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B Nuclear Binding Activity in Rat Testes1
Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET), Universidad de Buenos Aires, Argentina and
*
Departments of Nutrition and
Internal Medicine, University of California Davis, Davis, CA 95616
2To whom correspondence should be addressed. E-mail: oteiza{at}qb.ffyb.uba.ar
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B and AP-1] were assessed. AP-1 nuclear
binding activity did not differ among the groups, but NF-B nuclear
binding activity was lower in the low zinc group than in the control
groups (P < 0.05). We suggest that the reduction
in NF-B binding reflects an early response to zinc
deficiency–induced oxidative stress.
KEY WORDS: • zinc deficiency • oxidative stress • testes • nuclear factor-B • AP-1 • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The mechanisms underlying
zinc deficiency–associated alterations in testicular development and
function are considered to be multifactorial in nature, and include
impaired testicular steroidogenesis (Hamdi et al. 1997
,
Hunt et al. 1992
, Prasad 1991
), decreased
synthesis or activity of angiotensin-converting enzyme
(Stallard and Reeves 1997
) and free radical–mediated
damage to select cell components (Oteiza et al. 1995, 1996 and 1999
).
We reported previously that the induction of zinc deficiency in
developing male rats results in reduced testes growth and evidence of
oxidative stress (Oteiza et al. 1995,
1996
and 1999
). We
observed that after 14 d of zinc deficiency, the tissue was
characterized by a high ratio of 2-thiobarbituric-reactive substances
(TBARS)3
/peroxidation index, an indicator of lipid oxidation; high
concentrations of protein-associated carbonyls and low glutamine
synthetase activity, indicators of protein oxidation; and a high
concentration of 8-oxo-2'-deoxyguanosine, a marker of DNA oxidation
(Oteiza et al. 1995
). In addition to the above, we have
reported that young zinc-deficient rats are characterized by
alterations in the activities and concentrations of several enzymes and
components of the oxidant defense system. For example, after 14 d
of zinc deficiency, the activities of CuZn superoxide dismutase
(CuZnSOD) and glutathione reductase were higher in zinc-deficient
rats than in controls, and an altered ratio of reduced/total ubiquinol
was observed (Oteiza et al. 1996
). Using this animal
model, we reported that zinc deficiency increases the susceptibility of
the testes to cadmium-mediated oxidative damage, as evidenced by
higher TBARS levels and lower glutamine synthetase activity, in
zinc-deficient and cadmium-treated rats than in control and
cadmium-treated rats (Oteiza et al. 1999
).
Oxidative stress can trigger intracellular responses that modulate the
expression of select genes. The transcription factors AP-1 and nuclear
factor (NF)-B are sensitive to oxidants, antioxidants and conditions
that affect the intracellular redox state [see Ginn-Pease and Whisler (1998)
, Li and Karin (1999)
,
Schulze-Osthoff et al. (1997)
, Sen and Packer (1996)
, for reviews]. Consistent with the hypothesis that zinc
deficiency results in an early oxidative stress, we observed that the
exposure of 3T3 cells to zinc-deficient media induces oxidative
stress, and a high AP-1 and low NF-B nuclear binding activity
compared with zinc-adequate cells (Oteiza et al. 2000
).
To investigate whether oxidative stress was an early event in the
testicular pathology associated with zinc deficiency, in the present
study, we fed control and zinc-deficient diets (25 and 0.5
µg Zn/g, respectively) to weanling male rats for 7 d.
We evaluated indices of oxidative stress (oxidative damage to proteins
and lipids, and changes in select components of the oxidant defense
system) and the activation of two transcription factors (NF-B and
AP-1), which are sensitive to conditions that affect the redox state of
cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Sprague-Dawley rats (17 d old; Charles River, Wilmington, MA)
weighing 28–32 g were housed individually in suspended stainless steel
cages in a temperature (22–23°C)- and photoperiod (12
h/d)-controlled room. They were given free access to a control (25
µg Zn/g) or a zinc-deficient (0.5
µg Zn/g) diet (Keen et al. 1989
), or
restricted access to the control 25 µg Zn/g diet at a
level of intake similar to that of rats fed the 0.5 µg
Zn/g diet (restricted group). For the restricted group, rats were given
a standardized amount of diet, based on historical food intake data
collected in our laboratory for rats of this age fed the
zinc-deficient diet.
Assurance of compliance with animal codes.
