 3 (b)].
Under these conditions, addition of 60 µM GSH (9) or
2
.5 µM compound 3 (2) alone decreased the amount of
benzoate hydroxylation by 10 and 30%, respectively.
When GSH was combined with compound 3 (1), benzoate
hydroxylation was almost completely prevented during
the first minute of peroxynitrite infusion. Since a total
of ∼30 µM peroxynitrite had been added during the first
minute, these data suggest a stoichiometry of 2 equiv of
GSH consumed per equivalent of added peroxynitrite.
Nitr ite F or m a tion d u r in g P er oxyn itr ite In fu sion .
As the spontaneous decay of peroxynitrite generates
nitrate, an increase in the yield of nitrite at the expense
of nitrate measures the amount of peroxynitrite reduction
F igu r e 3. Protection of compound 3 against hydroxylation of
benzoate caused by peroxynitrite. Peroxynitrite-mediated oxida-
tion reactions were monitored using benzoate as a model target
compound essentially as described previously (7, 23). Peroxyni-
trite was infused with a micropump to maintain a 0.8 µM
steady-state concentration under constant mixing with a mag-
netic stirrer at room temperature into a mixture containing
benzoic acid (1 mM) and DTPA (0.1 mM) in 0.5 M potassium
phosphate buffer (pH 6.8). Cumulative peroxynitrite addition
after 2 min yielded a concentration of 67 µM. The amount of
hydroxylation of benzoate was determined fluorometrically (λex
)
300 nm, λem ) 410 nm) 0, 0.5, 1, and 2 min after peroxynitrite
(7). In our experiments, basal nitrite levels after spon-
infusion with a fluorescence microcuvette. Under these condi-
tions, the effects of GSH (60 µM; 9), compound 3 (2.5 µM; 2),
and GSH and compound 3 combined (1) are compared with no
addition controls (b). In control, reverse-order experiments, the
various compounds used in this study did not significantly
interfere with the fluorescence determination of benzoate hy-
droxylation. Data are means ( SD (n ) 3-6).
taneous decay of 67 µM peroxynitrite infused over the
course of 2 min were ∼75 µM, most likely due to synthesis
or storage of peroxynitrite. The amount of nitrite forma-
tion was calculated as increases over the degree of
spontaneous generation of nitrite during the decay of
peroxynitrite (no addition) and is summarized in Table
2
. Addition of GSH alone (120 µM) did not significantly
Ta ble 2. F or m a tion of Nitr ite d u r in g Stea d y-Sta te
Exp osu r e to P er oxyn itr itea
increase the amount of nitrite formation after peroxyni-
trite infusion for 2 min (Table 2). While there was an
increase in nitrite formation with compound 3 alone
during peroxynitrite infusion (Table 2), when GSH and
compound 3 were combined, there was a dramatic
increase in nitrite formation, accounting for ∼55% of the
total peroxynitrite that was infused (Table 2).
formation of nitrite
addition
µMb
% of peroxynitrite additionc
none
0
0
GSH (120 µM)
1.4 ( 10.6
2.2 ( 15.9
13.5 ( 5.9
54.8 ( 6.9
compound 3 (2.5 µM)
9.0 ( 3.9
3 and GSH (120 µM) 36.6 ( 4.6
Ca ta lysis of Zin c Relea se fr om Meta lloth ion ein
by Or ga n otellu r id es in th e P r esen ce of ter t-Bu tyl
Hyd r op er oxid e. Figure 4 shows time-dependent zinc
release from MT in the presence of tert-butyl hydroper-
oxide, while Table 3 summarizes total zinc release as well
as the estimated initial zero-order rates of this release.
tert-Butyl hydroperoxide alone released 10% of total zinc
in 30 min [Table 3 and Figure 4 (b)]. With the exception
of the selenium-containing compound 7, all tested com-
pounds increased zinc release from MT in the presence
a
Experimental conditions are analogous to those described in
the legend of Figure 3. Peroxynitrite was infused with a micro-
pump to maintain a 0.8 µM steady-state concentration under
constant mixing with a magnetic stirrer at room temperature into
a mixture containing DTPA (0.1 mM) in 0.5 M potassium phos-
b
phate buffer (pH 6.8). Data are means ( SD (n ) 3-5). Spon-
taneous formation of nitrite was subtracted. c Cumulative peroxy-
nitrite addition after 2 min yielded a concentration of 67 µM.
