Selenium, Selenoenzymes, Oxidative Stress and Risk of Neoplastic Progression from Barrett's Esophagus: Results from Biomarkers and Genetic Variants

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Abstract
Clinical trials have suggested a protective effect of selenium supplementation on the risk of esophageal cancer, which may be mediated through the antioxidant activity of selenoenzymes. We investigated whether serum selenium concentrations, selenoenzyme activity, oxidative stress and genetic variation in selenoenzymes were associated with the risk of neoplastic progression to esophageal adenocarcinoma (EA) and two intermediate endpoints, aneuploidy and tetraploidy. In this prospective cohort study, during an average follow-up of 7.3 years, 47 EA cases, 41 aneuploidy cases and 51 tetraploidy cases accrued among 361 participants from the Seattle Barrett's Esophagus Research Study who were free of EA at the time of blood draw and had at least one follow-up visit. Development to EA was assessed histologically and aneuploidy and tetraploidy by DNA content flow cytometry. Serum selenium concentrations were measured using atomic absorption spectrometry, activity of glutathione peroxidase (GPX) 1 and GPX3 by substrate-specific coupled test procedures, selenoprotein P (SEPP1) concentrations and protein carbonyl content by ELISA method and malondialdehyde concentrations by HPLC. Genetic variants in GPX1-4 and SEPP1 were genotyped. Serum selenium was not associated with the risk of neoplastic progression to EA, aneuploidy or tetraploidy (P for trend = 0.25 to 0.85). SEPP1 concentrations were positively associated with the risk of EA [hazard ratio (HR) = 3.95, 95% confidence intervals (CI) = 1.42-10.97 comparing the third tertile with the first] and with aneuploidy (HR = 6.53, 95% CI = 1.31-32.58), but not selenoenzyme activity or oxidative stress markers. No genetic variants, overall, were associated with the risk of neoplastic progression to EA (global p = 0.12-0.69). Our results do not support a protective effect of selenium on risk of neoplastic progression to EA. Our study is the first to report positive associations of plasma SEPP1 concentrations with the risk of EA and aneuploidy, which warrants further investigation.
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Selenium, Selenoenzymes, Oxidative Stress and Risk of
Neoplastic Progression from Barrett’s Esophagus: Results
from Biomarkers and Genetic Variants
Yumie Takata
1
, Alan R. Kristal
1
, Regina M. Santella
2
, Irena B. King
1,3
, David J. Duggan
4
,
Johanna W. Lampe
1
, Margaret P. Rayman
5
, Patricia L. Blount
6
, Brian J. Reid
1,6
, Thomas L. Vaughan
1
,
Ulrike Peters
1
*
1 Public Health Science Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 2 Department of Environmental Health Sciences,
Columbia University, New York, New York, United States of America, 3 Department of Internal Medicine, University of New Mexico, Albuquerque, New Mexico, United
States of America, 4 Division of Genetic Basis of Human Disease, Translational Genomics Research Institute, Phoenix, Arizona, United States of America, 5 Faculty of Health
and Medical Sciences, University of Surrey, Guildford, United Kingdom, 6 Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United
States of America
Abstract
Clinical trials have suggested a protective effect of selenium supplementation on the risk of esophageal cancer, which may
be mediated through the antioxidant activity of selenoenzymes. We investigated whether serum selenium concentrations,
selenoenzyme activity, oxidative stress and genetic variation in selenoenzymes were associated with the risk of neoplastic
progression to esophageal adenocarcinoma (EA) and two intermediate endpoints, aneuploidy and tetraploidy. In this
prospective cohort study, during an average follow-up of 7.3 years, 47 EA cases, 41 aneuploidy cases and 51 tetraploidy
cases accrued among 361 participants from the Seattle Barrett’s Esophagus Research Study who were free of EA at the time
of blood draw and had at least one follow-up visit. Development to EA was assessed histologically and aneuploidy and
tetraploidy by DNA content flow cytometry. Serum selenium concentrations were measured using atomic absorption
spectrometry, activity of glutathione peroxidase (GPX) 1 and GPX3 by substrate-specific coupled test procedures,
selenoprotein P (SEPP1) concentrations and protein carbonyl content by ELISA method and malondialdehyde
concentrations by HPLC. Genetic variants in GPX1-4 and SEPP1 were genotyped. Serum selenium was not associated
with the risk of neoplastic progression to EA, aneuploidy or tetraploidy (P for trend = 0.25 to 0.85). SEPP1 concentrations
were positively associated with the risk of EA [hazard ratio (HR) = 3.95, 95% confidence intervals (CI) = 1.42–10.97 comparing
the third tertile with the first] and with aneuploidy (HR = 6.53, 95% CI = 1.31–32.58), but not selenoenzyme activity or
oxidative stress markers. No genetic variants, overall, were associated with the risk of neoplastic progression to EA (global
p = 0.12–0.69). Our results do not support a protective effect of selenium on risk of neoplastic progression to EA. Our study
is the first to report positive associations of plasma SEPP1 concentrations with the risk of EA and aneuploidy, which warrants
further investigation.
Citation: Takata Y, Kristal AR, Santella RM, King IB, Duggan DJ, et al. (2012) Selenium, Selenoenzymes, Oxidative Stress and Risk of Neoplastic Progression from
Barrett’s Esophagus: Results from Biomarkers and Genetic Variants. PLoS ONE 7(6): e38612. doi:10.1371/journal.pone.0038612
Editor: DunFa Peng, Vanderbilt University Medical Center, United States of America
Received Febr uary 2, 2012; Accepted May 7, 2012; Published June 8, 2012
Copyright: ß 2012 Takata et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Supported by the National Cancer Institute (grant number: P01 CA91955 and K22 CA118421 and K05CA124911) and by the National Institute of Health
(grant number: DK58763). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: upeters@fhcrc.org
Introduction
Results from a few clinical trials and observational studies
support a protective effect of selenium on the risk of esophageal
cancer [1–5]. Two trials in China found non-significant reduced
risks of esophageal cancer (primarily squamous cell carcinoma)
with supplementation of selenium in combination with other
micronutrients for more than five years [1–3]. Case-cohort
analysis within one of these trials found a statistically significant
44% lower risk of esophageal cancer among supplemented
participants whose pre-trial serum selenium concentrations were
in the highest (.82
mg/L) rather than the lowest (,60 mg/L)
quartile [4]. In the U.S., secondary analysis of the Nutritional
Prevention of Cancer Trial showed a non-significant 70% lower
risk of esophageal cancer in the group supplemented with selenium
alone than in the placebo group after 6.4 years of follow-up, but
included only eight esophageal cancer cases [5]. Further, two small
case-control studies showed an inverse association between serum
selenium concentrations and the risk of esophageal cancer, but the
lower selenium status observed may have been caused by the
disease [6,7]. The only previous study of genetic variants in
selenoenzymes and risk of esophageal adenocarcinoma (EA)
reported null finding [8].
