Nutrient-Gene Expression

Gene Expression Profiling of Low Selenium Status in the Mouse Intestine:

Transcriptional Activation of Genes Linked to DNA Damage,
Cell Cycle Control and Oxidative Stress1
Lin Rao, Birgit Puschner* and Tomas A. Prolla2
Departments of Genetics and Medical Genetics, University of Wisconsin-Madison, Madison, WI, 53706
and *California Animal Health and Food Safety Laboratory System, Toxicology Laboratory,
University of California, Davis, CA 95616

KEY WORDS:

gene expression

selenium

selenoprotein

The essential trace mineral Se has been associated with

reduced cancer risk in several epidemiologic studies (13).
Various Se compounds of natural and synthetic origin have
also been shown to inhibit tumor development in animal
studies in a wide range of dosages (4). Although most animal
studies have employed pharmacologic doses of Se (2 mg/kg)
in cancer chemoprevention (4), Se deficiency has also been
shown to enhance mammary (5) and UVB-induced skin carcinogenesis (6). A recent double-blind, randomized cancer
prevention trial in humans involving physiologic doses (0.2
mg) of Se demonstrated a remarkable reduction in incidence
of lung, prostate and intestinal cancers (7). These observations
suggest that Se may be one of the most powerful cancer
chemopreventive agents in the human diet. Several hypotheses have been proposed to account for the Se-mediated inhibition of tumorigenesis. As a constituent of selenoproteins, Se
has an enzymatic role as an antioxidant (8). Studies have also
demonstrated stimulation of apoptosis and enhanced carcinogen detoxification after Se supplementation at pharmacologic
doses (9 12). However, despite decades of research in the
mechanisms of action of Se, there is currently no consensus on

oxidative stress

mice

how this remarkable micronutrient prevents tumorigenesis.

Possibly, low Se status associated with decreased dietary Se
intake and supplementation with pharmacologic doses of Se
affect tumor susceptibility by distinct mechanisms.
To gain a better understanding of the effect of Se deficiency, we used Affymetrix high density oligonucleotide arrays
representing 6347 murine genes to determine the transcriptional profile associated with low Se status in the intestines of
C57Bl/6J mice. Our observations demonstrate that under such
conditions, low Se status can induce multiple transcriptional
pathways suggestive of oxidative stress, DNA damage and
alterations in cell-cycle progression, providing a framework for
understanding the multiple roles of Se in human health (13).
MATERIALS AND METHODS
Animals and diets. C57BL/6J mice were originally provided by
Dr. W. Dove, from the colony at the McArdle Laboratory (University
of Wisconsin-Madison) and were raised from birth in our animal
facility. Guidelines for the ethical care and treatment of animals from
the Animal Care and Use Committee at the University of Wisconsin-Madison were strictly followed. Mice were housed in groups of
three in microisolator cages under fluorescent lighting on a 12-h
cycle. Tap water was available continuously during the experiment
and was replaced weekly. Diets were stored in the dark at 4C and
fresh diet was added to feeders twice weekly. Diets were randomly
allocated and were consumed ad libitum by weanling (3-wk-old)
mice. Mice were fed either the Se-deficient diet (Table 1) containing
0.01 mg/kg of Se or a high Se diet that represents the Se-deficient

1
Supported by a National Institutes of Health postdoctoral fellowship to L.R.,
and NIH grant RO1 CA78723 to T.A.P. T.A.P is the recipient of the Shaw Scientist
(Milwaukee Foundation) and Burroughs Wellcome Young Investigator Award in
the Toxicological Sciences.
2
To whom correspondence should be addressed.
E-mail: taprolla@facstaff.wisc.edu

0022-3166/01 $3.00 2001 American Society for Nutritional Sciences.

Manuscript received 21 February 2001. Initial review completed 19 March 2001. Revision accepted 5 September 2001.
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ABSTRACT The essential trace mineral selenium (Se) has been shown previously to inhibit intestinal, prostate,
lung and liver tumor development and associated mortality in both experimental animals and humans. Although Se
is likely to be one of the most powerful cancer chemopreventive agents in the human diet, its mechanism of action
is unknown. To better understand the biological consequences of alterations in Se status, the gene expression
profile associated with low Se status in the intestine of C57Bl/6J mice was analyzed. Mice were fed either a high
fat (14%), torula yeast based, Se-deficient diet (0.01 mg/kg) or the same diet supplemented with a high level of
dietary Se (1 mg/kg, as seleno-L-methionine) for 90 d. Use of high density oligonucleotide arrays representing 6347
genes revealed that low Se status results in a differential gene expression pattern indicative of activation of genes
involved in DNA damage, oxidative stress and cell cycle control, and a decrease in the expression of genes
involved in detoxification. These results suggest that suboptimal intake of a single trace mineral can have broad
effects on gene expression patterns, providing a framework for understanding the multiple beneficial effects of Se
in cancer chemoprevention and human health. J. Nutr. 131: 31753181, 2001.

RAO ET AL.

3176

TABLE 1
Composition of Se-deficient basal diet1,2
Ingredient

Amount
g/kg

Sucrose
Torula yeast
Corn oil
DL-Methionine
Mineral mix3
Vitamin mix4

538.6
300.0
140.0
3.0
15.4
3.0

1 Harlan Teklad (Madison, WI) TD 98248.

2 By analysis, the basal diet contains 0.01 mg of Se/kg.
3 Mineral mix components (g/kg diet): calcium carbonate, 2.02 g;

