Advances in Biological Chemistry, 2011, 1, 15-23 ABC
doi:10.4236/abc.2011.12003 Published Online August 2011 (http://www.SciRP.org/journal/abc/).
Published Online August 2011 in SciRes. http://www.scirp.org/journal/ABC
The role of vitamin C in alteration of enzymes responsible of
energy metabolism induced by administration of tamoxifen
to mouse*
Zaina Zuhair, Hasan ALamri
Girls College of Education, King Abdulazia University, Jeddah, Saudi Arabia.
Received 29 April 2011; revised 2 June 2011; accepted 13 June 2011.
ABSTRACT
Tamoxifen is a synthetic non-steroidal ant estrogen. It
was suggested to study the role of vitamin C in alte-
ration of enzymes responsible of energy metabolism
induced by administration of tamoxifen to mouse. The
effect of tamoxifen and tamoxifen with vitamin C on
some activity of enzymes in the mice represe nti ng g ly-
colytic, gluconeogenic and glycogenolytic pathway and
also, liver function enzymes represented by aspartate
aminotransferase (AST), alanine amino-transferase
(ALT), acid phosphatase (ACP) and alkaline phos-
phatase (ALP) were studied. The present results
showed that a significant (p < 0.001) increase in
glycolytic enzymes (HK, PK, GPI and PFK), glu-
coneogenic enzymes, G-6-Pase, acid phosphatase
(ACP), alkaline phosphatase (ALP) and glucose, were
observed in treated groups, while LDH, glycogen phos-
phorylase, AST and ALT enzymes activities showed
significant (p < 0.01) reduction. The present results
also, showed that significant reduction in glycogen,
total protein, total cholesterol, uric acid, urea, and
creatinine in treated mice as compared to the normal
healthy control group. However, normal control mice
treated with tamoxifen and vitamin C showed no side
effects of most parameters compared to the normal
healthy control group. It was concluded that vitamin C
may prevent tamoxifen-induced testes toxicity in mice.
Keywords: Adult Male Albino Mice; Tamoxifen
Vitamin C; Liver Tissue Homogenates
1. INTRODUCTION
Tamoxifen is one of the most effective synthetic non-
steroidal ant estrogenic compounds, it is widely used in
the treatment of advanced hormone-dependent breast
cancer which is the most worldwide common form of
cancer in women [1] by binding to estrogen receptors
and suppressing epithelial proliferation [2,3] and as ad-
juvant therapy following surgery in early stages of the
disease. Tamoxifen is also, proposed for the prevention
of cancer amongst high risk women [4]. Such an ap-
proach requires objective and accurate evolution of the
side effects which could result from the administration
of this drug.
Some studies showed that tamoxifen has adverse side
effects on the cardiovascular system, bone metabolism
and liver. Also, tamoxifen caused cytotoxicity on pri-
mary cells from human multiple organs: Kidney, liver
and lung [5].
Vitamin C is an antioxidant agent that limits the injury
produced by drugs. Vitamin C is an essential nutrient
that functions as a non-enzymatic antioxidant in the cy-
tosol. The various experimental studies indicated that
this vitamin is effective in preventing the oxidative renal
damage and stress [6].
It is well known that the liver is one of the major tar-
get organs affected by drug, where the most metabolic
processes are usually located. The most important me-
tabolic pathways are glycolysis, gluconeogenesis and
glycogenolysis contributions of these pathways elucidate
the metabolic relationship between glucose, glycogen
and energy release. The characteristic pattern of change
in some enzymes activity representing glycolytic path-
ways as (hexokinase (HK), pyruvate kinase (PK), phos-
phofructokinase (PFK), lactate dehydrogenase (LDH),
and glucose phosphate isomerase (GPI); glycogenolytic
pathways as glycogen phosphorlase, glucose-6-pho-
sphatase (G-6-Pase); gluconeogenic pathways as fruc-
tose-1-6 diphosphatase (F-D-P ase), phosphoenolpyru-
vate carboxykinase (PEPCK). as well as glycogen con-
tent in soft tissue and glucose in serum as bioenergetics
parameters of critical importance in reflecting the phy-
siological alteration of animals under the stress [7].