Here we investigated the association of serum selenium with the
risk of neoplastic progression to EA. Persons with Barrett’s
esophagus (BE), a premalignant metaplasia of the lower esopha-
geal epithelium, develop EA at a rate of 6–7 per 1,000 person-
years, which is substantially higher than in the general population
PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e38612
[9]. Thus, this cohort provides an opportunity to investigate
longitudinally the neoplastic progression to EA, which is a very
rare cancer in the general population. In a previous cross-sectional
analysis of BE patients in this cohort, we observed an inverse
association between serum selenium concentration and various
markers of neoplastic progression to EA [10]. This study further
investigates these results, by longitudinally examining the associ-
ations of selenium, selenoenzymes/selenoproteins and oxidative
stress with subsequent risk of neoplastic progression to EA,
aneuploidy, and tetraploidy. Because selenium exerts its antiox-
idant property through selenoenzymes, we also examined whether
the activity of, as well as genetic variations in, selenoenzymes were
associated with the risk of progression.
Methods
Study population
This prospective cohort study was conducted through the
Seattle Barrett’s Esophagus Research Study, a dynamic cohort of
persons diagnosed with BE [11]. The study was initially approved
by the Human Subjects Division of the University of Washington
in 1983 and was renewed annually thereafter with reciprocity from
the Fred Hutchinson Cancer Research Center (FHCRC) from
1993 to 2001. Since 2001, the study has been approved by the
Institutional Review Board of the FHCRC with reciprocity from
the Human Subjects Division of the University of Washington. All
participants provided written informed consent. The study began
in 1983 with endoscopic surveillance and was expanded as of
February 1, 1995 to include the collection of blood, interview and
anthropometric data. Participants underwent an extensive baseline
interview, after which they had shorter follow-up interviews at
subsequent endoscopies. The structured baseline interview took
approximately 45 minutes to complete and collected detailed
information on various lifestyle exposures, medical and medication
history, and anthropometric measurements (height, weight, and
circumference of waist, hips, thighs, and abdomen). At follow-up
interviews, updated information was obtained on anthropometric
measurements, lifestyle and current medication use, and a blood
sample was collected.
Endoscopic biopsy protocols and evaluations used in the Seattle
Barrett’s Esophagus Research Study have been published previ-
ously [11–13]. Briefly, for those without high-grade dysplasia,
four-quadrant biopsies were taken for histologic evaluation every
two centimeter throughout the length of the Barrett’s segment; for
those with a history of high-grade dysplasia, every one centimeter
throughout the length of the Barrett’s segment. Persons entering
the cohort with an outside diagnosis of high-grade dysplasia were
further evaluated by an intensive protocol of four-quadrant
biopsies every one centimeter two additional times within the first
four months of participation, after which they were followed up
approximately every six months. Those without high-grade
dysplasia or a history of high-grade dysplasia were followed up
every two to three years. Participants’ histology was classified
according to the highest grade of dysplasia present and study
pathologists were blinded to participant information. Overnight
fasting blood samples were collected prior to endoscopy and serum
and EDTA-treated plasma aliquots were prepared and stored at
270uC.
A total of 361 participants free of EA at the time of blood draw
and with at least one follow-up visit were included in analyses
based on serum selenium. For the analysis for aneuploidy and
tetraploidy, 37 and 36 participants, respectively, were excluded
from the analysis due to the diagnosis of the respective endpoint at
the time of blood draw. As a result, analyses for EA, aneuploidy
and tetraploidy had 2647.2, 2378.2 and 2290.0 person-years,
respectively. For the subset of 198 participants who had given
sufficient blood at the first or second follow-up visit for the
laboratory assays, selenoenzyme activity, or selenoprotein concen-
tration and two measures of oxidative stress were made. In
addition, single nucleotide polymorphisms (SNPs) in selenoen-
zyme/selenoproteins were genotyped in this subset.
Outcome measures
EA was assessed histologically, and aneuploidy and tetraploidy
were assessed as previously described [14]. Briefly, DNA content
was measured by flow cytometry using the computer program
Multicycle (Phoenix Flow Systems, San Diego, CA) [13,15]. A
diagnosis of aneuploidy was made if discrete peaks on the
histogram showed aneuploid and diploid cell populations and if
the aneuploid peak included at least 2.5% of cells in the biopsy
sample [13]. A diagnosis of tetraploidy (4N) was made if .6% of
cells had DNA content between 3.85 and 4.10 N.
Laboratory measures
Serum selenium concentrations were measured by atomic
absorption spectrometry (Perkin Elmer, Fremont CA) according
to the standard protocol [16]. This was conducted at the
Harborview Medical Center and the FHCRC. To ensure the
comparability of the assays conducted at the two locations, 57
samples were run at both locations, yielding a mean coefficient of
variation (CV) of 6.0% (range: 0.24–18.4%, pair: 57). The overall
mean CV from blinded duplicate samples run at either or both
locations was 15.1% (range: 0.24–39.0%, pair: 72). For a large
fraction of eligible participants, selenium was measured at baseline
and follow-up visits, yielding a total of 647 values for 369
participants. Due to the high CVs (.10%) observed in two pairs,
we excluded 82 selenium values that were analyzed on the same
day as these pairs. After the exclusion, the CV (mean 6 standard
deviation) of selenium values from the same participants measured
in blood samples collected at different time points was 8.866.4%.