diet supplemented with 1.0 mg/kg of Se as seleno-L-methionine

(Sigma Chemical, St. Louis, MO). The Se composition of diets was
determined by chemical analysis to be 0.01 mg/kg and 0.97 mg/kg
in the Se-deficient and high Se diet diets, respectively. The Sedeficient diet contained no added Se and 15 g/100 g total fat (Table
1). Mice in our study were deficient only in Se and received adequate
doses of vitamins and other trace minerals.
Glutathione peroxidase assay. Glutathione peroxidase (GPX)3
activities in tissue homogenates were determined by the indirect,
coupled test procedure (14). Briefly, the oxidized glutathione
(GSSG) produced from GPX enzyme activity was immediately reduced by NADPH and glutathione reductase. Therefore, the rate of
NADPH consumption was monitored as a measurement for the rate
of GSSG formation during the GPX reaction. The final concentrations of the reagents in the reaction mixture were 50 mmol/L potassium phosphate, pH 7.0, 1 mmol/L EDTA, 1 mmol/L NaN3, 0.15
mmol/L NADPH, 4 U glutathione reductase, 1 mmol/L glutathione
(GSH), and 0.15 mmol/L H2O2 at 25C. The rate of decrease in
absorption of NADPH at 340 nm was followed, and the GPX activity
was defined as nmol of NADPH consumption per min per mg of tissue
protein at 1 mmol/L GSH. GPX activity was calculated using a
mmol/L extinction coefficient for NADPH of 6.22. One enzyme unit
(U) was defined as 1 nmol of reduced glutathione oxidized per
minute. Tissue protein was determined by the Bradford method (15).
Tissue Se analysis. Total Se was analyzed by inductively coupled
argon plasma (ICP) atomic emission using hydride generation (16)
(Model ACCURIS ICP; Fison Instruments, Dearborn, MI). Tissue
(1 g) was digested for 3 h at 330C in a mixture of 1.0 mL concentrated sulfuric acid, 3.0 mL concentrated nitric acid, and 1.0 mL
concentrated perchloric acid to convert all Se species to selenate.
The selenate was reduced to selenite with 5 mol/L hydrochloric acid
at 95C. The selenite was quantitatively reduced to hydrogen selenide by acidic (10 mol/L HCl) sodium borohydride and then
determined by ICP atomic emission at 196.090 nm. The method
detection limit for a 1-g sample is 0.005 mg/kg. For acceptance of
data, the recovery rates had to fall within a range of 80 120% of the
certified value. A spiked sample was prepared using 0.100 mL of 10
mg/kg Se in 1 g of tissue, and spike recovery was within 80 120% for
3

Abbreviations used: apo, apolipoprotein; EST, expressed sequence tag; FC,

fold change; GPX, glutathione peroxidase; GSH, glutathione; GSSG, oxidized
glutathione; ICP, inductively coupled plasma; ID-1, Type 1 iodothyronine deiodinase; MM, mismatched; PM, perfectly matched; STAT, signal transducers and
activators of transcription; VEGF, vascular endothelial growth factor.

FC

SId SIa
1 if SId SIa
the smaller of either SIa or SId

FC

SId SIa
1 if SId SIa
the smaller of SId or SIa

where SId is the average signal intensity from a gene-specific probe

family from a mouse in the Se-deficient diet and SIa is that from a
mouse in the high Se diet. Alternatively, if the Qfactor, a measure of
the nonspecific fluorescence intensity background, is larger, the
smallest of either SIa or SId, the FC is calculated as:
FC

SId SIa
Qfactor

The Qfactor is automatically calculated for different regions of the

microarray and therefore minimizes the calculation of spurious FC.

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sodium chloride, 2.6; potassium citrate (monohydrate), 7.7; potassium

sulfate, 1.82; magnesium oxide, 0.84; ferric citrate, 0.21; manganous
carbonate, 0.12; zinc carbonate, 0.056; chromium potassium sulfate,
0.019; cupric carbonate, 0.011; potassium iodate, 0.0004.
4 Vitamin mix components (mg/kg diet): choline bitartrate, 2800;
niacin, 30; calcium pantothenate, 16; pyridoxine HCl, 7; thiamin HCl, 6;
riboflavin, 6; folic acid, 2; biotin, 0.2; vitamin B-12 (0.1% in mannitol),
25; dl--tocopheryl acetate (500 U/g), 100; vitamin A palmitate (500,000
U/g), 8; cholecalciferol (500,000 U/g), 0.4; phylloquinone, 3.

acceptability. In addition, acceptable drift was no more than 10%

over the entire analytical run, and reslope drift correction was no
more than 5% of the slope of the original calibration curve.
Tissue preparation and high density oligonucleotide array hybridizations. Weanling C57BL/6J male mice (21 d of age) were fed
either a Se-deficient diet containing Se at 0.01 mg/kg or a high Se
diet containing 1.0 mg/kg Se as seleno-L-methionine. Mice were
killed at 111 d of age by cervical dislocation after avertin anesthesia.
The intestine was flushed twice with saline solution and the small
intestine was measured and divided in three equal segments. A 3-cm
region of the middle segment of the small intestine corresponding to
the jejunum (300 mg of tissue) was cut and rinsed again with
physiological saline to completely remove contents, flash frozen in
liquid nitrogen and stored at 80C. We analyzed mRNA from each
mouse independently. Total RNA was extracted from frozen tissue
using the guanidinium isothiocyanate method using TRIZOL (Life
Technologies, Grand Island, NY), and poly(A) RNA was purified
using an oligo-dT-linked Oligotex resin (Qiagen, Valencia, CA).
Poly(A) RNA (1 g) was converted into double-stranded cDNA
using a Superscript Choice System (Life Technologies) and used to
synthesize biotin-labeled cRNA using a T7 Megascript kit (Ambion,
Austin, TX). Biotin-labeled cRNA was purified using a RNeasy
affinity column (Qiagen) and hybridized to the Affymetrix Mu6500
GeneChip (Affymetrix, Santa Clara, CA) as described (17). After
hybridization, the gene chips were washed and stained in a fluidic
station (Model 800101; Affymetrix). The gene chips were read at a
resolution of 6 m using a Hewlett-Packard GeneArray Scanner
(Model 900154; Affymetrix). Data collected from two scanned images were used for the analysis.
Data analysis. Detailed protocols for data analysis of Affymetrix
microarrays and extensive documentation of the sensitivity and quantitative aspects of the method have been described (18). The Affymetrix GeneChip MU6500 (Affymetrix) set was derived from selected genes and expressed sequence tags (EST) from the August 15,
1996 release of GenBank. Briefly, each gene is represented by the use
of 20 perfectly matched (PM) and mismatched (MM) control
probes. The MM probes act as specificity controls that allow the
direct subtraction of both background and cross-hybridization signals.
The number of instances in which the PM hybridization signal is
larger than the MM signal is computed along with the average of the
logarithm of the PM:MM ratio (after background subtraction) for
each probe set. These values are used to make a matrix-based decision
concerning the presence or absence of an RNA molecule. All calculations are performed by Affymetrix software. To determine the
quantitative RNA abundance, the average of the differences representing PM minus MM for each gene-specific probe family is calculated, after discarding the maximum, the minimum and any outliers
beyond 3 SD. To make comparisons between data-sets, the average
intensity differences for each gene are normalized to the total fluorescence intensity of the array. This is similar to the concept of
normalizing signal to a reference mRNA, such a -actin in a typical
Northern blot. To calculate fold changes (FC) between data sets
(after normalization) obtained from mice in the Se-deficient diet (d)
vs. mice in the high Se diet (a), the following formulas are used by the
software:

