Selenium, Lycopene, α-Tocopherol, β-Carotene, Retinol, and
Subsequent Bladder Cancer
Kathy J. Helzlsouer, George W. Comstock and J. Steven Morris
Cancer Res 1989;49:6144-6148.
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[CANCER RESEARCH 49. 6144-6148. November 1, 1989]
Selenium, Lycopene, a-Tocopherol, ß-Carotene,Retinol, and Subsequent
Bladder Cancer1
Kathy J. Helzlsouer, George W. Comstock,2 and J. Steven Morris
Department of Epidemiology, School of Hygiene and Public Health, Johns Hopkins University. Baltimore, Maryland 21205 ¡K.J. H., G. W. C.¡,and the Research
Reactor Facility. L'niversity of Missouri, Columbia, Missouri 65211 ¡J.S. M.J
ABSTRACT
To examine the association between serum nutrients and the develop
ment of bladder cancer we measured selenium, a-tocopherol, lycopene,
/3-carotene, retino!, and rctinol-binding protein in serum collected from
25,802 persons in Washington County, Ml), in 1974. Serum samples
were kept frozen at —¿70°C.
In the subsequent 12-year period, 35 cases
of bladder cancer developed among participants. Comparisons of serum
levels in 1974 among cases and two matched controls for each case
showed that selenium was significantly lower among cases than controls
(P = 0.03), lycopene was lower among cases at a borderline level of
significance (P = 0.07), and a-tocopherol was nonsignificantly lower (P
= 0.13). For selenium there was a nearly linear increase in risk with
decreasing serum levels (/' = 0.03). When examined by fertiles, the odds
ratio associated with the lowest fertile of selenium compared to the
highest fertile was 2.06. Serum levels of retino!, retinol-binding protein,
and /9-carotene were similar among cases and controls. These results
support a role for selenium in the prevention of bladder cancer.
INTRODUCTION
Bladder cancer is the most common urological cancer and is
the sixth most common cancer in the United States. An esti
mated 46,400 new cases will develop and 10,400 deaths will
occur in Americans in 1988 (1). Approximately one-third of
these tumors are found to be invasive at the time of diagnosis.
The other two-thirds are initially localized and noninvasive but
are responsible for considerable discomfort, disability, and ex
pense, especially since they have rates of recurrence reported to
be as high as 80%, and many become invasive (2, 3).
A number of environmental risk factors for bladder cancer
have been identified. These include cigarette smoking, radia
tion, schistosoma! infections, and certain aromatic amines and
amides (4, 5). The roles of saccharin (6, 7) and coffee (8, 9) in
human bladder cancer are uncertain at best. Foods are known
to contain possible cancer preventive factors (10). Most studies
of the preventive factors for bladder cancer have focused on
retino! (vitamin A) and related compounds such as /3-carotene
(a provitamin A compound) and synthetic retinoids (11-23). aTocopherol (vitamin E) and selenium are nutrients that act as
antioxidants and have been found to have protective associa
tions with other cancers (24-28).
The present study was designed to assess the associations of
prediagnostic serum levels of retino!, carotenoids, «-tocopherol,
and selenium with the development of bladder cancer.
basic demographic information, smoking history, including number of
cigarettes smoked per day for current smokers in 1974, and information
on all medications, including vitamin supplements, taken during the 48
h prior to blood sampling. A private census that included an estimated
90% of the county residents was conducted in the summer of 1975 to
ascertain the extent of participation in the blood collection drive.
Serum samples from 25,802 participants were collected and frozen.
Of these participants, 20,305 were identified as Washington County
residents through linkage to the private census and were included in
the cohort. The participation rate for eligible county residents was 30%.
Table 1 displays the degree of participation by age, sex, smoking status,
and education. Participation was better among women, nonsmokers,
and those with higher levels of education. While the age range of
participants was 11-98 years, the highest participation rate was in the
age group 55-64 years with few participants at the extremes of the age
range.