Hence, among the remaining selenium values, the analysis
included the blood sample that was collected during the earliest
study visits to maximize the time to event. Included in the analysis
were 349 samples from baseline, eleven from first follow-up and
one from the second follow-up.
The activity of glutathione peroxidase (GPX) 1 in white blood
cells and of GPX3 in plasma were measured by applying our
standardized protocol using OXItek commercial kit [ZMC catalog
# 0805002, ZeptoMetric Corporation, Buffalo NY,] based on
Paglia and Valentine’s method [17] and using cumene hydroper-
oxide as the substrate. Quality controls (QCs) of known activity
were run at the beginning of the assay each day to ensure the
quality of assay internally. The respective mean CV of GPX1 and
GPX3 activity from all samples run as duplicates was 2.1% and
3.2%. Both GPX1 and GPX3 assays were conducted at FHCRC.
Selenoprotein P (SEPP1) concentration was measured in plasma
samples using a sandwich ELISA method as previously described
[18]. N22 was used as a capture antibody and N11 as a
biotinylated antibody since each antibody recognizes different
proportions of the N terminus of the human protein. The CV
(mean 6 standard deviation) of blinded QCs from two plasma
samples, each measured seven times, was 6.8% (4.8360.33
mg/L)
and 17.1% (5.1460.88
mg/L). The SEPP1 assay was conducted at
the Vanderbilt University School of Medicine.
To assess lipid peroxidation, malondialdehyde (MDA) was
assayed in EDTA-treated plasma using HPLC as previously
described [19]. This assay was conducted at FHCRC. For the
assessment of protein oxidation, protein carbonyl content (PCC) in
Selenium, Selenoenzymes and Neoplastic Progression
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plasma was analyzed by the non-competitive ELISA method using
dinitrophenylhydrazine as an antibody that was developed
previously [20]. QCs with known PCC were included in each
plate. The CV (mean 6 standard deviation) from internal QCs in
all six plates was 10.0% (0.4060.04
mmol/L) and the CVs of
blinded QCs from two plasma samples measured each seven times
were 16.1% (0.3765.96
mmol/L) and 12.8% (0.3965.01 mmol/
L). This assay was conducted at the Columbia University.
A set of tagging SNPs (tagSNPs) in each of five selenoenzyme/
selenoprotein genes (i.e., GPX1, GPX2, GPX3, GPX4 and SEPP1)
was selected because these genes are associated with oxidative
stress and expressed in the gastrointestinal tract [21–23]. We first
sequenced these genes in European American subjects in the
HapMap population [24] and selected all SNPs in the selenocys-
teine insertion sequence and all nonsynonymous SNPs in exons
[24]. Secondly, additional tagSNPs were selected according to the
criteria of r
2
$0.8 and minor allele frequency $5% [25] based on
our sequencing data [24] on European American HapMap
samples [26]. A total of 34 tagSNPs were genotyped using
Matrix-assisted Laser Desorption/Ionization Time-of-Flight on
the Sequenom MassARRAY 7K platform (Sequenom, Inc., San
Diego CA) at the Translational Genomics Research Institute.
Each plate included blinded duplicates from 5% of study samples
as QC. Twelve SNPs were excluded for the following reasons: call
rate ,90% for three SNPs (rs75404373, rs2277501 and
rs4807542); P-value for Hardy-Weinberg equilibrium ,0.01 for
four SNPs (rs2293627, rs6888691, rs3763011 and rs757229);
concordance of the blinded QCs (10 pairs) ,90% for two SNPs
(rs2074452 and rs7579) and minor allele frequency #5% for three
SNPs (rs8179164, rs17883875 and rs4807543).
Statistical Analyses
Cox proportional hazards regression models were used to assess
the association of selenium, selenoenzyme activity, selenoprotein
concentrations, oxidative stress and genetic variants in selenoen-
zymes with time to neoplastic progression to EA, aneuploidy and
tetraploidy. The time of entry was time of the blood draw for
selenium, selenoenzyme and oxidative stress marker analyses and
time of baseline interview for genetic analysis. The time of exit was
defined as the date of endpoint diagnosis or date of the last follow-
up visit as of September 31, 2009, whichever came first. Serum
selenium concentration was analyzed as continuous, grouped into
tertiles based on the distribution in the entire cohort, and classified
into low and high concentrations based on the cut-off of 118
mg/L
used in our previous study (defined as the 75th percentile cut-off)
[10]. For MDA concentrations, nine outliers defined as being
outside of the upper and lower three interquartile ranges were
excluded. GPX1 activity and MDA concentrations were log-
transformed. Selenoenzyme activity and selenoprotein concentra-
tion as well as oxidative stress markers were categorized by tertile.
Tests for linear trend across tertiles were based on median values
in each tertile.
All analyses were controlled for gender, baseline values of age (5
categories), waist to hip ratio (quartiles), smoking status (never,
former or current) and the use of non-steroid anti-inflammatory
drugs (NSAIDs; never, former or current). Genetic analysis was
also controlled for Caucasian ethnicity (European American or
other). All covariates each affected the hazard ratio (HR) of at least
one of the outcomes by a minimum of 10%.
For genetic analysis of five selenoenzyme/selenoprotein genes,
the overall variation within a gene was first evaluated by global
gene test, which compared the log likelihood ratio statistics
between the model with and without all SNPs in a given gene [27].
If the test was significant (P,0.05), significant association of
individual SNPs was considered. The number of the minor allele
was coded as log-additive (i.e., 0 for homozygote common allele, 1
for heterozygotes and 2 for homozygote rare allele) to test for
trend. The effect of individual SNPs was tested by including one
SNP per model.
To assess if prevalence of high-grade dysplasia, aneuploidy or
tetraploidy influenced the results, a sensitivity analysis was
conducted in two ways: 1) excluding participants with high-grade
dysplasia and 2) excluding those with aneuploidy and/or
tetraploidy at baseline and considering two outcomes (EA and
aneuploidy and/or tetraploidy). The results from the sensitivity
analysis were compared with those from the overall analyses. All
statistical analyses were conducted by SAS 9.2 (Carey, NC) and
STATA 11 (College Station, TX).
