10

SELENIUM METABOLISM AND MECHANISMS

OF CANCER PREVENTION

Howard E. Ganther

University of Wisconsin
Madison, Wisconsin, 53706

INTRODUCTION

It has been 50 years since the surpriSIng discovery in 1949 that

selenium (Se) had cancer preventing properties in animals given a chemical
carcinogen (I). Numerous studies have confirmed the anticarcinogenic
effects of supranutritional levels of Se in animals and more recently in
humans given supplemental Se, as described by Combs (2). Proposed
mechanisms of action for Se, as reviewed elsewhere in more detail (3), are
based on (i) the formation of selenoproteins and (ii) the generation of low
molecular weight Se metabolites by intermediary metabolism of Se in
animals. A collaborative project (4,5) to investigate mechanisms and develop
improved forms of Se for cancer prevention has provided considerable
evidence that methylated metabolites of Se are important for Se
anticarcinogenic action in animal model systems. The objective of this article
is to summarize the findings that led to this concept and describe applications
using methylated selenoamino acids that occur naturally in plants as a
practical delivery mechanism for chemoprevention.

Nutrition and Cancer Prevention, edited under the auspices of AICR 119
Kluwer Academic / Plenum Publishers, New York, 2000.
120 H. E. Ganther

SELENIUM METABOLISM
Methylation is a major fate of Se in animals (5). The garlic odor in
breath of animals given inorganic Se salts was attributed to dimethyl selenide
over 100 years ago (6). Animals synthesize selenoproteins from hydrogen
selenide, a key intermediate in Se metabolism (Fig. I). It is formed by
reduction of inorganic salts such as sodium selenite, or by liberating Se from
organoselenium compounds by scission of C-Se bonds. The metabolism of
selenomethionine requires conversion to selenocysteine (transsulfuration
pathway) followed by selenocysteine lyase to release inorganic Se. Hydrogen
selenide is quite toxic but only miniscule amounts are formed to provide Se
for selenoprotein synthesis. Animals deal with any excess by methylation,
forming less toxic metabolites. Monomethylated forms are the major
excretory products at normal Se intakes. Methylselenol (CH3SeH) can be
formed directly in one step from Se-methylselenocysteine (7), a major
component of certain plants and selenized garlic (8). In this case the
inorganic pool is largely bypassed, although sufficient demethylation to
inorganic Se occurs to meet the needs for selenoprotein synthesis.
Hydrogen selenide is methylated by S-adenosylmethionine in three
successive reactions, catalyzed by different enzymes. Steps 1 and 2 are
catalyzed by an arsenite-sensitive microsomal enzyme called thiol S-
methyltransferase (9; 10), forming dimethyl selenide; step 3 is catalyzed by
thioether S-methyltransferase (10). The rate of methylation decreases with
increasing degree of methylation, and the third step may become rate limiting.
As a result, dimethyl selenide may escape and be excreted in the breath (10,
11). At normal levels of Se intake monomethylated forms are the major
metabolites (11). When Se is fed to animals at chemopreventive levels, the
urine contains much higher amounts of monomethylated forms of Se plus
trimethylselenonium ion. At the highest Se intake, dimethyl selenide (breath)
equals urinary monomethyl Se and exceeds urinary trimethylselenonium ion
(11).

EFFICACY, BIOAVAILABILITY, AND TOXICITY

Selenomethionine or inorganic Se salts such as sodium selenite were
used in most of the initial studies of cancer prevention because they were
readily available (4). Cancer prevention activity in animals is lowest for
selenomethionine, partly because it is incorporated into proteins in place of
methionine. Selenite is more active and Se-methylselenocysteine is about
twice as active as selenite (12). Other methylated forms of Se that bypass the
inorganic pool also show good chemopreventive activity (12). These results
suggested that activity might arise from some downstream metabolite of
H2Se. In that case, some of the potential toxic effects attributed to H2Se,
Selenium Metabolism and Mechanisms of Cancer Prevention 121

such as generation of superoxide radical (13), might be avoided by delivering

the Se in pre-methylated forms, bypassing H2Se.

t.
body proteins

/
I plants (Se yeast, garlic)
I
~

• / •
Se-methionine Selenoproteins H2SeO 3 Se-methyl

t
Se-cysteine

Se-cysteine

•
se-Pholhate GSSeSG

lyase
... ......

