Journal of Neuro-Oncology 46: 125–133, 2000.

© 2000 Kluwer Academic Publishers. Printed in the Netherlands.

Laboratory Investigation

Selenium causes growth inhibition and apoptosis in human brain tumor cell lines

Narayan Sundaram1 , Amit K. Pahwa1 , March D. Ard2 , Nan Lin2 , Eddie Perkins1 and Alfred P. Bowles, Jr.1
1
Department of Neurosurgery, 2 Department of Anatomy, University of Mississippi Medical Center, Jackson,
MS, USA

Key words: selenium, human glioma cells, mitochondria, apoptosis, fibroblasts, ultrastructure, MTT

Comments

There is in general a lack of knowledge concerning the role of trace elements in glial tumors, their influence on
tumor growth and cytotoxicity of high, or low, levels in tumor environment. This paper demonstrates the cytotoxi-
city of selenium in glioma cells and in normal low-passage fibroblasts. These results are not new but they confirm
a few previously published studies. The novel finding in this paper is the resistance of high-passage fibroblasts to
selenium. The reason for this lack of response is obscure, however, it points to a differentiated response in cells of
different stages of development. The induction of apoptosis by selenium is also demonstrated and confirmed by
the authors using other techniques than previously was done. In general, this paper contributes to the knowledge of
a differentiated effect of selenium on glial tumor cells compared to normal cells. Although the clinical relevance
of selenium in glial tumors is still unclear it is important to acknowledge all contributions to the understanding of
glial tumors and their response to different potentially usable substances.

Dr. A. T. Bergenheim

Summary
We examined the effect of the trace element selenium on human glioma cell lines: T98G, U373MG, and U87MG, in
addition to dermal fibroblast cells. Cultures were incubated with sodium selenite, and the following parameters were
studied: cell growth, mitochondrial function, and ultrastructure. Cell growth was assayed by counting the number
of viable cells after treatment with selenium. Mitochondrial function was analyzed using the MTT (tetrazolium salt
reduction) assay. Apoptosis was determined by evaluating nuclear chromatin condensation by electron microscopy.
The results indicated that selenium had a significant inhibitory effect on the growth of the tumor cells but had
little effect upon dermal fibroblasts which had been passaged numerous times. Selenium also induced mitochondrial
damage as shown by MTT assay in two brain tumor cell lines and in minimally passaged fibroblasts, but it had little
effect upon the high-passage fibroblasts. Ultrastructurally, mitochondria had electron-dense inclusions resulting
from selenium treatment. High rates of apoptosis were induced by selenium in the tumor cell lines and in the
minimally passaged fibroblasts, whereas the fibroblasts with a high number of passages had some resistance to
selenium treatment. This study correlates the adverse effects of selenium on mitochondrial function, inhibition
of cell growth, and apoptosis and shows that selenium similarly affects three different brain tumor cell lines and
minimally passaged fibroblasts. Further, the results with fibroblasts show that some types of cells after repeated
passages can develop resistance to selenium damage.