GENE EXPRESSION PATTERN OF LOW SELENIUM STATUS

Averages of pairwise comparisons are made between study groups,

each composed of three mice using Excel software. As an example,
each tissue from Se-deficient mice (n 3) is compared with high Se
mice (n 3), generating a total of nine pairwise comparisons.
Pearson correlation coefficients were calculated between individual mice in the same diet groups. No correlation coefficient between
two individual mice in the same diet group was 0.92. Specific
intragroup correlation coefficients were as follows: sd1/sd2 0.97;
sd1/sd3 0.96; sd2/sd3 0.94; sh1/sh2 0.98; sh1/sh3 0.92;
sh2/sh3 0.93 (sd, Se-deficient group; sh, high Se group fed 1.0
mg/kg Se). Supplementary information, including a complete list of
genes displaying increased (19) or decreased (20) gene expression,
including average signal intensity, is available. Data from mice fed
the two diets were compared by Studentss t test.

RESULTS

TABLE 2
Activities of selenium-dependent glutathione peroxidase
(GPX) and Se concentration in tissues of mice fed low
(0.01 mg/kg) or high (1.0 mg/kg) Se diets1
Organ
Liver
Kidney
Intestine

Dietary Se

GPX activity,
u/mg protein

Se, mg/kg
wet tissue

Low
High
Low
High
Low
High

81.0 38.3
859.0 201.4*
195.0 22.1
1005.0 146.6*
93.0 16.4
170.0 24.1*

0.09 0.03
2.17 0.15*
0.47 0.05
2.85 0.35*
ND
ND

1 Values are means SD; n 4 for all tissues except liver, (n 6).
* Different from 0.01 mg Se/kg, P 0.001. (Students t test).
ND Not determined.

chaperones HSP27 and HSP40 (21). Also induced were the

mitogen- and stress-activated protein kinase AMPK, and
metallothionein-I, a free radical scavenger implicated in oxidative damage protection (22). MDM2, an oncogene, which
functions mainly to modulate p53 tumor suppressor activity by
increasing its susceptibility to proteolysis (23) and is induced
directly by p53 under conditions of stress (24), was also induced by low Se status.
Low Se status was also associated with changes in the
expression of genes involved in cell proliferation, which accounted for 25% of genes that were upregulated in Se deficiency (Table 3). These include genes involved in cell cycle
control, such as M-phase inducer phosphatase 2, G2/mitoticspecific cyclin B2, cyclin-dependent kinase 1, cyclin-dependent kinase inhibitor, serine/threonine protein phosphatase
2A, protein-tyrosine-phosphatase IA-2 and a dual specificity
protein phosphatase PAC-1 (25,26). We also observed the
concomitant induction of signal transducers and activators of
transcription (STAT), including STAT3 and STAT5A proteins, which are involved in STAT signaling and oncogenesis
by stimulating cell proliferation and preventing apoptosis (27).
Additionally, low Se status resulted in changes in the
expression of genes that participate in angiogenesis and tumor
metastasis, such as keratin, arachidonate 12-LOX and PIGPEN (28). We also observed the parallel induction of vascular
endothelial growth factor (VEGF) and the tyrosine-protein
kinase receptor RSE, both of which play important roles in
vasculogenesis and angiogenesis (29). Genes involved in cell
adhesion and attachment, which are required for tumor
growth and invasion, were also induced by Se deficiency,
including laminin -chain 1 and plakoglobin (30). Our observations are in agreement with a recent report that low Se
status induces metastasis of melanoma cells in mice and increases the production of VEGF in rat mammary carcinomas
(31,32).
Decreased expression of genes encoding selenoproteins,
xenobiotic and lipid metabolism in Se deficiency. Se deficiency downregulated the mRNA levels of the Se-dependent
enzymes glutathione peroxidase (GPX1) and Type 1 iodothyronine deiodinase (ID-1) (Table 4) by 3.0- and 2.4-fold,
respectively (corresponding to 66 and 59% reductions in
mRNA levels). GPX1 is an important enzyme in cellular
antioxidant defense systems, detoxifying peroxides and hydroperoxides, and its expression is controlled at the mRNA
level by dietary Se (33). These observations are in agreement
with reports that GPX1 mRNA levels can decrease to 10%
of original levels in Se-deficient rat liver (34), that Se regulation of GPX1 mRNA requires a functional selenocysteine
insertion sequence in the 3-UTR that functions to stabilize
the transcript (35) and that feeding a Se-deficient diet to rats
leads to a 50% reduction in ID-1 mRNA levels in liver (35).
Genes involved in the cellular detoxification of both xenobiotic and endobiotic compounds accounted for 8% of the genes
decreased in Se-deficient mice, including genes that encode
the phase I detoxification enzymes cytochrome P450 3A1, 2B9
and the phase II enzymes epoxide hydrolase and a glutathione
S-transferase- homolog. Other Se-dependent genes that were
represented in the DNA chip and expressed in the intestine,
but which did not display reduced mRNA levels as a result of
low Se status, were selenoprotein-P, phospholipid hydroperoxide glutathione peroxidase (GPX4), plasma glutathione peroxidase (GPX2) and a poorly characterized Se-binding liver
protein (SLP-56) that has been shown to be expressed in
multiple tissues (36).

