Lipid Peroxidation and Antioxidant Status in Human Cervical...

Disease Markers 15 (1999) 283–291IOS Press

0278-0240/99/$8.00 © 1999 – IOS Press. All rights reserved

283

Maha I. Ahmed1, Salah T. Fayed2,#,Hanan Hossein1 and Fathy M. Tash1

1Department of Biochemistry, Ain ShamsFaculty of Medicine, Abassia, Cairo, Egypt2Department of Obstetric and Gynaecology,Ain Shams Faculty of Medicine, Ain ShamsUniversity Hospitals, Abbassia, Cairo, Egypt

ABSTRACT: Reactive oxygen species (ROS),represented by superoxide, hydrogen peroxide andhydroxyl radicals, have been implicated in manydiseases including cancer. ROS have been known toplay an important role in the initiation and promotionof multistep carcinogenesis. The cellular antioxidantsplay a crucial role in protection against neoplasticdisease. However, very little is known about theantioxidant defense in cervical carcinoma. This isaddressed in the present study. Lipid peroxides,glutathione content and the activities of antioxidantenzymes, together with vitamin C and E content, wereestimated in patients who had carcinoma of the cervix,and the values were compared with those of normalwomen. The results showed a remarkable reduction inthe content of glutathione, vitamin E and C. Activitiesof glutathione peroxidase and superoxide dismutasewere also reduced in cervical cancer compared tonormal controls (P < 0.001). This reduction was moremarked in late stages (III, IV) than in early stages (I,II) (P < 0.001). Glutathione was reduced more inpoorly differentiated tumors (grade III) than in welland moderately differentiated ones (grade I, II)(P < 0.05). Levels of lipid peroxides were found to besignificantly higher in malignant than in normal tissuesamples and their levels were correlated withadvanced clinical stage (P < 0.001). Our resultssuggest impaired antioxidant status in carcinoma of

# Correspondence: Dr. Salah T. Fayed, MD., Department ofObstetrics and Gynaecology, Ain Shams UniversityHospitals, Abbassia, Cairo, 11566, Egypt, Fax: +20 2 2859928, E-mail: [email protected]

the cervix. This impairment is related to tumorprogression.

KEYWORDS: Cervical carcinoma, LP, GSH, SOD,GSH-Px, vitamin C, vitamin E

INTRODUCTION

The close relationship between free radicalactivity and malignancy has been well-documented [10]. An increase in reactive oxygenspecies (ROS) in the cell due to overproductionand/or inability to destroy them may lead tosevere damage of cell molecules and structures.Among the biological consequences of thisdamage are mutations, chromosomal aberrationsand carcinogenesis [9]. The range of antioxidantdefences available within and outside the cellshould be adequate to protect against oxidativedamage. Therefore, a critical balance betweenfree radical generation and antioxidant defencesis required [3]. Intracellular antioxidants includethe enzymes glutathione peroxidase (GSH-Px),superoxide dismutase (SOD) and catalase.Sacrificial antioxidants present in cell membranesinclude tocopherols (vitamin E), beta-carotenesand ubiquinone. Many that circulate in the bloodare derived from diet, for example ascorbate ion(vitamin C) and lipophilic tocopherols [31].Lipid peroxidation has a very important role inthe initiation and promotion of cancer [5] and hasbeen implicated in adverse tissue changes incancer [19]. Thus, measuring lipid peroxides(LP) as thiobarbituric acid reactive substances(TBARS) provides an indirect measure ofantioxidant deficit [31]. Tumor cells have beenshown to have abnormal levels of antioxidantenzyme activities when compared with normalcells. However, enzyme activities also differamong individual tumors [27]. The current study

Lipid Peroxidation and Antioxidant Status in HumanCervical Carcinoma

M.I. Ahmed et al. / Antioxidants and Cervical Cancer284

investigates the alterations of LP and theactivities of GSH-Px and SOD together with theendogenous concentration of glutathione (GSH),one of the most important antioxidants, as well asvitamin E and C in human cervical carcinoma.