Incident bladder cancer cases were identified through discharge rec
ords from Washington County Hospital, the only general hospital in
the county, and from death certificates of county residents. Thirty-five
newly diagnosed cases of bladder cancer who had no other history of
cancer were identified during 1975-1986. Washington County is a
stable community with outmigration rates estimated at 1%/year. Com
pleteness of ascertainment was assessed by comparison of the observed
number of cases to the expected number derived from application of
sex- and age-specific bladder cancer rates for Connecticut State 19781981 to the cohort. The observed to expected ratio was 0.87. Histológ
ica! confirmation of bladder cancer was obtained from pathology re
ports. Eighty-six % were transitional cell carcinomas.
Two controls were selected for each case from a birth date listing of
the cohort. To be eligible, controls had to be alive and free of diagnosed
cancer on the date of diagnosis of the case. Cases and controls were
matched on sex, race, and within 2 h for the interval between blood
collection and the previous meal. Controls were the two persons who
met these eligibility and matching criteria and were nearest in age to
the matched case.
Laboratory Assays. Serum samples were stored in two Nunc tubes at
—¿70°C
until thawed for this study and were minimally exposed to
yellow incandescent light during thawing. Sera from each case and its
matched controls were assayed as sets on the same day. All assays were
done in duplicate and laboratory personnel were masked with regard
to the case-control status.
Serum retino! and «-tocopherol levels were measured by high-per
formance liquid chromatography using a slightly modified version of
the method as described by Driskell et al. (29). Serum carotenoids were
also measured by high-performance liquid chromatography using a
modification of the method by Katrangi et al. (30). Serum selenium
levels were determined by neutron activation analysis (31). Retinolbinding protein was assayed by radial immunodiffusion. The coeffi
cients of variation for assays were 3.4% for «-tocopherol, 3.7% for
MATERIALS AND METHODS
retinol, 6.9% for selenium, 4.5% for /3-carotene, and 7.4% for lycopene.
The coefficients of variation for concurrently analyzed duplicate assays
In the fall of 1974, a project was conducted in Washington County,
were less than 5% in all assays.
MD, to collect blood for a serum bank for cancer research. Brief
Statistical Analysis. Serum nutrients were treated as both continuous
questionnaires were obtained from the participants, and their blood
and categorical exposure variables. Because the distributions for the
pressures were taken as a service to them. The questionnaires included
carotenoids and «-tocopherolwere skewed to the right, the natural logtransformed values for these nutrients were used for statistical tests. A
Received 3/31/89; revised 7/21/89; accepted 8/4/89.
The costs of publication of this article were defrayed in part by the payment
paired t test, accounting for the matched design, was used to assess the
of page charges. This article must therefore be hereby marked advertisement in
mean difference in serum nutrients between cases and controls. Con
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ditional logistic regression analyses were used to assess and adjust for
' Supported by Grant CA36390 from the National Cancer Institute.
3To whom requests for reprints should be addressed, at Training Center for
potential confounders of the association between serum nutrients and
Public Health Research. Box 2067. Hagerstown. MD 21742-2067.
the risk of bladder cancer. Analyses were performed using the SAS
6144
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SEROLOGICAL
PRECURSORS
Table 1 Participation rates for Washington County residents, by age, sex,
smoking status, and years of schooling as estimated by linkage to the 1975
private census
OF BLADDER CANCER
Table 2 Comparison afeases and controls according to matching and other
potentially confounding characteristics in 1974
Controls
Cases
</V=35)
(N = 70)
Characteristic
who
census
donated
blood19.729.934.837.235.926.413.725.833.531.234.226.122.926.832.336.129.9
population10.28212,27410.03910,8839,1706,0813,49329,25432,86126,42512,97222,23512,69511.75723,97012,95562,222"%
CharacteristicsAge18-2425-3435-4445-5455-6465-7575+SexMaleFemaleSmoking
Mean ±SD
Age (yr)
Yr of schooling
Hours from last meal
58.9 ±7.8
11.4 ±3.6
3.5 ±2.4
58.9 ±7.8
10.5 ±3.2
3.3 ±2.4
89
81
23
43
39
29
23
11
19
14
20
16
26
80
11
66
% of total
statusNever
smokedExsmokersCurrent
smokersYr
schooling0-89-111213+Total1975
of
Married
Smoking history in 1974
Cigarettes
Never smoked
Formerly smoked
Current smoker
1-20 cigarettes/day
20+ cigarettes/day
Cigars
Ever smoked
Pipes
Ever smoked
Ever smoked cigarettes,
pipes or cigars
Month
participationAugust
of study
SeptemberOctoberNovemberVitamin
and
" Not all of above categories add to this total because of missing data on some
records.
