Vol. 10, 385–390, April 2001
Cancer Epidemiology, Biomarkers & Prevention
Interactions of Selenium Compounds with Other Antioxidants in DNA
Damage and Apoptosis in Human Normal Keratinocytes1
Chwan-Li Shen, Woosun Song, and Barbara C. Pence2
Texas Tech University Health Sciences Center, Lubbock, Texas 79430
Abstract
Selenite (SeL) or selenomethionine (SeM) are the most
common selenium (Se) compounds taken as dietary
antioxidants to reduce oxidative stress. Because the public
may frequently supplement Se compounds at high doses,
the possible pro-oxidant effect of Se becomes a concern.
SeL and SeM have entirely different pharmacokinetic
effects based on dose-related cytotoxicity. Our laboratory
has shown previously that high doses of SeL resulted in
cytotoxicity and induction of 8-hydroxydeoxyguanosine
(8-OHdG) in DNA of primary human keratinocytes
(NHK), compared with those treated with the same doses
of SeM. Besides Se compounds, other dietary
antioxidants, such as vitamin (Vit) C or Vit E, are often
supplemented and taken together with Se compounds.
However, the cellular effects of these interactions of Se
with antioxidants are still unknown. In addition, copper
is commonly present in drinking water, food, soil, or the
environment to increase the possibility of subchronic
toxicity. Copper has been shown to inhibit SeL-induced
cytotoxicity and apoptosis in human colonic carcinoma
cells. The present study was designed to investigate the
interactive effects of SeL or SeM plus Vit C, trolox (a
water-soluble Vit E), or copper sulfate (CuSO4) on cell
viability and induction of 8-OHdG adduct formation in
DNA of NHK. NHK cells were treated with no Se, SeL
(126.6 ␮M Se), or SeM (316.6 ␮M Se) plus two doses each
of Vit C (2.27 and 4.45 ␮M), trolox (40 and 80 ␮M), or
CuSO4 (7.85 and 15.7 ␮M) for 24 h. Coincubation of Vit
C or CuSO4 with SeL appeared to protect NHK against
SeL-induced cytotoxicity. However, synergistic effects
were observed between SeL and trolox resulting in
enhanced cytotoxicity. On the other hand, SeM ⴙ Vit C,
SeM ⴙ trolox, and SeM ⴙ CuSO4 did not affect cell
viability. In the absence of Se supplementation, Vit C,
trolox, or CuSO4 alone did not induce 8-OHdG adduct
formation, regardless of dose. When NHK cells were
coincubated with SeL (126.6 ␮M Se) and Vit C or CuSO4,
they protected NHK from SeL-induced DNA damage
Received 11/2/00; revised 1/11/01; accepted 1/30/01.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
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1
Supported by NIH Grant CA76675.
2
To whom requests for reprints should be addressed, at Department of Pathology,
Texas Tech University Health Sciences Center, Room 2B106, 3601 Fourth Street,
Lubbock, TX 79430. Phone: (806) 743-2556; Fax: (806) 743-2656; E-mail:
[email protected].
with a reduction in 8-OHdG generation. In contrast,
treatment of SeL ⴙ trolox elevated generation of 8OHdG. Furthermore, treatments of SeM plus trolox or
CuSO4 elevated 8-OHdG adduct formation. In terms of
apoptosis measured as internucleosomal DNA
fragmentation, copper protected NHK against SeLinduced apoptosis in cultured NHK. These data suggest
that the use of CuSO4 may play a protective role in SeLinduced cytotoxicity, DNA oxidative damage, and
apoptosis and that there may be potentially deleterious
interactions among common high-dose antioxidant
supplements taken by the public.
Introduction
Se3 is an essential dietary nutrient for all of mammalian species,
including humans. The mechanisms of action of Se compounds,
either via a pro-oxidant pathway, as seen in cytotoxicity and
apoptosis, or via an antioxidant pathway as proposed in cancer
chemoprevention, are still unclear but intriguing. Because the
public may frequently supplement Se and other antioxidant
compounds at high doses, the possible pro-oxidant effect of Se
becomes a concern. Previous studies (1– 6) have shown that
SeL and its metabolites at high doses resulted in cytotoxicity,
DNA fragmentation (7, 8), cellular apoptosis (1– 4), and DNA
oxidative damage in cells (6). The mechanism of the cytotoxicity of SeL and other redoxing Se compounds is believed to
derive from its pro-oxidant ability to catalyze the oxidation of
thiols and to produce superoxide simultaneously (5, 6). Our
previous studies also support the findings that high doses of
SeL supplementation resulted in cytotoxicity and induction of
8-OHdG lesions, an indicator of estimating DNA oxidative
damage, in mouse skin cells (6) as well as in primary NHK (9).
