[CANCER RESEARCH 44, 2803-2806,
July 1984]
Effect of an Inorganic and Organic Form of Dietary Selenium on the
Promotional Stage of Mammary Carcinogenesis in the Rat1
Henry J. Thompson,2 L. David Meeker, and Stephen Kokoska
Departments of Animal and Nutritional Sciences [H. J. T.] and Mathematics [L. D. M., S. K.], University of New Hampshire, Durham, New Hampshire 03824
ABSTRACT
The relative effectiveness of either sodium selenite or selenomethionine in the inhibition of mammary carcinogenesis was
studied in virgin female Sprague-Dawley rats. In one experiment,
rats were given 50 mg of 1-methyl-1-nitrosourea per kg of body
weight s.c. at 50 days of age. Beginning 7 days post-1 -methyl1-nitrosourea, they were assigned to a basal diet containing 0.1
ppm of selenium or basal diet supplemented to contain either 4,
5, or 6 ppm of selenium as sodium selenite or 5 or 6 ppm of
selenium as selenomethionine. Selenium treatment was contin
ued until termination of the study 135 days after 1-methyl-1nitrosourea treatment. Sodium selenite, at the 5-ppm level, was
the most effective chemopreventive agent. The highest level of
selenomethionine (6 ppm) caused grossly apparent liver damage.
No liver damage was noted in sodium selenite-treated rats. In a
second experiment, rats were given 5 mg of 7,12-dimethylbenz(a)anthracene at 50 days of age. Beginning 7 days after
7,12-dimethylbenz(a)anthracene
treatment, rats were assigned
randomly to the control group or to one of two selenium treat
ment groups receiving either 3.4 ppm of selenium as sodium
selenite or 3.4 ppm as selenomethionine in their drinking water.
Selenium supplementation was continued throughout the study
until its termination at 111 days postcarcinogen. Sodium selenite
significantly reduced cancer incidence and the average number
of cancers per rat. Treatment with selenomethionine was less
effective and caused severe liver damage. Although both sodium
selenite and selenomethionine can inhibit some aspect of the
postinitiation stage(s) of mammary carcinogenesis, selenium pro
vided as sodium selenite was the more effective and less toxic
of the two chemicals. Increasing the dose of sodium selenite
above 5 ppm did not enhance the inhibitory activity of selenium.
selenium reported to inhibit mammary carcinogenesis, no alter
ations in the circulating levels of estrogen and progesterone have
been observed, and inhibition of mammary carcinogenesis by
selenium has been reported in the presence or absence of the
ovaries (7). These data taken collectively have resulted in con
siderable interest in the potential of selenium as an anticarcinogen.
Very little work has been conducted to determine the effect(s)
of feeding organic forms of selenium on chemically induced
neoplasia. That which has been reported has been done primarily
with high-selenium yeast in which the forms in which selenium
exists are not defined completely (20). It was therefore decided
to determine the anticarcinogenic efficacy of selenomethionine
in the MNU3- and DMBA-induced mammary carcinogenesis sys
tems. This choice was made since selenomethionine has been
reported to be one of the predominant organic forms of selenium
in cereals, vegetables, and grains and, hence, is a form of
selenium that is ingested commonly (10, 31). Furthermore, it has
been shown that the selenium in selenite and selenomethionine
is absorbed to a similar degree and enters the same metabolic
pool in the body (24). In addition, despite the fact that the forni
of selenium at the active site of glutathione peroxidase appears
to be 1-selenocysteine, sodium selenite and selenomethionine
have about equal potency for maintaining and restoring glutathi
one peroxidase in rats, and equal selenium retention in the body
results from feeding either compound (2, 15, 19). It was antici
pated that this study would also provide mechanistic insights
into the mode of action of selenium as an antipromoter, since
the initial steps in the metabolism of the 2 compounds differ.
MATERIALS AND METHODS
All experiments
INTRODUCTION
Experimental evidence continues to accumulate supporting
the hypothesis that selenium can inhibit one or more stages of
the carcinogenic process in several target tissues, including the
mammary gland (7, 13, 16, 20, 25, 28). The anticancer effect(s)
of selenium has been found to be exerted against chemically
induced cancers in the rat and mouse and against virally induced
lesions in the mouse (7, 13, 20, 25). In certain mouse tumor
systems, selenium has been reported to exert its greatest pro
tective effect on early stages of the disease process, and contin
uous selenium treatment has been observed to be necessary to
sustain anticancer activity (13, 14). At the levels of dietary
1 Supported by USPHS Grant CA 28109 from the National Cancer Institute.
Scientific Contribution 1228 from the New Hampshire Agricultural Experiment
Station.