All animal care procedures met the NIH guidelines (NRC 1985
) and were
administered under the auspices of the Animal Resource Services of the
University of California, Davis, which is accredited by the American
Association for the Accreditation of Laboratory Animal Care.
Experimental protocols were approved before implementation by the
University of California, Davis Animal Use and Care Administrative
Advisory Committee, and were administered through the Office of the
Campus Veterinarian.
Tissue sampling.
Seven days after the initiation of the dietary regimen, the rats were
deprived of food for 6 h and then killed by overexposure to
CO2. Blood was collected by cardiac puncture into
heparinized syringes (Sarstead, Princeton, NJ) and centrifuged at 1700
x g for 15 min. The plasma was removed and stored
at -20°C until analyzed. The testes were quickly excised, weighed
and placed in ice-cold saline. One testis from six rats per group
was freeze-clamped and stored at -80°C for subsequent
glutathione determination. Testes were decapsulated and homogenized in
10 volumes of 50 mmol/L HEPES buffer (pH 7.4), 125 mmol/L KCl. TBARS in
total homogenates were measured immediately; total homogenate aliquots
were also stored at either -20 or -80°C for later analysis of
enzyme activities and zinc, copper and iron concentrations. One aliquot
of the homogenate was centrifuged at 15,000 x g
for 30 min, and the supernatant fraction was removed and stored at
-80°C for determination of the activities of glutamine synthetase
(Miller et al. 1978
) and glucose-6-phosphate
dehydrogenase (Olive and Levy 1975
), and the
concentration of protein carbonyls (Levine et al. 1990
).
The preparation of total and nuclear extracts for Western blotting and electrophoretic mobility shift assay (EMSA) was carried out in fresh tissue samples following the procedure described below.
TBARS determination.
Testes homogenates (10 mg wet tissue) were incubated in 50 mmol/L HEPES
buffer (pH 7.4), 125 mmol/L KCl in a 0.5 mL reaction volume. TBARS were
measured before incubation without additions, or after 60 min of
incubation at 37°C in the presence of 50 µmol/L
FeSO4. The incubation was terminated by the addition of 0.1
mL of 40 g/L BHT in ethanol, and lipid peroxidation products were
evaluated as TBARS using the fluorometric method of Fraga et al. (1988)
. TBARS values are expressed as malondialdehyde
equivalents.
Determination of enzyme activities.
Alkaline phosphatase activity was measured in total homogenates as
described by Mordente et al. (1987)
. For the
determination of Mn superoxide dismutase (MnSOD), CuZnSOD, glutathione
reductase and glutathione peroxidase activities, testes homogenates
were sonicated for 5 s on ice, centrifuged at 10,000 x g for 30 min at 4°C and the assays were conducted on
the supernatant. The activities of MnSOD and CuZnSOD were determined by
the method of Marklund and Marklund (1974)
. Plasma
extracellular SOD was measured as described by Olin et al. (1995)
. Data are shown as units of SOD/L plasma or mg of
tissue; one unit of SOD activity is defined as the amount of sample
needed to obtain 50% inhibition of pyrogallol oxidation.
The activity of glutathione peroxidase was determined by the method of
Lawrence and Burk (1976)
. The activity of glutathione
reductase was measured as described by Rogers and Augusteyn (1978)
. One unit of glutathione peroxidase and glutathione
reductase activity is defined as 1 nmol NADPH oxidized/(min · L).
Data are expressed as U/mg protein. Protein concentrations
were determined according to Bradford (1976)
using
bovine serum albumin as the standard.
Glutathione assay.
Testes kept at -80°C were thawed and immediately homogenized in 0.2
mol/L citrate buffer (pH 5.0), 5 mmol/L EDTA. After centrifugation at
10,000 x g for 2 min, the supernatant fraction was
added with 0.5 volumes of 100 g/L sulfosalicilic acid, and proteins
were precipitated by further centrifugation at 15,000 x g for 1 min. The concentration of total glutathione in
the supernatant was measured using the recycling assay in the presence
of 5,5'-dithiobis-(2-nitrobenzoic acid) and glutathione reductase
(Tietze 1969
)
Electrophoretic mobility shift assay.