Results
The majority of study participants were Caucasian and male
(Table 1), reflecting the typical distribution of these characteristics
in BE patients. More than half of the participants had at least some
college education. Participants who developed EA, aneuploidy or
tetraploidy tended to use NSAIDs less often than those who did
not. EA cases had smoked substantially more than aneuploidy or
tetraploidy cases or the total cohort. The proton pump inhibitor or
H2 blocker medication was used by almost all participants who
had not gone through anti-reflux surgery. All participants had
gastroesophageal reflux disease symptoms prior to or at the time of
the study enrollment. EA cases were most likely to have high-grade
dysplasia at baseline, followed by cases with aneuploidy and
tetraploidy. At baseline, both aneuploidy and tetraploidy were
common among EA cases compared with the overall cohort or
tetraploidy or aneuploidy cases. Among the study participants,
there were 33 participants who developed high-grade dysplasia,
but not EA during the study period. There were 10 participants
who had all three conditions (aneuploidy, tetraploidy and high-
grade dysplasia) at baseline. There was a broad range in serum
selenium concentrations, which varied from 67.1 to 213.2
mg/L.
Serum selenium concentrations were not associated with the risk
of neoplastic progression to EA (Table 2). The adjusted HRs
[95% confidence intervals (95% CI)] for EA, aneuploidy and
tetraploidy with each 50
mg/L increase in serum selenium
concentrations were 1.16 (0.60–2.28), 1.64 (0.79–3.42) and 1.06
(0.54–2.06), respectively. Analysis based on tertiles of selenium
concentrations also showed no association; the corresponding P for
trend for EA, aneuploidy and tetraploidy were 0.39, 0.25 and 0.85.
Compared with the participants with serum selenium concentra-
tion ,118
mg/L, the concentration $118 mg/L was not associ-
ated with risk of neoplastic progression to EA (EA: HR = 1.23,
95% CI = 0.58–2.60; aneuploidy: HR = 2.15, 95% CI = 0.83–
5.58; tetraploidy: HR = 1.38, 95% CI = 0.63–3.00, respectively).
Excluding participants with high-grade dysplasia or aneuploidy
and/or tetraploidy at baseline did not materially change results
(data not shown).
SEPP1 concentrations were positively associated with the risk of
EA and aneuploidy, but not tetraploidy (Table 3). Each one unit
(
mg/L) increase in SEPP1 concentration was associated with 46%
increase in EA risk (HR = 1.46, 95% CI = 1.05–2.05). Those in the
highest tertile of SEPP1 concentrations had a 3.95-time higher risk
of EA (HR = 3.95, 95% CI = 1.42–10.97, P for trend = 0.006) and
a 6.53-time higher risk of aneuploidy (HR = 6.53, 95% CI = 1.31–
32.58, P for trend = 0.02) compared with those at the lowest. None
of the other selenoenzymes (GPX1 and GPX3) or oxidative stress
markers (i.e., MDA and PCC) was associated with the risk of
neoplastic progression to EA. Excluding participants with high-
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grade dysplasia or aneuploidy and/or tetraploidy at baseline did
not alter the observed positive associations of SEPP1 concentra-
tions with the risk of EA and aneuploidy, which lost statistical
significance, but were in the same direction (data not shown). We
further examined the cross-sectional association of SEPP1
concentrations and the risk of high-grade dysplasia, aneuploidy
or tetraploidy and found no association [high-grade dysplasia:
odds ratio (OR) = 0.99, 95% CI = 0.70–1.40; aneuploidy:
OR = 0.76, 95% CI = 0.49–1.16; tetraploidy: OR = 1.05, 95%
CI = 0.73–1.53].
Two SNPs in the GPX3 gene were statistically significantly
associated with the risk of EA (P for trend = 0.03 for rs4958872
and P for trend = 0.04 for rs3792797); however, the overall genetic
variation was not significant (global P = 0.33) (Table 4). None of
the other SNPs in selenoenzyme genes, when combined within a
gene and assessed individually, was associated with the risk of
neoplastic progression to EA (global P = 0.12–0.69; P for
trend = 0.08–0.99).
Discussion
Our study found no evidence of an association of serum
selenium concentration with the risk of neoplastic progression to
EA, aneuploidy or tetraploidy. However, SEPP1 concentration
was strongly and significantly positively associated with risk of EA
and aneuploidy, though not tetraploidy. None of the overall
variation in GPX1-4 and SEPP1 genes was significantly associated
with the risk of neoplastic progression to EA.
Our null finding on serum selenium is consistent with the case-
cohort study in The Netherlands that observed no association
between toenail selenium and the risk of progression from BE to
EA or high-grade dysplasia [28]. However, our current prospec-
Table 1. Characteristics of the Study Population*.
