Indian Journal of Experimental Biology Vol. 37, September 1999, pp. 859-864

Glutathione level and its relation to radiation therapy in patients with cancer of uterine cervix

H Mukundan & A K Bahadur

Department of Radiotherapy, Maulana Azad Medical College and associated Lok Nayak Hospital , New Delhi 110002, India

and

A Kumar, S Sardana, S L D Naik, A Ray & B K Sharma*

Institute of Cytology and Preventive Oncology (ICMR), Maulana Azad Medical College Campus , New Delhi 110002, India.

Received 2 December 1998; revised 9 June 1999

Glutathione functions as an important antioxidant in the destruction of hydrogen peroxide and lipid peroxides ' by pro­viding substrate for the glutathione peroxidase and also promotes the ascorbic acid. Glutathione plays a vital role in detoxi­fication of xenobiotics, carcinogens, free radicals and maintena:1ce of immune functions . The study was aimed to determine plasma glutathione as well as erythrocyte glutathione and glutathione peroxidase in patients with invasive cervical carci­noma (n=30) before initiation and after completion of radiotherapy and subsequently, at the time of tirst three monthly fo l­low-up visit. The levels of plasma glutathione, erythrocyte glutathione and glutathione peroxidase activity were found to be lower in all cervical cancer patients as compared to age matched normal control women. The study indicates a change in an­tioxidant status in relation with the glutathione system among patients with invasive carcinoma of the uterine cervix. This study also demonstrates the effect of radiation therapy on this antioxidant system.

Cancer of the uterine cervix is the leading malignancy affecting women in developing countries and is the commonest malignancy among Indian women. Al­though a number of risk factors have been identified like early sexual activity (before 18 years of age), multiple sexual partners, cigarette smoking and low socio-economic status, the aetiology of cervical can­cer is not yet clearly understood. Numerous c1inico­epidemiological studies have implicated venereally transmitted agents, viz. HSV -2 and, more recently, HPV (Human papiiloma virus, mainly types 16 and 18) in the genesis of cervi~al neoplasm.

Cancer of the uterine cervix does not arise de novo, but are preceded by a spectrum of abnormal epithelial changes known as dysplasia which is the important precancerous lesion. Several studies have also sug­gested the role of antioxidant elements in diet in pre­vention of cervical cancer i

,2. Diet deficient in vitamin A, vi tamin C (ascorbic acid) and folic acid appears to playa role in the aetiology of this disease. Perox ida­tion of lipids exposed to oxygen is responsible for

* Correspondent author: Telephone: 011-323 1880,323 8950. Fax: 011-323 3406.

damage to tissues in vivo and has been implicated as a probable cause of cancer. To control and reduce lipid peroxidation, nature invokes the use of antioxidants. Glutathione (GSH) functions as an important antioxi­dant in the destruction of hydrogen peroxide and lipid peroxides by providing substrate for the glutathione peroxidases (GPX) . Glutathione also functions as an antioxidant by promoting formation of the reduced forms of other antioxidants such as ascorbic acid and detoxification of xcnobiotics, carcinogens and free radicals and maintenance of immune functions .1.

Radiation therapy is the most commonl y used therapeutic modality for inoperable cervical ca ncers. The effects of rad iation therapy are mediated by the production of free radical s. Since gl utathione is the most important cellular antioxidant and a quencher of free radicals, it can be postulated that those cell sys­tems containing a high ratio of glutathione, as re­flected by the plasma and erythrocyte glutathione lev­els, may be found to be relatively rad iores istanr-+.5, Studies on blood GSH in several malignancies have reported an altered GSH redox system (,.g Bhuvara­hamurthy et at. 9 studi ed the effect of radiotherapy and chemoradiotherapy on circulating antioxidant system

860 INDIAN 1 EXP BIOL, SEPTEMBER 1999

of human uterine cervical cancer and observed that reduced levels of GSH and GPX become normalized after treatment.

The present study was aimed to determine plasma GSH, erythrocyte GSH and GPX activity as well as their variations in patients with invasive cervical car­cinoma before and after radiotherapy .