of tert-butyl hydroperoxide. Compound 3 increased the
extent of total zinc release in the presence of tert-butyl

Antioxidant Protection by Organotellurides
Chem. Res. Toxicol., Vol. 13, No. 1, 2000
7
Ta ble 3. Zin c Relea se fr om Meta lloth ion ein -2 in th e
P r esen ce a n d Absen ce of ter t-Bu tyl Hyd r op er oxid e a n d
a
Or ga n otellu r iu m a n d Or ga n oselen iu m Com p ou n d s
rate of zinc release in the
presence of tert-butyl
hydroperoxide
basal rate of
zinc release
compound
no addition
ebselen
selenocystamine
bis(4-aminophenyl)
telluride
%b ×10-10 M s-1 c
%b
×10-10 M s-1 c
2.2 ( 0.6
4.0 ( 0.5
5.8 ( 0.3
10.3 ( 1.0
4.9
1.5
1.5
2.5
1.5
10.4 ( 3.5
17.2 ( 1.1
25.4 ( 2.6
49.4 ( 3.0
6.8
13.7
5.9
1
2
3
4
5
6
7
8
9
6.8
4.9
5.9
6.8
5.9
4.9
4.9
7.8
15.6
2.0
1.0
1.5
1.5
1.0
1.5
1.5
1.5
2.5
42.3 ( 1.1
48.5 ( 0.6
56.6 ( 1.0
36.1 ( 1.0
30.2 ( 1.7
47.5 ( 1.2
10.4 ( 1.1
35.4 ( 1.2
43.3 ( 2.0
9.5 ( 0.5
11.5 ( 0.5
13.3 ( 0.3
8.0 ( 0.5
5.7 ( 0.6
10.5 ( 0.5
1.5 ( 0.5
8.3 ( 0.8
8.5 ( 0.9
a
Data for zinc release in the presence of tert-butyl hydroper-
b
oxide are means ( SD (n ) 3). See also Figure 4. % release
calculated after 30 min. c Initial rates calculated from 2 to 12 min.
Discu ssion
Tellu r iu m in Ch em istr y a n d Biology. Selenium
and tellurium both belong to the metalloid family of
elements and therefore share some similar chemical
characteristics; however, there are some differences that
make tellurium compounds of interest. In general, orga-
notellurium compounds tend to be more nucleophilic than
their corresponding selenium and sulfur derivatives (33).
The order of reactivity (telluride > selenide > sulfide)
correlates with oxidation potentials and is also in accord
with the decreasing electronegativity of the elements as
one traverses the group of chalcogens. The reaction of
diorganyl chalcogenides with oxidants such as peroxyni-
trite and hydrogen peroxide involves nucleophilic attack
on oxygen. It is therefore not surprising that organotel-
luriums are better antioxidants than “classical” sulfur-
and selenium-based antioxidants. Like selenium, inor-
ganic tellurium is also toxic to mammalian species (see
refs 34 and 35 for a review), an effect that may be
partially mediated by binding to selenium-containing
residues on proteins (36). It is proposed that substituted
organotellurium compounds, like those tested here (Fig-
ure 1), may avoid potentially toxic leakage of tellurium
in vivo.
