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2
*
Department of Nutritional Sciences, University of Wisconsin, Madison, Madison, Wisconsin 53706 and
Department of Experimental Pathology, Roswell Park Cancer Institute, Buffalo, New York 14263
2To whom correspondence should be addressed at the Department of Experimental Pathology, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, NY 14263. E-mail: Clement.Ip{at}roswellpark.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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60%. The magnitude of
inhibition was dependent on the amount of thioredoxin in the assay and
was reversible by dialysis, suggesting that a competitive type of
inhibition occurs in vitro. Although thioredoxin reductase can be
inhibited by high levels of selenium in a cell-free system, it
should be noted that such a condition is unlikely to be attainable in
vivo. Caution needs to be exercised in interpreting the in vitro
results.
KEY WORDS: • thioredoxin reductase • methylated selenium compounds • in vivo study • in vitro study • rats.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Because it provides the reduced form of
thioredoxin for many processes necessary for cell growth and protection
against oxidant damage, the enzyme is likely to be an important
regulatory protein for both normal and transformed cells
(2)
. Additional roles independent of thioredoxin, such as
the recycling of ascorbate, have also been suggested
(2
,3)
. Despite the well known
function of selenium as an essential component of more than a dozen
selenoproteins, many of them normally attain maximal activities in
tissues at nutritional levels of selenium and show little or no
increase as selenium is increased to supranutritional levels
(4)
. An exception is the study by Berggren et al.
(5)
, in which they examined the change in thioredoxin
reductase in lung, kidney and liver of rats fed 1 µg selenite Se/g
diet and reported transient increases of enzyme activity in these
tissues. Because high levels of selenium inhibit carcinogenesis
(6
,7)
, this observation is of considerable interest given
the pervasive impact of thioredoxin reductase on modulation of cell
behavior. The present investigation was designed to revisit this issue
by elucidating the effects of methylated selenium compounds on
thioredoxin reductase activity after dietary administration in vivo or
their direct addition in vitro. The use of this class of selenium
reagent was based on a number of previous reports showing that it is
able to modify various biological processes, including cancer
prevention (8
,9)
, inhibition of cell growth and
proliferation (10
11
12)
, induction of apoptosis
(9)
and suppression of angiogenesis (13)
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Se-methyl-L-selenocysteine was obtained from Selenium
Technologies (Lubbock, TX). Methylseleninic acid was synthesized as
described elsewhere (13)
and used as a neutral aqueous
solution of the potassium salt. Dimethyl selenenylsulfide
(CH3-Se-S-CH3) was synthesized by reacting
methylselenenyl bromide (prepared by reacting dimethyl diselenide with
bromine) with methane thiol. Analysis of the reaction mixture by C18
reversed phase HPLC (75% methanol) using a diode array detector showed
successive peaks with UV spectra corresponding to S-S, Se-S and
Se-Se derivatives (14)
. The dimethyl selenenylsulfide
peak was isolated free of the symmetrical products by preparative HPLC
in 75% methanol. Dimethyl diselenide and dimethyl disulfide were
obtained from Aldrich Chemical (Milwaukee, WI) and dissolved in 75%
methanol.
Animals.
Pathogen-free Sprague-Dawley female rats were purchased from
Charles River Breeding Laboratories (Raleigh, NC) at 45 d of age.
They were fed the standard AIN-76A basal diet
(8
). The AIN-76 mineral mix provides 0.1 µg Se
(as sodium selenite)/g diet. The first feeding study consisted of three
dietary groups: basal, methylselenocysteine and methylseleninic acid.
Each of the two selenium compounds was added to the basal diet at a
final concentration of 2 µg Se/g diet. Rats (n = 6/group) were killed at 3, 6 or 10 wk later. Livers were excised,
immediately frozen in liquid nitrogen and stored at -80°C until
enzyme analysis.
The second feeding study consisted of the same three dietary groups
except that all rats were administered an intraperitoneal injection of
methylnitrosourea (50 mg/kg body) for the induction of mammary tumors.
These rats were actually prepared for a selenium mammary cancer
prevention experiment as reported in detail in a recent publication
(9)
. The rats were killed at 22 wk after carcinogen
dosing. Livers (n = 6/group) were excised and saved
from the control rats (no selenium supplementation) as well as from the
tumor-free selenium-treated rats. The purpose of this study was to
assess any changes in thioredoxin reductase activity due to high levels
of selenium after long-term administration. Although the present
design made use of tissue samples from a mammary cancer chemoprevention
experiment, there was no attempt to extrapolate the liver thioredoxin
reductase activities to the occurrences in the mammary gland.