All
Esophageal
adenocarcinoma Aneuploidy Tetraploidy
Number 361 47 41 51
Women 68 (18.8%) 6 (12.8%) 13 (14.6%) 10 (19.6%)
Age at baseline 61.1611.7 64.2610.6 61.0610.7 63.4611.6
Age at last follow-up visit 68.4611.5 67.5610.7 67.4610.0 71.1610.7
Waist to Hip ratio at baseline 0.9560.07 0.9660.07 0.9660.07 0.9660.08
Ethnicity
Caucasian 337 (93.4%) 46 (97.9%) 39 (95.1%) 47 (92.2%)
Others 24 (6.6%) 1 (2.1%) 0 (0%) 1 (2.0%)
Educational attainment
Grade school 6 (1.7%) 2 (4.3%) 0 (0%) 1 (2.0%)
High school 91 (25.3%) 14 (29.8%) 12 (33.3%) 16 (31.4%)
Technical/vocational school 21 (5.8%) 4 (8.5%) 1 (8.1%) 4 (7.8%)
College 242 (67.2%) 27 (57.4%) 28 (55.6%) 30 (58.8%)
NSAID use at baseline
Cumulative use (pill-months) 75.96163.5 61.16131.8 61.76120.1 59.86101.6
Never 149 (41.6%) 24 (51.1%) 21 (51.2%) 20 (39.2%)
Former 74 (20.7%) 10 (21.3%) 10 (24.4%) 11 (21.6%)
Current 135 (37.7%) 13 (27.6%) 10 (24.4%) 20 (39.2%)
Smoking at baseline
Pack-years 18.8624.0 25. 8624.0 15.4616.7 17.6621.8
Never 127 (35.2%) 10 (21.3%) 13 (31.7%) 17 (33.3%)
Former 197 (54.6%) 35 (74.5%) 24 (58.5%) 32 (62.7%)
Current 37 (10.2%) 2 (4.2%) 4 (9.8%) 2 (3.9%)
Serum selenium concentration (
mg/L) 135.0621.0 136.2619.4 138.7620.0 136.9619.3
GPX1 activity (U/g T protein)** 43.1621.8 45.6620.0 41.3612.3 43.0616.5
GPX3 activity (U/L)** 7296121 7046134 6996111 6916126
SEPP concentration (
mg/L)** 5.7861.12 6.2561.12 5.8861.09 5.7861.27
Malondialdehyde (
mmol/L)** 1.0961.15 1.2661.29 1.0661.00 1.0660.95
Protein carbonyl content (nmol/mg protein)** 0.3660.06 0.3660.06 0.3560.06 0.3760.06
High-grade dysplasia at baseline 66 (18.3%) 34 (72.3%) 46 (46.3%) 19 (37.3%)
Aneuploidy at baseline 37 (10.2%) 19 (40.0%) 0 (0%) 24 (47.1%)
Tetraploidy at baseline 36 (10.0%) 19 (40.0%) 13 (31.7%) 0 (0%)
*The mean 6 standard deviation or number (percentage) is provided.
**Up to 198 of the participants were included.
doi:10.1371/journal.pone.0038612.t001
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tive analysis did not replicate findings from our previous cross-
sectional analysis that showed an inverse association of serum
selenium [.1.5
mM (equivalent to 118 mg/L)] with aneuploidy,
high-grade dysplasia and 17p loss of heterozygosity, all measures
of neoplastic progression to EA [10]. This may be partly explained
by the different outcomes investigated in the two analyses and by
prevalent and incident cases. Further, our sensitivity analysis
excluding participants who had high-grade dysplasia or aneuploi-
dy and/or tetraploidy at baseline did not differ from the overall
finding, suggesting that the prevalent condition did not affect our
null finding. Hence, an inverse association observed in our
previous cross-sectional analysis is most likely due to reverse
causality or the fact that high serum selenium in our cohort might
be associated with other factors that increase the risk of neoplastic
progression to EA.
Within the general population, clinical trials of selenium
supplementation with or without other micronutrients in China
and the U.S. reported non-significant 6% to 70% decreased risk of
esophageal cancer with supplementation, although their 95% CI
were large, likely due to their small sample sizes [1,2,5]. Two small
case-control studies and a case-cohort of the trial in China, which
reported a significant 44% lower risk of esophageal cancer with
low selenium concentration (.82
mg/L vs. ,60 mg/L), also
support a protective effect of serum selenium on esophageal
cancer [4,6,7]. In addition, a recent case-cohort study in The
Netherlands reported a significant inverse association between
baseline toenail selenium and the risk of EA among women and
never smokers, but not among all participants [29]. The
discrepancy between our results and findings from these trials
and observational studies may be explained in part by the
difference in selenium status and the common subtype of
esophageal cancer in each study population. Selenium concentra-
tions were lower in those populations [1,2,4,5] than those in our
cohort; the mean serum selenium concentration ranged from 72 to
116
mg/L in the trials [4,5] and observational studies [6,7] and the
median toenail selenium concentration in the case-cohort study in
The Netherlands was 0.55
mg/g [29] and was substantially lower
than in U.S. populations (e.g., 0.84 [30] and 1.52
mg/g [31]). By
contrast, our population was selenium-replete (mean = 135
mg/L
and range = 67 to 213
mg/L) and included only five participants
with serum concentrations below 90
mg/L, the proposed threshold
of the antioxidant activity for GPX [32,33]. Accordingly, in our
cohort, selenium concentrations may be in a range where higher
concentrations have no further benefit. In addition, the difference
in the subtype of esophageal cancer needs to be addressed since
risk estimates were not reported separately by subtype in trials in
China [1,3,4], which most likely would have included predomi-
nantly squamous cell carcinoma given the high prevalence of this
subtype in the area. By contrast, the outcome in our study was EA.
Risk profiles for these two subtypes differ substantially [34], which
suggests different etiologies between the two cancer subtypes and
may also have contributed to the discrepant finding between our
study and trials in China.
Few studies have investigated associations of selenoenzyme
activity or selenoprotein concentrations with cancer [35–39] or BE
risk [40]. Lower GPX1 activity was observed in prostate cancer
cases than controls [36], while GPX1 activity was not associated
with the risk of colorectal [35] or breast cancer [37]. In two
previous studies that compared human esophageal tissue samples,
higher GPX2 expression and lower GPX3 expression were
observed in BE patients than that in healthy controls [40] and
the expression of GPX3 was lost in EA patients [39].
To our knowledge, our study is the first to report a positive
association of SEPP1 concentrations with the risk of progression
from BE to EA and aneuploidy. Aside from being a carrier of
selenocysteines, SEPP1 itself has antioxidant properties [41,42]
and we hypothesized that SEPP1 would be associated with a lower
risk of neoplastic progression to EA, which is in contrast to our
finding. Nonetheless, SEPP1 was positively associated with C-
reactive protein concentrations [43] and as a peroxynitrite
scavenger, could be induced by elevated peroxinitrite (ONOO
2
)
[44], especially at the relatively high selenium status of our cohort.
Peroxynitrite and its precursors, superoxide (O
2
2
N
) and nitric
oxide (NO) [45,46], have been hypothesized to induce the
progression to BE and EA [45,47,48] and it is possible that
SEPP1 may act as a marker of elevated peroxynitrite production in
aneuploidy and EA. However, there was no cross-sectional
association between SEPP1 concentrations and aneuploidy in
our study. The fact that such an association would have been
expected to be stronger than a prospective association does not
support the hypothesis that SEPP1 is a peroxynitrite scavenger in
Table 2. Association between Serum Selenium and the Risk of Neoplastic Progression to Esophageal Adenocarcinoma.