Both SeL and SeM are two commercial forms of Se
compounds that are available to and supplemented by the
public. Our laboratory has shown previously that SeL and SeM
have entirely different pharmacokinetic effects based on doserelated cytotoxicity in mouse skin cells (6) and in NHK (9). In
general, on an equal Se dose basis, SeM is not as active as SeL,
because SeM is a nonredoxing Se compound (6, 10). However,
the human recommended daily allowance for Se intake is 55–70
␮g/day, regardless of Se form, and the nontoxic dose limit for
any Se supplement is considered 400 ␮g/day by the NIH Office
of Dietary Supplements. For the purposes of this study, we have
chosen to use 10 ␮g/ml (126.6 ␮M) Se as SeL and 25 ␮g/ml
(316.6 ␮M) Se as SeM, but it is difficult to correlate these to
plasma levels obtained from the various Se dietary supplements.
Besides Se, other dietary antioxidants, such as Vit C or Vit
3
The abbreviations used are: Se, selenium; 8-OHdG, 8-hydroxydeoxyguanosine;
NHK, normal human keratinocytes; SeL, selenite; SeM, selenomethionine; Vit,
vitamin; HPLC, high-pressure liquid chromatography; 2-dG, 2-deoxygluanosine.
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Antioxidant Interactions with Selenium
E are commonly supplemented and taken together with Se.
Both Vit C and Vit E have been identified as antioxidant
nutrients that may reduce the risk of cancer (10 –12). Trolox, a
commercial name for a water-soluble Vit E analogue, 6hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, has
been shown to protect mammalian cells from oxidative damage
in vitro (13) as well as in vivo (14).
However, no studies have demonstrated the cellular interactions that would occur in cells supplemented with Se and
other dietary antioxidants. The hypothesis to be tested in this
study is that the antioxidants would interact with Se compounds
and result in enhanced cellular protection. Thus, the present
study was designed to investigate the cellular effects of SeL or
SeM in combination with antioxidants (Vit C or trolox) by
determining: (a) induction of cytotoxicity as measured by cell
viability; and (b) generation of oxidative damage as seen in
8-OHdG lesions in DNA of NHK.
In addition, copper (II) is one of the major transition
metals in the biological environment that is an active catalyst of
DNA damage in vitro and in vivo (15). In general, cupric sulfate
commonly presents in drinking water, soil, food, or the environment to increase the possibility of subchronic toxicity in
humans (16). Interestingly, Davis et al. (2) have reported that
copper as CuSO4 protects human colonic cancer cells (HT-29)
against SeL-induced cytotoxicity and apoptosis and acts as an
antioxidant. In this study, we investigated the interaction between Se compounds and CuSO4 in NHK, as well as the
interactive effects with Vit C and trolox.
Materials and Methods
Cell Culture. NHK (Cascade Biologicals, Portland, OR) were
grown in culture at 37°C in 5% CO2/95% air using CM154
medium supplemented with 1% human keratinocyte growth
supplement (Cascade Biologicals). Stock cells were plated and
maintained under a culture density of 80% confluence (⬃106
cells/100-mm plate) to minimize the proportion of cells mitotically arrested.
Treatment of NHK Cells. NHK were treated with no Se, SeL
(126.6 ␮M Se), or SeM (316.6 ␮M Se) plus two doses each of
Vit C (2.27 and 4.54 ␮M), trolox (40 and 80 ␮M), or CuSO4
(7.85 and 15.7 ␮M) for 24 h. The 24-h time point was chosen
from unpublished preliminary data indicating this was the optimal length of incubation with Se compounds for determination of the three end points, cytotoxicity, apoptosis, and oxidative DNA damage. We chose two Se compounds (SeL and
SeM) for their abilities to induce cytotoxicity and apoptosis.