MATERIALS AND METHODS

Patients

The study was conducted on patients sufferingfrom cervical carcinoma treated in Obstetrics andGynaecology Department, Ain Shams UniversityHospitals in the period from October 1996 throughDecember 1998. Blood samples and tissuespecimens were collected from 27 patients withcervical carcinoma in different stages. Controlblood and tissue samples were obtained from 20patients undergoing hysterectomy for non-malignant conditions. All patients were examinedunder anaesthesia to assess tumor size, parametrialor vaginal extension of the growth. The clinicalstage was determined according to FIGO staging.Patients were treated either surgically, byradiotherapy or both. Patients with bulky tumorsreceived neoadjuvant chemo-therapy before defi-nitive treatment whether surgical or radiotherapy.Serum and surgically resected tissue samples wereobtained before therapy and kept at í�� ºC till thetime of analysis.

Processing of tissue samples

Tissues were rinsed in saline to remove bloodthen homogenized (20% w/v) in ice cold KCL(0.15 M) using an ulteraturax T-25 homogenizerwith 3–5 bursts 30s, each with 1 min pause, onice. One aliquot of the homogenate was keptaside for estimation of LP, SOD activity and totalproteins. The rest of homogenate wascentrifuged at 100,000 x g for one hour at 4 ºCusing a Beckman ultracentrifuge L7 (Brae, USA).The supernatant (cytosolic fraction) was separa-ted and used for estimation of GSH, GSH-Px andtotal proteins. The pellet (membrane fraction)was reconstituted in KCL and used for estimationof vitamin E.

Biochemical analysis

Lipid peroxide levels in the homogenate wereestimated by determining the TBARS followingthe procedure of Esterbauer et al [11]. Thesamples were heated with thiobarbituric acidunder acidic conditions and the pink color formedwas read at 532 nm. The lipid peroxide contentwas expressed as n moles of malondialdehyde(TBARS) per gm tissue.

Protein concentrations in cytosols and homo-genates were estimated according to Bradford[4].

Total reduced glutathione (GSH) wasdetermined in the cytosol based on the reactionwith 5,5'-dithiobis-2 nitrobenzoic acid (DTNB)reagent to give a compound that absorbs light at412 nm [26]. GSH was expressed as �J�PJ

protein.The activities of SOD in the homogenate and

GSH-Px in the cytosol were assayed using RanselKIT (Randox, Lab. Ltd., Ardmore, Crumlin, Co.Antrim, UK, BT29 4QY). Their activities wereexpressed as U/min/mg protein. The method formeasurement of SOD activity [28] in tissuehomogenate employs xanthine and xanthineoxidase to generate superoxide radicals whichreact with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (I.N.T) to formformazon dye. The SOD activity is thenmeasured by the degree of inhibition of thisreaction.

GSH-Px activity in tissue cytosols is based onGSH oxidation by cumene hydroperoxide cata-lysed by cytosolic GSH-Px activity. In thepresence of GSH-reductase and NADPH theoxidized GSH (GSSG) is immediately convertedto the reduced form with concomitant oxidationof NADPH to NADP+ [23]. The decrease inabsorbance at 340 nm is measured.

Vitamin E in membranes was estimated by themethod of Hashim and Schultringer [13] wheredipyridyl, and ferric chloride were added to analiquot of the upper heptane to produce an orangecolor.

Ascorbic acid (vitamin C) in the serum wasdetermined by the method of Aye-Kyaw et al [1].The method is a simple precipitation of proteins

M.I. Ahmed et al. / Antioxidants and Cervical Cancer 285

and extraction of vitamin C by acid phosphotung-state and the blue color developed was measuredat 700 nm. Vitamin C was expressed as �J�PO

serum.

Statistical analysis

Biochemical values were expressed asmean ± standard deviation for patients andcontrol separately. The statistical difference wasanalysed using student ‘t’ test and ‘P’ valueswere expressed. One way analysis (ANOVA)was done to compare values of differentparameters at different stages and grades ofcarcinoma. Correlation analysis was alsoperformed. All analyses were performed usingStatistical Package for the Social Sciences(SPSS) software.