P r otection a ga in st P er oxyn itr ite-Med ia ted Oxi-
d a tion a n d Nitr a tion Rea ction s by Or ga n otellu -
r iu m Com p ou n d s. The reactivity of diaryl tellurides
critically depends on the substituents on the benzene ring
(23). Electron-donating substituents increase the reactiv-
ity of these compounds with electrophiles, while electron-
withdrawing groups decrease it. Compound 3, an electron-
rich telluride, is the best new peroxynitrite-scavenging
agent tested in this study. It is of interest that alkyl aryl
telluride 3 was as good a reductant of peroxynitrite as
bis(4-aminophenyl) telluride and bis[4-(dimethylamino)-
phenyl] telluride (23). Apparently, a single electron-
donating aryl group bonded to tellurium is enough to
ensure high antioxidant activity. These findings open the
field to a new class of diorganyl telluride antioxidants
where one of the substituents can be used to confer water
solubility. It is also of interest that one of the dialkyl
tellurides (compound 6) was effective at preventing
oxidation and nitration reactions by peroxynitrite. Ali-
F igu r e 4. Effect of organotellurium compounds and seleno-
cystamine on zinc release from metallothionein-2 in the presence
of tert-butyl hydroperoxide. Measurement of zinc release was
performed as described previously (38). Zinc release from 0.5
µM metallothionein-2 in 20 mM Hepes (pH 7.4) in the presence
of 500 µM tert-butyl hydroperoxide and various organotellurium
and organoselenium compounds (100 nM) was determined for
3
0 min using 100 µM 4-(2-pyridylazo)resorcinol (PAR) as a zinc
indicator. The effects of compound 3 ([), bis(4-aminophenyl)
telluride (1), selenocystamine (2), and ebselen (9) are compared
with zinc release caused by tert-butyl hydroperoxide alone in
the absence of catalyst (b). Data are means ( SD (n ) 3); lines
represent best fit analysis using second-order linear regression.
See Table 2 for summary data.
hydroperoxide to ∼60% [Table 3 and Figure 4 ([)]
and the initial rate more than 5-fold compared to that
with tert-butyl hydroperoxide alone (Table 3). Similar
results were obtained with bis(4-aminophenyl) telluride
[Table 3 and Figure 4 (1)]. All tellurium-containing
compounds enhanced total zinc release from MT in the
presence of tert-butyl hydroperoxide better than the
selenium-containing compounds, selenocystamine [Table
3
and Figure 4 (2)], ebselen [Table 3 and Figure 4 (9)]
and compound 7 (Table 3). Of the selenium-containing
compounds, selenocystamine [Table 3 and Figure 4 (2)]
was the most effective at enhancing zinc release and ∼2-
fold more effective than ebselen [Table 3 and Figure 4
(
[
9)]. While the electron-rich tellurides 2 (Table 3) and 3
Table 3 and Figure 4 ([)] caused higher extents of zinc
release and greater rates than compound 4 (Table 3), the
electron-poorer compound 5 released less zinc at a slower
rate (Table 3). Selenocystamine and compound 9 slightly
enhanced zinc release at a concentration of 100 nM in
the absence of tert-butyl hydroperoxide (Table 3).

8
Chem. Res. Toxicol., Vol. 13, No. 1, 2000
J acob et al.
Sch em e 1. P r op osed Mech a n ism s for Or ga n otellu r id e (Com p ou n d 3)-Ca ta lyzed Red u ction of
P er oxyn itr ou s Acid (or P er oxyn itr ite) to Nitr ou s Acid (or Nitr ite) Usin g Glu ta th ion e a s th e Stoich iom etr ic
Red u cta n t (P a n el A) a n d Oxid a tion of MT Zin c-Su lfu r Clu ster s Usin g ter t-Bu tyl Hyd r op er oxid e a s th e
Oxid a n t (P a n el B)
phatic diorganyl tellurides have the additional advantage
that they cannot be nitrated by peroxynitrite. Compound
effective than the water-insoluble bis(4-aminophenyl)
telluride. Structure-activity trends observed for peroxy-
nitrite protection were also seen for zinc release from MT.
The catalytic efficiency of water-soluble diorganyl tel-
lurides, especially of compound 3, makes this class of
compounds interesting agents for destruction of zinc-
sulfur complexes in the presence of hydroperoxides.
Catalyzed zinc release, which in itself could trigger zinc-
dependent signal transduction cascades (e.g., upregula-
tion of thionein production), is another mode of action
compound 3 might employ in vivo (37).
5
, which contains an electron-withdrawing substituent,
is less reactive than the unsubstituted compound 4 and
hence a poor scavenger of peroxynitrite. Similar observa-
tions are made with compound 9 which contains a
thiophene instead of a phenyl substituent on Te. Com-
pound 7 and compound 4 allow the comparison between
the reactivities of two structurally analogous organotel-
lurium and organoselenium compounds. In all in vitro
assays that were employed, the tellurium compound was
at least 5-fold more effective than the selenium analogue.
