Carcinogenesis vol.19 no.9 pp.1603–1607, 1998
Synthesis and excretion profile of
1,4-[14C]phenylenebis(methylene)selenocyanate in the rat
Karam El-Bayoumy1, Pramod Upadhyaya, Ock-Soon
Sohn, José G.Rosa and Emerich S.Fiala
American Health Foundation, 1 Dana Road, Valhalla, NY 10595, USA
1To
whom correspondence should be addressed
1,4-Phenylenebis(methylene)selenocyanate (p-XSC) inhibits
chemically induced tumors in several laboratory animal
models. To understand its mode of action, we synthesized
p-[14C]XSC, examined its excretion pattern in female CD
rats and also the nature of its metabolites. p-[14C]XSC was
synthesized from α,α-dibromo-p-[ring-14C]xylene in 80%
yield. The excretion profile of p-[14C]XSC (15.8 mg/kg body
wt, 200 µCi/rat, oral administration, in 1 ml corn oil) in vivo
was monitored by measuring radioactivity and selenium
content. On the basis of radioactivity, ~20% of the dose was
excreted in the urine and 68% in the feces over 3 days. The
cumulative percentages of the dose excreted over 7 days were
24% in urine and 75% in feces, similar to excretion rates of
selenium. According to selenium measurement, <1% of the
dose was detected in exhaled air; radioactivity was not
detected. Only 15% of the dose was extractable from the
feces with EtOAc and was identified as tetraselenocyclophane (TSC). Most of the radioactivity remained tightly
bound to the feces. Approximately 10% of this bound
material converted to TSC on reduction with NaBH4.
Organic soluble metabolites in urine did not exceed 2% of
the dose; sulfate (9% of urinary metabolites) and glucuronic
acid (19.5% of urinary metabolites) conjugates were
observed but their structural identification is still underway.
Co-chromatography with a synthetic standard led to the
detection of terephthalic acid (1,4-benzenedicarboxylic acid)
as a minor metabolite. The major urinary conjugates contained selenium. Despite the low levels of selenium in the
exhaled air, the reductive metabolism of p-XSC to H2Se
cannot be ruled out. Identification of TSC in vivo indicates
that a selenol may be a key intermediate responsible for the
chemopreventive action of p-XSC.
Introduction
Chemoprevention refers to the administration of either synthetic or naturally occurring chemical agents that minimize or
prevent tumor initiation (mutational) and/or tumor promoting
events during the process of carcinogenesis (1). The list of
agents with chemopreventive activity is growing rapidly (2).
One important class of chemopreventive agents is that of
organic and inorganic forms of selenium (3). Studies in our
laboratory have focused on the design of synthetic organoselenium compounds with improved chemopreventive efficacy
and lower toxicity than inorganic selenium compounds, and
Abbreviations: AAS, atomic absorption spectrometry; BSC, benzyl selenocyanate; HPLC, high performance liquid chromatography; NMR, nuclear
magnetic resonance; p-XSC, 1,4-phenylenebis(methylene)selenocyanate; THF,
tetrahydrofuran; TMSe, trimethylselenonium; TSC, 1,4-tetraselenocyclophane.
© Oxford University Press
the naturally occurring selenoamino acids (e.g. selenomethionine) (3). Ideally, such agents would be used as dietary
supplements to inhibit tumor development caused in different
organs by various classes of chemical carcinogens. We have
demonstrated repeatedly that one of the most effective of these organoselenium compounds, 1,4-phenylenebis
(methylene)selenocyanate (p-XSC), inhibits tumors in the
mammary glands, colon, lung and tongue of laboratory
animals (4,5).
Our results combined with other observations described in
the literature make it clear that the chemopreventive efficacy
of selenium as an anticarcinogen depends on the chemical
form in which it is administered, suggesting that metabolism
is a prerequisite for the cancer preventive potential of selenium
compounds (6). Understanding the metabolism of organoselenium compounds is essential to determine whether the
parent compound or any of its metabolites are responsible for
the chemoprevention of tumorigenesis. To this end, and as an
initial investigation, the synthesis of p-[14C]XSC, the most
potent chemopreventive organoselenium agent, was performed,
its excretion profile in female CD rats was studied and some
of its metabolites were identified. We anticipate that more
detailed knowledge of p-XSC metabolism will provide
important leads to the development of even more effective
chemopreventive organoselenium compounds.
Materials and methods
Apparatus
Chromatographic analyses were done with a Waters Associates Model ALC/
GPC 204 high-performance, high-speed liquid chromatograph equipped with
a Model 510 solvent delivery system, a Model U6K septumless injector, a
Model 440 UV/visible detector and a Model 680 automatic gradient controller.