RESULTS

Patients with cervical cancer were clinicallystaged by the FIGO staging system, they weredistributed as following: 6 stage I, 7 stage II, 8stage III, and 6 stage IV. Histopathologicalexamination of cervical carcinoma tissuesrevealed squamous cell carcinoma in 21 cases(77.8%), and adenocarcinoma in 6 cases(22.2%). Clinicopathological, and biomedicaldata are listed in Table 1.

Table 2 depicts the levels of lipid peroxides(LP), glutathione (GSH), the activities ofantioxidant scavenging enzymes, glutathioneperoxidase (GSH-Px) and superoxide dismutase(SOD) and vitamin E and C contents in patientswith cervical carcinoma and in controls. Ourdata revealed significant reduction in tissuecontent of GSH, and vitamin E and also in theactivities of GSH-Px and SOD in patients withcervical carcinoma versus controls (P < 0.001).Serum levels of vitamin C were also reduced inpatients with carcinoma compared to the controlgroup. Lipid peroxides were significantlyelevated in cervical carcinoma compared tocontrol group (P < 0.001). Considering thedifferent stages of cervical carcinoma, GSHcontent and the activities of GSH-Px and SOD

exhibited progressive decrease while LPexhibited progressive increase through stages I,II, III and IV (Tables 3 and 4). Moreover, GSHlevels showed significant reduction in poorlydifferentiated tumors, grade III (mean ± SD:5.1 ± 0.95) compared to well and moderatelydifferentiated, grade I, II (mean ± SD: 7.4 ± 3.0)as evidenced by student t test (t = 2.58,P = 0.016). In stage I patients, membranevitamin E remained unaltered. However, thevitamin showed progressive decrease in stages II,III and IV (Table 5). Vitamin C was not altered inearly stages (I, II) of cervical carcinoma and itslevel decreased in stages III and IV (Table 5). Incervical carcinoma, LP levels were inverselycorrelated with GSH-Px activity (r = í��P = 0.03) and vitamin E content (r = 0.71,P < 0.001). GSH content was correlated withGSH-Px activity (r = 0.73, P < 0.001) and withboth vitamin E (r = 0.67, P < 0.001) and vitaminC (r = 0.76, P < 0.001). Vitamin C wascorrelated with GSH-Px activity (r = 0.071,P < 0.001) and with vitamin E content (r = 0.67,P < 0.001). Vitamin E was correlated with GSH-Px activity (r = 0.67, P < 0.001).

DISCUSSION

Reactive oxygen species (ROS) production byoxidative toxins from many sources may accountfor at least some of their carcinogenicactions. ROS can oxidize any vulnerablemolecular species, and since DNA is one ofthese, oxidative stress can lead to cancer. Thevariability of individual response could be due todifferent antioxidant status at the time, or overthe period of exposure as well as genetic factors[31]. Various studies in the literature havedemonstrated that tumors from different organshave wide variations in their antioxidant status.Moreover, the link between lipid peroxidation(LP) and ROS has also given rise to muchcontroversy. On the other hand, LP has beenreported to be elevated in neoplastic tissues[5]. The increase in LP has been related to thepathogenesis of many diseases such as aging,astherosclerosis and carcinogenesis [7,32]. In the

Tab

le 1

Cli

nico

path

olog

ical

par

amet

ers,

and

bio

chem

ical

dat

a of

the

stud

ied

grou

ps28

3

Vit

Eµ g

/g ti

ssue

VIT

Cµ g

/ml s

erum

SOD

U/m

g pr

otei

nG

SH P

XU

/mg

prot

ein

GSH

µ g/m

g pr

otei

nM

DA

nM/g

tiss

ueG

rade

Stag

eP

atho

logy

Typ

eA

geN

o

10.9

817

.30

0.48

0.47

11.5

029

.20

..