Introduction of glutathione peroxidase, an enzyme important in

preventing oxidative damage [1,2,3]. It has also been
Selenium is a metalloid element that occurs naturally in linked to the enzyme iodothyronine deiodinase and the
the earth’s soil. Selenium has been linked to the activity ubiquitous protein, selenoprotein P, whose function has
126
yet to be determined, and to other anti-oxidant enzymes
[4,5]. Studies have shown that humans with cancer
often have lower serum concentrations of selenium
than those without cancer [6,7]. El-Yazigi et al. (1984),
reported lower levels of selenium in the CSF of patients
with malignant brain tumors than those with benign
tumors [8]. Many studies have illustrated a reduction in
carcinogenesis through diets high in selenium [9,10].
In countries such as Finland, which have a policy of
selenium supplementation in the food supply because
of low concentration in the soil, reduction in cancer
rates has been observed [11]. On the other hand, many
studies have analyzed the effect of excess selenium in
the diet, especially studies of selenosis, or selenium
poisoning in parts of China, where selenium content
in the soil is high [4]. Selenium poisoning can lead to
many adverse reactions such as loss of hair and nails,
skin lesions, ‘garlic-breath,’ and neurologic impair-
ment. Several bench studies have been conducted to
determine the molecular mechanism and effects of sele-
nium on various cancer cells (e.g., 1,11). However,
there is little information about the effect of selenium
on brain tumor cells; some have postulated that sele-
nium induces apoptosis in these cells [12,13]. In this
study, we sought to further analyze apoptotic effects of
selenium on brain tumor cell lines and to compare the
response of these cells to that of non-cancerous der-
mal fibroblasts. Specifically, we measured cell number,
mitochondrial function, and nuclear chromatin conden-
sation as revealed by electron microscopy (EM). We
studied proliferation, or change in cell number, to ver-
ify past research in addition to testing new brain tumor
cell lines upon which the effects of selenium have not
been studied. Mitochondrial analysis was conducted in
an attempt to correlate loss in cell number with loss of
mitochondrial activity and to determine the time course
of selenium’s inhibitory effect upon the ‘engine of the
cell,’ the mitochondria. EM studies confirmed that sele-
nium induced apoptosis, or programmed cell death, in
brain tumor cell lines and in fibroblasts.
Materials and methods
T98G, U373MG, and U87MG human glioma tumor
cell lines were obtained from American Type Culture
Collection (ATCC). Dermal fibroblasts were obtained
from human skin and maintained in culture for either
fewer than 10 (low-passage fibroblasts) or more than
100 passages (high-passage fibroblasts). The form
of selenium used was sodium selenite (Na2 SeO3 ),
obtained from Sigma Chemical Company.
Cell proliferation
To evaluate cell proliferation, cells were plated onto 24-
well cell culture plates at 10,000 cells per well in 1 ml of
culture medium: Dulbecco’s Modified Eagle’s Medium
(DMEM) with 10% fetal bovine serum, 1% glutamine,
and penicillin–streptomycin antibiotics. Cells were
incubated in 5% CO2 and 95% air at 37◦ C. Prior to treat-
ment cells were allowed 24 h to adhere to the bottom of
the plate. After 24 h of incubation, a concentrated stock
solution of selenium (100 mg/ml) was prepared, then
diluted so that the concentration delivered to the cells
was 2 µg/ml or 4µg/ml. Controls were treated with the
diluent, sterile water. On days 3, 6 and 9 of incuba-
tion at 37◦ C, the cells were harvested by suspension in
0.025% trypsin in 0.02% EDTA solution for 15 min.
Cell counts were then performed in duplicate using a
hemacytometer, with trypan blue exclusion to identify
viable cells. Growth curves were generated for each cell
line. Statistical significance of the difference between
treated samples and controls was determined by Stu-
dent’s t-test. P values less than 0.05 were considered
to be significant. Growth curves were generated from
the results obtained.
Mitochondrial activity (MTT assay)
To evaluate cellular mitochondrial activity, the 3-
[4, 5-dimethyl-thiazol-2-yl]-2, 5-diphenyltetrazolium-
bromide (MTT) assay was used [14]. Ten thousand
cells per well of each cell type were plated in 96-
well cell culture plates with 100 µl culture medium and
incubated for 24 h to attach to the bottom of the well.
After incubation, cells were treated with 2 µg/ml and
4 µg/ml dosages of selenium, delivered in a 10 µl vol-
ume to each well. Controls were treated with 10 µl ster-
ile water. Plates were incubated for 0, 2, or 3 h; then
10 µl (10% of culture volume) of MTT solution (5 mg
tetrazolium salt with 1 ml sterile water) was added to
each well, and incubation was continued at room tem-
perature in the absence of light. The media and MTT
then were removed and 100 µl of 0.4 N HCl in 99%
isopropanol was added to each well to dissolve the for-
mazan crystals resulting from mitochondrial enzymatic
activity on the MTT substrate. The cells were incubated
for another hour in the absence of light on a platform
shaker to completely dissolve all formazan crystals.