Therefore, higher doses of Se compounds were chosen to establish clear-cut end points that could be seen and modulated.
The doses of SeL and SeM were determined by the limiting
toxicity. Doses of Vit C, trolox, and Cu were chosen based on
results with these compounds in our previous studies (2, 4).
After 24 h, cells were detached with 0.025% trypsin/0.01 M
EDTA and neutralized with trypsin neutralizer solution containing 0.5% calf serum (Cascade Biologicals).
Determination of Cytotoxicity. Cells were counted using a
hemocytometer, and cell viability was measured by the method
of trypan blue exclusion (1, 2). Cell pellets were resuspended in
PBS and stored at ⫺80°C for 8-OHdG analyses.
DNA Isolation and DNA Digestion. Cell DNA was isolated
using a DNA purification kit (Qiagen, Valencia, CA). Cell
DNA was enzymatically hydrolyzed according to a modified
method adapted from Stewart et al. (4). Briefly, for 200 ␮l of
DNA extract, 25 ␮l of 0.5 M sodium acetate (pH 5.1) was added
along with 2.25 ␮l of 1 M magnesium chloride and mixed well.
The DNA sample was boiled for 5 min and then placed on ice
for 8 min. The DNA was digested to the nucleotide level by
incubation with 1 unit of Nuclease P1 (Sigma, St. Louis, MO)
at 37°C for 1 h. The DNA sample was optimized for alkaline
phosphatase activity by adding 8 ␮l of 1 M Tris-base (pH 7.8).
One unit of alkaline phosphatase (Sigma) was added to the
DNA sample and incubated at 37°C for 1 h. Finally, 5 ␮l of
glacial acetic acid:ddH2O (1:2, v/v) was added to the DNA
sample. The DNA sample was filtered through a 0.2-␮m syringe filter and prepared in an autosampler vial for HPLC
injection.
Detection of 8-OHdG Adducts. The HPLC system consisted
of a Waters Model 600 pump (Waters Associates, Milford,
MA), a Waters Model 717 plus autosampler, a reverse-phase
Rainin Microsorb-MV C18 column (4.6 mm ⫻ 25 cm; 5-␮m
particle size), a Waters Model 486 UV absorbance detector, a
Waters amperometric Model 464 electrochemical detector with
a glassy carbon electrode, and a Waters millennium chromatography software (version 2.0). The UV detector was equipped
with a 254-nm lamp for the determination of 2-dG in DNA. A
potential of 600 mV was applied to the electrochemical detector
for the measurement of 8-OHdG adducts in DNA. The mobile
phase was prepared containing 23 mM citric acid, 42 mM sodium acetate, 50 mM sodium hydroxide, and 1 ml/liter glacial
acetic acid. The final running buffer was 20% mobile phase,
20% HPLC grade methanol, and 60% HPLC grade dd-H2O,
and the flow rate was 1.0 ml/min. Standards of 2-dG (Sigma)
and 8-OHdG (ESA, Chelmsford, MA) were prepared in mobile
phase. 2-dG and 8-OHdG standard curves were constructed
based on peak heights and concentrations in a concentrationdependent manner and used for quantitation of 2-dG and
8-OHdG, respectively, in eluted DNA samples. The results
were calculated based on the ratio of 8-OHdG (fmol):2-dG
(fmol) and reported as numbers of 8-OHdG residues/105 2-dG
residues.
Determination of DNA Fragmentation. DNA fragmentation
assays were only performed on the combined supplementation
with SeL and CuSO4 because previous results showed little
DNA fragmentation with SeM, and cell viability was too low
with SeL and trolox.
Cells were treated with SeL at doses of 0 and 126.6 ␮M Se
plus CuSO4 (7.85 and 15.7 ␮M) for 24 h. Internucleosomal
DNA fragmentation was measured by a cell death detection
ELISAplus kit (Boehringer Mannheim, Germany), as described
previously (17). Briefly, after cells were treated with trypsin,
cell pellets were resuspended in medium. The cytoplasmic
fractions of a sample containing 104 cells were prepared following the manufacturer’s instructions. This assay is designed
to measure the cytoplasmic histone-associated DNA fragments
(mononucleosomes and oligonucleosomes) contained in the
cells that bind to an immobilized anti-histone antibody. Results
were reported as a fragmentation ratio: the absorbance of the
sample divided by the absorbance of the corresponding control
(no Se and no CuSO4 added), indicating apoptosis as X-fold
internucleosomal DNA fragmentation.