Nor

mal

421

11.5

313

.90

0.42

0.54

15.4

426

.40

..

Nor

mal

362

8.20

17.3

00.

350.

5216

.07

37.9

5.

.N

orm

al45

39.

6314

.75

0.35

0.45

9.90

22.5

0.

.N

orm

al48

411

.03

20.2

5

0.

60.

4511

.50

34.1

0.

.N

orm

al42

56.

9820

.40

0.63

0.57

13.5

621

.20

..

Nor

mal

496

12.5

022

.10

0.42

0.54

13.7

234

.40

..

Nor

mal

437

9.78

16.0

00.

420.

4916

.07

37.7

0.

.N

orm

al42

812

.60

22.2

00.

710.

5318

.55

21.3

0.

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orm

al58

97.

5419

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0.61

0.54

12.0

930

.24

..

Nor

mal

3710

7.14

21.9

00.

620.

5714

.89

22.3

0.

.N

orm

al72

1111

.95

15.1

20.

350.

5112

.78

29.4

0.

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orm

al54

1211

.54

20.0

00.

630.

5315

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21.9

0.

.N

orm

al65

1310

.53

19.6

00.

510.

5310

.03

23.0

0.

.N

orm

al45

147.

5514

.90

0.54

0.48

10.9

622

.00

..

Nor

mal

5015

7.13

14.4

50.

550.

5111

.30

24.1

0.

.N

orm

al56

1610

.50

19.9

50.

360.

4316

.40

25.2

0.

.N

orm

al43

179.

4919

.15

0.53

0.53

14.8

031

.20

..

Nor

mal

4518

10.7

916

.18

0.49

0.53

11.3

032

.00

..

Nor

mal

4819

8.49

18.1

30.

510.

4911

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34.0

0.

.N

orm

al51

206.

9317

.08

0.28

0.37

9.34

38.1

01

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CC

ance

r39

218.

1014

.00

0.22

0.49

7.60

40.8

01

IA

CC

ance

r45

227.

6723

.00

0.39

0.55

12.6

042

.16

2I

AC

Can

cer

5223

10.6

713

.55

0.39

0.31

6.90

35.4

01

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ance

r53

247.

4517

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0.40

0.38

10.4

638

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17.3

50.

240.

437.

8236

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SCC

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cer

4126

6.33

20.0

00.

200.

306.

4041

.98

1II

SCC

Can

cer

3827

7.95

12.4

00.

240.

449.

9538

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2II

AC

Can

cer

4728

7.34

14.4

00.

280.

3411

.75

36.5

12

IISC

CC

ance

r50

296.

2013

.60

0.37

0.33

6.85

47.1

72

IISC

CC

ance

r65

307.

6914

.80

0.27

0.41

5.65

34.1

03

IISC

CC

ance

r55

316.

8013

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0.29

0.36

9.90

35.0

51

IISC

CC

ance

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326.

7016

.80

0.27

0.35

9.58

40.1

92

IIA

CC

ance

r50

335.

7512

.90

0.23

0.32

6.60

44.3

23

III

SCC

Can

cer

4634


Tab

le 1

(con

tinue

d)28

4

Vit

Eµ g

/g ti

ssue

VIT

Cµ g

/ml s

erum

SOD

U/m

g pr

otei

nG

SH P

XU

/mg

prot

ein

GSH

µ g/m

g pr

otei

nM

DA

nM/g

tiss

ueG

rade

Stag

eP

atho

logy

Typ

eA

geN

o

5.00

12.4

90.

140.

243.

4053

.19

2II

ISC

CC

ance

r55

355.

257.

830.

180.

333.

7037

.10

3II

ISC

CC

ance

r50

366.

3811

.00

0.08

0.27

5.80

38.0

01

III

AC

Can

cer

6537

5.55

11.8

00.

160.

324.

6043

.79

1II

ISC

CC

ance

r60

385.

459.

400.

170.

284.