The isolation of the nuclear fraction was done with minor modifications
to a procedure described previously (Dignam et al. 1983
,
Osborn et al. 1989
). The buffers used contained 0.5
mmol/L phenylmethylsulfonyl fluoride (PMSF), 1 mg/L leupeptin, 1
µg/mL pepstatin, 1.5 mg/L aprotinin, 2 mg/L bestatin
and 0.4 mmol/L sodium pervanadate. The tissue (50 mg) was added with
100 µL of buffer A [10 mmol/L HEPES (pH 7.9), 1.5
mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L dithiothreitol
(DTT), 0.1% Igepal (Sigma Chemical, St. Louis, MO)], disrupted by
pulling it up six times through a pipette tip followed by a brief (1 s)
sonication. Samples were incubated for 10 min at 4°C, and centrifuged
for 30 s at 12,000 x g. The supernatant
fraction was removed and the pellet was washed in 200
µL of buffer A. After centrifugation, the nuclear
pellets were resuspended in 40 µL of buffer B (10
mmol/L HEPES, pH 7.9, 1.5 mmol/L MgCl2, 420 mmol/L NaCl,
0.5 mmol/L DTT, 0.2 mmol/L EDTA, 25% glycerol). Samples were incubated
for 15 min at 4°C and centrifuged at 10,000 x g
for 10 min at 4°C. The supernatant fraction was transferred to a new
tube, protein concentration was determined (Bradford 1976
) and samples were stored at -80°C.
For the EMSA, the oligonucleotides containing the consensus sequence
for AP-1 and NF-B were end labeled with
[
-32P] ATP using T4 polynucleotide kinase
(Promega, Madison, WI) and purified using Chroma Spin-10 columns
(CLONTECH Laboratories, Palo Alto, CA). The labeled oligonucleotides
were incubated with the nuclear fractions for 20 min at room
temperature in 50 mmol/L Tris-HCl buffer (pH 7.5) containing 20%
glycerol, 5 mmol/L MgCl2, 2.5 mmol/L EDTA, 2.5 mmol/L DTT,
250 mmol/L NaCl and 0.25 g/L poly(dI-dC). The products were separated
by electrophoresis in a 4% nondenaturing polyacrilamide gel using 0.5X
TBE (45 mmol/L Tris/borate, 1mmol/L EDTA) as the running buffer. The
gels were dried and exposed to an X-ray film for 12 h and the
bands were quantitated by densitometric analysis.
Western blot analysis.
A portion of the testes homogenates was combined with an equal volume of 50 mmol/L HEPES (pH 7.4), 125 mmol/L KCl, which contained protease inhibitors and 2% Igepal. The final concentration of the inhibitors was 0.5 mmol/L PMSF, 1 mg/L leupeptin, 1 mg/L pepstatin, 1.5 mg/L aprotinin, 2 mg/L bestatin and 0.4 mmol/L sodium pervanadate. Samples were sonicated briefly, incubated at 4°C for 30 min and centrifuged at 15,000 x g for 30 min. The supernatant was decanted and protein concentration was measured as previously described. Proteins (50 µg per sample) were separated by reducing 10% polyacrylamide gel electrophoresis and electroblotted to PVDF membranes (Bio-Rad, Hercules, CA). Molecular weight standards (Santa Cruz Biotechnology, Santa Cruz, CA) were run simultaneously. Membranes were blotted overnight in 5% nonfat milk, incubated in the presence of the specific antibody for p65 (1:1000 dilution) (Santa Cruz Biotechnology) for 90 min at 37°C. After incubation in the presence of the secondary antibody (HRP-conjugated) (1:10000 dilution), the conjugates were visualized using a chemiluminescence detection (ECL Western blotting system, Amersham Pharmacia Biotech, Piscataway, NJ).
Mineral analysis.
Plasma and testes samples were wet-ashed with 16 mol/L nitric acid
(Baker’s Instra-analyzed: J. T. Baker, Philipsburg, NJ),
evaporated and diluted with 0.1 mol/L nitric acid (Baker’s
Instra-analyzed) as previously described (Oteiza et al. 1995
). Concentrations of copper, zinc and iron were determined
by flame atomic absorption spectrophotometry (model 551; Thermo Jarrel
Ash. Wilmington, MA). Certified reference solutions (1000 mg metal/L;
Fisher Scientific, Santa Clara, CA) were used to generate standard
curves for each element. A sample of National Bureau of Standards
bovine liver (SRM 1577; U.S. Department of Commerce, National Bureau of
Standards, Washington D.C.) was included with the samples to ensure
accuracy and reproducibility.
Statistics.