After an hour, formazan was measured by reading
absorbance at a wavelength of 570 nm with a reference
setting of 630 nm on a microplate spectrophotometer.
Statistical significance of differences between treated
samples and controls was determined by Student’s t-
test. P values less than 0.05 were considered to be
significant.
Electron microscopy (apoptosis)
Five cell types (low-passage fibroblasts, high-passage
fibroblasts, T98G, U373MG, and U87MG) were plated
on separate 60 mm petri dishes and incubated for 24 h
to attach to the bottom of the dishes. Cells were then
treated with 2 µg/ml and 4 µg/ml concentrations of
selenium. Controls received saline. After 24 h, cells
were scraped off the dish and fixed in a 1 : 1 solution
of culture medium: 2% glutaraldehyde in 0.1 M PO4 ,
pH 7. Cells were transferred to microcentrifuge tubes
and sedimented. The pellet was resuspended in 2% glu-
taraldehyde, then washed twice in 0.1 M PO4 , pH 7,
then incubated in a solution of 1% OsO4 in the same
buffer for 15 min, followed by five rinses in deion-
ized water. Cells were stained for 10 min in 2% uranyl
acetate, rinsed three times in deionized water, and dehy-
drated in successive concentrations of ethanol, ending
with a 1 : 1 100% ethanol : propylene oxide solution.
Cells were then infiltrated with 100% propylene oxide;
1 : 3, Epon : 100% propylene oxide; 3 : 1, Epon : 100%
propylene oxide; and finally 100% Epon. Following
this, cells were embedded in fresh 100% Epon at 60◦ C
overnight. All Epon steps contained EM Corp. Internal
Lubricant. Cell blocks were thin sectioned on a Leica
Ultracut UCT ultramicrotome. Sections were mounted
on formvar coated copper slot grids and stained with
lead citrate.
The cells were observed using a Leo 906 transmis-
sion electron microscope and scored as apoptotic or
normal based on chromatin condensation [15]. Each
section was scanned systematically at low magnifica-
tion (approximately 3000×), and the first 100 cells
encountered were scored, using increased magnifica-
tion where necessary. Examples of scoring criteria are
presented in Figure 4. In addition, mitochondria were
examined for the presence of electron-dense inclu-
sions, exemplified in Figure 3. Examination of mito-
chondria was carried out using electron micrographs
of 4–7 cells from each grid, selected as representatives
of chromatin scoring. Results were analyzed using the
127
Mann–Whitney statistical test for data from ordered
categories [16], using selenium dose as the ordinal.
Results
In control cultures without selenium, cell lines and
fibroblasts grew at a sustained rate, as determined by
counting cells (Figure 1). Selenium slowed the rate
of growth in all cell types studied with the notable
exception of the high-passage fibroblasts (Figure lA).
In T98G, U373MG, and U87MG brain tumor cell
lines the selenium treatment drastically reduced cell
numbers compared to controls after the sixth day
(Figure 1B,C,D). In contrast to the brain tumor cell
lines, the growth of the high-passage dermal fibrob-
lasts was not inhibited and may even have been slightly
enhanced by selenium (Figure lA). Suppression of cell
number by selenium was dose-dependent in all three
brain tumor cell lines, with cell numbers most reduced
at the higher concentration of selenium. The time
course of the effect varied among cell lines. Differences
in growth between selenium treated brain tumor cell
lines and control were statistically significant accord-
ing to Student’s t-test. There was no statistically sig-
nificant difference between treated and control cells of
the high-passage fibroblasts.
The MTT assay of mitochondrial enzyme activity
is often used in conjunction with assay of apopto-
sis. Results from this analysis suggest that mitochon-
drial function was compromised by both 2 µg/ml and
4 µg/ml dosages of selenium in the T98G and U373MG
glioma cell lines (Figure 2C,D,E), whereas the effect
on both low- and high-passage fibroblasts occurred
only at the higher dose (Figure 2A,B). Susceptibility
of the cells to selenium damage varied according to
cell type. The maximum effect of selenium was seen at
2 h of treatment, and recovery of hydrogenase activity
was evident at 3 h, showing cell viability. The T98G
and U87MG cell lines had better recovery of hydroge-
nase activity after the 2 µg dose than the 4 µg dose of
selenium.
Mitochondrial damage was also evident in EM
observations of selenium treated cells. Electron-dense
inclusions were found in mitochondria of all cell types
following 24-h incubation with selenium (Figure 3).
The association of inclusions with the dosage level of
selenium was highly significant (P < 0.001), as shown
by the Mann–Whitney test [16] (Table 1). The lim-
ited number of observations made in this part of the
study did not permit analysis by individual cell type;
however, cells from each type at all treatment levels
128

Figure 1. In vitro growth curves of three human glioma cell lines and high-passage dermal fibroblasts after treatment with selenium. Each
line represents data from three separate experiments. Selenium treatment resulted in a statistically significant decrease in cell number
compared to control in all three glioma cell lines (B–D), but not in high-passage fibroblast cultures (A), as determined by Student’s t-test.
(B) At 2 µg/ml selenium, P < 0.05; at 4 µg/ml, P < 0.009. (C) The difference was significant on day 9 only: at 2 µg/ml selenium,
P < 0.01; at 4 µg/ml, P < 0.002. (D) The difference was significant on days 6 and 9 only: at 2 µg/ml selenium, P < 0.002; at 4 µg/ml,
P < 0.000003.