Mass spectra were obtained on a Hewlett-Packard Model HP5988A mass
spectrometer. 1H NMR studies were performed on a Bruker AM 360-MHz
spectrophotometer, in DMSO-d6 and DMSO-d6/D2O. A Beckman Model
LS9800 scintillation counter was employed for measuring radioactivity in a
scintillation cocktail and radiochromatography was done with a radioactivity
flow detector (Radiomatic Instruments and Chemical Co. Inc., Tampa, FL).
Atomic absorption spectrometry (AAS) was employed for selenium determination, i.e. an AAS, Varian AA-1475 series (Varian Tectron, Springvale,
Australia) was used. This instrument was equipped with a deuterium arc
background correction and a Model GTA-95 graphite tube atomizer with
sample dispenser. With argon (99.996%) as the sheath and purge gas, the
parameters described by Voth-Beach and Shrader (7) were utilized for operating
the furnace. A hollow cathode lamp was used with background correction in
the ‘on’ position, lamp current, 10 or 12 mA; wavelength, 196 nm; slit, 1 nm.
In most cases, samples were dissolved or diluted in 14% HNO3, and for each
injection 40 µl of sample was introduced into the pyrolytically-coated graphite
tube along with 10 µl of reduced Pd matrix modifier (3.33 g of PdCl2/l, 2%
hydroxylamine hydrochloride and 2% HCl) by the sample dispenser (8). The
peak height of absorbance was recorded, and the analyses were based on the
mean of duplicate injections. Ganther et al. (9) have described the difficulties
encountered in the analysis of trimethylselenonium ion (TMSe) in the urine
by AAS. In order to address this problem, the iodine salt of TMSe was
obtained commercially (Organometallics Inc., East Hampstead, NH) and
dissolved in H2O. Indeed, we found that TMSe was underestimated by our
method upon using a final concentration of 0.35% of HNO3. However, with
HNO3 concentration of 7% or even higher (14%), which is used in the current
study, we achieved 100% recovery. In the case of rat urine spiked with TMSe,
the recovery was ~80% at 7% HNO3. The reason for this difference is not
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K.El-Bayoumy et al.
Fig. 1. Electron impact mass spectrum of 1,4-tetraselenocyclophane.
yet clear. However, when urine samples that had been spiked with TMSe
were digested using the microwave digestion system as described previously
(8), full recovery was achieved. This experimental approach was used for the
analysis of urine samples from rats treated with p-XSC.
Chemicals
p-XSC was obtained as described previously (10). α,α-Dibromo-p-[ring14C]xylene was obtained commercially (Chemsyn Science Laboratories,
Lenexa, KS; 100 mCi/mmol, radiochemical purity .95%). Briefly, the
synthesis of p-[14C]XSC was as follows. As for the synthesis of [14C]benzyl
selenocyanate (BSC) (11,12), and for the unlabeled p-XSC (10), the bromo
compound was added dropwise to a solution of KSeCN in acetone at room
temperature and was stirred for 12 h. The reaction mixture was diluted with
CH2Cl2 and washed with H2O. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The pure p-[14C]XSC was purified by
HPLC; Rt 5 22–23 min, with an elution gradient of 20% CH3OH–H2O to
100% CH3OH in 60 min at a flow rate of 3 ml/min, using a semi-preparative
Vydac-C18-10 µ reverse phase column (1.3325 cm, Separations Group,
Hesperia, CA). The yield was 80% and the purity of the compound was
.96% as ascertained by HPLC and radioflow monitored radioactivity.
Synthesis of 1,4-tetraselenocyclophane (TSC)
To a solution of p-XSC (0.314 g, 1 mmol) in dry THF (34 ml), a solution of
KOH (0.06 g, 1 mmol) in CH3OH (34 ml) was added over a period of 4 h at
room temperature under N2. During this time, the solution turned cloudy and
then yellowish. The reaction mixture was stirred overnight at which point
TLC (100% CH2Cl2) showed the complete disappearance of p-XSC (Rf 0.58).
The polymer by-product was collected by filtration as a yellow powder
(0.2162 g). The filtrate was concentrated in vacuo to yield the crude TSC.