•t
[H 2Se ] GSSeH

lyase
CH 3SeH

Figure 1. Selenium metabolism in animals (5). Plants supply Se to animals in the form of
methylated selenoamino acids. Conversion to inorganic Se is necessary for synthesis of
selenoproteins. Se-methylselenocysteine does not get incorporated into proteins and is directly
converted to methyl selenol by cysteine conjugate ~-lyase. At normal levels of Se intake the
major urinary metabolites are monomethylated compounds (represented by methyl selenol).
These can undergo further methylation (see text).

Dimethyl selenide and trimethylselenonium ion have low tOXICIty,

partly because of the diminished reactivity of Se bound to two or three carbon
atoms, and partly because these metabolites are readily excreted. Dimethyl
selenide was not tested directly, but a non-volatile presursor, dimethyl
selenoxide, was used to generate it. This compound had relatively low
chemopreventive activity and was rapidly eliminated as dimethyl selenide
(12). Trimethylselenonium chloride had little or no chemopreventive activity
at dietary Se levels as high as 80 ppm, even though at 40 ppm Se it had

Table 1. Anticancer efficacy vs. bioavailability of selenium compounds

Compound Dietary Se for 50% Tumor GSH Peroxidase
Inhibition (% of Control)

Se-Meselenocysteine 2 ppm 105 @0.5 ppm

(CH3)3Se+CI- No effect at 80 ppm 105 @ 40 ppm

(C6H5»Se+Cr 10-20 ppm I @ 30 ppm

122 H. E. Ganther

bioavailability sufficient for full synthesis of the selenoenzyme, glutathione

peroxidase (14) (Table I). In contrast, the novel organoselenium compound,
triphenylselenonium chloride, had chemopreventive activity at 5 ppm Se but
had no measurable bioavailability for synthesis of the selenoenzyme at 30
ppm Se (15); moreover, it produced no toxic effects at a level of 200 ppm Se.
From these results it can be concluded that (i) selenoprotein synthesis can be
independent of anticancer efficacy, and (ii) anticarcinogenic efficacy can be
obtained independent of selenoprotein synthesis. In the case of
triphenylselenonium chloride, there appears to be a rather distinct separation
of anticancer efficacy from bioavailability and overt toxicity.
Additional evidence that cancer preventive activity could be
independent of the inorganic Se pool was obtained using arsenite as a probe
(16). A low level (5 ppm As) of arsenite alone had little cancer preventing
activity. When this level of arsenite was fed together with a chemopreventive
level of inorganic selenite, the chemopreventive activity was largely
eliminated. However, when methylated forms of Se were used in
combination with arsenite, chemopreventive activity was enhanced (16).
Arsenite is known to antagonize many of the effects of inorganic selenium,
and its failure to inhibit the chemopreventive activity of methylated forms of
Se suggests that the formation of inorganic Se is not involved in the
chemopreventive effects obtained with methylated Se compounds.
Chemopreventive efficacy for the final products of Se metabolism is
shown diagramatically in Fig. 2. Activity is lower for HZSe and reaches a
peak at the monomethylated stage. Further methylation diminishes activity
and increases excretability. This model predicts that with blockage (bars) of
HzSe methylation by inhibitors (arsenite) or genetic mutation, the absence of
a functional methyl transferase activity would prevent the formation of
monomethylated Se, thus giving lower chemopreventive activity with
inorganic forms of Se. If Se is delivered at the monomethylated level from a
suitable methylated precursor, however, there is no need for Step 1
mPthylation, and activity is as high or even higher because inactivation of
monomethylSe by further methylation to dimethyl selenide would decrease if
thiol S-methyltransferase is inhibited. In a human population where genetic
polymorph isms are known to affect methylating abilities (17), individuals
having low activity for enzymes catalyzing the initial stages of HZSe
methylation might respond poorly to chemopreventive forms of Se such as
inorganic salts or selenomethionine that are metabolized through the HZSe
pool, but would likely show a response with Se compounds delivering Se in
monomethylated forms.
Selenium Metabolism and Mechanisms of Cancer Prevention 123