This is in agreement with the general reactivity differ-
ences between tellurium and selenium compounds.
Ca ta lytic Ma in ten a n ce of An tioxid a n t Activity of
Or ga n otellu r iu m Com p ou n d s. Protection by organo-
tellurium compounds against oxidation of DHR-123 and
nitration of HPA are indices of the relative rates with
which these compounds react with peroxynitrite. How-
ever, of equal importance as far as the organism is
concerned (5) is the “catalytic” performance of the com-
pounds. In addition to reacting rapidly with peroxynitrite,
compound 3 can be readily recycled in the presence of
GSH. Further, the extent of nitrite formation is increased
under these conditions, indicating that the major part of
this reaction is a two-electron reduction of peroxynitrite
Im p lica tion s. The performance of organotellurium
compounds in assays involving peroxynitrite, hydroper-
oxide, glutathione, and metallothionein clearly indicates
that tellurides are more reactive than selenides in
reactions involving reactive oxygen species. As a conse-
quence, these compounds have a potential to effectively
counteract oxidative stress by quenching or catalytic
reduction of reactive oxygen species in the presence of
thiols in vivo.
Ack n ow led gm en t. This study was supported by the
Deutsche Forschungsgemeinschaft, SFB 503, Project B1,
the National Foundation for Cancer Research (Bethesda,
MD), the Swedish Research Council for Engineering, and
the Swedish Natural Science Research Council and
Cancerfonden. C.J . holds a BASF-Forshungsstipendium
of the Studienstiftung des Deutschen Volkes. G.E.A. is
a Research Fellow of the Alexander von Humboldt
Foundation (Bonn, Germany).
(see Scheme 1, panel A), analogous to previous findings
with ebselen and selenoproteins (7, 9, 11).
The release of zinc from MT is a useful tool for
determining the relative rates at which the tested
compounds can be recycled. The catalytic mechanism
most likely involves tert-butyl hydroperoxide oxidation
of the telluride and telluroxide-induced oxidation of MT
thiols with the regeneration of the active divalent dior-
ganyl telluride (see Scheme 1, panel B). Under these
conditions, the ability of the tellurium compounds to
effect peroxidation of MT clearly is superior to those of
the selenium analogues, compound 3 and compound 6
again being the most efficient. Both of them are more
Refer en ces
(1) Beckman, J . S., Beckman, T. W., Chen, J ., Marshall, P. A., and
Freeman, B. A. (1990) Apparent hydroxyl radical production by
peroxynitrite: implications for endothelial injury from nitric oxide
and superoxide. Proc. Natl. Acad. Sci. U.S.A. 87, 1620-1624.
(
2) Beckman, J . S. (1996) The physiological and pathophysiological
chemistry of nitric oxide. In Nitric oxide: principles and actions
(Lancaster, J ., Ed.) pp 1-82, Academic Press, San Diego, CA.

Antioxidant Protection by Organotellurides
Chem. Res. Toxicol., Vol. 13, No. 1, 2000
9
(
3) Zhu, L., Gunn, C., and Beckman, J . S. (1992) Bactericidal activity
of peroxynitrite. Arch. Biochem. Biophys. 298, 452-457.
4) Ischiropoulos, H. (1998) Biological tyrosine nitration: a patho-
physiological function of nitric oxide and reactive oxygen species.
Arch. Biochem. Biophys. 356, 1-11.
tellurides as inhibitors of lipid peroxidation in biological and
chemical systems. Free Radical Res. 20, 401-404.
(
(
22) Wieslander, E., Engman, L., Svensj o¨ , E., Erlansson, M., J ohans-
son, U., Linden, M., Andersson, C.-M., and Brattsand, R. (1998)
Antioxidative properties of organotellurium compounds in cell
systems. Biochem. Pharmacol. 55, 573-584.
23) Briviba, K., Tamler, R., Klotz, L.-O., Engman, L., Cotgreave, I.