Carbohydrates and amino acids are one of the impor-
tant parameters which could be used as another indicator

Z. Zuhair et al. / Advances in Biological Chemistry, 2011, 1, 15-23 16
of the stress by molluscicides [8].
The aim of the present work is to cast more light on
the toxic effect of tamoxifen on some liver enzymes in
mice representing metabolic pathways are glycolysis,
gluconeogenesis and glycogenolysis and the role of vita-
min C in minimizing the toxicity induced by tamoxifen.
2. MATERIALS AND METHODS
2.1. Experimental Animals
Adult male albino mice in the present investigation were
obtained from Schistosome Biological Supply Program
(SBSP), Theodor Bilharz Research Institute, Cairo, Egypt.
They were 3 months old and of an average weigh of 25 -
30 g. They were fed ad labium with a standard diet. They
were kept in cages and acclimatized in the laboratory for
7 days prior to experimentation.
2.2. Applied Drugs
Tamoxifen and vitamin C were purchased from Che-
mical Industries Development (CID) Company, Al-Harm,
Giza, Egypt as tablets for oral administration, each tab-
let contains 40 mg and 500 mg of the active ingradi-
ents, respectively. The therapeutic dose of each drug
for mice was calculated according to the table given by
Paget and Barnes [9].The therapeutic doses of ta-
moxifen and vitamin C were calculated as 0.1 mg/kg
and 1.25 mg/kg for mice, respectively. The doses were
given orally and estimated according to the body weight
of the mouse.
2.3. Experimental Design
The mice were divided into three equal groups. The 1st
group served as control and received distilled water. The
2nd group was daily administrated 0.1 mg tamoxifen. the
3th group were given daily 1.25 mg vitamin C simulta-
neously with the dose of 0.1 mg tamoxifen. All the doses
were given daily for 28 days and sacrificed after month.
Animals’sacrifice and examination stared 4 weeks post
treatment; it was done on successive days. In each day
only two animals from each group were sacrificed. Each
liver was then taken and divided into 0.25 g portions.
The liver portion were taken and covered with aluminum
foil and stored at –4˚C until used for homogenization
and biochemical assays. All mouse were subjected to
determine HK, PK, GPI, LDH, PFK, FD Pase, PEPCK,
G-6-Pase, AST, ALT, ADP, ALP and glycogen phos-
phorylase enzyme activities as well as, glycogen and
protein content in liver tissue. Glucose, total cholesterol
uric acid, urea, and creatinine in sera.
2.4. Preparation of Liver Tissue Homogenates
On the day of each the following enzyme parameter assay,
one portion weighing 0.25 g from each liver aluminium
package was taken and homogenized in 2.5 ml of the spe-
cific recorded solution to give 10% concentration and then
used for assay. Similar periods elapsed between homog-
enization and enzyme was assayed in two livers from
each group on the same days.
2.5. Bleeding and Preparation of Serum
Blood samples were collected, after 4 weeks post-treat-
ment. Collected blood samples were centrifuged at 3000
rpm for 10 minutes and sera were stored immediately at
–80°C until time of analysis.
2.6. Enzyme Assays
All physiological parameters determined in this study
were determined spectrophotometrically, using reagent
kits purchased from BioMerieux Company, France.
Hexokinase (HK) was assayed according to the me-
thod of Uyeda and Raker [10]. Pyruvatekinase (PK) ac-
cording to the method of McManus and James [11].
