Role of Oxidative Stress by Lipid Peroxidation in Developing
Urinary Bladder Cancer and Correlation between
Malondialdehyde and Trace Elements Copper and Zinc
Thanaa M. Juda
Mufeed J.Ewad
Mohammed R.Judy
Kadum J.Alhamdani
Babylon University,College of Medicine, Hilla- P.O. Box 473, Iraq.
MJ B
Abstract
This study was applied on fifty patients having urinary bladder cancer, proved by histopathological
study for the biopsy taken from the bladder by cystoscopy performed under general anesthesia.
In this study the type of cancer was transitional cell carcinoma of urinary bladder of different stages.
In this study changes in malondieldehyde as biomarker of lipid peroxidationhave been studied the
result revealed significant elevation in patient groups compared with control, and glutathione level
(GSH) which considered as antioxidant defenses mechanism are significantly reduced in patients
groups in comparison to control groups.
The changes in level of trace elements copper and zinc are study in this work, the result revealed
increase level of copper and reduced level of zinc in patients groups in comparism with control groups
‫لدرا دس ذذتل‬
‫الخالصة‬
‫بتذذتللذذنل ذ‬.‫ عذخلإصذذب عةهلر ذذسابنلدرل‬. ‫بتذذتخلعذذهلد عذذذبسههلرةذ الدرا دس ذذتل‬.‫هذ الدرا دس ذذتلعلذذخلس ذ ل ل ذينللسيضذذبلر ذذسابنلدرل‬
‫بتتلدر يلعهلإج دسلؤالعحخلدرع ايسلدرعبهلخ ل‬.‫لتبظ سلدرل‬
‫بتتللنل‬.‫درت يجذتلر عيتبخلدرعيلعهلأ هبللنلرابتتلدرل‬
‫بتتلفيللسدحللل ع فتخ ل‬.‫لنله الدرا دس تلكبنلت علدر سابنله ل سابنلدرت يجلدرا ئيلدالتعقبريلر ل‬
‫ كبتخلدرتعيجذتلدسعفذبعلل حذ يلفذيلل ذع ىلدرلبر تايةبيذالستذاللسضذ ل‬,‫فيله الدرا دس تلعهلقذبسلل ع ىلدرلبر تايةبيالكادرتلألك اةلدراه نل‬
‫بتتللقبستتللعللجل ستلدرضبراتخ ل‬.‫سابنلدرل‬
‫بتذتللقبستذتلستذال‬.‫بي نلدرل عز لدر يلذعع سلكلضبادخلافبسذتلضالدألك اةلفقالكبتخللت فضتلستاللسضذ ل ذسابنلدرل‬.‫ألبلل ع ىلدرك عب‬
‫لجل ستلدرضبراتخ ل‬
‫لدرعتبص ذ لس هيلحص ذسدلدرتحذذبسل لدر بسصذذينل لكبتذذخلدرتعيجذذتلدسعفذذبعلفذذيلل ذذع ىل‬
‫فذذيله ذ دلدرعلذذللعذذهلا دس ذذتلدرعسي ذسدخلفذذيلل ذذع ىلرع ذ‬
‫بتتللقبستتلرلجل ستدرضرط لخ ل‬.‫لفيلل ع ىلدر بسصينلستاللسض ل سابنلدرل‬
‫درتحبسل لدت فب‬
‫ذذذذذذذذذذذذذذذذذذذذذذذذذذذذذذذذذ ل‬
Introduction
U
rinary bladder cancer (UBC) is
the fifth most common cancer
in the United States and
western countries where it is a
disease of white men over the
age of sixty five [1].
The incidence of bladder cancer
increases with age ,few cases of occur
under the age of forty, the male to
female ratio ranges from 2:I -4:1,and
the incidence is more common in large
urban centers due to the increasing
increase exposure to risk factors[2].
Development and progression of
bladder cancer is derived by the
malfunction of specific gene i.e. due to
over expression of oncogen or
inactivation of tumor suppressor genes.
The oncogen that has been associated
with bladder cancer is
(ras)gene
family mainly, including p21 ras
oncogen.
The
most
important
suppressor gene isP53 [3].