Materials and Methods . Patients-30 patients of hi stopathologically

proven carcinoma of the uterine cervix (Stages Ib­IIIb) were randomly selected from patients attending the Radiot~erapy Department of Maulana Azad Medical College and associated L.N. Hospital , New Delhi.

Controls-30 age-matched females also were se­lec ted from the Obstetrics and Gynaecology Depart­ment of Maulana Azad Medical College, New Delhi . The controls had no history or evidence of malig­nancy, pelvic inflammatory disorders, or any systemic illness and had 2 consecutive PAP smears negative for cervical abnormality . .

A detail ed clinical history of the subjects was col­lected by personal interview and clinical examina­tions were perfOimed. The cervical tumour grading was done as per FIGO guideline 10. Blood samples at three time points were collected from all patients in­cluded in the study and only one sample was drawn from the control women. For patients , first blood sample was drawn at the time of presentation in the Department of Radiotherapy prior to initiation of therapy. Second blood sample was collected at the completion of treatment. Subsequently, the third sam­ple was collected on the first three monthly foll ow-up visit. All patients were treated as per treatment proto­col being followed at the Radiotherapy Department of Maulana Azad Medical College by external rad iation therapy with Theratron 780 machine and/or intra­cavitary irradiation by Selectron LDRlMDR brachytherapy unit.

Biochemical analysis-5 ml of peripheral venous blood was collected in heparinised tubes. The plasma was separated by centrifugation and used for GSH assay. Total plasma GSH was estimated according to the method of Tietze". Briefly, an aliquot of plasma was mixed with precipitating solution (1.67% glacial metaphosphoric acid, 0.20% EDTA, 30% NaCI) and the mixture was kept on ice for 10 min . After cen­trifugation at 800 g for 10 min, the supernatant was separated out and used for the assay. The assay was

carried out in 3 ml of reaction mixtu re. The react ion mixture contained 1.0 ml of 1M Tris (pH 7.2) ; IOOJ..lI of 12 mM NADPH; 60 J..II of DTNB (40 mg/d l) and 100 J..II of supernatant. The reaction was initiated by adding 0.3 V/ml of glutathione reductase enzyme. The linear changes in absorbance were monitored at 412nm.

After separation of plasma from the whole blood, erythrocytes were washed twice in cold normal sa line (0.9% NaCl solution) . Washed e rythrocytes were lysed in di stilled water, and were ce nt rifuged at 800 g for 15 min at 4°C. Supernatant was used to determine enzyme activities of the se lenium dependent GPX according to the method of Pag li a and Valentine l2

.

Reduced GSH content in erythrocytes was measured spectrophotometrica lly using DTNB as colouring reagent as per the method described by Beutler e!

al.l3. Haemoglobin was determined by cyanmethemo· globin method .

Statistical analysis-The data of the stl:ldy were subjected to univariate and bi va riate analysis . In order to test the significance of observed re lationships be­tween various variables, the Student's t test was em· ployed.

Results The mean age of the patients included in the study

was 48 .8 years (SD ± 11 .4), while control group of women had a mean age of 49 .0 years (±1 3.4) . Amongst the cases, the mean age at menarche was observed to be 14.56 ± 1.22 years, whereas the con­trol women had a mean age of 14.06 ± 0.72 at men­arche. The mean age at marriage for pati ents was 16.1 ± 1.04 years and 15.88 ± 1.77 years for controls. A v­erage parity of the patient group was 5 ± 2, whereas the control group had onl y 3 ± I. Our of total 30 pa­tients , 17 patients belonged to postmenopausa l and 13 patients were premenopausal.

Out of the tota l 30 pati ents stud ied, 83 % cases (n=25) had a histopathological d iagnosis of squamous cell carcinoma of the cervix and the rest were adeno­carcinoma. Of the pati ents with squamous cell carci­noma, 9 patients had well differenti ated tumour, 13 had moderately differentiated and the res t 3 cases belonged to poorly differenti ated squamous ce ll car­cinoma. With regard to cli nica l staging of the pa­tients, 3 patients were of stage J, whil e 10 and 17 pa­tients were of stage II and stage III re ·pecti ve ly.