Data were analyzed using one-way ANOVA. Fisher’s Least Significance Difference test was used to look for differences between group means. A P-value < 0.05 was considered significant. Data are shown as means ± SEM
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cumulative food intakes over the 7 d were 51.5 ± 0.6, 36.7 ± 0.1 and 32.9 ± 1.6 g for the ad libitum control, restricted and low zinc rats, respectively. Total body weight was significantly lower (P < 0.01) in the zinc-deficient and restricted controls than in the ad libitum group (42 ± 1, 47 ± 1 and 67 ± 2 g, respectively). Testes weights did not differ among the groups. Testes/body weight ratios were significantly greater (P < 0.05) in the restricted and low zinc rats (6.2 ± 0.3 x 10-3 and 6.7 ± 0.3 x 10-3, respectively) than in the ad libitum controls (5.1 ± 0.3 x 10-3).
Plasma zinc concentrations were significantly lower (P < 0.001) in the low zinc group (5.5 ± 0.8 µmol/L) than in the ad libitum (24.7 ± 1.1 µmol/L) and restricted (23.2 ± 1.0 µmol/L) controls. Plasma copper concentrations did not differ among the groups. Testes zinc concentrations were significantly lower (P < 0.05) in the zinc-deficient group (0.220 ± 0.013 nmol/g wet tissue) than in the ad libitum (0.264 ± 0.009 nmol/g wet tissue) and restricted (0.267 ± 0.015 nmol/g wet tissue) controls. Testes copper and iron concentrations did not differ among the groups. Alkaline phosphatase activity was significantly lower (P < 0.05) in the low zinc group (0.024 ± 0.004 mU/mg protein) than in the ad libitum and restricted controls (0.049 ± 0.017 and 0.048 ± 0.002 mU/mg protein, respectively).
Lipid and protein oxidative damage.
Testes endogenous TBARS levels did not differ among the groups.
Fe2+-stimulated TBARS production was
significantly lower (P < 0.05) in testes obtained from
rats fed the low zinc diet than in testes from the ad libitum controls
(Table 1
)
|
. Antioxidant defenses.
Testes glutathione peroxidase and glutathione reductase activities did
not differ among the three dietary groups (Table 2
). Testes MnSOD activity was significantly higher (P < 0.05) in the restricted control and low zinc groups than in ad libitum
controls. Testes CuZnSOD activity was significantly higher
(P < 0.05) in the zinc-deficient group than in the
two control groups (Table 2)
. Plasma extracellular superoxide dismutase
activity was lower (P < 0.05) in the restricted and
zinc-deficient rats (48 ± 5 and 37 ± 3
kU/L, respectively) than in the ad libitum controls (75
± 10 kU/L). Testes total glutathione concentrations
were 25% lower (P < 0.05) in the low zinc and
restricted groups than in the ad libitum group (Table 2)
.
|
B nuclear binding activity.
Figure 1
depicts the testes nuclear binding activity of the transcription factor
AP-1. A positive control (HeLa extract, Promega) indicates the position
of the AP-1-oligonucleotide complex (Fig. 1)
. The specificity of the
binding was assessed by competition with a 100-fold molar excess of
unlabeled oligonucleotide containing the consensus sequence for AP-1
(Fig. 1)
. The nuclear binding activity of AP-1 was not different among
the groups. The intensity of the bands in arbitrary units was 153
± 41, 190 ± 26 and 109 ± 53 for the ad libitum,
restricted and zinc-deficient groups, respectively.
|
depicts an EMSA analysis for NF-B of testes nuclear extracts
obtained from the three dietary groups. The position and specificity of
the NF-B-oligonucleotide complex was assessed by competition with a
100-fold molar excess of unlabeled oligonucleotides containing the
consensus sequence for either NF-B or SP-1 (Fig. 2)
. NF-B binding
activity was significantly lower (38–45%, P < 0.05)
in testes nuclear extracts isolated from the low zinc rats than in the
two control groups (Fig. 2)
. The concentration of p65, an ubiquitous
component of NF-B, was similar in the three dietary groups (Fig. 2B
).