were included in the overall statistical analysis. Asso-
ciation of mitochondrial inclusions with chromatin
condensation in cells was of minimal significance
(P = 0.034).
Observation of nuclear chromatin patterns by EM
confirmed that cells damaged by selenium were under-
going apoptosis. One hundred cells were examined
in each category defined by cell type and treatment
level (Table 2). One set of low-passage fibroblasts,
high-passage fibroblasts, and U373MG cultures were
examined, and two sets of T98G and U87MG cultures
grown at different times were embedded and studied.
In each sample, nuclei were scored 0, 1, 2, or 3 by
morphologic criteria, as demonstrated in Figure 4.
Nuclei scored 0 or 1 were considered normal; those
scored 2 or 3 were clearly undergoing apoptosis. The
Mann–Whitney test was used to assess association
between the percent of apoptotic nuclei encountered
and the dose of selenium applied (0, 2, or 4 µg/ml for
24 h). Selenium treatment was significantly associated
with apoptosis in a dose-dependent manner in each
cell type (Figure 5). In accordance with the results of
the MTT assay, apoptotic susceptibility to selenium
appeared to be lower in the high- passage fibroblasts
than in the low-passage fibroblasts or the cell lines. For
unknown reasons, inconsistent results were obtained
in the two different experiments with cells of the T98G
cell line (Figure 5B).
 129

Figure 2. Graphs representing the outcome of mitochondrial function assays in human glioma cells and human dermal fibroblasts after
treatment with selenium. At the 2 h time point, MTT reduction was decreased by the 4 µg/ml selenium treatment in all cells except U87MG
(P ≤ 0.016). The 2 µg/ml selenium treatment decreased MTT reduction only in the T98G (P = 2 × 10−8 ) and U373MG (P = 0.047)
glioma cell lines but not in the low- or high-passage fibroblasts. Significance of these differences was determined by Student’s t-test.

Figure 3. Mitochondria in selenium-treated cells contained electron-dense inclusions (arrows). In (C) the apoptotic nucleus of the treated
cell is also visible. Original magnification = 10,000×. m, mitochondrion; Nuc, nucleus.
130

Table 1. Mitochondrial inclusions induced by selenium treat- Discussion

ment
Chromatin Selenium Number of cells Mitochondrial Our data are consistent with results from previous stud-
score (µg/ml) examined (n) inclusions ies on other tumor types. Like the studies by Zhu et al.
‘yes’/n (%) [17] and Zhang et al. [1], we found that selenium inhib-
0 28 6/28 (21) ited the growth of brain tumor cell lines, but did not
0 2 17 8/17 (47) affect the growth of the high-passage dermal fibrob-
4 6 3/6 (50) lasts. Different glioma cell lines differed somewhat in
0 19 5/19 (26) their responses to selenium treatment.
1 2 13 7/13 (54) This study correlated cells’ ability to metabolize
4 9 4/9 (44) MTT with apoptosis. Loss of ability to reduce MTT
0 2 1/2 (50) may be an indication of the preliminary stages of
2 2 15 6/15 (40) apoptosis. Hence, in cells used in this study selenium
4 13 9/13 (69) decreased mitochondrial MTT reduction and caused
0 3 1/3 (33) apoptosis as defined by changes in nuclear chromatin.
3 2 10 5/10 (50) Mitochondrial electron-dense inclusions were visi-
4 14 8/14 (57)
ble indicators of damage in cultures embedded for EM.
Electron-dense inclusions in mitochondria were significantly Whereas selenium deficiency has long been known to
associated with selenium treatment at P < 0.001, by Mann– result in mitochondrial swelling (e.g., 18), examples
Whitney test. For association between mitochondrial inclusions
of ultrastructurally observed inclusions in response to
and chromatin score, P = 0.034.
excess selenium are rarer [19,20]. In our study, the
inclusions were highly correlated with selenium treat-
ment across all cell types studied. However, there
Table 2. Chromatin condensation in response to selenium
treatment
was not a strong association between the presence of
mitochondrial inclusions and nuclear chromatin con-
Cell type Treatment Nuclear chromatin
densation. Inclusions occurred in cells with normal-
(µg/ml) score
appearing nuclei. This result would be predicted if
0 1 2 3 mitochondrial damage is preliminary to apoptosis, so
that at the one time point studied by EM, 24 h, some
0 70 28 2 0
Low-passage fibroblasts 2 3 1 27 69
cells sustaining mitochondrial damage had not yet
4 1 0 48 51 undergone apoptosis.
0 82 17 0 1
This study also correlated diminished mitochondrial
High-passage fibroblasts 2 67 29 2 2 activity with cell loss. Mitochondrial damage after
4 17 28 41 14 treatment with selenium appeared early (after 2 h) in
0 67 33 0 0 T98G and U373MG brain tumor cell lines and in fibrob-
T98G(98) 2 56 41 3 0 lasts. This led to a reduction in cell number in these
4 39 52 9 0 cultures over a 9-day period. Lower cell counts could
0 67 32 1 0 result from either slowed proliferation or loss of cells
T98G(99) 2 27 42 28 3 through apoptosis, or both.
4 9 49 39 3 Fibroblast were used as primary human cells for
0 74 22 0 4 comparison to the glioma cell lines. Although astro-
U373MG(99) 2 37 21 26 16 cytes were considered as an alternative more closely
4 32 11 21 36
related to the glioma cells, primary astrocytes can be
0 38 52 10 0 cultured with rapid growth only from fetal tissue within
U87MG (98) 2 40 37 13 10
a limited age window. Unavailability of such tissue on
4 3 13 40 44
a regular basis led to the choice of fibroblasts.
0 69 23 8 0
The high-passage fibroblasts, in contrast to the other
U87MG (99) 2 15 23 29 33
4 0 22 51 27 cell types, showed resistance to selenium treatment.
Selenium at 2 µg/ml did not affect the mitochondrial
One hundred cells were examined in each treatment group. function or morphological characteristics of these cells.