Glucose phosphate isomerase (GPI) according to the
method of King [12], Phosphofructokinase (PFK) ac-
cording to the method of Zammit et al. [13] and Lactate
dehydrogenase (LDH) activity according to the method
of Cabaud and Wroblewski [14]. Phosphoenolpyruvate
carboxykinase (PEPCK) according to the method of
Suarez et al. [15] and Glucose-6-phos-phatase according
to the method of Swanson [16]. Fructose-1,
6-diphosphatase (FD Pase) according to the method of
Sand et al. [17] and Glycogen phosphorylase according
to Hedrick and Fischer [18]. Aspartate and alanine ami-
notransferases according to the method of Reitman and
Frankel [19]. Acid phosphatase and alkaline phosphatase
activities according to Fishman and Ferner [20] and
King and King [21] respectively. Total protein according
to the method of Lowry et al. [22]. Glycogen according
to the method of Nicholas et al. [23]. Glucose according
to the method of Trinder [24].
Sera were used for measuring concentrations of glu-
cose, total cholesterol, uric acid, urea and creatinine.
2.7 Statistical Analysis
Data were analyzed by comparing values for different
treatment groups with the values for individual controls.
Results are expressed as mean ± S.D. The significant
differences among values were analyzed using analysis by
student’s t-test for comparing the means of experimental
and control groups [25].
3. RESULTS
The present results in the Tab le 1 and Figure 1 showed
that very highly significant (p < 0.01) reduction in Lac-
tate (LDH) enzyme activity in mice treated with Ta-

Z. Zuhair et al. / Advances in Biological Chemistry, 2011, 1, 15-23 17
moxifen (32.5  2.3) as compared to the normal control
(42.32  2.6), while significant (p < 0.001) increase was
noticed in other glycolytic enzymes hexokinase (HK),
pyruvatekinase (PK), phosphofructokinase (PFK) and
glucose phosphate isomerase (GPI) as compared to the
normal healthy control. The enzymes activities in treated
mice were 0.141  0.13, 7.8  2.1, 26.2  2.2 and 1201 
2.1) and in control mice were (0.085  0.05, 5.8  2.1,
18.21  1.4 and 88.6  8.2) µ mol/min/mg protein, re-
spectively. Moreover, treatment of mice with the ta-
moxifen and vitamin C recorded no significant differ-
ence in all glycolytic enzymes as compared to control
group. A noticeable remark on the effect of tamoxifen
with vitamin C pointed out to that there is no side effects
on all glycolytic enzymes (LDH, HK, PK & GPI) as
compared to the normal healthy control group.
The present results (Table 2 and Figure 2) showed
that the effect of tamoxifen on some gluconeogenic en-
zymes. Significant increase (p < 0.001) in the levels of
FDPase and PEPCK was noticed in treated group as
compared to the normal group. The percentage of in-
creases were 39.66% and 48.57%, respectively Figure 1,
Ta ble 2 showed a very highly significant increase (p <
0.001) in G-6-Pase, while a highly significant reduction
(p < 0.001) in glycogen phosphorylase was noticed in
treated group as compared to the normal healthy control
group. The percentage of increases was 35.15% and
51.54% respectively.
Moreover, treatment of mice with the tamoxifen and
vitamin C recorded no significant difference in all gly-
colytic, gluconeogenic and glycogenolytic enzymes as
compared to control group. A noticeable remark on the
effect of tamoxifen with vitamin C pointed out to that
there is no side effects on all glycolytic, gluconeogenic
and Glycogenolytic enzymes as compared to the normal
healthy control group.
Ta ble 3 and Figure 3 showed that highly significant
reductions (p < 0.01) in aspartate aminotransferase (AST)
and alanine aminotransferase (ALT) enzymes in mice
treated with tamoxifen while significant (p < 0.01) in-
creases were observed in acid phosphatase (ACP) and
alkaline phosphatase (ALP) levels as compared to the
normal healthy control group.
Moreover, treatment of mice with the tamoxifen and
vitamin C indicated that no significant difference in the
level of liver function enzymes as compared to control
group. Treatment of the normal healthy mice with the
tamoxifen and vitamin C showed no side effects on the
level of liver function enzymes.