|
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Burke and Fenton 1985
, Kraus et al. 1997
, Oteiza et al. 1995,
1996
and 1999
,
Sullivan et al. 1980
, Tate et al. 1999
,
Virgili et al. 1999
), protein and DNA oxidation
(Olin et al. 1993
, Oteiza et al. 1995
)
have been described in different models of zinc deficiency. In the
testes from rats fed zinc-deficient diets for 14 d, we found
several indicators of oxidative stress, including increased oxidation
of cell constituents and alterations in select components of the
oxidant defense system (Oteiza et al. 1995,
1996
and 1999
). In this study, rats fed a zinc-deficient diet for
only 7 d were characterized by low testes zinc content and a low
activity of the zinc-dependent enzyme alkaline phosphatase compared
with ad libitum and restricted controls. However, markers for lipid
(TBARS) and protein (protein carbonyls, glutamine synthetase and
glucose-6-phosphate dehydrogenase activities) oxidative damage were not
different between the zinc-deficient and the control groups. These
results indicate that feeding a zinc-deficient diet for 7 d
does not cause overt signs of oxidative damage to cell components in
developing rats. Indeed, a lower peroxidizability of testes lipids was
observed in the low zinc group compared with the ad libitum controls.
We reported previously (Oteiza et al. 1996
) that in rats
fed a low zinc diet for 14 d, a lower tissue peroxidizability can
be associated with a lower peroxidation index, mainly as a result of a
lower than normal content of the fatty acid 20:4 in tissues from
zinc-deficient animals. It is important to note that testes iron
concentrations were not influenced by the 7-d deficiency regimen. This
is in marked contrast to the sharp increases observed in testes iron
concentration after 14 d of zinc deficiency (Oteiza et al. 1995
). The tissue oxidative damage that occurs with zinc
deficiency can be attributed in part to zinc deficiency–induced
changes in iron metabolism, and subsequent iron-induced free
radical formation (Oteiza et al. 1995,
1996
and 1999
).
When the activities of enzymes that constitute part of the oxidant
defense system were measured, we observed that glutathione reductase
and glutathione peroxidase activities did not differ among the groups
after the 7-d dietary treatment. We reported previously that when rats
are fed zinc-deficient diets for 14 d, the activity of testes
CuZnSOD was significantly higher in the zinc-deficient rats than in
controls (Oteiza et al. 1996
). In that study, MnSOD
activity was also elevated in the zinc-deficient rats relative to
the ad libitum controls. Similarly, in 3T3 cells exposed to
zinc-deficient media, elevated activities of CuZnSOD and MnSOD were
also observed, as well as enhanced expression of the MnSOD gene
(Oteiza et al. 2000
). Recently Tate et al. (1999)
reported a higher than normal activity of total SOD in
zinc-deficient retinal pigment epithelial cells. Similarly, higher
than normal total SOD and CuZnSOD activities, without differences in
CuZnSOD gene expression, have been documented in duodenum and jejunum
from zinc-deficient rats (Virgili et al. 1999
). In
agreement with the above results, there were no changes in the
expression of CuZnSOD gene in 3T3 cells cultured in zinc-deficient
media. As previously proposed by Virgili et al. (1999)
,
the increased CuZnSOD activity apparent under zinc-deficient
conditions may be secondary to increased intracellular levels of
copper, which can activate an inactive superoxide dismutase proenzyme
(Galiazzo et al. 1991
). However, the small changes in
intracellular copper concentration that would be required to activate
CuZnSOD would not have been detectable by the technique used in the
current study.
In contrast to the activity of CuZnSOD, the increased MnSOD activity observed in the zinc-deficient rats may represent a protective response of the tissue to an increased intracellular concentration of oxidant species. Interestingly, there was also higher MnSOD activity in the restricted rats. This is consistent with the concept that on an acute basis, food restriction can impose a modest oxidative stress.
Several of the compounds or conditions that modulate AP-1 and NF-B
involve an increase in the steady-state concentration of oxidants
and alterations in the redox state of cells [see Ginn-Pease and Whisler (1998)
, Li and Karin (1999)
,
Schulze-Osthoff et al. (1997)
, Sen and Packer (1996)
for reviews]. Using 3T3 cells, we demonstrated that the
incubation of cells for only 24 h in a zinc-deficient medium
can markedly increase AP-1 nuclear binding activity and decrease
NF-B binding (Oteiza et al. 2000
). On the basis of
these results, we evaluated the activation of these transcription
factors in the present rat model as a means of assessing a possible
early response to oxidative stress before measurable alterations in
oxidative damage to cell components occurred. Consistent with our
observations in 3T3 cells, we found that NF-B testes nuclear binding
activity was significantly lower in the low zinc group than in the
control groups. However, in contrast to the 3T3 cells, zinc
deficiency–induced changes in AP-1 nuclear binding activity were not
documented; if anything, there was a trend for lower binding activities
in the low zinc group, a finding contrary to that seen in the
zinc-deficient 3T3 cells. This difference in response of AP-1
binding activity may be due to either a difference in the severity of
zinc deficiency in the two models or to a differential sensitivity of
the various cell types to zinc deficiency. The marked increase in
CuZnSOD and MnSOD activities (1.8- to 3.8-fold increase after 24 or
48 h incubation) in 3T3 cells exposed to low zinc media
(Oteiza et al. 2000
) may lead to an important
intracellular build up of
H2O2, a recognized signal
for AP-1 activation (Wenk et al. 1999
). In the present
model of short-term zinc deficiency, only a mild increase of
CuZnSOD and MnSOD activities (24 and 20%, respectively) was observed
in the low zinc group, which may not have increased
H2O2 to the critical levels
required to trigger AP-1 activation.