3837

.81

2II

ISC

CC

ance

r45

396.

4012

.20

0.14

0.30

5.82

46.2

13

III

SCC

Can

cer

5240

5.45

11.4

30.

150.

284.

5043

.38

2II

ISC

CC

ance

r47

414.

006.

950.

060.

273.

0043

.54

2IV

AC

Can

cer

5542

5.15

7.12

0.08

0.25

4.90

54.9

03

IVSC

CC

ance

r60

434.

006.

950.

070.

264.

8052

.00

3IV

SCC

Can

cer

5844

3.86

6.75

0.07

0.24

3.10

53.1

42

IVSC

CC

ance

r48

454.

976.

550.

090.

264.

6046

.24

3IV

SCC

Can

cer

5246

4.00

7.35

0.07

0.25

4.20

56.8

82

IVSC

CC

ance

r51

47

SC

C:

Squa

mou

s ce

ll c

arci

nom

aA

C:

Ade

noca

rcin

oma



Table 2LP and antioxidant status in uterine cervical tissues compared to control

Parameter studied Normal control (n = 20) Cervical carcinoma (n = 27)

LP (nM MDA/g tissue) 28.00 ± 5.7 42.13 ± 6.0*

GSH (µg/mg protein) 13.4 ± 2.4 6.72 ± 2.68*

GSH-Px (U/mg protein) 0.51 ± 0.04 0.33 ± 0.08*

SOD (U/mg protein) 0.5 ± 0.11 0.21 ± 0.1*

Vitamin E (µg/g tissue) 9.79 ± 1.0 6.37 ± 1.47*

Vitamin C (µg/ml serum) 18.13 ± 2.7 12.89 ± 4.07*

*P < 0.001: control versus carcinoma. The values are expressed as mean ± SD.

Table 3Lipid peroxides (MDA), and glutathione (GSH) content in control and different stages of uterine cervical carcinoma

Groups NumberMDA (nM/g tissue)

mean ± SDGSH (µg/mg protein)

mean ± SDControl 20 28.00 ± 5.7 13.39 ± 2.4Stage I 6 38.58 ± 2.51* 9.12 ± 2.1*

Stage II 7 39.02 ± 4.54* 8.58 ± 2.27*

Stage III 8 42.98 ± 5.39* 4.85 ± 1.12*a

Stage IV 6 51.12 ± 5.17*b 4.1 ± 0.85*a

*P < 0.001: each stage versus control; aP < 0.001:each stage versus stage I,II; bP < 0.001: each stage versus stage I, II, III.

Table 4Activity of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in control and different stages of

uterine cervical carcinoma

Groups Number GSH-Px (U/min/mgprotein)mean ± SD

SOD (U/min/mg protein)mean ± SD

Control 20 0.51 ± 0.04 0.51 ± 0.11Stage I 6 0.42 ± 0.09* 0.32 ± 0.08*

Stage II 7 0.36 ± 0.05* 0.27 ± 0.05*

Stage III 8 0.29 ± 0.03*b 0.157 ± 0.04*a

Stage IV 6 0.25 ± 0.01*a 0.07 ± 0.01*a

* P < 0.001:each stage versus control; a P < 0.001:each stage versus stage I,II; bP < 0.001: each stage versus stage I.

Table 5Levels of vitamin C and E in control and different stages of uterine cervical carcinoma

Groups NumberVitamin C (µg/ml)

mean ± SDVitamin E (µg/g tissue)

mean ± SDControl 20 18.13 ± 2.7 9.79 ± 1.88Stage I 6 17.15 ± 3.4 8.17 ± 1.31Stage II 7 15.06 ± 2.58 7.00 ± 0.67*

Stage III 8 11.13 ± 1.71*b 5.65 ± 0.5*

Stage IV 6 6.95 ± 0.28*a 4.33 ± 0.57*b

*P < 0.001: each stage versus control; aP < 0.001: each stage versus stage I,II; bP < 0.001: each stage versus stage I.