supplementsOther
statistical package, including PROC MCSTRAT and PROC PHGLM
procedures (32). Examining the serum nutrients as continuous vari
ables, the best-fitting model in predicting the outcome of bladder cancer
was determined and models were compared using likelihood ratio tests.
The categories of serum nutrients were based on the distributions of
serum levels in the controls, trichotomizing the distributions of sele
nium, a-tocopherol, /3-carotene, and lycopene by tenues. Serum retinol
and retinol-binding protein distributions were dichotomized by their
medians due to low numbers of cases in the middle tertile resulting in
unstable estimates. Odds ratios, representing the risk of bladder cancer
according to levels of serum nutrients, were calculated from the regres
sion coefficients, with the highest category of nutrient level as the
reference value. Monotonie trends for the risk estimates were evaluated
by the regression coefficients when the nutrients were examined in the
model as continuous variables. Interactions were assessed by multivariate conditional logistic regression. Ninety-five % confidence intervals
were used for significance testing. All P values are based on two-tailed
tests.
medications40402011693640242070
Table 3 Serum levels of nutrients in cases and matched controls
35)55.2=
±17.2*
(20.0-90.0)c5.3
70)56.4
=
(mg/dl)/3-Caroteneprotein
±0.9
(2.8-7.3)36.2
±0.9
(2.8-8.2)32.7
(jig/dl)Lycopene
±39.5
(1.0-194.0)41.8
±22.3
(5.0-138.0)44.8
(/ig/dl)a-Tocopherol
±28.8
(6.0-100.0)1.
±21.2
(10.0-100.0)1.11
(mg/dl)Selenium
00 ±0.34
(0.48-1.87)11.1
±0.34
(0.53-2.57)tl.7±
NutrientRetinol
(mg/dl)Retinol-binding
(¿ig/dl)Cases(N
RESULTS
±17.7
(23.0-103.0)5.4
±1.1
1.3
(8.1-13.5)Controls(N
(9.3-16.4)f0.550.640.250.070.130.03
" Based on matched t test.
As a result of matching, cases and controls had the same sex* Mean ±SD.
c Numbers in parentheses, range.
race composition (all cases were white), the same mean age,
and almost the same mean hours between blood collection and
the previous meal (Table 2). Cases were more likely to have between cases and controls, taking the matching into account,
showed lower levels in cases than controls for all serum nu
been married in 1974 and were slightly better educated than
trients, including /3-carotene. The mean case-control difference
controls. They were also somewhat more likely to have smoked
was statistically significant for serum selenium (P = 0.03) and
tobacco in some form in 1974. The distributions of months
when blood was collected were nearly the same for the two of borderline significance for serum lycopene (P = 0.07).
Current smokers had lower levels of /3-carotene and retinol;
groups, so that seasonal differences in the availability of fresh
local produce should not affect the comparisons. Seventy % of levels decreased with increasing number of cigarettes smoked.
the study population had taken some medication within the 48 Smoking status was not associated with selenium or lycopene
h prior to blood collection, although cases were less likely to levels in the control population. The history of ingestion of
have used vitamin supplements than controls. None of the vitamin supplements in the 48-h period prior to blood sampling
observed differences were statistically significant at the 95% was significantly and positively associated with a-tocopherol
levels (P = 0.02) but not with other nutrients. All odds ratios
level of confidence.