We evaluated the possibility that the low binding activity of NF-B
in the testes from the low zinc rats could be due in part to a
decreased protein synthesis. However, we observed that the
concentration of p65, one of the most ubiquitous components of NF-B,
was similar among the groups, a finding that does not support this
hypothesis. To test whether the low NF-B nuclear binding activity
could be due to low intracellular levels of zinc, we added zinc (10
µmol/L) to the binding assay mixture. As suspected,
because zinc is not involved in the binding of NF-B to DNA, the
addition of zinc did not restore NF-B binding in the nuclear
extracts from the low zinc rats (data not shown).
Even when both transcription factors are activated by oxidative
conditions, their binding to the DNA consensus sequence depends on the
presence of key cysteine residues located in the DNA-binding region
(Abate et al. 1990
, Toledano and Leonard 1991
, Xanthoudakis et al. 1992
). The modulation
of AP-1 and NF-B activation is thought to be regulated at least in
part by the thiol redox state through mechanisms that are complex and
only partially understood (Arrigo 1999
). Although the
total glutathione concentration in the testes from the rats fed the low
zinc diet was lower than in the ad libitum controls, values did not
differ between the low zinc and restricted groups. We suggest that the
observed reduction in NF-B nuclear binding activity is due in part
to zinc deficiency–induced alterations in the intracellular redox
state of the cell that led to the oxidation of the thiol groups
involved in the binding of the transcription factors to the DNA. The
testing of this hypothesis will be the focus of future work. However,
it is important to note that other aspects of the multiple steps
involved in NF-B activation could also be affected by zinc
deficiency. At the plasma membrane level, zinc can participate in the
binding of ligands to receptors, regulate the local rigidity of
membranes or be involved in ion channels. Any of these mechanisms could
be involved in the triggering of intracellular cascades, such as
NF-B or AP-1 activation. It was proposed previously (Bettger and O’Dell 1993
) that a decrease in membrane zinc
concentration could desensitize the cell to different stimuli. Such
desensitization could explain the low DNA binding activity of NF-B
and the trend (P = 0.10) for lower AP-1 binding
capacity observed in the testes nuclear fractions isolated from the
zinc-deficient rats.
An antiapoptotic role for NF-B has been proposed on the basis of
experimental evidence showing that NF-B activation rescues cells
from apoptotic death [see Soneshein (1997)
for a review].
Mice lacking Rel A (p65) show a massive death of liver cells by
apoptosis, suggesting that this NF-B subunit could be involved in
the antiapoptotic action of the transcription factor (Begg et al. 1995
). We propose that a low basal level of NF-B
activation in testes, as a consequence of zinc deficiency, may trigger
an increased cell death by apoptosis, which could contribute to zinc
deficiency–associated testes pathology.
In summary, a short-term (7 d) zinc deficiency does not cause overt
signs of oxidative damage to cell components in the testes from young
developing rats. However, lower nuclear binding activity of NF-B was
observed in the zinc-deficient group, which we suggest reflects an
early effect of zinc deficiency on the thiol redox status of cells.
These results provide additional evidence for the concept that
oxidative stress is an early effect of zinc deficiency, rather than a
simple reflection of zinc deficiency–induced tissue pathology.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
3 Abbreviations used: CuZnSOD, CuZn superoxide
dismutase; DTT, dithiothreitol; EMSA, electrophoretic mobility shift
assay; MnSOD, Mn superoxide dismutase; NF, nuclear factor; PMSF,
phenylmethylsulfonyl fluoride; TBARS, 2-thiobarbituric-reactive
substances.
Manuscript received February 28, 2000. Initial review completed April 7, 2000. Revision accepted September 18, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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