Statistical Analysis. The data were analyzed by one-way
ANOVA, and mean separations were performed by Duncan’s
multiple range test (P ⬍ 0.05). All of the analyses were performed on PC SAS (SAS Institute, Cary, NC).
Results
Cytotoxicity. Fig. 1, A-C, shows the viability of cultured NHK
treated with SeL or SeM plus antioxidants or copper for 24 h.
In general, compared with control (no Se or other compounds
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Cancer Epidemiology, Biomarkers & Prevention
In the absence of Se, supplementation of Vit C or trolox
alone did not affect viability of NHK (Fig. 1, A and B). CuSO4
alone appeared to decrease cell viability (Fig. 1C); however,
this does not appear to be biologically relevant.
When NHK cells were coincubated with SeL at 126.6 ␮M
Se and each of the other compounds, Vit C and CuSO4 protected NHK against SeL-induced cytotoxicity (P ⬍ 0.05; Fig. 1,
A and C). In contrast, the synergistic effect of SeL ⫹ trolox was
found in the increased induction of cytotoxicity (P ⬍ 0.05; Fig.
1B), compared with supplementation of SeL alone. Regarding
SeM plus other compounds, no changes in cell viability were
observed in those treated with SeM ⫹ Vit C, SeM ⫹ trolox, or
SeM ⫹ CuSO4 shown in Fig. 1, A, B, and C, respectively.
Induction of 8-OHdG Adduct Formation. Fig. 2 demonstrates the formation of 8-OHdG adducts in DNA of NHK
treated with SeL or SeM plus other compounds for 24 h.
Compared with the control (no Se or other compounds), SeL
alone elevated 8-OHdG induction in the DNA of human keratinocytes (P ⬍ 0.05), whereas SeM alone did not induce generation of 8-OHdG adducts.
Without Se supplementation, neither Vit C, trolox, nor
CuSO4 increased the formation of 8-OHdG adducts (P ⬍ 0.05;
Fig. 2, A–C). Protection of NHK against SeL-induced DNA
damage was observed in the treatments with SeL ⫹ Vit C and
SeL ⫹ CuSO4 (Fig. 2, A and C). However, SeL plus high dose
trolox (80 ␮M) enhanced the formation of 8-OHdG adducts,
compared with SeL added alone (Fig. 2B). When NHK cells
were coincubated with SeM at 316.6 ␮M Se and either trolox
(40 and 80 ␮M) or CuSO4 (15.7 ␮M), there was increased
induction of 8-OHdG adducts (Fig. 2, B and C).
Protective Effect on SeL Induced by Copper. In the present
study, we only measured DNA fragmentation in NHK treated
with SeL and CuSO4, as discussed in a previous section. Because there was only minimal inhibition of SeL-induced
8-OHdG with Vit C or trolox, we did not perform the apoptosis
assays with these compounds. Supplementation with SeL alone
significantly increased internucleosomal DNA fragmentation
(P ⬍ 0.05; Table 1). Supplementation of CuSO4, even at a dose
of 15.7 ␮M, did not cause DNA of cultured human keratinocytes to undergo cell apoptosis, as determined by internucleosomal DNA fragmentation (Table 1). Furthermore, the inhibition of SeL-induced apoptosis by CuSO4 supplementation was
observed.
Fig. 1. Viability of NHK treated with SeL or SeM plus Vit C (A), trolox (B), or
CuSO4 (C) for 24 h. Results of the cell viability are expressed as means; bars,
⫾SE; n ⫽ 3 for each treatment. Bars within Se doses sharing identical letters
(a– d) are not statistically significantly different at P ⫽ 0.05 by Duncan’s multiple
range test.
added), supplementation of SeL alone at a dose of 126.6 ␮M Se
significantly decreased viability of NHK (P ⬍ 0.05), whereas
treatments of SeM (316.6 ␮M Se) alone did not affect viability
of NHK.