A., and Sies, H. (1998) Protection by organotellurium compounds
against peroxynitrite-mediated oxidation and nitration reactions.
Biochem. Pharmacol. 55, 817-823.
24) J acob, C., Maret, W., and Vallee, B. L. (1998) Control of zinc
transfer between thionein, metallothionein, and zinc proteins.
Proc. Natl. Acad. Sci. U.S.A. 95, 3489-3494.
25) J acob, C., Maret, W., and Vallee, B. L. (1998) Ebselen, a selenium-
containing redox drug, releases zinc from metallothionein. Bio-
chem. Biophys. Res. Commun. 248, 569-573.
(26) Vas a´ k, M. (1991) Metal removal and substitution in vertebrate
and invertebrate metallothioneins. Methods Enzymol. 205, 452-
458.
(27) Engman, L., and Persson, J . (1993) 2:1 Complexes of anilines with
tellurium tetrachloride. Syntheses of amino-substituted diphenyl
tellurides. Organometallics 12, 1068-1072.
28) Kanda, T., Engman, L., Cotgreave, I. A., and Powis, G. (1999)
Novel water soluble diorganyl tellurides with thiol peroxidase and
antioxidant activity. J . Org. Chem. 64, 8161-8169.
29) Koppenol, W. H., Kissner, R., and Beckman, J . S. (1996) Syntheses
of peroxynitrite: to go with the flow or on solid grounds? Methods
Enzymol. 269, 296-302.
(
5) Arteel, G. E., Briviba, K., and Sies, H. (1999) Protection against
peroxynitrite. FEBS Lett. 445, 226-230.
(
(
6) Sies, H., and Masumoto, H. (1997) Ebselen as a glutathione
peroxidase mimic and as a scavenger of peroxynitrite. Adv.
Pharmacol. 38, 229-246.
(
(
(
7) Sies, H., Sharov, V. S., Klotz, L.-O., and Briviba, K. (1997)
Glutathione peroxidase protects against peroxynitrite-mediated
oxidations. A new function for selenoproteins as peroxynitrite
reductase. J . Biol. Chem. 272, 27812-27817.
8) Arteel, G. E., Mostert, V., Oubrahim, H., Briviba, K., Abel, J .,
and Sies, H. (1998) Protection by selenoprotein P in human
plasma against peroxynitrite-mediated oxidation and nitration.
Biol. Chem. Hoppe-Seyler 379, 1201-1205.
(
(
9) Arteel, G. E., Briviba, K., and Sies, H. (1999) Function of
thioredoxin reductase as a peroxynitrite reductase using seleno-
cystine or ebselen. Chem. Res. Toxicol. 12, 264-269.
(
10) Briviba, K., Roussyn, I., Sharov, V. S., and Sies, H. (1996)
Attenuation of oxidation and nitration reactions of peroxynitrite
by selenomethionine, selenocystine and ebselen. Biochem. J . 319,
(
(
(
1
3-15.
(
11) Masumoto, H., and Sies, H. (1996) The reaction of ebselen with
peroxynitrite. Chem. Res. Toxicol. 9, 262-267.
12) Salgo, M. G., Stone, K., Squadrito, G. L., Battista, J . R., and Pryor,
W. A. (1995) Peroxynitrite causes DNA nicks in plasmid pBR322.
Biochem. Biophys. Res. Commun. 210, 1025-1030.
13) Roussyn, I., Briviba, K., Masumoto, H., and Sies, H. (1996)
Selenium-containing compounds protect DNA from single-strand
breaks caused by peroxynitrite. Arch. Biochem. Biophys. 330,
(
30) Beckman, J . S., Ischiropoulos, H., Zhu, L., van der Woerd, M.,
Smith, C., Chen, J ., Harrison, J ., Martin, J . C., and Tsai, M. (1992)
Kinetics of superoxide dismutase- and iron-catalyzed nitration
of phenolics by peroxynitrite. Arch. Biochem. Biophys. 298, 438-
(
2
16-218.
4
45.
(
14) Saito, I., Asano, T., Sano, K., Takakura, K., Abe, H., Yoshimoto,
T., Kikuchi, H., Ohta, T., and Ishibashi, S. (1998) Neuroprotective
effect of an antioxidant, ebselen, in patients with delayed
neurological deficits after aneurysmal subarachnoid hemorrhage.