Enzyme assays.
Thioredoxin reductase activity was determined in the liver only because
the enzyme activity in the mammary gland is barely above background
value. Livers were homogenized at a 20 g/L concentration in
phosphate-buffered saline containing 1 mmol/L EDTA. The homogenate
was centrifuged at 13,000 x g for 30 min. The
supernatant was dialyzed against phosphate-buffered saline for
16 h to remove endogenous reduced glutathione. Because thioredoxin
reductase is heat stable, the dialysate was heated at 55°C for 10
min, cooled and centrifuged at 13,000 x g for 30
min to remove denatured protein. The standard assay for thioredoxin
reductase using insulin as the substrate (15)
was modified
slightly in our experiments by increasing the thioredoxin level to 10
µmol/L. The reaction was started by the addition of 200–250 µg
protein and allowed to continue for 10 min at 37°C. On termination of
the reaction, the absorbance at 412 nm was measured. The NADPH
reduction of insulin that was not dependent on the thioredoxin
reductase-thioredoxin system was determined by performing duplicate
reactions in the absence of thioredoxin. This
non–thioredoxin-dependent reaction was then subtracted in the
calculation of thioredoxin reductase activity, which is expressed as
A412 units x 1000/(min · mg
protein).
In addition, some experiments used a different assay method of Hill et
al. (16)
that does not involve thioredoxin. Thioredoxin
reductase is very sensitive to inhibition by gold. The NADPH-linked
reduction of 5,5'-dithiobis(2-nitrobenzoic acid)
(DTNB)3
was measured in the presence or absence of gold thioglucose, and the
difference was used to calculate thioredoxin reductase activity. The
concentrations of dialyzed liver supernatant protein, DTNB, gold
thioglucose and NADPH added to the reaction mixture were similar to
those described by Hill et al. (16)
. Heat treatment of the
dialysate was not included in the sample preparation step for this
assay.
For the in vitro thioredoxin reductase inhibition studies with the S-S, Se-S and Se-Se derivatives, the enzyme source was preincubated with each test compound (concentrations specified in Results) for 15 min before the start of the reaction. The final concentration of methanol (used to dissolve the diselenide and disulfide compounds) in the reaction mixture was never more than 4%. A solvent control was analyzed simultaneously to account for any interference by methanol alone.
Statistics.
Statistical evaluation of the data were done by analysis of variance
with post hoc comparisons among the different groups as described in
our previous publication (9)
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Liver thioredoxin reductase activities at different times after
treatment with selenium are shown in Table 1
. The 3-, 6- and 10-wk protocol duplicated that used by Berggren et al.
(5)
in their study of thioredoxin reductase modulation by
selenite. The activities varied little over this period, although the
values at 10 wk were somewhat higher than at earlier time points in all
three groups. At each time, there was no difference between the control
group and either of the two selenium groups (methylselenocysteine or
methylseleninic acid). The lack of an effect suggests that methylated
selenium compounds do not cause a transitory increase in liver
thioredoxin reductase like that observed in rats fed an inorganic form
of selenium (5)
. Thioredoxin reductase activities in the
liver of rats from the 22-wk study are also shown in Table 1
. Supplementation with either selenium compound had no detectable
effect on thioredoxin reductase activity, regardless of whether the
activity was measured by the thioredoxin/insulin method (data in Table 1
) or the DTNB/gold method (data not shown).
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We tested three methylated organoselenium reagents that could be
substrates for thioredoxin reductase or could form protein adducts of
the type P-S-Se-CH3 or
P-Se-Se-CH3 by reacting with cysteine or
selenocysteine, respectively (17)
. Table 2
compares the effects of methylseleninic acid, dimethyl diselenide,
dimethyl selenenylsulfide and dimethyl disulfide, on thioredoxin
reductase activity. None of the selenium compounds significantly
inhibited these enzyme levels of 
10 µmol/L. Significant inhibition
occurred with 25 µmol dimethyl diselenide and dimethyl
selenenylsulfide per L. At 50 µmol/L, all compounds were inhibitory,
but the magnitude of inhibition with dimethyl diselenide and dimethyl
selenenylsulfide (60%) was significantly greater than that observed
with methylseleninic acid and dimethyl disulfide (30%). Dimethyl
diselenide was therefore selected for additional studies of the assay
conditions that might influence thioredoxin reductase activity.