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Tissue Se and GPX Activity. To determine the effect of

the Se-deficient (0.01 mg/kg) and high Se (1.0 mg/kg) diets
(Table 1) on Se status, we determined GPX activity in liver,
kidney and intestine in mice at 111 d of age (Table 2).
Compared with mice fed the high Se diet (1.0 mg/kg), those
receiving the Se-deficient diet had significantly lower GPX
activity in all organs tested (Table 2). There was a 90%
reduction of activity in liver (P 0.0001), an 81% reduction
of activity in kidney (P 0.0013) and a 45% reduction of
activity in intestine (P 0.0008). Se levels (Table 2) were
96% lower in liver Se (P 0.0002) and 83% lower in kidney
(P 0.0007).
Increased expression of genes involved in oxidative stress,
genetic stability and cell cycle progression is associated with
low Se status. We examined the gene expression profile
associated with Se status using C57BL/6J mice. A comparison
of the small intestine from mice fed the Se-deficient diet or the
high Se diet revealed that Se status was associated with alterations in specific mRNA levels, which may reflect changes in
gene expression, mRNA stability or both. Of the 6347 genes
surveyed in the DNA microarray, only 48 (0.8%) displayed a
greater than twofold decrease in expression in response to low
Se status, whereas 84 (1.3%) displayed a greater than twofold
increase in expression. Functional classes were assigned to
genes displaying the largest alterations in expression.
Genes that displayed a greater than twofold increase in
expression in low Se dietfed mice were consistent with a
state of DNA damage, genetic instability and oxidative stress
(Table 3). These included alterations in expression of the
cell cycle arrest/DNA damage inducible genes GADD34,
GADD45, GADD and XP-E, as well as the molecular

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3178

TABLE 3
Genes upregulated in the intestine of C57B1/6J mice fed Se-deficient (0.01 mg/kg) diet1
Fold

SEM

Encoded protein

18.2
13.3
13.1
13.1
13.1
13.1
12.7
12.7
12.3
12.3
12.3
12.1

3.8
1.5
1.1
0.9
0.8
0.6
0.5
0.8
0.5
0.3
0.4
0.3

DNA-repair protein XP-E

5-AMP-activated protein kinase (AMPK-1)
DNA-damage inducible protein GADD45
GADD34
Mitogen and stress-activated protein kinase-2
Casein kinase 1 delta
DNAJ protein homolog (HSP40)
HSP27
Metallothionein-I (MT-I)
Nuclear factor B Inhibitor (IB)
Ubiquitin-conjugating enzyme (E2)
MDM2

DNA Excision repair

Energy homeostasis
Growth arrest/DNA damage response
Growth arrest/DNA damage response
Growth arrest
DNA repair
Chaperone
Chaperone
Oxidative stress induced response
NFB nuclear translocation inhibition
Ubiquitin-dependent pathway and stress
Oncogene

Cell cycle/growth control

AA020101
114.3
X66032
15.0
U06924
14.9
U00454
13.7
U77630
13.7
X06762
13.3
U27323
12.9
AA103691
12.8
AA000276
12.8
W49108
12.8
W16389
12.6
D63902
12.6
W82181
12.5
AA080176
12.5
U22399
12.4
M34094
12.4
U37465
12.3
U01841
12.3
W64759
12.2
U21103
12.1
X82457
12.1

5.2
2.1
3.5
0.6
2.3
1.1
0.7
0.6
0.3
0.5
0.5
0.4
0.4
0.8
0.6
0.9
0.4
0.4
0.3
0.7
0.5

Peanut-like protein 1
G2/mitotic-specific cyclin B2
Interleukin-6 receptor (STAT3)
Homeobox protein CDX-2
Adrenomedullin
Homeobox protein HOX-B7
M-phase inducer phosphatase 2
Cell division control protein 2
Dual specificity protein phosphatase PAC-1
Insulin-like growth factor 1 receptor
TGF-1-BP-1
Estrogen-responsive finger protein
Protein phosphatase inhibitor 2 (IPP-2)
Phospholipase C (PLC-III)
Cyclin-dependent kinase inhibitor 1C
Midkine homolog
Protein-tyrosine-phosphatase IA-2
Peroxisome proliferator activated receptor
Serine/Threonine protein phosphatase 2A
STAT5A
MLN 64 protein

Cell cycle control

Cell cycle control
Cell signaling/oncogenesis
Intestinal development/tumor-suppressor
Peptide hormone/tumor growth
Development/tumor progression
Cell cycle control
Cell cycle control
Cell cycle control
Tumor progression
Growth suppressor
Hormone response/tumor growth
Cell cycle control
Cell signaling
Cell cycle control
Growth factor
Cell cycle control
Nuclear steroid receptor/tumor development
Cell cycle control
Cell signaling
Steroid synthesis/tumor development

Angiogenesis/cell adhesion
AA003323
17.9
U43298
14.7
M28730
14.1
U43836
13.9
M90365
13.6
W34697
13.2
U39200
12.8
W07963
12.7
X03491
12.6
U60150
12.5
U18343
12.1

5.0
1.5
1.4
0.7
1.9
0.9
1.5
0.8
1.0
1.1
0.3

Filamin 1 (actin-binding protein-280)

Laminin -1 chain
Tubulin beta chain
Vascular endothelial growth factor
Junction plakoglobin
Myosin light chain 1
Arachidonate 12-LOX
RNA-binding protein FUS/TLS
Keratin (cytokeratin 4)
Associated membrane protein 2
Tyrosine-protein kinase receptor RSE

Cytoskeletal protein
Membrane attachment
Cytoskeletal protein
Angiogenesis
Cell adhesion
Cytoskeletal protein
Tumor metastasis
Angiogenesis
12-LOX trafficking
Organelle specific trafficking
Cell adhesion

ORF2
Stress response
W41070
AA008244
X54149
X51829
AA061016
W30116
AA061086
AA016411
V00835
U19799
U19854
X58876

Function

Of the 48 genes that decreased in expression in mice of low

Se status, 15% were genes that participate in lipid metabolism
(Table 4), especially in lipid transport, including apolipoprotein (apo)AI, AIV, CII, CIII, APOBEC-1, nonspecific lipidtransfer protein and fatty acid binding protein. The apoAIClll-AIV gene cluster is modulated by both gene- and clusterspecific cis-acting elements (37). Apo-Al is the major
determinant of the capacity of HDL particles to promote
cholesterol efflux and is associated with the inhibition of
atherosclerosis (38).