2 To whom requests for reprints should be addressed, at the Human Nutrition
Center, Colovos Road, University of New Hampshire, Durham, NH 03824.
Received March 10,1983; accepted March 29, 1984.
JULY
were conducted
with virgin female Sprague-Dawley
rats obtained from Taconic Farms, Inc., Germantown, NY. Animals were
fed a torula yeast diet, the composition of which is shown in Table 1. A
20% fat diet was selected because such high fat diets have been reported
to promote mammary carcinogenesis (3). Given that the antipromotional
activity of selenium was being investigated, diet conditions were chosen
to amplify the promotion phase of carcinogenesis. The diet was also
formulated to contain 0.5% DL-methionine, the most limiting amino acid
in torula yeast (5). It is important to note that this is far below the 2.0%
level of dietary methionine known to be toxic to the rat (6, 17). We also
felt it to be extremely important to provide sulfur-containing amino acids
sufficiently above requirement levels so that the selenomethionine would
be available for metabolic utilization as selenium and would not be used
to satisfy the requirement for methionine (22). Furthermore, increasing
the concentration of dietary methionine has been reported to reduce
selenium toxicity (11). Dt-Selenomethionine was used since it has been
reported to be as equally well utilized as sodium selenite for regeneration
of glutathione peroxidase activity in the rat (29).
Experiment 1. The purpose of this study was to confirm and extend
3 The abbreviations
used are: MNU, 1-methyl-1-nitrosourea;
DMBA, 7,12-di-
methylbenz(a)anthracene.
1984
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2803
H. J. Thompson et al.
previous observations of the effect of selenium in the experimental
mammary carcinogenesis system induced with 50 mg of MNU/kg of
body weight (25, 27). The study differed from those reported previously
in that tumor appearance was accelerated by feeding of a 20% (w/w) fat
diet. Although selenium has been reported to be effective in the DMBAinduced mammary tumor system promoted by high dietary fat (8), this
has not been shown for the MNU induction system. Furthermore, very
little selenium dose-response work has been done to date. Diminished
anticarcinogenic effectiveness with 4 ppm of selenite in the MNU system
has been reported (27), and no work has been done above 5 ppm of
selenite. We therefore examined the dose range from 4 to 6 ppm of
selenite, the upper limit being necessitated by the induction of selenium
toxicity above this level (9). Furthermore, we sought to examine the
effect of selenium supplied as selenomethionine incorporated to provide
the same final concentration of selenium provided by sodium selenite,
i.e., 5 or 6 ppm of selenium. Between 35 and 57 days of age, animals
were provided with distilled water and a lab chow diet (Purina 5001)
which contained 0.2 ppm of selenium. At 50 days of age, 120 rats
received a s.c. injection of 50 mg of MNU/kg of body weight (26). The
MNU was dissolved in acidified saline (pH 4.0) immediately prior to
injection. Seven days after MNU treatment, animals were randomized
into 1 of 6 groups and received the torula yeast diet containing 0.1 ppm
of selenium, to which was added either no additional selenium or suffi
cient selenium to make diets of 4, 5, or 6 ppm of selenium as sodium
selenite or 5 or 6 ppm of selenium as selenomethionine. Concentrations
of selenium in these diets were determined fluorometrically (18). Food
intakes of individually housed, 0.9% NaCI solution (saline)-injected ani
mals assigned to each treatment were determined during the first 8
weeks of the study and were used to estimate the amount of selenium
ingested per day. Animals were maintained under these conditions for
the remainder of the study until its termination 135 days after carcinogen
treatment.
Table 1
Compositionof the torula yeast diet
The diet used in both Experiments 1 and 2 was as described in 'Materials and
Methods."
IngredientCom
composition20
starch
Cerelose
20
Torula yeast"
30
Fiber6
5
Com oil0
20
Vitamin mix0
1.0
Mineral mix"
3.5
DL-Melhionme%
0.5
* USF XIX. Obtained from the Lakes States Division, St. Regis, Rhinelander,
Wl.
b Obtained from Sulkafolk, Brown Co.. Berlin, NH.
: Mazólacom oil obtained from Best Foods Co., Englewood, NJ.
' American Institute of Nutrition formulations obtained from Teklad, Madison,
Wl.
Experiment 2. This experiment was conducted in order to evaluate
the results of Experiment 1 in the different but analogous mammary
tumor system induced with DMBA. Given the strong promotional activity
of dietary fat in this system, a low dose of DMBA was used in order to
increase the sensitivity of the assay system for detection of differences
in tumor induction.