Nearly all UBC are arise from
transitional epithelial lining due to
exposure of urothelium to chemical
carcinogen, and the bladder is common
site of transitional cell tumor (TCT)
[4].The other bladder cancer are:1. Adeno carcinoma account for less
than 2%of all bladder cancer
2. Sequomous cell carcinoma (SCC)
account for 5-10%of all bladder
cancer, it is associated with history of
chronic
infection
[5].In
under
developed nations (SCC) is associated
with bladder infection by schistosoma
haematoma [6].
Staging is done to obtain
prognostic information and to guide
treatment selection, stage1 tumor
confined to epithelium, stage2
Tumor invasive to muscularis propria,
stage3
tumor
involvement
of
perivesical fat, stage4 distal metastasis
[7].
Tobacco smoking is the most
established modifiable risk factor for
bladder cancer [8].Cigarette smoking
induced oxidative stress and involved
in DNA damage, and impaired anti
oxidative defense mechanism [9].
Variety of industrial exposure risk for
bladder cancer as aromatic amines
present in product of dyes and
occupational hazard are painter,
drivers, hair dresser [10].
The
disturbance
between
prooxidant and antioxidant balance
results in oxidative stress which is
defined as imbalance between the
production of various reactive oxygen
species(ROS) and the ability of
antioxidant protective mechanism to
cope with these ROS and prevent its
adverse effect on biological system
[11].
These ROS have one or more
unpaired electrons in there's outer
shells. It is consequently has tendency
to accepted an electron from other
substance make it highly reactive,
these ROS including super oxide anion
radical O2.-, Hydroxyl radical
HO. ,
Hydrogen peroxide
H2O2,
Lipid
peroxide radical Roo. Singlet oxygen
1
O2, Hypochlorous HOCL, Ozon O3
[12].
The first sources for ROS are
mitochondria through respiratory chain
[13]. The second sources are enzymatic
action as oxidase,
Dioxygynase,
xanthenes
oxidase,
cytochrome P450 Nitric oxide synthase
[14]. Third source for ROS are
xenobiotics [15]. Interactions between
ROS and antioxidant are as follow
[16].
Figure 1 The prooxidant and antioxidant network
Lipid peroxidation is oxidative
degradation of lipid ,by which free
radicals steal electron from lipid in cell
membrane and resulting in cell damage
Poly unsaturated fatty acid (PUFA)
serve as excellent substrate for lipid
peroxidation [17].
Lipid peroxidation process yields
several cytotoxic product including
saturated
aldehydes
e.g.
Malondialdehyde
(MDA)
and
unsaturated aldehydes e.g. 4-hydroxy
–trans-2-nonenal and 4- hydroxy-2hexenal (4-HHE) and acrolein [18]
These aldehyde react with cellular
nucleophiles such as glutathione
(GSH) and cystiene, histidine, lysine
residues of protein which causes
destructive functional modification and
its effect on DNA resulting in
genotoxic effect that promotes cancer
[19].
Glutathione (GSH) reduced
form is a linear tripeptide of
L.glutamate, L-cysteine and glycine.
GSH has sulfhydryl (SH) group on
cysteinyl portion, which account for its
strong electron donating character. As
electrons are lost from the GSH the
molecule become oxidized and two
such molecules becomes linked by
disulfide bond to form glutathione
disulfide or oxidized glutathione
(GSSG) . This linkage is reversible
upon rereduction, so GSH is under
tight haemostatic control both intra
cellular and extra cellular .So a
dynamic balance is maintained
between GSH synthesis, its recycling
The first reaction catalyzed by the
from GSSG and metabolism.
action of gamma- glutamyl cysteine
The synthesis of GSH involves two
synthetase as follow:closely linked enzymatic controlled
reactions.
1. Glutamate +cysteine +ATP
gamma-glutamyl cysteine+ADP+P
The second reaction catalyzed by glutathione synthetase
2. Gamma glutamyl cysteine +glycine +ATP
glutathione +ADP+P[20].
Zinc is essential trace element for all
forms of life. It is absorbed from
duodenum, zinc is mostly transported
bound
to
albumen,
alpha-2macroglobulin and transferene, zinc
sequestration in enterocytes with
metalothionein, some of this transfer to
the plasma, the rest is lost when the
enterocytes are sloughed [21]. Zinc
plays important roles in growth and
development, the immune response,
neurological
function,
and
reproduction [22].