Overall, mean serum nutrient levels were lower in those who for the risk of bladder cancer are presented after adjustment for
went on to develop bladder cancer compared to controls, with a history of cigarette smoking and intake of vitamin supple
the exception of 0-carotene (Table 3). The elevated mean ß- ments. However, the crude, unadjusted odds ratios were not
carotene level in cases compared to controls was primarily due significantly altered by adjustment for these factors. Adjustment
to one outlier value among the cases. The mean differences
for a history of cigarette smoking at the time of blood collection
6145
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SEROLOGICAL PRECURSORS OF BLADDER CANCER
was examined by categories of current, former, and never smok
ers. Collapsing into categories of ever and never smokers did
not alter the observed associations. Cases and controls were
also compared with respect to a number of other factors: marital
status; years of education; number of medications taken; and
hormone use (in females). Adjustments for the effects of these
factors did not alter the associations between the serum nu
trients and the risk of bladder cancer.
The relative odds of developing bladder cancer associated
with serum levels of the six nutrients can be seen in Table 4.
There was no demonstrable association with retinol, retinolbinding protein, or 0-carotene. The risk of bladder cancer
increased with decreasing levels of lycopene, «-tocopherol, and
selenium. The trend for a-tocopherol was monotonically in
creasing but not statistically significant. That for lycopene was
almost significant at the 95% level of confidence but did not
appear to be linear. The odds ratio associated with lycopene
levels in the lowest tertile compared to the highest tertile was
2.02 with 95% confidence limits of 0.63 and 6.54. For selenium,
there was a significant increase in risk with decreasing serum
levels. The odds ratio associated with the lowest tertile of
selenium compared to the highest tertile was 2.06 with 95%
confidence intervals of 0.67 and 6.35.
Similar analyses of the associations of lycopene and selenium
were done treating nutrient levels as continuous variables. The
odds ratio for a 1-log unit decrease in Mg/dl of lycopene was
2.18 (P = 0.06); for a 1-Mg/dl decrease in selenium, it was 1.56
(P = 0.03).
Lycopene levels were significantly and positively correlated
with serum ß-carotenelevels (r = 0.58) and weakly but signifi
cantly correlated with retinol levels (r —¿
0.27). Selenium levels
were not correlated with cv-tocopherol levels (r = -0.11). Ad
justments for other serum nutrients did not alter the main
effects of either lycopene or selenium on the risk of developing
bladder cancer. No significant interactions were detected in this
relative small group of cases and controls.
Stratification by sex did not alter the direction of the observed
associations for /3-carotene, lycopene, or selenium. Lower reti
nol levels appeared to be protective among men and lower levels
of a-tocopherol were associated with an increased risk, whereas
the reverse was found among women. The numbers in each
stratum are small, and these findings are only suggestive at
best.
The observed associations between bladder cancer and serum
levels of lycopene and selenium were similar for transitional
cell cancers and nontransitional cell cancers.
Because of the possibility that cancer might have been present
at the time of blood collection in some cases and that some of
the observed effects could therefore be related to the presence
of cancer, the analyses were repeated omitting the five cases
whose cancers were recognized within 2 years of the time of
blood collection. The observed associations were not changed
to any meaningful extent. Case-control sets were also catego
rized into "early" and "late" according to the length of time
between date of blood collection and date of diagnosis of the
case. Results for "late" case-control sets, those occurring after
6 years from the time of blood collection, were not significantly
different from "early" cases. If anything the observed associa
tions were strengthened. The odds ratios for the middle and
high tertiles compared to the lowest tertile of selenium were
1.77 and 7.62, respectively. The odds ratios for the middle and
high tertiles compared to the lowest for lycopene were 1.33 and
2.62, respectively.