Purification by column chromatography on silica (initially eluted with hexane,
and gradually increasing CH2Cl2 until final elution with 40% CH2Cl2 in
hexane) yielded a pure product (0.017 g, 3%), Rf 0.85 (CH2Cl2). The mass
spectrum (Figure 1) showed the following fragmentation (m/z, relative
intensity) 528 (M1, 4%), 448 (M1-Se, 88%), 368 (M1-2Se, 12%), 288 (M13Se, 65%), 208 (M1-4Se, 30%); 1H NMR (CDCl3) δ 7.16 (s, 8H, ArH), 3.75
(s, 8H, -CH2); HPLC (254 nm) Rt was 65 min with an elution gradient of
20% CH3OH–H2O to 100% CH3OH over 60 min at a flow rate of 3 ml/min,
using a semipreparative Vydac-C18-10 µ reverse phase column.
The polymer by-product (0.05 g) was suspended in 2 ml of 4:1 THF:0.5 N
NaOH and 1 ml of CH3OH and then subjected to NaBH4 reduction in a N2
atmosphere. The reaction mixture was stirred for 10 min at 0°C and allowed
to warm up to room temperature over a period of 60 min under N2. The flow
of N2 was terminated and the reaction mixture continuously stirred overnight
at room temperature. At this time, CH2Cl2 (5 ml) was added and the reaction
mixture filtered. The filtrate was washed with H2O (4310 ml), and the organic
layer dried and evaporated under reduced pressure to give a slightly yellow
solid in 20% yield. This solid had HPLC characteristics identical to those of
the synthetic standard, TSC.
Enzymes and reagents
Arylsulfatase (from limpets, type V) and β-glucuronidase (from Escherichia
coli, type IX) were purchased from Sigma (St Louis, MO). Monofluor was
acquired from National Diagnostics (Somerville, NJ).
In vivo metabolism of p-[14C]XSC in the rat
Female CD rats, ~200 g, body wt each, obtained from Charles River Breeding
Laboratories (Wilmington, MA), were acclimated for 2 weeks prior to
metabolism studies. Four rats (one rat/chamber) were housed in Delmar–
Roth-type glass metabolism chambers (Bioserv Inc., Frenchtown, NJ) designed
for urine and feces collection, and for trapping volatile metabolites in the
exhaled air over three sequential traps with 8 N HNO3 (13). Rats were fed a
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modified AIN-76A semipurified high-fat diet (10) 1 week before and then
throughout the experiment; diet and water were given ad libitum. The
metabolism room was controlled at 22 6 1°C and 50% humidity, 12 h light/
dark cycle. p-[14C]XSC was administered by intragastric gavage in 1 ml corn
oil at a dose of 15.8 mg/kg body wt, 200 µCi/rat. Urine and feces were
collected every 24 h for 7 days and stored below 0°C until analysis. The
contents of the gas wash bottles were checked for [14C] radioactivity and
selenium content daily for 1 week following p-XSC administration.
Analysis of urinary and fecal metabolites of p-[14C]XSC
Rat urine was extracted with ethyl acetate or benzene. The extract was dried
(MgSO4) and concentrated to dryness, and the residue dissolved in THF and
analysed by HPLC using a linear gradient from 2% CH3COOH in H2O to
100% CH3OH in 60 min at a flow rate of 3 ml/min using a Vydac-C18-10 µ
reverse phase column (1.3325 cm). Fractions were collected and analysed
for selenium content using AAS. For the isolation of water-soluble metabolites,
urine was adjusted to pH 5.5 with citrate buffer and the mixture incubated
with β-glucuronidase or arylsulfatase for 4 h at 37°C. In a typical experiment,
10 000 U (3 mg) of β-glucuronidase, or 63 U (5 mg) of arylsulfatase, and
20 mg of saccharic acid 1,4-lactone were used for each 10 ml of pH-adjusted
urine sample. Control experiments were performed as described above but in
the absence of enzymes. Feces were suspended in 50% ethyl acetate–ethanol
and vortexed at 37°C for 30 min. The residue was filtered and the filtrate was
dried (MgSO4), concentrated and analysed by HPLC. The percentage of
recovery of radioactivity eluted from the HPLC column was determined to
be 96.7 6 5.2%.
AAS was used for selenium determination in urine and exhaled air (8,14).
Urine samples were diluted with 14% HNO3 prior to analysis by AAS.
Results
The time courses of excretion of radioactivity in urine and
feces after oral administration of p-[14C]XSC to female CD
rats are depicted in Figure 2. Approximately 20% of the dose
was excreted in urine and 68% was excreted in the feces over
the course of 3 days. The cumulative percentages of the dose
excreted over 7 days were 24% in urine and 75% in feces;
consistent with a previous report, the analysis of selenium
content by AAS yielded comparable results (14). Less than
1% of the dose, based on selenium measurement, was detected
in the exhaled air; the latter was free of radioactivity. Only
15% of the dose was extractable from the feces with EtOAc.