Discussion
Main Effects on Cytotoxicity by Se, Antioxidants, and Cu
Alone. Studies (5, 18, 19) have demonstrated that Se toxicity
to cells results from increased thiol oxidation, redox cycling,
and superoxide generation in cells in a Se concentration-dependent pattern. In the present study, we compared the effects of
two commercially available Se compounds, SeL and SeM, on
cytotoxicity to NHK. Our observations that SeL at a dose of
126.6 ␮M Se is cytotoxic to NHK are similar to those reported
in other cultured cell studies (2, 20, 21), probably because of its
capability of redox cycling and superoxide production. In the
present study, the finding that SeM in NHK culture, even at a
dose of 316.6 ␮M Se, is not cytotoxic to NHK is consistent with
those seen in other in vitro studies (6, 22). Because SeM is a
nonredoxing Se compound, it does not produce superoxide,
which would produce cytotoxicity in NHK.
Vits C and E have been represented as antioxidant nutrients that may reduce cancer risk (10 –12). Vit E is the most
important physiological membrane-associated lipid-soluble antioxidant in a biological model. Vit E has been reported to
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Antioxidant Interactions with Selenium
Table 1 Effects of SeL plus copper (CuSO4) on internucleosomal DNA
fragmentation in DNA extracted from NHK cellsa
Treatment
X-fold increase in
apoptotic fragmentation
Control (no Se, no CuSO4)
SeL (126.6 ␮M Se) only
CuSO4 (7.85 ␮M) only
CuSO4 (15.7 ␮M) only
SeL (126.6 ␮M Se) ⫹ CuSO4 (7.85 ␮M)
SeL (126.6 ␮M Se) ⫹ CuSO4 (15.7 ␮M)
1.0 ⫾ 0
15.6b ⫾ 0.5
NDe
NDe
6.0c ⫾ 0.3
1.7d ⫾ 0.1
Values are means ⫾ SE, n ⫽ 3/treatment.
Values with the same superscripts (b, c, d) are not significantly different
P ⬍ 0.05.
e
ND, not detectable.
a
b– d
Fig. 2. Numbers of 8-OHdG adducts/105 2-dG residues in DNA of NHK. Cells
were treated with SeL or SeM plus Vit C (A), trolox (B), or CuSO4 (C) for 24 h.
Results are expressed as means; bars, ⫾SE; n ⫽ 3 for each treatment. Bars within
Se doses sharing identical letters (a– d) are not statistically significantly different
at P ⫽ 0.05 by Duncan’s multiple range test.
inhibit lipid peroxidation generated by dietary fat during the
proliferation of various types of cells both in vivo (23, 24) and
in vitro (25–28). In this study, instead of Vit E, we used
water-soluble trolox (a Vit E analogue) acting as an antioxidant
in the aqueous medium of cultured cells (4). As we expected,
the antioxidants, Vit C and trolox, did not induce cytotoxicity
in cultured NHK cells. Vit C and trolox have been shown to
protect cultured mouse keratinocytes (4) as well as hairless
mice (Skh:HR-1) skin (29) from UV B radiation-induced damage. We have also confirmed the ability of Se compounds to
protect against UV-induced 8-OHdG in epidermis and incorporation into glutathione peroxidase in hairless mice.4
Copper (II) is one of the major transition metals in the
biological environment (15). Because humans may be exposed
to cupric sulfate through drinking water, food, soil, or the
environment, a high risk of subchronic copper toxicity may
occur (16). Copper (II) has been shown to catalyze DNA
damage extensively both in vitro and in vivo (15). Our finding
that CuSO4 alone induced cytotoxicity in NHK cells confirms
work from other laboratories (2, 15, 30) using different methodologies and cell lines.
Interactive Effects between Se and Antioxidants/Cu on Cytotoxicity. The present study was designed to investigate the
cellular interactions between Se and antioxidants or CuSO4 in
a cultured NHK model. Trypan blue exclusion was used for
determining cytotoxicity on the basis of cell viability. In the
present study, Vit C partially protected NHK cells from Seinduced cytotoxicity after 24 h of coincubation with SeL (Fig.