Methylated Se c===-? Cl\SeH

p~~ /

Efficacy HzSe

(Cij~e

\
(Cij)~e'"

Excretability

Figure 2. Profile of chemopreventive activity For Se metabolitrs. The bars indicate metabolic
steps where interference with Se methylation might influence relative chemopreventive
activities for inorganic forms of Se vs. methylated precursors (see text).

SE-METHYLSELENOCYSTEINE METABOLISM

Se-methylselenocysteine was one of the first naturally-occurring

forms of Se to be identified in plants (18). It is present at high levels in
certain species of Astragalus known as Se accumulators that tolerate high
levels of Se. It is also the major form of Se in selenized garlic (8). The
biosynthesis of Se-methylselenocysteine results from methylation of
selenocysteine by a specific Se-methyltransferase recently identified in
Astragalus that is inactive with cysteine but efficiently methylates
selenocysteine, thus diverting selenocysteine to a form that cannot be
incorporated into proteins (19).
Se-methylselenocysteine was readily metabolized to mono-, di-, and
tri-methylated forms of Se in animals (11, 20) and scission by a lyase to
methyl selenol was suggested (16). Cysteine conjugate p-Iyase, found in
kidney and other tissues, has no activity with S-methy1cysteine but shows
good activity with Se-methylselenocysteine and a number of other Se-
alkylselenocysteine derivatives (21). Such activity makes it feasable to use
Se-alkylselenocysteine derivatives as dietary chemopreventive agents that
release the Se-alkyl moiety upon metabolism in vivo. Using this approach,
the activity of Se-methyl-, Se-propyl-, and Se-allyl derivatives was compared
at a level of 2 ppm dietary Se in the rat methylnitrosourea model (22). The
Se-methyl and Se-propyl derivatives each gave about 50% reduction in tumor
124 H. E. Ganther

formation, whereas Se-allylselenocysteine gave almost 90% inhibition.

These results show that the presence of a terminal double bond in the 3-
carbon Se-allyl derivative confers greater activity compared to the saturated
analogue or methyl derivative. Additional studies with Se-allylselenocysteine
are in progress.
Since S-allykysteine and related allyl sulfides are major forms of
sulfur in garlic, it was anticipated that growth of garlic on a selenized
medium might result in biosynthesis of Se-allylselenocysteine. Selenized
garlic was shown to have very good chemopreventive activity, associated
with a water-soluble component (23, 24). Chemical speciation studies have
shown that Se-methylselenocysteine derivatives are the major forms of Se in
selenized garlic, and Se-allyl derivatives were not detected (25). The
chemopreventive activity of selenized garlic therefore is attributed to Se-
methylselenocysteine. Se-garlic is a convenient means to deliver this form of
Se using plant-based technology (23). The cancer preventing activity of Se-
garlic was shown to be about twice that of selenized yeast in the rat models.
This difference likely is related to Se-methylselenocysteine being the major
form in Se-garlic whereas selenomethionine is the major form in Se-yeast
(25).