present investigation, the observed increase inLP, the end product of lipid peroxidation, incervical carcinoma tissues compared to controlsmay be due to extensive tissue damage ordecrease in the antioxidant defence mechanisms.Increased levels of LP in tumor tissues and in thesera of patients with cervical carcinoma havebeen reported [2,3,8]. It has been suggested thatincrease in peroxidation would cause degenera-tion of tissues and that LP formed at the primarysites, would be transferred through the circulationto other tissues and provoke damage by pro-pagating the process of lipid peroxidation [18].The negative correlation between LP levels andvitamin E content as well as GSH-Px activity incervical carcinoma indicates that increased lipidperoxidation could be a result of poor antioxidantdefences in tumor cells.

Glutathione is a tripeptide which plays a vitalrole in the protection of cellular constituentsagainst oxidative damage. GSH alone or inconjunction with other proteins can protect thecells against lipid peroxidation [29]. The tissueGSH level was low in all stages of malignancycompared to controls which agrees with previousreports [2,3]. Corrocher et al. [9] have reportedthat a diminished level of GSH leads tooverproduction of free radicals and consequently,neoplastic transformation. It has been stressedthat supplementation of GSH to mice bearingexperimental hepatoma leads to a remarkableimprovement and finally disappearance of tumors[20,21]. Following this approach, Kawano et al.[16] successfully treated with a large dose ofGSH a patient with inoperable hepatoma.

Glutathione peroxidase (GSH-Px) is aselenium-dependant enzyme which catalyses thedetoxification of hydrogen peroxide and otheroxidants through glutathione [30]. GSH-Pxactivity was found to be decreased in cervicalcarcinoma and this decrease was correlated toadvanced stage. Decreased activities of GSH-Pxwere reported in experimental carcinogenesis[22,4], in human hepatoma [6,9] and in cervicalcarcinoma [2,3]. The low activity of GSH-Pxmight increase the accumulation of free radicalswhich, in turn, may induce the neoplasticprocess. Further, it is possible that the scavenger

system is impaired due to reduced synthesis ofglutathione peroxidase due to carcinogenesis.The low availability of its substrate, GSH, maybe a reason for the decreased activity of the en-zymes, given the significant correlation betweenthe two values. It has been also reported thatGSH-Px activity is inactivated by increasedproduction of free radicals [9].

Superoxide dismutase (SOD) catalyzes celldefence reactions, against the potentially harmfuleffects of superoxide anion generated by a widevariety of biological process [3]. We observedmarked reduction in SOD activity in carcinomaof the uterine cervix as compared to controltissues. The decrease in SOD activity might beattributed to reduced synthesis. Depressed SODactivity and other defense enzymes in tumorscould be responsible for the increasedperoxidation products generated by tumor cells[2]. These observations coincide with studiesreported for patients with malignant lymphomaand acute myeloid leukemia [25] and withcervical carcinoma [2,3]. Depressed SODactivities were also seen in different organs inEhrlich carcinoma bearing mice [17] and also infew examples of cell lines when compared tonormal tissues [12].

Vitamins C and E are powerful antioxidants,free radical scavengers and well-known inhibitorsof lipid peroxidation [15]. Jayashree and Sukla[14] have reported that serum ascorbic acid levelswere diminished in cancer patients andsupplementation of this vitamin could inhibit thegrowth of transplantable sarcoma. Diminishedlevels of vitamin E and C were also observed insera of patients suffering from cervical carcinomacompared to controls [3]. These findings havebeen supported in the present study as regardsvitamin E and C levels and the decreased anti-oxidants may be one of the factors responsiblefor progression of cervical carcinoma. Althoughthe precise mechanisms by which oxidative stresscan produce cancer are still difficult to establish,our findings suggest that oxidative stress in theform of elevated levels of lipid peroxidestogether with impaired antioxidant defencemechanisms may play a role in the etiology andprogression of cervical carcinoma.


Acknowledgement

The authors thank Prof. Ali Khalifa for hisunlimited support.

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