Zinc has antioxidant action [23]. And
anticancer action [24]. And has role in
vitamine A action Zinc is a component
of retinol binding protein, which is
necessary for transporting vitamin A in
the blood [25].
.Copper in the body shifts between the
cuprous (Cu+1) and cupric (Cu+2), the
ability of copper to easily accept and
donate electron explains it is important
role in oxidation- reduction (redox)
effect about 90% of copper bound to
ceruloplasmen which is copper
contains protein the copper containing
enzyme, while when copper first
absorbed by intestine it is transported
to
liver
bound
to
albumen.
Ceruloplasmen (ferroxidasel) and
(ferroxidasell), which have the
capacity to oxidize ferrous ion (Fe+2) to
ferric ion (Fe+3) the form of iron that
can be loaded onto the protein
transferrin for transport to the site of
red blood cell formation [26].
Aims of the Study
1. Studing the antioxidant defense
mechanism of reduced glutathione
(GSH) as main non enzymatic
antioxidant in patients with urinary
bladder cancer and compared with
control groups.
2. Studing the role of lipid
peroxidation through studing the level
of MDA as a biomarker of lipid
peroxidation in patient with bladder
cancer and control groups
3. Study the changes occuring in serum
level of trace elements: zinc and
copper in bladder cancer patients and
control.
Materials and Methods
1-Samples
Two groups of samples were included
in this study ,patients groups fifty
patients(forty six males and four
females) proved to have urinary
bladder cancer admitted to Al-Hilla
Teaching Hospital, and twenty five
healthy persons considered as control
groups. Five ml of blood was obtained
from each subject , and was pushed
slowly into plain disposable tubes with
out anticoagulant. Serum was obtained
by centrifuging 2500 rpm for
approximately 10-15 minutes.
2-Methods
. Determination of the serum reduced
glutathione according to the Burtis and
Ashwood method [27].
. Determination serum level of
Malondialdehyde as biomarker of lipid
peroxidation
by
reaction
with
thiobarbituric acid [28].
.Determination of serum level of zinc
and copper through reaction of zinc
and copper with chromagen reagent
and the color intensity is proportion
with zinc and copper concentration
which
determination
by
spectrophotometer [29].
Results and Discussion
In this work the mean serum level of
MDA among urinary bladder patients
is 11.07 μmol/l and control is 3.048
μmol/ l and the results are as follows:-
Table 1 The mean serum level of MDA in urinary bladder cancer patients and control
groups
groups
MDA μmol/l
SD
P value
patient
11.07
2.27
significant
control
3.048
0.65
Concentration of MDA (umol/L)
The differences between mean of
serum level of MDA among patients
and control groups as follows:12
10
8
Patients
6
Control
4
2
0
Patients
Control
Figure 2 Mean serum level of MDA between urinary bladder patients and control
The mean of serum MDA according to
the stages of bladder cancer are
represented in table 2:
Table 2 Mean of serum level of MDA according to different stages of bladder cancer
group
Stage l
Stage ll
Stage lll
In this work there is significant
difference in the level of MDA
between urinary bladder cancer
patients and control group.
The serum level of MDA in UBC
patients is 3.6 times more than that of
the control, and there is increase of
level of MDA with increase stage of
cancer, the greatest MDA level are in
stage lll
MDA μmol/l
8.5
11.3
13.9
MDA has a mutagenic and
carcinogenic role and causes mutation
in DNA ,and when its level increases
due to oxidative stress; this will lead to
mutagenic
and
carcinogenic
effects[30].
The mean serum level of GSH
among patients is 4 μmol/l and control
is 16 μmol/l ,and the results are as
follows:-
Table 3 The mean serum GSH in UBC patients and control
Group Mean serum GSH μmol/L SD
Patients 4
1.08
control 16
2.12
P value
significant
Concetration of GSH (umol/L)
The difference in mean of serum GSH
between UBC patients and control is
represented in the bellow figure:20
16
15
Patients
10
5
Control
4
0
Patients
Control
Figure 3 The mean serum level of GSH in UBC patients and control.
The mean of serum level of GSH
according to the stage of bladder
cancer are as follow.
Table 4 Mean serum GSH in UBC patients according to the stage of cancer.