The levels of plasma glutathione (GS H). erythro­cyte glutathione and glutathione peroxidase (GPX)

MUKUNDAN e/ al.: GLUTATHIONE & RADIATION THERAPY IN CERVICAL CARCINOMA 861

activity were found to be reduced in all patients with invasive carcinoma of the uterine cervix as compared to age matched control group. These differences in the levels of GSH and GPX were found to be statisti­cally significant (P<O.OO I) (Table I) . Further,. the mean value of plasma GSH in patients after rad.io­therapy was found to be lower than that of normal controls. Similarly, the mean value of erythrocyte GSH and GPX activity in post-radiotherapy cases were lower as compared to matched normal controls. Similar decreased levels of plasma GSH, erythrocyte GSH and GPX values were also observed in the pa­tients on follow-up (3 months after radiotherapy). However, no remarkable alterations in the levels of plasma GSH, erythrocyte GSH and GPX were found at the two different time points after treatment (Table I).

Clinical stage-wise analysis of the levels of GSH showe~ an increase in the mean values of plasma GSH levels in all patients who had completed the treatment, except amongst the follow-up patients with invasi ve cervical carcinoma stage III wherein mean

plasma GSH level was lower than the corresponding post-radiotherapy state (Table 2). The histological type of cervical carcinoma also revealed an interest­ing observation (Table 3). Significant difference (P<0.05) was observed in the plasma GSH values among patients with well differentiated squamous cell carcinoma and moderately differentiated squamous cell carcinoma before treatment. The differrence be­came non-significant after treatment.

Out of the total 30 patients studied II patients (stage I: 3 patients, stage II: 4 patients and stage III : 4 patients) showed complete response (>75 % regres­sion) whereas 6 patients (Stage II: 3 patients, stage III: 3 patients) revealed partial response (50% regres­sion) and the equal number of patients (s tage II : I patient, stage III : 5 patients) showed no response «50% regress ion). 7 patients left without completing their treatment. Among the patients who showed no response (n=6), 5 patients left afterr completing the treatment and did not attend the follow-up study. The changes of the levels of plasma GSH, RBC GSH and GPX activity of the patients with cervical carcinoma

Table I-Levels of plasma GSH, RBC GSH and GPX activity in patients with invasive carcinoma of the uterine cervix before (Pre RT) and after radiotherapy (Post RT) as well as on I sl follow up

[Values are mea!1 ±SD)

Mode of treatme!}t No.of patients Pl asma GSH (Ilg/ml) RBC GSH (Ilg/g Hb) GPX (1 U/g Hb)

Controls 32 209 .1 ± 55.15 389.8 ± 11 3.3 35 .5 ± 8.0 1 Pret RT 30 117.8 ± 44.54* 267.4 ± 86.18* 28 .23 ± 7.55* Post RT 23 125 ± 47.58* 252.3 ± 85.07* 26 .9 ± 5.53* Follow up 18 131 ± 58.49* 243.4 ± 84.05* 27.72 ± 6.78*

* P < 0.001

TaI:,le 2-Levels of plasma GSH, RBC GSH and GPX activity in various clinical stages of patients with carcinoma .of the uterine cervix before and after radiotherapy as well as on I st follow up

[Values are mean ± SD)

Clinical No. of Pre RT --~----~~~--~---

stage cases Plasma RBC No. of Post RT

GPX cases Plasma RBC No . of Follow up

GPX cases Pl asma RBC GPX GSH GSH GSH GSH GSH GSH

3 83.20 378.8 20.23 3 93 . 1 354.8 20.83 2 100.5 350.1 14.38 ± ± ± ± ± ± ± ± ±

21.82 99.07 10.73 25 .52 82.83 7.78 20.72 114. 1 6.32

II 10 128.1 259.9 28.49 8 145.7 252.9 26.13 7 168.1 275. 3 27.44 ± ± ± ± ± ± ± ± ±

56.14 70.54 5.63 56. 13 71.45 4.90 54.65 65.2 4.68

III 17 117.9 251 .2 29.56 12 120.9 226.4 28.87 9 109 174.9 30.9 ± ± ± ± ± ± ± ± ±

37.92 82.28 7.57 43 .79 78.66 4.42 54.7 6 1.74 5.01

862 INDI AN J EXP BIOL, SEPTEMBER 1999

Tabie 3--Levels of pl asma GSH, RBC GSH and GPX activity in patients with diffferent histological differenti ati ons of sq uamous cell carcinoma of the uterine cervix before and after radiotherapy as well as on I st follow up.