Esophageal
adenocarcinoma Aneuploidy Tetraploidy
Number of cases/all 47/361 41/324 51/325
cases HR (95% CI)* cases HR (95% CI)* cases HR (95% CI)*
Serum selenium
concentration
Per 50
mg/L 47 1.16 (0.60–2.28) 41 1.64 (0.79–3.42) 51 1.06 (0.54–2.06)
Tertile 1 (,126.3
mg/L) 12 reference 11 reference 14 reference
Tertile 2 (126.3–143.8
mg/L) 19 1.67 (0.79–3.53) 14 1.17 (0.51–2.66) 20 1.39 (0.37–2.88)
Tertile 3 (.143.8
mg/L) 16 1.40 (0.65–3.02) 16 1.60 (0.72–3.55) 17 1.60 (0.52–2.29)
P for trend ** 0.39 0.25 0.85
,118
mg/L 9 reference 5 reference 8 reference
$118
mg/L 38 1.23 (0.58–2.60) 36 2.15 (0.83–5.58) 43 1.38 (0.63–3.00)
*HRs were adjusted for age at time of blood draw (5 categories), waist: hip ratio (quartiles) at baseline, sex, smoking status and NSAID use (both for never, former or
current).
**P for trend wa s obtained by assigning median values of each tertile.
doi:10.1371/journal.pone.0038612.t002
Selenium, Selenoenzymes and Neoplastic Progression
PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e38612
neoplastic progression of BE to EA. Hence, we cannot rule out the
possibility of a chance finding.
Only a single previous study has investigated the association
between two potentially functional candidate variants in the GPX2
gene (rs4902346 and rs2737844, also known as gastrointestinal
GPX) and the risk of EA in a case-control study; however, no
association was found [8]. Consistent with that finding, our study
did not find an association of GPX2 with the risk of neoplastic
progression to EA, nor did we find such an association with GPX1,
GPX 4 and SEPP1 genes. Although two GPX3 variants were
individually significantly associated with the risk of EA, the overall
variation was not significant. Hence, this finding may be due to
chance. Our study was limited in sample size; in our post-hoc
power calculation while adjusting for alpha level to 0.0114 to
account for multiple comparisons of SNPs per gene, the powers of
detecting the risk estimate of 1.20 to 1.50 ranged from 1.4% to
3.2% for the observed minor allele frequency of 5% and from
2.4% to 11.9% for the observed minor allele frequency of 49%,
respectively for all three measures of the risk of neoplastic
progression. Hence, our study was almost certainly underpowered
to detect the type of weak associations found in genome-wide scans
for other cancers.
Strengths of our study include the prospective design, the long
follow-up (on average 7.3 years) and the high frequency of follow-
up visits (on average 5.7 visits) for biospecimen collection. We were
also able to evaluate flow cytometric abnormalities (i.e., aneuploi-
dy and tetraploidy) that reflect neoplastic progression, which
extended our ability to measure progression. Detailed exposure
Table 3. Association of Selenoenzyme Activity or Concentration and Oxidative Stress with the Risk of Neoplastic Progression to
Esophageal Adenocarcinoma.
Esophageal
adenocarcinoma Aneuploidy Tetraploidy
Number of cases/all 27/171 16/140 17/137
cases HR (95% CI)* cases HR (95% CI)* cases HR (95% CI)*
GPX1 activity
Per U/T g protein 27 1.26 (0.50–3.17) 16 1.80 (0.47–6.80) 17 0.69 (0.20–2.37)
Tertile 1 (,35.2 U/T g protein) 7 reference 5 reference 5 reference
Tertile 2 (35.2–44.5 U/T g protein) 9 1.09 (0.38–3.11) 3 0.44 (0.09–2.20) 3 0.33 (0.07–1.55)
Tertile 3 (.44.5 U/T g protein) 11 1.48 (0.56–3.94) 8 1.68 (0.50–5.74) 9 0.98 (0.31–3.14)
P for trend** 0.43 0.34 0.82
GPX3 activity
Per 10 U/L 27 0.88 (0.61–1.28) 16 0.83 (0.50–1.39) 17 0.70 (0.42–1.18)
Tertile 1 (,674 U/L) 8 reference 4 reference 7 reference
Tertile 2 (674–787 U/L) 11 1.86 (0.62–5.57) 8 1.89 (0.48–7.44) 6 1.17 (0.32–4.24)
Tertile 3 (.787 U/L) 8 1.52 (0.52–4.47) 4 1.37 (0.29–6.40) 4 0.85 (0.22–3.34)
P for trend** 0.48 0.70 0.83
SEPP1 concentra tion
Per
mg/L 27 1.46 (1.05–2.05) 16 1.31 (0.84–2.02) 17 0.85 (0.51–1.42)
Tertile 1 (,5.4
mg/L) 7 reference 2 reference 10 reference
Tertile 2 (5.4–6.1
mg/L) 6 1.89 (0.58–6.13) 5 4.08 (0.70–23.69) 2 0.22 (0.04–1.12)
Tertile 3 (.6.1
mg/L) 14 3.95 (1.42–10.97) 9 6.53 (1.31–32.58) 5 0.67 (0.21–2.19)
P for trend**
0.006 0.02
0.48
MDA
Per 0.1 mmol/L 27 1.10 (0.97–1.24) 16 1.13 (0.98–1.29) 17 1.04 (0.87–1.24)
Tertile 1 (,0.751
mmol/L) 5 reference 4 reference 5 reference
Tertile 2 (0.751–0.971
mmol/L) 13 2.80 (0.95–8.21) 7 1.29 (0.33–4.97) 8 2.05 (0.62–6.80)
Tertile 3 (.0.971
mmol/L) 9 2.04 (0.67–6.27) 5 1.14 (0.28–4.62) 4 0.71 (0.18–2.81)
P for trend** 0.33 0.91 0.54
PCC
Per 0.1 nmol/mg protein 27 1.21 (0.60–2.42) 16 0.64 (0.25–1.65) 17 1.19 (0.46–3.08)
Tertile 1 (,0.333 nmol/mg protein) 8 reference 8 reference 7 reference
Tertile 2 (0.333–0.384 nmol/mg protein) 8 1.11 (0.40–3.10) 4 0.43 (0.12–1.56) 5 0.75 (0.22–2.55)
Tertile 3 (.0.384 nmol/mg protein) 11 1.38 (0.55–3.49) 4 0.45 (0.13–1.56) 5 0.62 (0.19–2.08)
P for trend** 0.49 0.18 0.44
*HRs were adjusted for age at time of blood draw (5 categories), waist: hip ratio (quartiles), sex, smoking status, NSAID use (both for never, former or current) and serum
selenium concentrations (continuous).