Stage of cancer
Stage I
Stage II
Stage III
Mean of GSH μmol/l
6.5
3.9
2.4
In this study there is a significant
difference in mean of serum GSH
between UBC patients and control ,
p<0.01 , in bladder cancer the level of
GSH is significantly reduced compared
to control , the level of GSH is
approximately 25% in bladder cancer
patients compared to control and
decrease more with the increasing
stage of cancer.
Decrease in GSH in UBC patients is
due to continuous consumption of
GSH by tumor cell , because in cancer
state there is oxidative stress and
accumulation of reactive species
occuring and because GSH is concede
red a master of antioxidant in the body
through[31] .
The serum level of zinc in UBC
patients and control is as follows
Table 5 The mean serum level of zinc in UBC patients and control groups
groups Mean of zn μg/dl
Patients 64.14
control 99.6
The mean of serum level of copper in
UBC patients and control is as follow:-
SD
4.2
8.6
P value
0.01
significant
Table 6 The mean serum level of copper in UBC patients and control.
groups CU μg/dl
Patients 167
control 101
In this study
there is a
significant defference in zinc level
between patients and control groups in
UBC patients the serum level of zinc
is significantly lowered than that in the
control , p value < 0.01 , in contrasted
to this result the serum level of copper
is significantly elevated in UBC
patients in relation to control, p value
< 0.01 .
The result of this study has indicated
that the zinc is reduced and copper is
elevated in bladder cancer state
compared to control.
SD
20
10.4
P value
<0.01
significant
Copper is one of the metal
which has the ability to catalyzed
formation of OH. And other radicals
from H2O2 and O2.through Fenton
reaction [32].
Zinc has antioxidant property through
antagonism of redox active transition
metal such as copper [33].
Zinc
and
copper
are
antagonized each other by competition
between these metals in intestinal
mucosal cell[34].
The correlation between MDA versus
zinc and copper is as follows:
Table 7 The correlation between the level of lipid peroxidation MDA via zinc and
copper level in bladder cancer patients
The correlation of MDA Correlation coefficient (r)
μmol/L vs.
Copper Cu μg/dl
0.775
Zinc Zn μg/dl
-0.720
The correlation between MDA
and copper in bladder cancer patients is
positive significant (r = 0.775 ,p < 0.05
),while the correlation between MDA
and zinc level in bladder cancer
patients is negative significant (r = 0.720 ,p <0.05 )
which means
according to this statistical correlation
in bladder cancer patients there is an
increase level of copper which has
oxidative power and cause an increase
P value
Positive significant
Negative
significant
in lipid peroxidation with increase the
level of MDA end product of lipid
peroxidation , zinc level decrease in
bladder cancer patients ,and because
zinc has an antioxidant action this lead
to accumulation of free radical which
enhance the process of lipid
peroxidation.
The correlation between GSH versus
zinc and copper as follow
Table 8 The correlation between GSH via zinc and copper level in bladder cancer
patients
The
correlation
of Correlation coefficient (r)
P value
GSHμmol/L vs.
Copper Cu μg/dl
-0.681
significant
Zinc Zn μg/dl
0.700
significant
The correlation between GSH and
negative significant (r =-0.681,p < 0.05
copper in bladder cancer patients is
),while the correlation between GSH
and zinc level in bladder cancer
patients is positive significant (r = 0.700,p <0.05 )
Conclusion
1.There is a role for lipid peroxidation
in bladder cancer state due to oxidative
state , so the level of malondialdehyde
the biomarker of lipid peroxidation is
significantly elevated
2.There is decrease in antioxidant
defenses mechanism in bladder cancer
state mainly glutathione.
3. Copper and zinc levels are disturbed
in bladder cancer state copper level has
oxidative property is elevated in cancer
state while zinc level is decrease in
bladder cancer .
References
1. Boring C , Squires T 1994. cancer
statistics . J clin. Cancer,44: 7-26.
2. Lars D , Torben F 2006. cancer
epidemiology and prevention Cancer
res . 66(3): 1851-1858.
3. Shuya H , Kelsey D , Carmen 2005 .
Alteration p53 in human bladder
cancer .J cancer. 104 (9):1918-1923 .
4. Roderick NM , Macsween K ,
Whaley A 1996. Muris textbook of
pathology . 13th ed . oxford university
press , new York. p 942-945
5 . Emil A , Jack W . smith's general
urology 2000. 15th ed . Lebanon . p
357-358 .