DISCUSSION
Previous studies that have addressed the role of Se in
carcinogenesis have often employed pharmacologic doses of Se
(2 mg/kg), whereas the potential role of physiologic doses of
Se in human cancer has received far less attention. Because
pharmacologic and physiologic doses of Se may act through
different mechanisms, it is unclear whether studies employing
pharmacologic doses of Se can provide insights concerning the
mechanisms by which low dietary Se is inversely related to

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1 The fold increase shown represents the average of all nine possible pairwise comparisons among individual mice fed with Se-deficient (0.01
mg/kg) and 1.0 mg/kg Se (n 3 for each group). The SEM was calculated for the nine pairwise comparisons and was obtained by dividing the standard
deviation by the square root of 3. The criteria for inclusion of a gene in this table is that the observed fold change is larger than the SEM 1.3. GenBank
accession numbers are listed under ORF. For a more comprehensive list, including genes that did not fit into these classes, and average signal
intensities, see (19).
2 ORF, open reading frame.

GENE EXPRESSION PATTERN OF LOW SELENIUM STATUS

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TABLE 4
Genes downregulated in the intestine of C57B1/6J mice fed Se-deficient (0.01 mg/kg) diet1
ORF2

Fold

SEM

Detoxification
M60273
AA060704
Z37107
X60452

29.2
22.6
22.6
22.3

2.0
0.6
1.2
0.7

Cytochrome P450 2B9

Glutathione S-Transferase Mu homolog
Soluble epoxide hydrolase
Cytochrome P450 3A1

Xenobiotic
Xenobiotic
Xenobiotic
Xenobiotic

Encoded protein

Function

metabolism
metabolism
metabolism
metabolism

23.0
22.4

1.5
0.4

Glutathione peroxidase (GPX1)

Type 1 iodothyronine deiodinase

Peroxide degradation
Thyroid hormone biosynthesis

26.4
24.0
23.5
23.3
22.4
22.0
22.0

3.2
2.3
1.6
1.3
0.3
0.5
0.4

Fatty acid binding protein

Apolipoprotein A-IV precursor
Apolipoprotein C-III precursor
Apolipoprotein C-II
Nonspecific lipid-transfer protein
Apolipoprotein B mRNA editing protein
Apolipoprotein A-I

Intracellular lipid transport

(HDL) Plasma lipid transport
Lipid transport
(VLDL) Lipid transport
Steroidogenesis/fatty acids oxidation
mRNA processing
(HDL) Lipid transport

Angiogenesis/cell adhesion
M62860
W16201
U43194
X61435
AA035825
AA013604

24.6
23.3
22.5
22.5
22.4
22.4

3.0
0.3
0.6
0.5
0.5
0.5

Myelin P0 protein precursor

P21-activated kinase 2
GTP-rho binding protein 1
Neuronal kinesin heavy chain
Ankyrin (brain variant)
G25K GTP-binding protein

Membrane adhesion
Morphological signaling
Cytoskeletal protein
Organelle transport
Membrane attachment
Actin cytoskeleton

Cell cycle/growth control

U30482
D12885
L13593
AA166517
U35312
AA104472
L35549

23.2
23.1
22.6
22.6
22.4
22.1
22.0

1.4
0.7
1.0
0.5
0.4
0.1
0.3

Orphan nuclear receptor TR2

Growth hormone factor (PIT-1)
Prolactin receptor (PRL-R)
RAS-related protein RAP-1A
Nuclear receptor co-repressor N-CoR
Serine/threonine protein phosphatase alpha-1
Y Box transcription factor

Hormone receptor/repressor
Hormone regulation
Hormone regulation
Tumor suppressor
Hormone regulation/repressor
Cell cycle control
Cell proliferation regulation

1 The fold decrease shown represents the average of all nine possible pairwise comparisons among individual mice fed with Se-deficient (0.01
mg/kg) and 1.0 mg/kg Se (n 3 for each group). The SEM was calculated for the nine pairwise comparisons and was obtained by dividing the standard
deviation by the square root of 3. The criteria for inclusion of a gene in this Table is that the observed fold change is larger than the SEM 1.3. GenBank
accession numbers are listed under ORF. For a more comprehensive list, including genes that do not fit into these classes, and average signal
intensities, see (20).
2 ORF, open reading frame.

cancer incidence in human populations (13), dietary supplementation with modest doses of Se in humans inhibits cancer
development at multiple sites (7) and Se deficiency enhances
tumorigenesis in at least some tissues in rodents (5,6). In fact,
previous studies suggested that there are at least two roles for
Se in cancer chemoprevention, i.e., overt Se deficiency promotes tumorigenesis in the presence of high fat (5), whereas
pharmacologic levels of Se (2mg/kg diet) protect against
tumorigenesis relative to Se-adequate levels (4).
In the current study, mice receiving the Se-deficient diet
displayed both GPX activity and tissue Se concentrations that
were consistent with a state of Se deficiency, as previously
reported in mice fed Se-deficient torula yeast diets (39 41).
Interestingly, liver GPX activity decreased to only 9% of the
level found in mice fed high Se (Table 2), whereas liver GPX
activity was reported to decrease to 1% of Se- adequate
levels in mice fed comparable Se-deficient diets (42). Thus,
tissue GPX activities suggest that these mice may not have
been as deficient as those in some other animal studies. In
contrast, mice receiving the high Se levels had elevated liver
Se levels compared with a previous study that reported tissue
levels in mice receiving 0.5 mg/kg of Se as sodium selenite
(39). The elevated Se tissue levels in the mice receiving the
high Se diet in our study may have been due to the fact that