CELLULAR MECHANISMS OF ACTION

Initial studies using inorganic sodium selenite showed inhibitory

effects on growth, DNA synthesis, and many other cellular processes (4).
These effects were more pronounced with transformed cells compared to
normal cells, and greater in early stages of carcinogenesis (26). Apoptosis is
an important mechanism for chemoprevention because the deletion of
initiated cells prevents clonal expansion. Beginning with studies in
Thompson's laboratory (27), many studies have shown the importance of
apoptosis for Se chemoprevention (4). Apoptosis is one of several cellular
processes showing very different action of monomethylated forms of Se vs.
inorganic Se (4). Sinha and Medina observed altered cell cycle kinetics and
found cdk2 kinase activity in mammary tumor cells was decreased by Se-
methylselenocysteine, but not by inorganic selenite (29). Lu discusses
apoptogenic mechanisms as well as his recent discovery of differential anti-
angiogenesisis effects with monomethylated Se vs. inorganic Se in the
following paper (28).
If monomethylated forms of Se such as methylselenol are critical
chemopreventive metabolites, it would be expected that the direct addition of
simple compounds generating methylselenol in vitro would be effective
against transformed cells. With in vitro studies, systemic metabolism is not
involved. Better control of experimental conditions is achieved, and the
genetic makeup of cells can be varied. Direct addition of methylselenol is not
Selenium Metabolism and Mechanisms of Cancer Prevention 125

feasible because of its facile oxidation, but alternative forms that are
reducible to methylselenol can be used. Methylselenocyanate (CH3SeCN)
has substantial volatility and stench. Methylseleninic acid (CH3SeOZH) is
more convenient to use because it ionizes at neutral pH in aqueous solutions
to give the nearly odorless anion, and a number of studies are in progress
using this compound. It has been shown to inhibit growth and DNA synthesis
at low micromolar levels (30), and the recently described anti-angiogenic
effects (28) are especially interesting. It has oxidizing properties comparable
to selenite. A number of mechanisms based on formation of Se adducts with
cysteine sulfhydryl groups of proteins or catalysis of reactions involving
cysteine residues can be formulated. These mechanisms have been discussed
at length in a recent review (3).

CYSTEINE CLUSTERS IN PROTEINS AS TARGETS

Clusters of cysteine residues occur in catalytic centers, regulatory

sites, hormone binding domains, metal-binding sites, and in ion channel
proteins. The stoichiometry of the reaction of oxidized forms of Se with
sulfhydryl groups (up to four SH per Se) can be a basis for selectivity in
protein modifications. Gopalakrishna's group has shown that the catalytic
subunit of protein kinase C is inhibited by micromolar levels of selenite,
whereas proteins lacking such clusters are unaffected (31).
Methylseleninic acid reacts with thiols in a 1:3 stoichiometric ratio,
and modification of a protein cysteine cluster composed of three cysteine
residues is depicted in Fig. 3. A methylselenenylsulfide derivative is formed
with one cysteine, using reducing equivalents supplied by oxidation of two
cysteine residues to an intramolecular disulfide bond.

Figure 3. Proposed modification of protein cysteine cluster with methylseleninic acid.

126 H. E. Ganther

A parallel type of modification was proposed for the reaction of methyl

methanethiosulfonate with glucocorticoid receptor thiols, where the
formation of a methylthio mixed disulfide and an intramolecular disulfide in
the binding pocket for the steroid hormone inhibit steroid binding (32).
Benzeneseleninic acid recently was shown to release zinc from tightly-bound
zinc-sulfur clusters in metallothioneir.; the authors suggest that displacement
by Se of zinc in zinc fingers of transcription factors or proteins involved in
cell signaling could be a potential chemopreventive mechanism (33).
Formation of protein-Se adducts or catalysis of thiol/disulfide
reactions may affect redox signaling (3). Figure 4 depicts a model where
transcription factors, ion channel proteins, and other redox-regulated proteins
could be poised in two states, ON/OFF, differing by the oxidation state of
cysteine, and subject to time-limited activation. Se is proposed to catalyze
reactions that return the activated state to the basal state by forming more
reactive S-Se intermediates. The result is faster resetting of the basal state,
and less time in the activated state.

Figure 4. Se as a redox catalyst in modulation of redox-regulated proteins (3).