Figure 4. Apoptosis is characterized by condensation of chromatin in the cell nucleus. Normal nuclei observed by EM were scored 0
(A) or 1(B). Nuclei in early (C) or advanced (D) stages of apoptosis were scored 2 or 3, respectively. Vacuolization of cytoplasm with
apoptosis is apparent. Original magnification = 6000×.
It was interesting that the higher concentration of sele-
nium, 4 µg/ml, inhibited mitochondrial activity after
just 2 h of treatment. Cell numbers in these cultures
were reduced by selenium treatment at both 2 and
4 µg/ml concentrations during the first six days of treat-
ment but recovered to control levels by 9 days. From
our data it appears clear that repeated passaging of cells
can result in resistance to selenium damage, at least
at lower doses. These results are consistent with the
resistance to selenomethionine toxicity observed in a
fibroblast cell line by Redman et al. [21]. It is not known
whether some cell types in vivo may be resistant to sele-
nium poisoning; the low-passage dermal fibroblasts we
tested in vitro were highly susceptible.
Differences in cellular responses to selenium con-
ceivably could be exploited therapeutically, if a dosage
could be defined which causes apoptosis in tumor cells
131
while leaving normal cells intact. In this study, how-
ever, the low-passage fibroblasts which were tested as
a representative of normal cells had among the highest
rates of apoptosis after selenium treatment. Neverthe-
less, the doses used here were 10 to 90 times higher
than the reference values for normal serum, 0.046–
0.143 µg/ml [22]; further experimentation may evalu-
ate tumoricidal effects of lower doses applied over an
extended period of time. In vivo, too much selenium
is very toxic to human beings, leading to such compli-
cations as nerve and liver damage. On the other hand,
selenium is a cofactor for antioxidant enzymes, and
selenium deficiency leads to mitochondrial dysfunc-
tion. There may be an appropriate balance of dietary
selenium which is inhibitory to tumor growth. In this
study selection of selenium doses were determined
based on those that were reported in the literature and
132

Figure 5. Graphs representing the percentage of apoptotic nuclei in glioma and dermal fibroblast cells after treatment with selenium.
Each point represents 100 cells examined by transmission EM. Association of apoptosis with selenium dose was tested for significance
by the Mann–Whitney test. P < 0.001 in all cases except T98G 1998, P = 0.006 (B).

normal levels found in plasma and CSF. We selected a of Mississippi Medical Center Institutional Research
range that we considered therapeutic and not toxic and Support Grant to APB.
this was based on doses reported in the literature to be
effective in inhibiting tumor cell growth (13,22,23).
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Address for offprints: Alfred P. Bowles, Jr. Department of Neuro-
surgery University of Mississippi Medical Center, 2500 North
State Street Jackson, MS 39216-4505, USA; Tel.: 601 984 5700