1A). However, our findings, as shown in Fig. 1B (SeL ⫹
trolox), demonstrated that trolox enhanced SeL-induced cytotoxicity of NHK cells. Although the mechanism by which
trolox induces NHK cells to enhance SeL-induced apoptosis is
still unknown, several studies have shown that Vit E increases
the cytotoxic effects of antitumor drugs in both carcinoma cell
cultures (31–35) and laboratory animals (31, 34). Additionally,
Vit E not only acts as an antioxidant but may also intensify
antineoplastic activity of anticancer drugs by inducing apoptosis (31, 36) through an inhibition of protein tyrosine kinases
(36).
The observations that CuSO4 protected NHK cells from
SeL-induced cytotoxicity as illustrated in Fig. 1C are in agreement with those in (SeL ⫹ CuSO4)-treated HT-29 cells (2).
Davis and Spallholz (37) and others have suggested that Cu
may complex with SeL to form a CuSe complex. The proposed
complex of CuSe appears to inhibit the SeL-catalyzed generation of reactive oxygen species, which is known to induce
apoptosis and DNA damage. In a cell-free model, copper (II)
was shown to inhibit both SeL-catalyzed generation of superoxide and the conversion of SeL to elemental Se (37).
4
Unpublished data.
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Cancer Epidemiology, Biomarkers & Prevention
Lu et al. (38) reported that the pathways affecting cell
cycles including cell proliferation and cell death are dependent
on different metabolic products derived from the different Se
compounds. In the present study, the findings that the combination of SeM and other compounds did not affect cell viability
are consistent with those seen in mouse skin cells (6). Previous
data from our collaborators (37) showed that SeM is a nonredoxing Se compound in which SeM does not generate superoxide, and we have shown that SeM is not cytotoxic to cultured
mouse skin cells (6).
Main Effects on DNA Oxidative Damage by Se, Antioxidants, or Cu Alone. The formation of 8-OHdG-adducted
bases is an oxidative modification of DNA detected both in vivo
(39) and in cultured cells (4, 15, 39, 40) and is considered as a
consequence of oxidative stress (4, 39, 41). There is a strong
correlation between higher amounts of 8-OHdG adducts and
greater degrees of DNA strand breaks (15) or DNA damage (4).
Several investigators have reported that high doses of SeL in in
vitro studies resulted in DNA oxidative damage as determined
by 8-OHdG lesions (4, 9), induction of cytotoxicity (2, 4, 9),
and apoptosis as measured by DNA strand breaks or DNA
ladders (1, 8, 9). In this study, we used the Floyd et al. (42, 43)
method to measure 8-OHdG in keratinocyte DNA, and this
method has consistently produced a mean of 0.5–1.5 8-OHdG/
105 2dG in DNA in our control treatment (no Se as well or other
compounds added). We have compared the capability of two Se
compounds to induce oxidative stress in cultured NHK cells.
Our data showed that SeL at a dose of 126.6 ␮M Se significantly
induced oxidative DNA damage as 8-OHdG adducts in DNA of
NHK, compared with the control (no Se added; Fig. 2). Levels
of the 8-OHdG bases in NHK cells treated with SeL were seen
to be 15–20 times the number of 8-OHdG adducted bases in
control NHK cells. SeM-treated NHK cells expressed no more
8-OHdG bases than did the control NHK cells. In agreement
with previous findings (6), high dose SeL, acting as a prooxidant, induced cytotoxicity and DNA adducts in mouse skin
cells, whereas SeM did not.
Interactive Effects on DNA Oxidative Damage between Se
and Antioxidants/Cu. In the present study, we found Vit C or
trolox alone did not induce DNA oxidative damage in NHK
(Fig. 2, A and B). However, it is notable that coincubation of
SeL with Vit C in medium for 24 h significantly reduced the
levels of 8-OHdG, compared with those induced by SeL at a
dose of 126.6 ␮M Se alone (Fig. 2A). Other studies (4, 44) have
shown that the DNA oxidative damage, resulting as a consequence of UV irradiation, can be attenuated by supplementation
of antioxidant nutrients (such as Vit C or trolox), resulting in a
reduced number of 8-OHdG adducts in different cell lines.
However, in the present study, we showed that both Se compounds (SeL and SeM) plus trolox significantly increased the
amounts of 8-OHdG in DNA of NHK (Fig. 2B). Further investigation is needed to elucidate the interactions between Se
compounds and water-soluble trolox on oxidative DNA damage during cell growth, possibly related to generation by Se of
a Vit E radical enhancing the damage.