TD No.of cases

WD 9

MD 13

PD 3

AD 5

Plasma GSH

98.16

± 35. 16

128.3

± 47.05

124.6

± 44.76

123.9

± 55. 12

TD = tumour di fferentiation

Pre RT RBC GSH

241.3

± 75 .62

260.6

± 83.2 1

285.7

± 77.56

319.6

± 65.5

GPX

26.26

± 6.24

27.04

± 6.4

38.2

± 6.97

28.64

± 5.8

[Values are mean ± SD]

No. of Post RT ~P~la-s-m-a--~R~B~C~--~G~P~X~ cases

GSH GSH

7 109.6 240.5 26.09

± ± ± 44.42 77.97 6.06

130.7 225.3 26.36

± ± ± 5 1.43 77.79 5.58

3 141.2 257.6 30.55 ± ± ±

40.45 86.83 3.33

153.6 285.2 25.87 ± ± ±

46.01 . 68.5 3.89

No. of Follow up --P-I-as-ln-a~~R~B~C~--~G~P~X cases

7

7

3

GSH GSH

106

± 59.7

148.9

± 66.47

130. ;

137.6 ±

32.0:1

23 1.7

± 83.01

207.4

± 68.58

214.8

270.3 ±

9 1.22

26.36

± 5.7

27.82

± 6.47

32.90

30.55 ±

3.54

WD=Well differentiated squamous cell carcinoma (SCC); MD=Moderat ely di fferentiated SCC; PD=Poorly di fferen ti ated SCC; AD = Adenocarcinoma

Table 4--Levels of plasma GSH, RBC GSH and GPX activity in patients with in vasive carcinoma of the uterinc lcrvix during trcatmcnt response.

[Values are mean ± SD]

No.of cases*

Responder II

Partial responder 6

Non Responder 6**

* 7 did not complete treatment ** 5 left after treatment

Pl asma GSH

133.7

± 51.17

120.5 ±

46.77

100. 1

± 39. 19

PreRT RBC GPX GSH

269.9 28.0

± ± 84.90 8.28

221.6 27.75 ± ±

76.36 6.69

228.9 27.53

± ± 74.45 7.52

included in our study did not show any s ignificant relationship with the treatment response (Table 4).

Discussion The study has high lighted the alteration of GSH

and GPX in patien ts with invasive cervical carc inoma before, during and after radiotherapy treatment. This is in conformation with earlier reports on cervica l

Post RT Fo ll ow lip Plasma RBC GPX Plasma RB C GPX GSH GSH GSH GSH

139.7 251.2 28.58 146.7 257. 1 27.38 ± ± ± ± ± ±

51.85 86.08 5.95 58.3 1 93. 76 7.82

11 3.4 220.6 28.06 97.55 203.0 27.90 ± ± ± ± ± ±

43.53 77.76 5.6 1 52.59 48.94 5.68

106.7 219.6 27.78 130.1 2 14.8 32.90 ± ± ±

39.77 87.52 4.70

carcinogenesis in re lat ion with plasma GSH I~. The utili zati on of GSH may be mediated by the gl utat hi­one S- transferase (GST) or the g lutathi one perox idase system ts

. Red cell s are found to have very low oxi­dised glutathione content s ince oxid ised g lutathi one is known to be continually transported out ward from the red cells into the pl asma l6

. In humans , the level of g lutathi one is too low to make a distinct ion impracti-

MUKUNDAN et at.: GLUTATHIONE & RADIATION THERAPY IN CERVICAL CARCINOMA 863

cal between the reduced and oxidised forms. There­fore, only total GSH (oxidised + reduced) was esti­mated. Several reports have shown altered GSH con­tent in a number of cancers of human origin '7, '8. as well as of animal origin '9

. Increase in red cell GSH has been also reported in leukaemia20 and gastroin­testinal adenocarcinoma21

. The present study has re­vealed lower levels of plasma GSH in patients of in­vasive cervical carcinoma before and after radiother­apy as well as during follow-up in comparison to controls. Interestingly, difference between GSH lev­els in relation to histological differentiation were also observed. Contrary to the findings of Bhuvaraha­murthy et ai9, levels of GSH and GPX of our study did not change to the normal values (normal controls' values) even after treatment.