Ta ble 4 and Figure 4 showed that highly significant
reductions (p < 0.01) in glycogen and total protein levels
in treated mice with tamoxifen as compared to the nor-
mal healthy control group. The percentages of reductions
were 31.01% and 37.56% respectively. The table shows
a highly significant increase (p < 0.01) in glucose level
(28.1%). The present results in the Tabl e 4 showed that
very highly significant (p < 0.01) reduction in total cho-
lesterol, uric acid, urea and creatinine in mice treated
with tamoxifen as compared to the normal healthy con-
trol. The percentages of reductions were 25.71%, 34.2%,
27.42% and 19.32%, respectively. Moreover, treatment
of mice with the tamoxifen and vitamin C recorded no
significant difference in the level of glucose, total cho-
lesterol, uric acid, urea and creatinine as compared to
control group. Treatment of the normal healthy mice
with the tamoxifen and vitamin C showed no side effects
on the level of glucose, total cholesterol, uric acid, urea
and creatinine.
4. DISCUSSION
In the present study, significant increase in glycolytic
enzymes HK, PK, GPI and PFK were observed in
treated group with tamoxifen, while LDH enzyme activ-
ity showed significant reduction. The enhancement in
the activities of glycolytic enzymes in treated mice could
be attributed to increase metabolic activities of treated
liver tissues to compensate the inhibition of host Krebs’
cycle of host caused by treatment with tamoxifen [26,27].
LDH inhibition revealed the aerobic–anaerobic switch
induced by treatment with tamoxifen [27]. Kuser et al.
[28] indicated that lactate is accumulated and glycogen
depleted confirming inhibition of aerobic respiration and
stimulation of anaerobic glycolysis through hexokinase,
a rate limiting enzymes of glycolysis. Some authors re-
ported that tissue damage followed the release of cellular
enzymes such as LDH [29,30]. Besides in spite of the
decrease in LDH activity, there was insignificant change
in D-lactate and pyruvate level as compared to untreated
snails, as reported by Reddy et al. [31].
Concerning gluconeogenic enzymes activities (Fruc-
tose-1,6-diphosphatase and Phosphoenolpyruvate Car-
boxykinase), the present results showed significant ele-
vations in treated mice, where the significant increase in
gluconeogenic enzymes fructose-1,6-diphosphatase is
due to depletion of glucose in the treated mice, where the
ratio of glycogen to glucose levels in liver is known to
be regulated by the balance between glycogen synthesis
and degradation capacities. The increase influx of glu-
cose into glycolytic flux and enhanced glycogen stored
lead to stimulation of the enzymes [32]. The nature of
the end product formed is dependent on the competition
for PEP by the two enzymes pyruvate kinase (PK) and
phosphoenolpyruvate carboxykinase (PEPCK). Stimula-
tion of PK in treated animals ascertained the stimulation
of the gycolytic flux previously reported by Horemans et
al. [33] and Ahmed and Gad [26]. With respect to G-6-

Z. Zuhair et al. / Advances in Biological Chemistry, 2011, 1, 15-23 18
Table 1. Effect of tamoxifen and vitamin C on some glycolytic enzyme, in mice liver.
Glycolytic enzymes
HK PK PFK LDH GPI
Mean  SD %
change Mean  SD % Change Mean  SD% changeMean  SD% Change Mean  SD% Change
Contol 0.0 85  0.05 5.8  2.1 18.21  1.4 42.32  2.6 88.6  8.2
Mice treated
wth
tamoxifen
0.141  0.13*** 64.7% 7.8  2.1*** 34.48% 26.2  2.2***43.88% 32.5  2.3**23.2% 1201  2.1***35.44%
mice treated
with
tamoxifen &
vitamin C
0.0820.072 3.53% 5.6  1.5 3.4% 18.8  2.1 3.24% 40.12  3.45.2% 90.2  5.3 1.81%
Data are means S.D. of five mice in each test; All values are expressed as µ mol/min/mg protein; ** p < 0.01 & ***p < 0.001
Figure 1. Percentage change in the activity of glycolytic enzyme of mice treated with Tamoxifen
and Tamoxifen with vitamin C.
Table 2. Effect of tamoxifen and vitamin C on some gluconeogenic and glycogenolytic enzyme, in mice liver.