selenomethionine is incorporated nonspecifically into protein

and is therefore deposited nonspecifically in tissues at pharmacologic levels of dietary Se (42). In Americans, the daily Se
intake was recently estimated to be 108 g Se/d, whereas daily
Chinese Se intakes associated with overt deficiency disease are
20 g/d (43), and recent European daily Se intakes ranged
from 28 to 67 g Se/d (13). The amount of dietary Se required
to reach plateau levels of plasma GPX activity has been
estimated to be between 40 and 70 g Se/d (43,44). Therefore,
a large fraction of the human population may consume levels
of Se that are below the levels that result in plateau levels of
activity of Se-dependent enzymes.
It has been suggested that pharmacologic doses of Se influence tumorigenesis through the metabolism of Se, which leads
to the formation of monomethylated forms of Se (4), whereas
suboptimal intakes of Se in the human population lead to
reduced enzymatic activity of selenoproteins in multiple tissues
(45). Because the Se-dependent enzymes, GPX, thioredoxin
reductase, phospholipid hydroperoxide glutathione peroxidase, gastrointestinal glutathione peroxidase and selenoprotein P, function as antioxidants (46), it is plausible that low Se
status is associated with some forms of oxidative stress. Indeed,
symptoms of Se deficiency such as hepatic necrosis and muscular dystrophy are severely aggravated by a simultaneous

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Selenoproteins
AA038494
U49861
Lipid transport
AA087320
M64250
W17412
W62976
M91458
U21951
W14335

3180

RAO ET AL.

TABLE 5
Global view of transcriptional changes in intestine of
C57Bl/6J mice induced by low Se status
1 Stress response

2 Selenoproteins

Induction of:
Heat shock response
DNA damage-inducible genes
Oxidative stress-inducible genes

Reduced antioxidant capacity

2 Xenobiotic metabolism
Reduced detoxification systems

1 Cell cycle control/oncogenesis

2 Lipid metabolism

Upregulation of:
Protein phosphorylation
Signal transduction
Angiogenesis
Cell adhesion

Decreased lipid transport

proliferation (55). Importantly, our observations suggest a link

between genetic instability, oxidative stress and oncogene
activation at the transcriptional level, resulting from suboptimal intake of a single micronutrient.
ACKNOWLEDGMENTS
We thank P. J. Focke and S. A. Perdue, R. Kara and K. M.
Jolivette for technical assistance.

LITERATURE CITED
1. Salonen, J. T., Alfthan, G., Huttunen, J. K. & Puska, P. (1984) Association between serum selenium and the risk of cancer. Am. J. Epidemiol. 120:
342349.
2. Willett, W. C., Polk, B. F., Morris, J. S., Stampfer, M. J., Pressel, S.,
Rosner, B., Taylor, J. O., Schneider, K. & Hames, C. G. (1983) Prediagnostic
serum selenium and risk of cancer. Lancet 2: 130 134.
3. Virtamo, J., Valkeila, E., Alfthan, G., Punsar, S., Huttunen, J. K. & Karvonen, M. J. (1987) Serum selenium and risk of cancer. A prospective followup of nine years. Cancer 60: 145148.
4. Ip, C. (1998) Lessons from basic research in selenium and cancer
prevention. J. Nutr. 128: 18451854.
5. Ip, C. & Daniel, F. B. (1985) Effects of selenium on 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis and DNA adduct formation.
Cancer Res. 45: 61 65.
6. Pence, B. C., Delver, E. & Dunn, D. M. (1994) Effects of dietary
selenium on UVB-induced skin carcinogenesis and epidermal antioxidant status.
J. Investig. Dermatol. 102: 759 761.
7. Clark, L. C., Combs, G. F., Jr., Turnbull, B. W., Slate, E. H., Chalker, D. K.,
Chow, J., Davis, L. S., Glover, R. A., Graham, G. F., Gross, E. G., Krongrad, A.,
Lesher, J. L., Jr., Park, H. K., Sanders, B. B., Jr., Smith, C. L. & Taylor, J. R.
(1996) Effects of selenium supplementation for cancer prevention in patients
with carcinoma of the skin. A randomized controlled trial. J. Am. Med. Assoc. 276:
19571963.
8. Allan, C. B., Lacourciere, G. M. & Stadtman, T. C. (1999) Responsiveness of selenoproteins to dietary selenium. Annu. Rev. Nutr. 19: 116.
9. Sundaram, N., Pahwa, A. K., Ard, M. D., Lin, N., Perkins, E. & Bowles,
A. P., Jr. (2000) Selenium causes growth inhibition and apoptosis in human
brain tumor cell lines. J. Neurooncol. 46: 125133.
10. Stewart, M. S., Spallholz, J. E., Neldner, K. H. & Pence, B. C. (1999)
Selenium compounds have disparate abilities to impose oxidative stress and
induce apoptosis. Free Radic. Biol. Med. 26: 42 48.
11. Redman, C., Xu, M. J., Peng, Y. M., Scott, J. A., Payne, C., Clark, L. C. &
Nelson, M. A. (1997) Involvement of polyamines in selenomethionine induced
apoptosis and mitotic alterations in human tumor cells. Carcinogenesis 18: 1195
1202.
12. Ip, C. & Lisk, D. J. (1997) Modulation of phase I and phase II xenobiotic-metabolizing enzymes by selenium-enriched garlic in rats. Nutr. Cancer 28:
184 188.
13. Rayman, M. P. (2000) The importance of selenium to human health.
Lancet 356: 233241.
14. Flohe, L. & Gunzler, W. A. (1984) Assays of glutathione peroxidase.
Methods Enzymol. 105: 114 121.
15. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem. 72: 248 254.