HPLC analysis of the EtOAc extract showed a major and a
minor peak (Figure 3A); the major peak was identified as TSC
based on comparison of its chromatographic characteristics
with those of the synthetic standard. Although the synthesis
and characterization of other selenocyclophanes have been
described (15,16), this is the first report on the synthesis of
TSC. To determine the nature of the unextractable radioactivity,
the remaining dried pellets were suspended in 4:1 THF:0.5 N
NaOH and reduced with NaBH4 in CH3OH. The outcome of
this experiment indicates the formation of TSC in ~10% yield
(Figure 3B).
Approximately 1.8% of the dose was extractable with
Synthesis and excretion profile of p-XSC
Fig. 2. Excretion of radioactivity following administration of p-[14C]XSC to
female CD rats by gavage. For urine analysis, each time point represents the
average of four individual rats. For fecal analysis, each time point
represents one animal, since feces from three rats were pooled to obtain
ample materials for spectral analysis.
Fig. 4. Radiochromatogram obtained upon reverse phase HPLC analysis of
whole urine. On the basis of enzyme hydrolysis, peaks 1 and 4 were
identified as glucuronic acid and sulfate conjugate, respectively. Peak 3 was
identified as terephthalic acid (1,4-benzenedicarboxylic acid) based on
co-chromatography with the standard. Peaks 2, 5 and 6 remain to be
identified.
3) all other peaks contain selenium. On the basis of cochromatography, peak 3 was assigned as terephthalic acid; as
expected, it did not contain any measurable selenium. Enzymatic hydrolysis of water-soluble metabolites indicated the
presence of sulfate (9% of urinary metabolites) and glucuronide
conjugates (19.5% of urinary metabolites). The radioactivity
eluting between 29–31 min (peak 4) decreased drastically
following sulfatase hydrolysis. Radioactivity eluting between
10–12 min (peak 1) almost disappeared upon β-glucuronidase
hydrolysis. These results indicate that peaks 1 and 4 are
glucuronic acid and sulfate conjugates, respectively. Structural
determination of these two major urinary conjugates requires
further investigation.
Discussion
Fig. 3. Radiochromatogram obtained upon reverse phase HPLC analysis of
EtOAc extractable metabolite from feces (A); NaBH4 reduction of the
unextractable material in the feces (B).
EtOAc, but ,0.05% of the dose was extractable with benzene
from the urine. Figure 4 shows a typical radiochromatogram
obtained upon HPLC analysis of 24 h urine sample after
p-XSC administration. Qualitatively similar radiochromatograms were obtained following HPLC analysis of urine voids
collected after 48, 72 and 168 h. Analysis of urine fractions
collected, clearly showed that (with the exception of peak
The purpose of this study was to examine the excretion
profile and to provide some structural information of in vivo
metabolites of p-XSC in an animal model (female CD rats)
that is currently being used in efficacy studies for the evaluation
of organoselenium compounds as chemopreventive agents
against mammary cancer. Such knowledge is essential to
understand the mode of action of p-XSC and provide insights
as to those forms of selenium that are responsible for its
chemopreventive effects (4,17).
The dose of p-XSC was selected based on our previous
study (14) in which two doses (15.8 and 1.58 mg/kg body wt)
were employed. The results of the excretion kinetics were
highly comparable at the two dose levels (14). Therefore, for
comparative purposes, we used 15.8 mg/kg body wt in the
present investigation. In chemoprevention protocols a range
of doses (5, 10, 15 and 40 p.p.m. as selenium in the diet) was
used (10,17). Accordingly, one can estimate the daily dietary
intake of selenium. For example, at 15 p.p.m. selenium in the
diet, the daily consumption would amount to 300 µg selenium
per 200 g rat consuming 20 g diet/day; this is comparable
with a single oral administration of 1.58 mg/kg body wt which
was used in our previous study (14).
The results indicate that fecal elimination is the major route
of excretion; TSC was identified as a minor fecal metabolite.