Neurosurgery 42, 269-278.
15) Yamaguchi, T., Sano, K., Takakura, K., Saito, I., Shinohara, Y.,
Asano, T., and Yasuhara, H. (1998) Ebselen in acute ischemic
stroke: a placebo-controlled, double-blind clinical trial. Ebselen
Study Group. Stroke 29, 12-17.
(31) Szab o´ , C., Ferrer-Sueta, G., Zingarelli, B., Southan, G. J ., Salz-
man, A. L., and Radi, R. (1997) Mercaptoethylguanidine and
guanidine inhibitors of nitric-oxide synthase react with peroxy-
nitrite and protect against peroxynitrite-induced oxidative dam-
age. J . Biol. Chem. 272, 9030-9036.
(32) J acob, C., Maret, W., and Vallee, B. L. (1999) Selenium redox
biochemistry of zinc-sulfur coordination sites in proteins and
enzymes. Proc. Natl. Acad. Sci. U.S.A. 96, 1910-1914.
(
(
16) Ogawa, A., Yoshimoto, T., Kikuchi, H., Sano, K., Saito, I.,
Yamaguchi, T., and Yasuhara, H. (1999) Ebselen in acute middle
artery occlusion: a placebo-controlled, double-blind clinical trial.
Cerebrovasc. Dis. 9, 112-118.
(
33) Wada, M., Nobuki, S., Tenkyuu, Y., Natsume, S., Asahara, M.,
and Erabi, T. (1999) Bis(2,6-dimethoxyphenyl) sulfide, selenide
and telluride, and their derivatives. J . Organomet. Chem. 580,
2
82-289.
34) Taylor, A. (1996) Biochemistry of tellurium. Biol. Trace Elem. Res.
5, 231-239.
35) Larner, A. J . (1995) Biological effects of tellurium: a review. Trace
Elem. Electrolytes 12, 26-31.
(17) Engman, L., Stern, D., Pelcman, M., and Andersson, C.-M. (1994)
Thiol peroxidase activity of diorganyl tellurides. J . Org. Chem.
(
(
5
5
9, 1973-1979.
(
18) Engman, L., Persson, J ., Vessman, K., Ekstr o¨ m, M., Berglund,
M., and Andersson, C.-M. (1995) Organotellurium compounds as
efficient retarders of lipid peroxidation in methanol. Free Radical
Biol. Med. 19, 441-452.
(36) Garberg, P., Engman, L., Tolmachev, V., Lundqvist, H., Gerdes,
R. G., and Cotgreave, I. A. (1999) Binding of tellurium to
hepatocellular selenoproteins during incubation with inorganic
tellurite: consequences for the activity of selenium-dependent
glutathione peroxidase. Int. J . Biochem. Cell Biol. 31, 291-301.
(
(
(
19) Vessman, K., Ekstr o¨ m, M., Berglund, M., Andersson, C.-M., and
Engman, L. (1995) Catalytic antioxidant activity of diaryl tel-
lurides in a two-phase lipid peroxidation model. J . Org. Chem.
6
0, 4461-4467.
(37) Vallee, B. L., and Falchuk, K. H. (1993) The biochemical basis of
20) Andersson, C.-M., Hallberg, A., Brattsand, R., Cotgreave, I. A.,
Engman, L., and Persson, J . (1993) Glutathione peroxidase-like
activity of diaryl tellurides. Bioorg. Med. Chem. Lett. 3, 2553-
zinc physiology. Physiol. Rev. 73, 79-118.
(
38) Maret, W., J acob, C., Vallee, B. L., and Fischer, E. H. (1999)
Inhibitory sites in enzymes: zinc removal and reactivation by
thionein. Proc. Natl. Acad. Sci. U.S.A. 96, 1936-1940.
2
558.
21) Andersson, C.-M., Brattsand, R., Hallberg, A., Engman, L.,
Persson, J ., Mold e´ us, P., and Cotgreave, I. A. (1994) Diaryl
TX990156G