**P for trend wa s obtained by assigning median values of each tertile.
doi:10.1371/journal.pone.0038612.t003
Selenium, Selenoenzymes and Neoplastic Progression
PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e38612
assessments allowed us to adjust for important potential con-
founding in our analysis. Finally, we extended the evaluation of
common variants in selenoenzyme/selenoprotein genes by includ-
ing more genes than in a previous study of EA [8].
One very important limitation of our study is the relatively small
number of endpoints in our cohort. To some extent, this is
mitigated by the involvement of high-risk participants and the use
of valuable intermediate markers of neoplastic progression. Our
ability to detect an association between selenium and the risk of
neoplastic progression to EA also may have been limited by the
relatively high selenium concentrations in our cohort. We used a
single serum selenium measurement, which may not capture
participants’ selenium intake during the entire follow-up period.
Finally, serum selenium concentrations may not reflect tissue
concentrations, which may be the exposure of most importance.
In summary, we found no evidence of association of selenium
concentrations with the risk of neoplastic progression to EA. This
finding is inconsistent with our previous cross-sectional analysis
and suggests that findings from cross-sectional studies of selenium
and neoplastic progression need to be interpreted with caution.
Our study is the first to observe positive associations of plasma
SEPP1 concentrations with the risk of neoplastic progression to
EA, a finding that warrants further investigation.
Acknowledgments
We thank the study participants and research staff of the Seattle Barrett’s
Esophagus Research Study. We also thank Drs. Raymond Burk and
Kristina Hill at the Vanderbilt University School of Medicine for providing
the SEPP1 data.
Author Contributions
Conceived and designed the experiments: BJR TLV UP. Performed the
experiments: RMS IBK DJD PLB BJR TLV UP. Analyzed the data: YT.
Contributed reagents/materials/analysis tools: RMS IBK DJD PLB BJR
TLV. Wrote the paper: YT ARK JWL MPR PLB TLV UP. Directed the
overall study operation: BJR TLV UP.
Table 4. Association between SNPs in Selenoenzymes and Risk of Neoplastic Progression to Esophageal Adenocarcinoma*.
Esophageal
adenocarcinoma Aneuploidy Tetraploidy
Gene SNP HR (95% CI) P trend HR (95% CI) P trend HR (95% CI) P trend
GPX1 rs3448 0.79 (0.39–1.60) 0.51 1.95 (0.71–5.35) 0.20 0.98 (0.37–2.58) 0.96
rs1987628 0.56 (0.30–1.19) 0.14 0.62 (0.24–1.59) 0.32 1.61 (0.72–3.58) 0.25
Global P 0.12 0.37 0.48
GPX2 rs4902347 1.45 (0.66–3.21) 0.36 1.21 (0.39–3.75) 0.74 1.48 (0.47–4.70) 0.50
rs4902346 1.37 (0.71–2.63) 0.35 1.18 (0.46–3.03) 0.73 1.72 (0.74–3.98) 0.21
rs2071566 1.05 (0.58–1.92) 0.87 0.89 (0.38–2.07) 0.79 1.44 (0.74–2.79) 0.23
rs10121 0.82 (0.27–2.50) 0.73 0.91 (0.21–3.93) 0.89 1.62 (0.51–5.15) 0.41
Global P 0.17 0.26 0.60
GPX3 rs3763013 1.34 (0.76–2.34) 0.31 0.95 (0.41–2.19) 0.90 1.25 (0.61–2.55) 0.54
rs3805435 1.14 (0.34–3.85) 0.84 1.24 (0.29–5.30) 0.77 0.35 (0.04–3.01) 0.34
rs8177406 0.77 (0.34–1.75) 0.54 1.09 (0.40–2.98) 0.86 1.06 (0.44–2.55) 0.90
rs4958872 2.08 (1.07–4.05) 0.03 1.00 (0.39–2.56) 0.99 0.60 (0.21–1.71) 0.34
rs736775 1.47 (0.76–2.75) 0.22 1.00 (0.43–2.32) 0.99 0.77 (0.35–1.69) 0.52
rs3792797 2.22 (1.04–4.76) 0.04 1.30 (0.45–3.77) 0.62 0.69 (0.21–2.27) 0.54
Global P 0.33 0.65 0.69
GPX4 rs8178974 0.83 (0.37–1.85) 0.65 0.46 (0.14–1.51) 0.20 0.90 (0.29–2.76) 0.85
rs8178977 1.71 (0.91–3.21) 0.10 1.71 (0.68–4.33) 0.26 0.41 (0.14–1.19) 0.10
rs713041 0.69 (0.40–1.20) 0.19 1.32 (0.61–2.86) 0.48 1.98 (0.93–4.23) 0.08
rs2074451 0.66 (0.38–1.15) 0.14 1.47 (0.64–3.40) 0.37 1.84 (0.85–3.97) 0.12
Global P 0.46 0.45 0.34
SEPP1 rs11959466 0.58 (0.15–2.24) 0.43 0.70 (0.19–2.58) 0.59 0.47 (0.06–3.67) 0.47
rs12055266 0.86 (0.46–1.61) 0.63 0.94 (0.43–2.06) 0.88 1.06 (0.50–2.28) 0.87
rs3797310 0.97 (0.53–1.77) 0.91 1.24 (0.59–2.62) 0.57 1.03 (0.48–2.21) 0.93
rs230819 1.03 (0.61–1.74) 0.93 1.12 (0.58–2.16) 0.73 0.68 (0.34–1.34) 0.26
rs13168440 1.02 (0.50–2.09) 0.95 0.75 (0.27–2.09) 0.58 0.38 (0.11–1.35) 0.14
rs3877899 1.34 (0.71–2.51) 0.37 0.75 (0.29–1.95) 0.56 0.47 (0.16–1.39) 0.17
Global P 0.65 0.14 0.46
*HR and 95% CIs were based on additive model and adjusted for age at baseline (5 categories), waist: hip ratio (quartiles), sex, smoking status, NSAID use and Caucasian
ethnicity; Global p is based on the log likelihood ratio statistics comparing the model with and without all SNPs in a given gene.