6. Montiel j , Smith DL , Sandler H
2004. clinical oncology, 3rd ed,p 20592084 .
7. Robert W,Schrier MD, Carl
W,Goottschalk MD1996.disease of
kidney.6th ed..p.810-813.
8. Zeegers NP , Keller E , Buntex F
2004. Association between smoking
consumption , diet and bladder cancer .
World J . Urol . 21: 392-401 .
9. Sinko I , Monika M , Istevan R
2005. effect of smoking on
DNA
Damage .J Reproductive Toxicology .
20(1):65-71 .
10.Zeegers m , Swean G , Goldbohm P
2001. Occupational risk factors for
bladder cancer .J. occup. Environ.
Med.;58(9):590 596 .
11.Ripple M , Ray R , wilding G 1997.
prooxidant and antioxidant Shift in
human cancer j. Natl. cancer
Inst.;89,10;40-8
12.Droge W 2001 . free radical and
physiological condition of cell. J
Physiol. Rev.;82:74-95 .
13. Han D , William E2001 .
mitochondrial
respiratory
chain
Generating super oxide anion .
biochem .J .;353: 411-416 .
14. Yokoyama Y , Becham j 1990.
xanthine oxidase potentiated ischemic
injury . Am.J.physiol.;258:564-570 .
15. Amie k , Steven l , Petrson G 2005.
Redox modulation and Xenobiotics
action
of
ROS
formation
.
toxicological,Science .;88(1):265-273 .
16. Peter G , Winnie j , Gorden P 2005.
Molecular
and
biochemical
Mechanism of reactive oxygen species
.
toxicology
and
Applied
pharmacology.;207:354-366 .
17.Alexander n , spector A . 1994poly
unsaturated fatty acid increase
lipid
radical .J lipid res.;35:1773-1785 .
18. Niederhofer j , Daniel S , Green R
2003. MDA a product of lipid
Peroxidation mutagen to human cell .
J.biological
chemistry;278933:314330.
19. Nimet V , Rucken T 2004. Effect
of stress indused lipid Peroxidation on
cell function . International cell
biology;28(7):517-521 .
20- Ulrike L , Terrance J , Kavanagh C
2001. gonadotrophine and Regulation
of glutathione synthesis . reproductive,
Toxicology .;15(5):495-504 .
21.Smith AF , Beckett GJ , Walker
SW , Rae P, 1998. lecture notes On
clinical biochemistry .6thed .Blackwell
science ;p140.
22.Trung-tran AQ , Chai F , Z alewski
PD 2000. role of zinc and cellular
function . J.Nutr.;130:1432-1436 .
23.Bray TM , better WJ . 1990the
physiological role of zinc 24.Emily H
2004. zinc deficiency , DNA damage
and cancer risk .J of nutri. Biochem
.;15(10):572-578 . as an Antioxidant
Free radic Biol.Med.;8:281-291.
25. Christian P , West KP 1998;.
interaction between zinc and Vitamin
A .Am. J. clin. Nutr. 68:435-441 .
26.Caballereo b , Cousins RJ ,
Turnland JR 2006. copper .modern
Nutrition in health and disease
.10thed.philadelphia.
lippincotts
Williams&wilkins.;p.289-299
27.Burtis CA , Ashwood ER 1999.
Tietz
text
book
of
clinical
Biochemistery
3rded.Saunders
company , Tokyo.; p.1034-1054 .
28.Burtis CA , Ashwood ER1999..Tetz
text book of clinical biochemistry 3rd
ed. Saunders company , Tokyo,
29.colorimetric determination of zinc
and copper (kit)
30.Saad M , Nashed W , Husen M,
Giebry M 2006. urinary telomere
activity and MDA as marker of bladder
cancer .Turkish J. of Biochem
.;31(4):164-168 .
31. Guogao W , Sheng Y , Nancy D ,
Joan R 2004.glutathione and it is
implication for health . J. of nutrition
.;134:489-492
32. MichealD , Jonathan H. 2004.
Copper
and
oxidative
state
.Am.J.physiol.cell.;286:293-301 .
33.BrayTM , B etter WJ 1990. zinc az
antioxidant
.free
radic.Biol.Med.;8:281-291
34- KimikoH.Akria Y 1998.frre radical
and trace elements.j. of trace element
in experm.med.;11(20;209-217