Between the ages of 35 and 111 days, animals were provided ad
libitum with distilled water and an adequate selenium diet, the composi
tion of which is shown in Table 1. At 50 days of age and following an
18-hr fast, rats received 5 mg of DMBA p.o. The carcinogen was
dissolved in sesame oil an delivered in a volume of 1 ml. Seven days
after carcinogen treatment, animals were randomized into 1 of 3 groups
of 21 rats each. At this time, selenium treatment via the drinking water
was initiated. Animals either continued to receive distilled water or were
given water containing 3.4 ppm of selenium supplied as either sodium
selenite or selenomethionine, a level of selenium reported to be effective
in other mammary tumor systems (28). The selenium solutions were
prepared and given to the rats fresh every other day. Selenium concen
trations were determined fluorometrically (18). Water intakes of individ
ually housed, solvent-intubated rats assigned to each treatment were
determined during the first 8 weeks of the experiment and were used to
estimate the amount of selenium ingested per day. Animals were main
tained under these conditions for the remainder of the study.
In both experiments, rats were weighed each week and palpated for
the detection of mammary tumors twice weekly. At necropsy, the skin
of each rat was transillumina ted. and all grossly observable tumors were
removed and processed for histopathological evaluation according to the
criteria of Young and Hallowes (30). Only tumors confirmed to be
mammary carcinomas are reported.
Statistical analyses of the data were performed as follows. The tumor
counts were transformed using the square root transformation as rec
ommended by Snedecor and Cochran (21). The transformed counts
were then tested for response to selenium dose by regression analysis.
Differences in tumor incidence were tested using the x-square test
without the overly conservative continuity correction (1). The groups
were tested for a hypothesized delay in time to tumor appearance with
increased levels of dietary selenium by the nonparametric test of Mantel
(12).
RESULTS
The inhibitory activity of graded levels of dietary selenium
against mammary cancer occurrence in animals treated with
MNU is shown in Table 2. In comparison to the control, supple
menting diets with 4 and 5 ppm of selenite resulted in a dosedependent reduction in the number of cancers per rat. The
protective effect of selenite fed at 6 ppm decreased sharply 110
days postcarcinogen, despite the fact that rats ingested more
Table 2
Effect of selenium administered in the diet on MNU-inducedmammary carcinogenesis
All rats were given MNU (50 mg/kg of body weight) as described in "Materials and Methods." Twenty rats were assigned to each treatment group. All rats survived
until the study was terminated 135 days after treatment with MNU.
Selenium
(ppm)'Basal
selenium6Selenite
of
sele
nium intake (>ig/
day)2
cancer in
cidence
(%)90
no. of can
cers/cancer-bearing
no. of
cancers/rat423.2a
rat*?3.5-2.8'
cancer-free
time(days)61±4.9* *
61*
247 ±4.44.0
Selenite
4047
90
2.7'
71 '
241 ±2.6'
5.0
Selenite
95
4.5-4f£
4.7-5'1*
71'
249 ±5.3"
Selenite
6.0
51
90
252 ±3.15.0
Selenomethionine
50
95
66?
3.3-Mean
4.1"Median
72'Bodywt(g)c259
SelenomethionineEstimated 63Final
6.0Source
80Mean
241 ±5.1
a Concentration of selenium in the diet. The basal diet was supplementedwith sodium seleniumto contain 0.1 ppm of selenium as confirmed by analysis.
Selenite was supplied as the sodium salt.
0 Group mean body weights at the time the study was terminated.
- Statistically different from r; p < 0.05.
8 Mean ±S.E.
2804
CANCER
RESEARCH
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VOL. 44
Selenium Inhibition of Mammary Cancer
Tables
Effect of selenium administered in drinking water on DMBA-induced mammary carcinogenesis
All rats were given 5 mg of DMBA as described in "Materials and Methods." Twenty-one rats were assigned to each treatment group. All rats survived until the study
was terminated at 111 days post-DMBA.
Selenium
(ppmf0.0
selenium"Basal
of
selenium
intake (fig/day)2
cancer inci
dence
(%)38'
no. of can
cers/rat01.5(32)''9
no. of cancers/
rat withcancer"4.0'2.d1.0*Body (g)e253
wt
±5.2'' "
diet
0.1 (2)'
235 ±5.4'
3.4
Selenite
5059Final
33'Mean
0.3 (7)'Mean
SelenomethionineEstimated
3.4Source
227 ±4./
8 Amounts of selenium with which the drinking water was supplemented. All rats were fed the basal diet which was supplemented with sodium selenite to contain 0.1
ppm of selenium, as confirmed by analysis.
" Selenite was supplied as the sodium salt.