DISCUSSION
In this prospective study of bladder cases and matched con
trols, the most convincing association was with serum selenium
levels. Only one other study has reported specifically on this
association. Nomura et al. (28) studied 29 bladder cases among
men of Japanese ancestry in a nested case-control design similar
Table 4 Adjusted" relative odds of developing bladder cancer associated with
to
ours. The odds ratio for the subsequent development of
specified levels of serum nutrients
bladder cancer was 3.1 in the lowest quintile of serum selenium
compared to the highest quintile (P = 0.10). However, the test
half*1.019341.01632Middle
half0.77(0.18-3.16360.96
NutrientRetinolCasesControlsRetinol-binding
for trend was not significant. Another study among Finnish
men and women reported on 27 newly diagnosed cancers of the
urogenital tract but did not specify how many were bladder
cancer cases (27). They failed to demonstrate a statistically
(0.41-2.1938Lower
proteinCasesControlsfJ-CaroteneCasesControlsLycopeneCasesControlstt-TocopherolCasesControlsSeleniumCasesControlsUpper
significant difference between cases and controls. Of interest in
this connection is the fact that serum selenium levels in this
Finnish study were approximately one-half of those found in
the United States.
third''0.58(0.18-1.90)730.98
third1.60(0.50-5.19)17232.02
third1.011241.010231.09241.0724Upper
Additional support for a possible role for selenium in pro
tecting against bladder cancer comes from a study of glutathione-related enzymes in the transitional epithelium of the uri
nary bladder in rabbits (33). The authors postulated that the
(0.30-3.22)8231.63(0.51-5.27)13231.94(0.57-6.61)1423LevelsLower
(0.63-6.54)17241.75(0.37-8.32)13232.06
vulnerability of bladder transitional epithelium to chemical
carcinogens might be a result of the low levels of these seleniumdependent enzymes in bladder tissue. Selenium is believed to
operate as a biological antioxidant primarily as a component
of glutathione peroxidase (34).
Lycopene is the carotenoid responsible for the red pigment
(0.67-6.35)142328)c25)Ptrend0.350.060.180.03
in fruits and vegetables such as tomatoes. Its role in health and
disease has received virtually no attention since it does not
appear to be essential for nutrition. Other carotenoids, such as
" Adjusted for effects of cigarette smoking and vitamin supplements.
canthoxanthin, which have no provitamin A activity have anti* Upper half: retinol 254 mg/dl: retinol-binding protein ^5.6 mg/dl.
c Numbers in parentheses, 95% confidence intervals.
tumorigenic effects attributable to the antioxidant properties of
''Middle third, /¿-carotene.21-38 mg/dl, inclusive; lycopene, 35-51 mg/dl,
carotenoids (35). Support for a protective effect of lycopene
inclusive; a-tocopherol, 0.85-1.24 mg/dl. inclusive: selenium. 11.0-11.9 „¿i:
ill.
against some cancers comes from the fact that low levels of
inclusive.
6146
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SEROLOGICAL PRECURSORS OF BLADDER CANCER
lycopene have been found to be associated with the subsequent
development of cancer of the pancreas (36) and cancer of the
rectum.3 Lycopene, however, was not associated with subse
quent prostate cancer in this population.4
The majority of studies of the relationship of bladder cancer
to nutrients have focused on the retinoids, which include vita
min A and its chemical analogues. These nutrients have been
of particular interest in bladder cancer because of their role in
normal growth and differentiation of epithelial tissues. A pro
spective study of the relationship between cancer and dietary
intake of vitamin A and 0-carotene in retirement community
residents reported lower incidence rates of bladder cancer for
the higher two tenues of dietary ß-caroteneintake and total
vitamin A intake compared to the lowest tertile but only in
women (17). A few case-control studies of the association
between serum retino! and carotenoid levels and bladder cancer
have been reported. A study examining both dietary intake and
serum levels of retinol, retinol-binding protein, and the carotenoids among transitional cell carcinoma patients and controls
showed no difference in serum levels or dietary intakes of
vitamin A (18). Two other case-control studies have examined
the association between serum retinol and carotene levels and
bladder cancer but have limited relevance to our patient popu
lation due to the presence of parasitic infections in the cases as
well as a predominance of squamous cell type. One study of 70
bilharzial bladder cancer cases included 20 cases with transi
tional cell type. Serum retinol and /3-carotene levels were lower
in cases than controls but the difference was not statistically
significant (19). The other study reported on 10 bladder cancer
cases associated with schistosomiasis; only one case was tran
sitional cell type. Mean serum retinol levels and total carotene
levels were significantly lower compared to 10 normal controls
(20).