Downloaded from jn.nutrition.org at MICHIGAN STATE UNIVERSITY on April 27, 2015

deficiency of vitamin E, an inhibitor of lipid peroxidation

(47,48). However, the link between specific Se-dependent
enzymes and oxidative stress remains unclear because GPX1deficient mice are healthy under normal conditions and are
actually protected against -irradiationinduced DNA damage
(49). Additionally, simultaneous deficiency in both Se and
Vitamin E, but not Se deficiency alone, leads to elevated levels
of tissue and plasma F2-isoprostanes, a marker of lipid peroxidation (50).
DNA damage in the intestine of Se-deficient mice consuming a high fat diet is supported by our observation that low Se
status resulted in the induction of the DNA-damage inducible
genes GADD34 and GADD45 (51) (Table 3). Interestingly,
peroxynitrite, a strong oxidant, has recently been shown to
induce the expression of both GADD34 and GADD45 in
human neuroblastoma cells (52). Other oxidative stress and
DNA damageinducible transcripts induced by low Se status
include metallothionein-I, a free radical scavenger implicated
in oxidative damage protection (22) and XP-E, a DNA repair
protein. Importantly, we note that the link between the observed changes in gene expression and oxidative stress, cell
cycle alterations and DNA damage can be definitively established only through biochemical analysis of the tissues under
study. Interestingly, a previous study established that after Se
deficiency, loss of GPX activity is rapid in mice, followed by a
slower, concerted increase in the activity of several enzymes
involved in drug detoxification, including GSH transferases
and reductases (40). We did not observe the upregulation in
expression of any gene involved in drug detoxification in the
intestine of Se-deficient mice. The previously observed effect
may be liver specific, or is mediated at the protein level, as
opposed to increases in mRNA abundance.
The data presented here provide the first global assessment
of gene expression patterns in response to a nutritional deficiency in mammals. Importantly, the observed effects may be
limited to the chemical form of Se used in our study, selenoL-methionine, and may not reflect the events that follow
supplementation with pharmacologic doses of Se as used in
many experimental studies in rodents. We also note that the
reported alterations in gene expression might well require both
overt Se deficiency and a high fat diet because only in rats that
were fed a diet high in polyunsaturated fat did Se deprivation
result in a marked enhancement of mammary tumorigenesis
(5). Additionally, the gene expression profile of Se deficiency
in the intestine is complex and reflects multiple cell types,
such as those found in the intestinal epithelium, muscularis
and lymphatic nodules (53). Although several animal models
of cancer and human clinical trials indicate that Se is inversely
associated with risk of cancer, the mechanism of action of this
trace element is unknown. Here we show that the suboptimal
intake of a single micronutrient can have complex effects on
several pathways related to tumorigenesis, a finding that suggests that the mechanisms of Se-mediated cancer prevention
may be multiple. Clearly, identifying optimal intakes of Se in
the human population is likely to have a broad effect in human
health.
Taken as a whole, our results suggest that in mice of low Se
status fed high dietary fat, there is an induction of a stress
response at the transcriptional level (Table 5). This response
may be due to oxidative stress, DNA damage or both, and
could be related to a reduction in the activity of selenoproteins
and detoxification enzymes. The gene expression profile also
suggests that other responses to the low Se status may be the
induction of genes involved in cell cycle regulation and oncogenesis (54), a finding that may be linked to the previous
observation that oxidative stress is associated with cellular

GENE EXPRESSION PATTERN OF LOW SELENIUM STATUS

37. Groenendijk, M., Cantor, R. M., de Bruin, T. W. & Dallinga-Thie, G. M.

(2001) The apoAI-CIII-AIV gene cluster. Atherosclerosis 157: 111.
38. Fruchart, J. C., De Geteire, C., Delfly, B. & Castro, G. R. (1994) Apolipoprotein A-I-containing particles and reverse cholesterol transport: evidence
for connection between cholesterol efflux and atherosclerosis risk. Atherosclerosis. 110: Suppl: S35S39.
39. Reiter, R. & Wendel, A. (1983) Selenium and drug metabolism-I:
multiple modulations of mouse liver enzymes. Biochem. Pharmacol. 32: 3063
3067.
40. Reiter, R. & Wendel, A. (1984) Selenium and drug metabolism-II:
independence of glutathione peroxidase and reversibility of hepatic enzyme modulations in deficient mice. Biochem. Pharmacol. 33: 19231928.
41. Fergunson-Kohout, N. & Sunde, R. A. (1997) Dietary selenium regulation of glutathione peroxidase (GPX1) and phospholipid hydroperoxide glutathione peroxidase (GPX4) in mice. FASEB J. 11: A359 (abs.).
42. McAdam, P. A. & Levander, O. A. (1987) Chronic toxicity and retention
of dietary selenium fed to rats as D- or L-selenomethionine, selenite, or aelenate.
Nutr. Res. 7:601 610.
43. Anonymous (2000) Selenium. In: Dietary Reference Intakes for Vitamin
C, Vitamin E, Selenium and Carotenoids, pp. 284 324. Food and Nutrition Board,
National Academy Press, Washington, D.C.
44. Duffield, A. J., Thomson, C. D., Hill, K. E & Williams, S. (1999) An
estimation of selenium requirements for New Zealanders. Am. J. Clin. Nutr. 70:
896 903.
45. Thomson, C. D., Steven, S. M., van Rij, A. M., Wade, C. R. & Robinson,
M. F. (1988) Selenium and vitamin E supplementation: activities of glutathione
peroxidase in human tissues. Am. J. Clin. Nutr. 48: 316 323.
46. Holben, D. H. & Smith, A. M. (1999) The diverse role of selenium within
selenoproteins: a review. J. Am. Diet Assoc. 99: 836 843.
47. Fraga, C. G., Arias, R. F., Llesuy, S. F., Koch, O. R. & Boveris, A. (1987)
Effect of vitamin E- and selenium-deficiency on rat liver chemiluminescence.
Biochem. J. 242: 383386.
48. Hong, C. B. & Chow, C. K. (1988) Induction of eosinophilic enteritis
and eosinophilia in rats by vitamin E and selenium deficiency. Exp. Mol. Pathol.
48: 182192.
49. Esworthy, R. S., Mann, J. R., Sam, M. & Chu, F.-F. (2000) Low
glutathione peroxidase activity in GPX1 knockout mice protects jejunum crypts
from gamma-irradiation damage. Am. J. Physiol. 279: G426 G436.
50. Awad, J. A, Morrow, J. D., Hill, K. E., Roberts, L. J., II & Burk, R. F. (1994)
Detection and localization of lipid peroxidation in selenium- and vitamin E-deficient rats using F2-isoprostanes. J. Nutr. 124: 810 816.
51. Hollander, M. C., Zhan, Q., Bae, I. & Fornace, A.J.J. (1997) Mammalian
GADD34, an apoptosis- and DNA damage-inducible gene. J. Biol. Chem. 272:
1373113737.
52. Oh-Hashi, K., Maruyama, W. & Isobe, K. (2001) Peroxynitrite induces
GADD34, 45, and 153 VIA p38 MAPK in human neuroblastoma SH-SY5Y cells.
Free Radic. Biol. Med. 30: 213221.
53. Burgess, A. W. (1998) Growth control mechanisms in normal and
transformed intestinal cells. Phil. Trans. R. Soc. Lond. B Biol. Sci. 353: 903909.
54. Wagner, H. P. (1998) Cell cycle control and cancer. Indian J. Pediatr.
65: 805 814.
55. Irani, K., Xia, Y., Zweier, J. L., Sollott, S. J., Der, C. J., Fearon, E. R.,
Sundaresan, M., Finkel, T. & Goldschmidt-Clermont, P. J. (1997) Mitogenic
signaling mediated by oxidants in Ras-transformed fibroblasts. Science (Washington, DC) 275: 1649 1652.