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88%) compared with
the highest level of 10 µmol/L (54%). In other studies (not
shown), time course measurements of the reaction over a period of 3–15
min with or without 50 µmol dimethyl diselenide/L showed that the
degree of inhibition was similar at all times. In addition, the enzyme
was inhibited to the same extent when preincubation time with dimethyl
diselenide was varied between 5 and 60 min, suggesting that the
interference with enzyme activity did not increase with the length of
preexposure to the inhibitor, as might be expected if the formation of
an irreversible complex between enzyme and inhibitor were occurring in
a time-dependent manner.
These results were consistent with an inhibitory mechanism based on
competition of the selenium reagent for reducing equivalents, thus
decreasing the amount of reduced thioredoxin available for reduction of
insulin. Extracts that had been preincubated with 50 µmol dimethyl
diselenide/L and then dialyzed to remove the reagent recovered almost
all of the thioredoxin reductase activity, further supporting a
reversible type of enzyme inhibition (Table 3
). A possible alternative explanation was that a covalently bound adduct
was formed during the preincubation of thioredoxin reductase with
dimethyl diselenide but was subsequently cleaved during the assay by
reduced thioredoxin generated by residual active enzyme. To exclude
this possibility, the same samples were assayed by the DTNB/gold
method, which does not use thioredoxin. The results of the assays were
similar (Table 3)
; thus, the reversible type of inhibition observed is
consistent with a competitive mechanism.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
Although increased activity could be useful in controlling oxidative
damage, increased reduction of thioredoxin would be expected to benefit
transformed cells by preventing apoptosis (2)
. A number of
studies have found substantially increased activities of thioredoxin
reductase after selenium supplementation to epithelial cancer cell
cultures (18
,19)
or in tissues of animals fed a
selenium-adequate diet compared with those fed a
selenium-deficient diet (15)
. However, only one study
has demonstrated an increased activity in vivo in rats fed a
supranutritional level of selenite compared with a nutritionally
adequate level (5)
. Our present observation of no increase
in thioredoxin reductase activity after 3, 6 or 10 wk of treatment with
either Se-methylselenocysteine or methylseleninic acid (at 2 µg
Se/g diet) contrasts with the results obtained by Berggren et al.
(5)
with selenite (1 µg Se/g diet), in which they
reported a transitory increase in liver enzyme activity after 3 wk of
feeding. In both studies, the thioredoxin-dependent reduction of
insulin was measured, which is specific for the selenium redox center
of the enzyme. We also measured thioredoxin reductase activity after
the long-term administration of Se-methylselenocysteine
or methylseleninic acid. Again, we found no difference in thioredoxin
reductase activity determined at the end of the 22-wk experiment.
Bioavailability of selenium in Se-methylselenocysteine or
methylseleninic acid for synthesis of selenoenzymes is very good
(9
,20)
. Thus, differences in bioavailability between
inorganic selenite and the methylated forms of selenium may not explain
the contrasting results of Berggren et al. (5)
and our
study in regard to the effects on thioredoxin reductase activity.
Inhibitory effects of selenium on thioredoxin-dependent processes
have been demonstrated in a purified mammalian thioredoxin
reductase/thioredoxin system (17)
. In the present study,
the direct addition of methylated selenium compounds to the crude liver
assay system inhibited thioredoxin-mediated reduction of insulin
only at relatively high selenium levels. The inhibition was reversed by
dialysis, which is consistent with a competitive type of mechanism
(17)
. However, it should be noted that the high
concentrations of selenium (25–50 µmol/L) needed to achieve enzyme
inhibition in vitro were unlikely to be attained in vivo. In rats fed 2
µg Se/g diet as either Se-methylselenocysteine or methylseleninic
acid, the concentration of selenium in the liver was 6 µg/g, or 75
µmol/L (9)
, with most of the selenium bound to proteins.
Thus, the downregulation of thioredoxin reductase activity by high
levels of selenium in a cell-free system may have little relevance
in situ.
In summary, the present evidence suggests that tissue thioredoxin reductase activity in animals fed high levels of selenium does not differ from that in animals fed a nutritionally adequate level of selenium. In this regard, thioredoxin reductase behaves like most selenoproteins, which show little response to supranutritional selenium supplementation. Our results do not exclude the possibility that thioredoxin reductase activity might be modulated by high levels of selenium at a localized site in tissues.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviation used: DTNB, 5,5'-dithiobis(2-nitrobenzoic acid).
Manuscript received June 8, 2000. Initial review completed June 30, 2000. Revision accepted November 21, 2000.
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