Three intervention trials of the effect of a vitamin A analogue,
etretinate, on the prevention of recurrent superficial bladder
tumors have been reported (21-23). All trials had significant
problems with side effects from etretinate, primarily mucous
membrane dryness, requiring dose reductions and resulting in
significant dropout rates. Two of the three studies reported a
reduction in the incidence of recurrent tumors in treated pa
tients (21, 22). These trials should be viewed as preliminary
because of the small numbers of patients enrolled, short followup, and difficulties with the side effects of the preventive agent.
Serum a-tocopherol levels were lower in cases than controls
but the dose-response trend was not statistically significant.
Despite its biological synergism with selenium, no evidence of
an interaction was demonstrated in this study.
One other study has reported an association between serum
vitamin E and the risk of cancer among Finnish men. Twentysix cases of cancer of the urinary organs, which include an
unspecified number of bladder cancer cases, occurred in the
cohort. Although there was no difference in the mean level of
vitamin E between cases and controls, the relative risk between
the two highest quintiles and the three lowest quintiles was 0.26
(95% confidence interval, 0.07-0.99). No monotonie dose re
sponse was observed (37).
Alternative explanations should be considered for our find
ings. In any cohort study the possible effect of a bias introduced
by losses to follow-up should be considered. However, the
Washington County population has a low rate of outmigration
and case ascertainment is estimated to be reasonably complete.
3T. L. Bush, personal communication.
4 A. W. Hsing, personal communication.
Therefore, we think it is unlikely that significant bias is occur
ring in this study. The observed inverse associations between
the risk of bladder cancer and serum selenium and lycopene
levels may not be a direct protective effect of these nutrients.
The relationships may be due to other protective factors in the
diet associated with the intake of selenium or carotenoids, or
other life-style factors associated with a specific diet. Neverthe
less, a few of the criteria for attributing causality to these
associations have been met. While the odds ratios for cancer
risk associated with low levels of selenium and lycopene are not
high, even low odds ratios can have great public health signifi
cance. It could be that high odds ratios would be expected only
if the study population contained sufficient numbers of persons
with serum levels that approached or reached clinical defi
ciencies. Furthermore, the effectiveness of these nutrients as
antioxidants might well be diluted by the presence of other
antioxidants, known or unknown. Against the latter argument,
however, is the fact that we observed no protective association
with /3-carotene which is also postulated to act as a biological
antioxidant (38). Both selenium and lycopene showed trends
that were consistent with dose-response effects. Biological plau
sibility for an effect of selenium is suggested by one investiga
tion of selenium-dependent glutathione enzymes and the tran
sitional bladder epithelium in rabbits (33).
Despite the small size and limited power of the present study
its findings strengthen the case for conducting controlled trials
of selenium as a possible protective agent against bladder can
cer. They also signal the desirability of seeing if our findings
with respect to lycopene can stand the test of replication.
ACKNOWLEDGMENTS
We are indebted to Marie Foard, Johns Hopkins School of Hygiene
and Public Health, for the vitamin A, «-tocopherol and carotenoid
determinations; to Gail Hehring and the stall at the Johns Hopkins
Training Center for Public Health Research for technical and clerical
assistance; to Drs. Diamond and Piantadosi for review of statistical
considerations; and to the residents and health care providers of Wash
ington County, MD.
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