doi:10.1371/journal.pone.0038612.t004
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PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e38612
    • "By executing the first preliminary experiments on the potential mechanisms between HFRS and selenium, we tried to estimate how selenium concentrations could potentially have their effect. At first we looked at human factors in the relation between selenium and HFRS by performing a case-control study in HFRS cases and comparing the plasma concentration and activity of the major selenium binding proteins GPx and GPx-3 [36]. We thereby hypothesized that deficiency in selenium could either increase the hosts' susceptibility to hantavirus infection, leading to more infections, or increase the host response to the hantavirus infection, which would lead to a more severe disease and thus an increase of reported HFRS cases. "
    [Show abstract] [Hide abstract] ABSTRACT: Hemorrhagic fever with renal syndrome (HFRS) caused by hantaviruses and transmitted by rodents is a significant public health problem in China, and occurs more frequently in selenium-deficient regions. To study the role of selenium concentration in HFRS incidence we used a multidisciplinary approach combining ecological analysis with preliminary experimental data. The incidence of HFRS in humans was about six times higher in severe selenium-deficient and double in moderate deficient areas compared to non-deficient areas. This association became statistically stronger after correction for other significant environment-related factors (low elevation, few grasslands, or an abundance of forests) and was independent of geographical scale by separate analyses for different climate regions. A case-control study of HFRS patients admitted to the hospital revealed increased activity and plasma levels of selenium binding proteins while selenium supplementation in vitro decreased viral replication in an endothelial cell model after infection with a low multiplicity of infection (MOI). Viral replication with a higher MOI was not affected by selenium supplementation. Our findings indicate that selenium deficiency may contribute to an increased prevalence of hantavirus infections in both humans and rodents. Future studies are needed to further examine the exact mechanism behind this observation before selenium supplementation in deficient areas could be implemented for HFRS prevention.
    Full-text · Article · Jan 2015
    • "By overexpressing MMPs and uPA, GPX1 can promote cell invasion and metastasis [30][31][32][33]. Abnormal expression of GPX1 in Barrett's esophagus cases have been reported [27], but the mechanism and significance of this remains unclear. In ESCC, especially in the invasion, migration, cisplatin resistance and proliferation aspects, GPX1 is understudied target. "
    [Show abstract] [Hide abstract] ABSTRACT: Esophageal cancer is one of the most common cancers worldwide. Despite recent progress in the development of novel therapies, esophageal carcinoma remains an aggressive cancer associated with a poor prognosis. The glutathione peroxidase 1 (GPX1) gene located on chromosome 3p21.3 is associated with the cancer of several organs. According to available information, GPX1, a gene downstream of NF-κB, is considered to exert adverse effects on tumour progression and enhance malignancy in some cancers but has not been reported in esophageal cancer. It is also reported that vitamin D (Vit. D), a widely used drug in the clinical setting, could suppress GPX1 expression through the NF-κB pathway. Thus, it is speculated that Vit. D could reduce malignancy in esophageal cancer by altering the NF-κB pathway. In this study, we confirmed our speculation by finding that Vit. D, through the inhibition of GPX1, decreased the migratory, invasive and proliferative capabilities, as well as cisplatin resistance, in esophageal cancer cells. Furthermore, when invasion and migration were reduced in the GPX1-inhibited cells, the expression of urokinase type plasminogen activator (uPA) and matrix metalloproteinase-2 (MMP2) was also suppressed correspondingly. Therefore, we believe that, in esophageal cancer cells, the expression of GPX1 can promote invasion, migration, proliferation and cisplatin resistance, and Vit. D can reduce the associated malignancy through the NF-κB pathway. The Vit. D- and NF-κB-mediated decrease in GPX1 expression resulted in a decrease in MMP2- and uPA-mediated invasion and migration.
    Full-text · Article · Sep 2014
    • "If the impact of genetic variation is largest in subjects with low selenium concentrations, we expect that our power to identify selenium-related genetic variants would be improved if we had included more subjects with low serum selenium concentrations. About 94% of the WHI participants were cancer cases and their selenium levels may not represent those from women without cancers, although we did not observed difference between cancer cases and matched controls [38,56]. Also, there was no difference in circulating selenium concentrations (nature log scale) between individuals with and without cancer (p value of t test = 0.16) in our study. "
    [Show abstract] [Hide abstract] ABSTRACT: Selenium is an essential trace element and circulating selenium concentrations have been associated with a wide range of diseases. Candidate gene studies suggest that circulating selenium concentrations may be impacted by genetic variation; however, no study has comprehensively investigated this hypothesis. Therefore, we conducted a two-stage genome-wide association study to identify genetic variants associated with serum selenium concentrations in 1203 European descents from two cohorts: the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening and the Women's Health Initiative (WHI). We tested association between 2,474,333 single nucleotide polymorphisms (SNPs) and serum selenium concentrations using linear regression models. In the first stage (PLCO) 41 SNPs clustered in 15 regions had p < 1 × 10-5. None of these 41 SNPs reached the significant threshold (p = 0.05/15 regions = 0.003) in the second stage (WHI). Three SNPs had p < 0.05 in the second stage (rs1395479 and rs1506807 in 4q34.3/AGA-NEIL3; and rs891684 in 17q24.3/SLC39A11) and had p between 2.62 × 10-7 and 4.04 × 10-7 in the combined analysis (PLCO + WHI). Additional studies are needed to replicate these findings. Identification of genetic variation that impacts selenium concentrations may contribute to a better understanding of which genes regulate circulating selenium concentrations.
    Full-text · Article · May 2013
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