0 Total number of cancers divided by the number of rats per group.
d Total number of cancers induced divided by the number of rats bearing a cancer.
" Group mean body weights at the time the study was terminated.
' Statistically different from /; p < 0.01.
3 Numbers in parentheses, total number of cancers induced in each group.
" Mean ±S.E.
selenium per day. The reason for this is unclear. Median cancerfree time was marginally extended at both 5 and 6 ppm of
selenite. Selenomethionine also suppressed cancer occurrence,
but it appeared that 6 ppm organic selenium was not as effective
as was 4 ppm selenite. At necropsy, hard nodular cirrhosis was
observed in 50% of the animals receiving the highest level of
Selenomethionine. Livers were shrunken in size. Histologically,
extensive necrosis, hemorrhage, and fibrosis were observed.
Comparable effects were not noted in animals on the other
dietary treatments. The rate of body weight gain was decreased
similarly to the same extent among the various selenium-treated
groups. This implies that a difference exists in the marginal
toxicity of selenite and Selenomethionine, as indicated by re
duced weight gain and the gross liver damage observed in the
animals fed 6 ppm of selenium as Selenomethionine.
In Table 3, data is presented which summarizes the effects of
selenium administered in the drinking water on the promotion
stage of DMBA-induced mammary carcinogenesis. Selenium
provided as either selenite or Selenomethionine significantly re
duced cancer induction in comparison to animals receiving no
supplemental selenium. However, as reflected by the comparison
of estimated dose of selenium ingested from each diet group,
selenite was more protective than was Selenomethionine, both
in terms of reducing the number and in incidence of mammary
carcinomas. The protective effect of selenium against promotion
was, however, actually eliminated by increasing the dose of
DMBA to 15 mg in animals that were fed the high-fat diet.4 Hard
nodular cirrhosis was also noted in 35% of the animals receiving
the Selenomethionine in the drinking water, whereas the livers of
the selenite-treated group were within normal limits. Body
weights were depressed similarly in both groups in comparison
to the control.
DISCUSSION
This investigation provides evidence that dietary selenium in
the form of sodium selenite provides greater inhibition of mam
mary carcinogenesis than does an equivalent amount of selenium
in the form of Selenomethionine. Similar differences between
Selenomethionine and sodium selenite have been observed with
transplantable tumors (16). As has been reported by Sunde ef
al. (22), selenium provided from Selenomethionine can either be
incorporated into tissue protein in place of methionine or cata4 H. J. Thompson, unpublished observation.
JULY
bolized into the selenite pool for subsequent utilization or elimi
nation. The extent to which the former pathway is favored is, in
part, determined by the adequacy of dietary methionine. As noted
above, this experiment was designed to minimize the diversion
of Selenomethionine to satisfy the requirement for methionine
and, hence, permit a better comparison of the protective activity
of selenium from 2 defined sources.
It has been reported previously that feeding the levels of
dietary selenium in the dose range used in this study results in
a 10 to 15% reduction in the rate of body weight gain. This
observation was again confirmed. However, it has been reported
that this degree of growth retardation cannot account for the
protective activity of selenium (7, 23, 25). The unanticipated
finding of hard nodular cirrhosis at the highest dose of Seleno
methionine fed in diet or drinking water illustrates the toxicity of
the compound and the narrow dose range over which the
transition from marginal to overt toxicity occurs. This observation
supports the need for additional research to clarify those aspects
of selenium toxicity that are characteristic of Selenomethionine
per se (9).
Although the fact that selenium can act as an anticarcinogen
is well established, little progress has been made toward eluci
dating the mechanism(s) by which selenium inhibits the neoplastic process. The results of this investigation suggest that no
advantage is gained from the use of Selenomethionine rather
than selenite for cancer prophylaxis. The observation of Seleno
methionine toxicity accompanied by a diminished protective ef
fect against mammary carcinogenesis raises an important ques
tion. Why is the same amount of selenium essentially nontoxic
and highly protective when supplied as selenite but more toxic
and less protective when supplied as Selenomethionine? One
answer could be that selenite is more effectively detoxified than
is Selenomethionine. This possibility suggests that the chemopreventive activity of selenium may occur, at least in part, as a
consequence of its effective detoxification.
ACKNOWLEDGMENTS
We wish to thank Anne M. Roñanfor her technical assistance and Karen Savard
for her assistance in the preparation of this manuscript.
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CANCER
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VOL. 44
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Effect of an Inorganic and Organic Form of Dietary Selenium on
the Promotional Stage of Mammary Carcinogenesis in the Rat
Henry J. Thompson, L. David Meeker and Stephen Kokoska
Cancer Res 1984;44:2803-2806.
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