Downloaded from jn.nutrition.org at MICHIGAN STATE UNIVERSITY on April 27, 2015

16. Maas, J., Galey F. D., Peauroi, J. R., Case J. T., Littlefield, E. S., Gay,
C. C., Koller, L. D., Crisman R. O., Weber, D. W. & Warner, D. W. (1992) The
correlation between serum selenium and blood selenium in cattle. J. Vet. Diagn.
Investig. 4: 48 52.
17. Lipshutz, R. J., Fodor, S. P., Gingeras, T. R. & Lockhart, D. J. (1999)
High density synthetic oligonucleotide arrays. Nat. Genet. 21: 20 24.
18. Lee, C. K., Klopp, R. G., Weindruch, R. & Prolla, T. A. (1999) Gene
expression profile of aging and its retardation by caloric restriction. Science
(Washington, DC) 285: 1390 1393.
19. http://www.wisc.edu/genetics/CATG/prolla/data/selenium/table-5.pdf.
20. http://www.wisc.edu/genetics/CATG/prolla/data/selenium/table-6.pdf.
21. Takekawa, M. & Saito, H. (1998) A family of stress-inducible GADD45like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK.
Cell 95: 521530.
22. Chubatsu, L. S. & Meneghini, R. (1993) Metallothionein protects DNA
from oxidative damage. Biochem. J. 291: 193198.
23. Momand, J., Wu, H. H. & Dasgupta, G. (2000) MDM2master regulator of the p53 tumor suppressor protein. Gene 242: 1529.
24. Mendrysa, S. M. & Perry, M. E. (2000) The p53 tumor suppressor
protein does not regulate expression of its own inhibitor, MDM2, except under
conditions of stress. Mol. Cell. Biol. 20: 20232030.
25. Goldberg, Y. (1999) Protein phosphatase 2A: who shall regulate the
regulator? Biochem. Pharmacol. 57: 321328.
26. Camps, M., Nichols, A. & Arkinstall, S. (2000) Dual specificity phosphatases: a gene family for control of MAP kinase function. FASEB. J. 14: 6 16.
27. Bowman, T., Garcia, R., Turkson, J. & Jove, R. (2000) STATs in
oncogenesis. Oncogene 19: 2474 2488.
28. Nie, D., Tang, K., Diglio, C. & Honn, K. V. (2000) Eicosanoid regulation
of angiogenesis: role of endothelial arachidonate 12-lipoxygenase. Blood 95:
2304 2311.
29. McMahon, G. (2000) VEGF receptor signaling in tumor angiogenesis.
Oncologist 5: 310.
30. McCarthy, J. B., Basara, M. L., Palm, S. L., Sas, D. F. & Furcht, L. T.
(1985) The role of cell adhesion proteinslaminin and fibronectinin the movement of malignant and metastatic cells. Cancer Metastasis Rev. 4: 125152.
31. Yan, L., Yee, J. A., Li, D., McGuire, M. H. & Graef, G. L. (1999) Dietary
supplementation of selenomethionine reduces metastasis of melanoma cells in
mice. Anticancer Res. 19: 13371342.
32. Jiang, C., Jiang, W., Ip, C., Ganther, H. & Lu, J. (1999) Seleniuminduced inhibition of angiogenesis in mammary cancer at chemopreventive levels
of intake. Mol. Carcinog. 26: 213225.
33. Moriarty, P. M., Reddy, C. C. & Maquat, L. E. (1998) Selenium deficiency reduces the abundance of mRNA for Se-dependent glutathione peroxidase 1 by a UGA-dependent mechanism likely to be nonsense codon-mediated
decay of cytoplasmic mRNA. Mol. Cell. Biol. 18: 29322939.
34. Bermano, G., Nicol, F., Dyer, J. A., Sunde, R. A., Beckett, G. J., Arthur,
J. R. & Hesketh, J. E. (1995) Tissue-specific regulation of selenoenzyme gene
expression during selenium deficiency in rats. Biochem. J. 311: 425 430.
35. Weiss, S. L. & Sunde, R. A. (1998) Cis-acting elements are required for
selenium regulation of glutathione peroxidase-1 mRNA levels. RNA 4: 816 827.
36. Bansal, M. P., Mukhopadhyay, T., Scott, J., Cook, R. G., Mukhopadhyay,
R. & Medina, D. (1990) DNA sequencing of a mouse liver protein that binds
selenium: implications for seleniums mechanism of action in cancer prevention.
Carcinogenesis 11: 20712073.

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