Erythrocyte glutathione peroxidase acts as an anti­oxidant enzyme. However, it has been observed in the present study that the erythrocyte glutathione peroxi­dase activity among patients of cervical cancer was significantly lower in comparison to normal healthy controls which is in conformation of the findings on CIN and invasive cervical carcinoma, as reported by Kumar et ai. 22 and Bhuvarahamurthy et ai.9 respec­tively. Balasubramaniyam et ai. 2

.1 reported that the activities of glutathione peroxidase were significantly lower in stage ill and IV patients of carcinoma of human uterine cervix than that of normal controls. Similar findings were also observed in CIN ill as well as in invasive cervical cancer by Kumar et al. 22. It has also been reported that the reversion of erythrocyte GPX activity to normal level occurred following ra­diotherapy in patients with uterine cervical carci­noma9

. These findings reflect that GSH detoxification system, one of the defense systems against exogenous as well as endogenous factors (i.e., free radicals, car­cinogens and other peroxides, etc.) is greatly influ­enced during the process of cervical carcinogene~is. As such, lower level of GPX together with the lower GSH contents may favour an overproduction of free radicals and lipid peroxides which in tum may induce the damage to cell membrane and cellular molecules (DNA, RNA) leading to neoplastic changes. It is pos­sible that GSH scavenger system may be impaired as a consequence of reduced synthesis of the enzymes due to the carcinogenic process .

Ionizing radiation is toxic to organisms because it induces deleterious stuctural changes in essential macromolecules. These changes can be the result of direct interaction with radiation. However, it is gen-

erally considered that, because of the abundance of water in living organisms, a more important mecha­nism is the interaction with free radicals formed by photolysis of water. GSH plays a key role in protect­ing cells against electrophiles and free radicals24

. Thi s is due to the nucleophilicity of the SH group and to the high reaction rate of thiols with free radicals . Ra­diation resistance of many cells is associated with high intracellular levels of GSH25

.26

. Administration of radioprotective agents like WR-272 I has shown an elevation of the blood GSH levels27

. Cells containing low levels of GSH were found to be much more sen­sitive to the effect ~f irradiation than controls28

, al­though Backer et ai?9 reported that loss of GSH does not affect radiosensitization. However, in normal cells, GSH or non-protein cellular thiol s are present at levels far in excess of that required for the repair of radiation-induced damage3o

. Further Cholon et al. .1 1

observed that ionizing radiation decreased the leve ls of total GST activity.

Numerous studies have shown a correlation be­tween the erythrocyte glutathione peroxidase activity and the radiation response. Glutathione peroxidase is an important ' scavenger' enzyme which has an incor­porated selenium molecule to which it owes its activ­ity. Bewick et ai. 32 observed the deficiency of this enzyme in erythrocytes of lymphoma patients. len­dryczko et al. 33 observed an inverse correlation be­tween the activity of this enzyme and the extent of the disease in patients with colon cancer.

In a study conducted by ladhav et al . .1 ~, a good cor­relation was detected between the degree of GSH de­pletion and the tumour response. However, the pres­ent study did not reveal any significant relationship between the treatment response and the changes of the levels of either glutathione in blood plasma and RBC or glutathione peroxidase in RBC of cervical cancer patients.

The data of the present study clearly suggest the changes in antioxidant status with special reference to the glutathione system among patients with invasive €arcinoma of the uterine cervix in comparison with the normal controls. This study also demonstrates the effect of radiation therapy on this antioxidant system. However, study on larger sample size, with a longer follow-up period and measuring other related antioxi­dant levels also, may yield more meaningful data on the role of antioxidant system in the clinical course of cervical tumours.

864 INDIAN J EXP BIOL, SEPTEMBER 1999

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