Gluconeogenic
enzymes
Glycogenolytic
enzymes
FD Pase PEPCK G-6-Pase Glycogen
phosphorylase
MeanSD % change MeanSD % Change
Contol 11.6.1  0.21 3.5  1.8 18.212.1 1.3  0.3
mice Treated with
tamoxifen 16.2  1.8 *** 39.66% 5.2  0.22***48.57% 24.6  3.1 ***35.15% 0.63  0.32***51.54%
mice Treated with
tamoxifen & vitamin C 12.1  2.2 4.31% 3.6  0.62 2.86% 18.621  0.72 2.31% 1.26  2.1 3.08%
Data are means S.D. of five mice in each test; All values are expressed as µ mol/min/mg protein; **p < 0.01 & ***p < 0.001.

Z. Zuhair et al. / Advances in Biological Chemistry, 2011, 1, 15-23 19
-60
-40
-20
0
20
40
60
%change
FD PasePEPCKG-6-PaseGly.p h o sph o ryl ase
G luc oneogenic and G lyc ogenoly t ic e nzyme s
M ice T reate d with tam o xi fen Mice T reate d with tam o xi fen & vitam in C
Figure 2. Percentage change in the activity gluconeogenic and glycogenolytic enzyme of mice
treated with Tamoxifen and Tamoxifen with vitamin C.
Table 3. Effect of tamoxifen and vitamin C on liver function enzymes in mice.
Enzyme activity  mol/min/mg protein
Aspartate amino transferase
(AST))
Alanine amino transferase
(ALT) Acid phosphatase (ADP) Alkaline phosphatase (ALKP)
Parameters
Treatment Mean  SD % change Mean  SD % ChangeMean  SD % change Mean  SD % change
Control 31.6  3.1 21.8  2.11 5.8  1.1 3.5  0.52
Mice treated with
tamoxifen 20.5  1.03** –35.13 % 12.8  0.75***–41.28% 8.6  0.31***48.28% 5.6 ± 0.08*** 60%
Mice treated with
tamoxifen &
vitamin C
30.7  0.04 –2.85% 22.1 + 0.86* –1.38% 6.2  0.38 6.9% 3.7 0.12 5.71%
*p < 0.05, **p < 0.01 & ***p < 0.001
Figure 3. Percentage change in the activity of liver function enzymes of mice treated with
Tamoxifen and Tamoxifen with vitamin C.

Z. Zuhair et al. / Advances in Biological Chemistry, 2011, 1, 15-23 20
Table 4. Effect of tamoxifen and vitamin C on biochemical parameters in mice.
Control Mice treated with tamoxifen Mice treated with tamoxifen & vitamin C
Treatment
parameters
Mean  SD Mean  SD % Change Mean  SD % change
Total protein 31.6  3.1 21.8  2.11** –31.01% 30.5  3.1 –3.48
Glycogen 20.5  1.03 12.8  0.75*** –37.56% 19.5  3.31 –5.85%
Glucose 30.7  0.04 22.1 + 0.86** –28.1% -29.8  2.38 –2.93%
Total Cholesterol 2.1  1.2 1.6  0.13** –25.71% 1.98  0.06 –5.71%
Uric Acid 52.13  0.4 34.3  4.1** –34.2% 50.8  4.3 –2.55%
Urea 6.2  0.4 4.5  1.1** –27.42% 6.1  0.73 –1.62%
Creatinine 35.1  2.1 28.4  3.6* –19.32 34.6  1.8 –1.42%
-40
-35
-30
-25
-20
-15
-10
-5
0
% change
Total
protein G lucoseu r ic acidcr eat i ni ne
Mice treated with tamoxifen Mice treated with tamoxifen & vitamin C
Figure 4. Percentage change in biochemical paramters of mice treated with Tamoxifen and Ta-
moxifen with vitamin C.