SELENOPROTEINS AND CANCER

CHEMOPREVENTION

All of the known functions of Se as an essential nutrient involve

selenoproteins. The presence of selenocysteine in proteins enables unique
chemistry and high amplification mechanisms of action for Se. Although
many believe that anticancer effects observed with Se at near nutritional
levels must involve selenoproteins, the experimental animal models have not
shown evidence th?t selenoproteins are increased by chemopreventive levels
of Se. As discussed at greater length elsewhere, newly discovered
selenoproteins such as thioredoxin reductase or novel selenoproteins having
unidentified functions must be considered, along with classic Se-dependent
peroxidases (3). Although chemopreventive activity is seen with Se
compounds in which Se is unavailable for selenoprotein synthesis,
selenoproteins hold promise for roles in cancer prevention, along with the
better established roles for low molecular weight Se metabolites.
Selenium Metabolism and Mechanisms of Cancer Prevention 127

Lu has recently proposed an attractive model that integrates both

concepts, with particular emphasis on the microvascular system (28). He
proposes that Se interferes with angiogenesis, so that vascularization needed
for growth and progression of small, early stage tumors is precluded. Such
avascular masses of cells are suggested to develop a conditioned Se
deficiency, so that synthesis of selenoproteins is impaired, even though Se
supply elsewhere may be sufficient to allow the synthesis of normal levels of
selenoproteins. His model predicts that higher Se intakes are required in
order to drive sufficient Se into the avascular areas.
Based on physicochemical considerations of Se delivery, it might be
speculated that some of the beneficial effects of methylated Se metabolites
are exerted through provision of a bioavailable source of Se in the form of
low molecular weight, readily diffusible forms of Se that can traverse
avascular areas more readily than larger, more polar forms such as
glutathione derivatives or inorganic Se. Production of methylated
metabolites rises markedly with the attainment of chemopreventive levels of
dietary Se (II). The greatest relative increase in any single metabolite was
the IOO-fold increase observed for dimethyl selenide. This volatile, neutral
compound has sufficient lipophilic properties to move easily through
membranes but also has appreciable water solubility. It is known that
compounds such as dimethyl selenoxide, that generate mainly dimethyl
selenide, provide sufficient Se for synthesis of selenoproteins (14).
Moreover, anticarcinogenic activity of selenobetaine methyl ester, a
compound undergoing direct and extensive metabolism to dimethyl selenide
(34), is comparable to Se-methylselenocysteine and greater than that for
inorganic selenite (35). The concept that methylation of Se might have
implications for delivery of Se in vivo, where the degree of vascularization of
tissues can influence the supply of nutrients, helps bridge the gap between in
vivo studies suggesting efficacy even for extensively methylated forms of Se,
and in vitro studies using cell cultures, where such compounds generally lack
activity. Moreover, it is helpful in connecting the established efficacy of
methylated Se metabolites with the tradional role of Se in selenoproteins.
Multiple chemopreventive mechanisms, as in the integrated model of Lu, are
likely to be involved.
It is concluded that animals have a long, comfortable relationship
with methylated forms of Se. They consume methylated selenoamino acids
in the diet, metabolize them to release methylated or inorganic forms, and
readily methylate any excess inorganic Se for excretion. Through such
metabolic processes, chemopreventive methylated Se metabolites are
generated, with a peak in activity at the monomethylated stage. The
appearance of dimethylselenide in breath may be a useful marker for
attainment of maximal levels of Se methylation. There is evidence that Se
selectively affects transformed cells compared to normal cells, and has
greater effectiveness in early stages of carcinogenesis. Multiple
chemopreventive actions are known, including growth inhibition, altered cell
128 H. E. Ganther

cycle kinetics, and induction of apoptosis independent of DNA damage and

functional p53. Se-methylselenocysteine is a good precursor for generating
active monomethylated Se metabolites in vivo. It is more active than
selenomethionine, gives a lower body burden of Se, and is rapidly eliminated
upon cessation of intake. It delivers a steady stream of monomethylated Se in
one step, beyond the hydrogen selenide pool. It occurs naturally in plants and
can be delivered through food sources or provided in synthetic form.

ACKNOWLEDGMENT
This contribution was supported by grant no. CA 45164 from the
National Cancer Institute, NIH.

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