However, detectable amounts of the parent compound, pXSC, were not observed. Although the major portion of the
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K.El-Bayoumy et al.
radioactivity excreted in the feces remained unknown, it
appeared that a high molecular weight metabolite(s) containing
an Se–S bond may be present. This notion is based on the fact
that this material was not extractable in organic solvents, but
yielded TSC upon NaBH4 treatment. Collectively, these results
indicate that the facile reaction of p-XSC with proteins and
amino acids containing a thiol (-SH) functionality can yield
intermediates with an Se–S bond. This type of reaction has
been demonstrated with organoselenium compounds (12,18–
20). Moreover, compounds containing functional groups other
than selenocyanate (e.g. ebselen) were postulated to react
with proteins containing SH groups to form Se–S-containing
intermediates (21). Reductive cleavage of the Se–S bond of
such intermediates will lead to the formation of selenols that
will be highly susceptible to oxidation to form diselenide
containing the Se–Se functionality. In the present study, the
reductive metabolism of such an intermediate metabolite(s)
derived from p-XSC, led to the formation of selenols and,
finally, to TSC. The formation of selenols is of particular
significance since it is now well established that the anti-tumor
properties of selenium compounds are strongly influenced by
their metabolism and, furthermore, selenols appear to be
important intermediates in cancer prevention by selenium
compounds (6,12,22–24).
We have demonstrated previously the formation of benzyl
selenol, benzyl seleninic acid and dibenzyl diselenide as
metabolites of BSC (12). We hypothesize that selenols derived
from aryl selenocyanate may be acting as chemopreventive
intermediates in a similar fashion as methyl selenol derived
from sodium selenite (6,20,21). Therefore, the role of TSC
and possibly that of selenols derived from p-XSC in efficacy
studies will be evaluated shortly in our laboratories.
The anti-tumor properties of selenite are strongly influenced
by its metabolism. After absorption, selenite is reduced by
glutathione via selenodiglutathione to the highly toxic H2Se
(6,25). The latter can be incorporated as selenocysteine into
selenoproteins, such as glutathione peroxidases (26–29), type
I iodothyronine deiodinase (30,31), or the 57 kDa plasma
selenoprotein and selenoprotein P (32). H2Se is methylated to
mono-, di- and tri-methylated derivatives before excretion.
Several experiments with compounds that are directly converted into methyl selenol indicate that the latter mediates the
anticarcinogenic effect of selenite (6,23,24). In the case of
p-XSC metabolism, ,1% of the selenium dose was detected
in the exhaled air. Despite the low levels of selenium in the
exhaled air, the reductive metabolism of p-XSC to H2Se cannot
be ruled out. In fact, Ip et al. (17) have shown that p-XSC
restores glutathione peroxidase activity in selenium-deficient
rats which supports the formation, although minor, of inorganic
selenium.
Our results indicate that urine is a minor route of excretion;
water-soluble metabolites constitute most of the radioactivity
excreted in urine after oral administration of p-[14C]XSC. We
did not detect TMSe as a urinary metabolite. Moreover,
unmetabolized p-XSC was not detected in the organic-extractable metabolites of urine that accounted for ,2% of the dose.
The lack of selenium in peak 3 and its chromatographic
characteristics support the identity of this metabolite as terephthalic acid. Cleavage of selenium from p-XSC leading to the
formation of terephthalic acid in the urine is a minor pathway;
an observation which is in line with the low levels of selenium
found in the exhaled air as described above. Although this
constitutes only a minor urinary metabolite, its formation is
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expected since, in our previous investigation, we were able to
identify benzoic acid as a metabolite from the monofunctional
selenocyanate compound BSC (12).
Selenols are highly nucleophilic in nature and their biotransformation to glucuronic acid conjugates has been suggested
with other organoselenium compounds (21). On the basis of
enzyme hydrolysis, we demonstrated that peak 1 is a glucuronic
acid conjugate; the same approach was also used to assign peak
4 as a sulfate conjugate. Unequivocal structural identification of
these conjugates requires further investigation.
In the present investigation, we did not measure levels of
selenium in various organs since we have already reported
(14) that ,5% of selenium was distributed among all major
organs of the rat following the oral administration of p-XSC
at a dose equal to that used here. However, in a separate study
(unpublished data) and under chemoprevention protocols (15
p.p.m. as selenium in the diet), we found that the liver had
the highest selenium content, followed by the kidney and the
target organ (mammary) had the lowest selenium content. The
method employed for selenium analysis in these organs cannot
provide structural information on the form of selenium (14).
Collectively, on the basis of the present investigation and
results described in the literature, we believe that the major
focus in cancer prevention by organoselenium compounds may
be directed toward the design of agents that lead to the facile
formation of selenols in vivo. Currently, our efforts focus on
testing this hypothesis.
Acknowledgements
We thank Mrs Patricia Sellazzo for preparing, and Mrs Ilse Hoffmann for
editing, this manuscript. This work was supported by grant CA 46589 from
the National Cancer Institute. This is paper no. 24 in the series ‘Selenium in
the Chemoprevention of Carcinogenesis’.
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Received on March 9, 1998; revised on May 22, 1998; accepted on May
29, 1998
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