Pase as glycogenolytic enzyme, it showed an enhanced
activity in treated mice which was attributed to either
synthesis and/or degradation of glycogen [34], inhibition
of translocase (T1) the glucose-6-phosphate transport
protein [35] and to the elevation of cytosolic calcium
that can trigger the conversion of the enzyme phos-
phorylase b (inactive form) to phosphorylase a (active
form) which degrades glycogen into glucose [36].
Concerning AST and ALT enzymes activities, signifi-
cant reduction was observed in both treated mice. The
present result indicated that decrease in AST and ALT
attributed to the hepatocellular damage where the
transaminases level showed an intimate relationship to
cell necrosis and/or increased cell membrane permeabil-
ity led to discharge of the enzyme to blood stream
[37,38]. The decrease in transaminases level providing
additional support for the side effect of the toxic sub-
stance on mitochondria of the hepatic cells as it is the
subcellular localization of transaminases.
In the present study, acid phosphatase (ACP) and al-
kaline phosphatase (ALP) showed that significant eleva-
tion in treated mice . Higher levels of acid phosphatase
and alkaline phosphatase (ALP) in tissue was observed
by El-Aasar et al. [37] and Abdel-Rahman et al. [39]
which was attributed to the irritation of liver cells by
toxins or due to increase loss of intracellular enzyme by
diffusion through cell membrane which appear to act as
a stimulus to the synthesis of more enzyme.
Tamoxifen was reported to alter the glutathione me-
tabolizing enzymes [40]. Tamoxifen induces free radi-
cals production in renal tissue, and at the same time de-
creases its ability to detoxify reactive oxygen species.
TM intoxication leads to disruption of the activity of
glutathione metabolizing and antioxidant enzymes [5].
There is no side effects on all glycolytic, gluconeo-
genic and glycogenolytic and liver function enzymes of

Z. Zuhair et al. / Advances in Biological Chemistry, 2011, 1, 15-23 21
mice treated with tamoxifen with vitamin C as compared
to the normal healthy control group. The results of the
present study indicated that the exogenously adminis-
tered vitamin C may prevent tamoxifen-induced testes
toxicity in mice. The protective effect of vitamin C is
probably due to a counter action of free radicals by its
antioxidant nature. Vitamin C may be recommended as
an adjuvant therapy with certain anticancer. Protective
effects of vitamin C against chemically-induced damage
in various rodent organs have been demonstrated by
many investigators [41,42]. Vitamin C was found to be
effectively protecting chemically-induced oxidative re-
nal damage in animals [42-44] reported that mega-dose
of vitamins rendered significant protection of renal
damage induced by anticancer and the effect of vitamin
C was higher than that of vitamin E. The protective ef-
fects may be partially mediated by preventing the renal
antioxidant status.
Increasing the glucose concentration stimulated gly-
cogen synthesis and decreased the activity of glycogen
phosphorylase. An inverse relationship was shown be-
tween the actual glycogen content and the rate of glyco-
genesis. So there is a substrate cycling that occurred
between glucoses-6- phosphate and glycogen content, i.e.
glucose was incorporated into glycogen during period of
net glycogen breakdown, and vice versa; glycogen deg-
radation occurred during periods of net glycogen synthe-
sis which depends on glucose concentration [27]. Our
data recorded enhancement levels of glucose-6-phos-
phatase and glucose.
The present results showed that significant reduction
in total protein content in treated mice which could be
attributed to cellular damage caused by toxins [30]. The
main fraction of total protein content is albumin in turn
may result from decrease anabolism or increase catabo-
lism [45]. The significant decrease in total protein is
mainly due to increase in messenger RNA degradation
which is the possible cause for the hypoalbuminemia
[46]. Also, the present results showed that very highly
significant reduction in total cholesterol, uric acid, urea
and creatinine in mice treated with tamoxifen as com-
pared to the normal healthy control.
5. CONCLUSIONS
Normal control mice treated with tamoxifen showed side effects of
most parameters compared to the normal healthy control group.
Moreover, normal control mice treated with tamoxifen and vitamin C
showed no side effects of most parameters compared to the normal
healthy control group. Hence, vitamin C may prevent tamoxifen-in-
duced testes toxicity in mice.
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