Interactive Effects on Apoptosis between Se and Copper.
Metal ions, such as copper (II), act as a transition metal that can
catalyze extensive DNA damage to induce the generation of
oxidative 8-OHdG adducts in various in vitro models (15, 40).
In this study, we measured the number of 8-OHdG residues
after treating NHK cells with CuSO4 alone for 24 h as shown
in Fig. 2C. In our cultured NHK cells, CuSO4 alone (even at a
dose of 15.7 ␮M) did not enhance 8-OHdG formation, although
Table 2
Summary of effects of intercellular interaction between Se
compounds and antioxidantsa
Treatments
Selenium
Control (no Se, no antioxidants)
SeM
SeM
SeM
SeM
SeL
SeL
SeL
SeL
End points
Antioxidants Cell viability
0
Vit C
Trolox
CuSO4
0
Vit C
Trolox
CuSO4
No change
No change
No change
No change
2 48%
2 60%
2 20%
No change
8-OHdG
No change
No change
1 7.6-fold
1 3.3-fold
1 22.7-fold
1 14.5-fold
1 23.8-fold
No change
a
The above information is based on the data from Figs. 1 and 2. All of the
treatments are compared with the control (no Se, no antioxidants supplemented).
this is not consistent with results reported previously (15) by
others using supercoiled plasmid DNA.
In the present study, we chose only to measure apoptosis
in NHK treated with SeL plus CuSO4, because no significant
interaction was seen in NHK treated with SeM plus antioxidants. In addition, the magnitude of differences seen in
8-OHdG generation by SeL ⫹ Vit C or trolox was not substantial enough to expect induction of apoptosis. In this study,
SeL treatment caused a 15-fold increase in internucleosomal
DNA fragmentation as measured by an ELISA kit, compared
with the control (no Se added; Table 1). Supplementation of
CuSO4 alone did not cause cells to undergo apoptosis in our
cultured NHK cells. However, compared with SeL supplementation alone, treatment of SeL ⫹ CuSO4 resulted in higher cell
viability (Fig. 1C), fewer 8-OHdG adducts (Fig. 2C), and a
lesser degree of internucleosomal DNA fragmentation (Table
1). We have reported similar results with SeL and CuSO4 in
HT-29 cells (2) and in cell-free systems (37).
A summary of effects of intercellular interaction between
Se and other antioxidant compounds is shown in Table 2. Our
results confirm that cytotoxicity and oxidative DNA base modification are directly interrelated in cultured NHK. We have
shown previously (9) a significant correlation between 8-OHdG
formation, DNA strand breaks, and apoptosis by Se compounds. Our present data suggest that Vit C appears to scavenge
some of the superoxide radicals generated by SeL metabolism,
thus producing fewer 8-OHdG in NHK DNA, when compared
with those treated with SeL alone. In terms of Se ⫹ trolox, it
appears that SeL interacts with trolox synergistically to increase
the pro-oxidant effects of SeL. We speculate that trolox may be
functioning as a Vit E radical in conjunction with SeL. However, the possibility also exists that trolox may exert unknown
indirect effects as well. Regarding SeL ⫹ CuSO4, CuSO4
appears to play a protective role in SeL-induced cytotoxicity,
oxidative damage, and apoptosis of NHK, when compared with
SeL alone. It is apparent from these data that significant interactions occur at the cellular and molecular level among antioxidant supplements; however, the impact from dietary combinations cannot be predicted from these in vitro studies.
Additional studies are also needed to investigate the implications of these interactions for chemoprevention with SeL and
possibly other Se compounds.
References
1. Lu, J., Kaeck, M., Jiang, C., Wilson, A. C., and Thompson, H. J. Selenite
induction of DNA strand breaks and apoptosis in mouse leukemic L1210 Cells.
Biochem. Pharmacol., 47: 1531–1535, 1994.
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Downloaded from cebp.aacrjournals.org on June 16, 2017. © 2001 American Association for Cancer Research.
Interactions of Selenium Compounds with Other Antioxidants
in DNA Damage and Apoptosis in Human Normal
Keratinocytes
Chwan-Li Shen, Woosun Song and Barbara C. Pence
Cancer Epidemiol Biomarkers Prev 2001;10:385-390.
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