CANCER

ELSEVIER Cancer Letters 94 ( 1995) 107-l 11

LETTERS

Decreased plasma glutathione in cancer of the uterine cervix

Ajay Kumara,*, S. Sharma, C.S. Pundirb, A. Sharmac

aBiochemistry Division, Institute of Cytology of Preventive Oncology (ICMR), Maulana Azad Medical College,
B. Shahzafar Marg, New Delhi 110 002, India
bDepartment of Biosciences, M.D. University, Rohtak I24 001, India
‘Malaria Research Centre, 22- Sham Nath Marg, Delhi 110 054. India

Received 12 September 1994; revision received 27 April 1995; accepted 28 April 1995

Abstract

Plasma total glutathione (GSH) content (reduced plus oxidized) was estimated in varying grades of cervical intraepithelial
neoplasia (CIN) and in invasive cervical cancer. The values were compared with age-matched control women. The results
show significantly lower level of plasma GSH in CIN III and invasive cancer compared to controls (0.724 versus 1.082 and
0.622 versus l.O82,~moI/ml of plasma, P < 0.05). Further, the odds ratio analysis showed high plasma GSH content was
found to be protective against the development of cervical cancer. The results suggest a plausible association of plasma GSH
with cervical carcinogenesis. The quantitative changes occurring in plasma total glutathione during cervical carcinogenesis is
a useful finding and might represent a systemic biochemical marker for precancerous and cancerous lesions of the uterine
cervix.

Keywords: Total glutathione (reduced + oxidized); Cervical intraepithelial neoplasia (GIN); Cervical carcinogen&s

1. Introduction cals [ 11. Free radicals are toxic to the cells and are
thought to be contributory factors in carcinogenesis.
Cancer of the uterine cervix is the leading malig- Glutathione (GSH), the principle non-protein thiol
nancy afflicting developing countries [ 171. In India, in mammalian cells, has an important role in cellular
about one lakh women develop cervical cancer an- detoxication processes [4]. Among its functions is to
nually which constitutes the 16% of the global inci- quench endogenousperoxides and free radicals [ 131.
dence [22]. The etiology of this disease is thought to Changes in the GSH content have been reported in
be multifactorial [9]. Viruses (HPVs, HSVs) and several malignancies [7,8,16]. Cancer of the uterine
bacteria are thought to be important risk factors for cervix hasbeen largely neglected in relation to GSH
the development of cervical carcinogenesis [ 18,231. related parameters. Few studies have reported de-
It has also been speculated that as a consequence of creased levels of GSH in cervical neoplasia [2,19].
these infections, phagocytosis may produce free radi- But these studies have been carried out from areas
where the frequency of this malignancy is very low.
Therefore, it is worthwhile to examine the role of
* Corresponding author. GSH in cervical carcinogenesis among Indian women

0304-3835/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved

SSDI 0304-3835(95)03832-H
where the prevalence of cervical cancer is the highest
in the world (221. Since measurement of tissue GSH
content is tedious and requires an invasive procedure,
we have estimated plasma GSH content to find out
whether plasma GSH is related to cervical carcino-
Lrencsis
This present case-control study has estimated the
piasrna total GSH (GSH + GSSG) content in women
with various grades of cervical intraepithelial neo-
plasia (CIN) and invasive cancer, and the values were
compared with controls.
2. Material and methods
Glutathione, reduced (GSH); 5,5’-dithiobis (2-
nitrobenzoic acid) (DTNB); NADPH; glutathtone
reductase (GR) from yeast extract (90 U/ml), were
purchased from Sigma Chemical Co., USA. Other
chemicals were obtained locally and were of analyti-
::a1 grade.
2 2. Subjects
We have examined a group of womenmparticipat-
ing in a case-control study of cervical cancer carried
out in North Indian women. Cases were defined as
women with two consecutive positive Pap smears for
precancerous and cancerous lesions of the uterine
cervix. Colposcopically directed biopsies were inter-
preted as various grades of CIN, i.e. GIN l and IT
(N - Xl), CIN III (n= 50) and invasive cancer
{ri =: 30). Controls (n = 60) were age-matched women
with two consecutive negative or intlammatory pap
smears without any known gynecological dysfunc-
tion. Women taking oral contraceptives or having
smoking habits were excluded from this study.
pernatant was separated out and used for the assav.
i
The assay was carried out in 3 ml. The reaction con-
tained I:0 ml of 1 M Tris (pH 7.2); 100~1 of 12 mM
NADPH; 60 ,LI1 of DTNB (40 mg/dl) and 100 ,u 1 of
supernatant. The reaction was started by adding
0.3 U/ml of GR enzyme. The linear change in absor-
bance was monitored at 412 nm. Values were ex-
pressed as /Amol/ml plasma.
2.4. Statistical analysis
The group means among the various study groups,
categorized by the cervical morphologies, were com-
pared using one-way analysis of variance (ANOVA).
‘To determine the significant difference in the mean
values of the pairs of the various study groups, the
studentized range test procedure was used after ob-
taining the critical difference value (CD) at the 0.05
level. If the difference between the mean values of
two groups was found to be greater than the calcu-
lated CD value, the two groups were considered to be
significant!y different for that parameter. The odds
ratio (OR) values were calculated using first quartile
of plasma GSH content as reference group.
3. Resutts
The mean age among al1 the study groups was
found to be non-significantly different (P = 0.1010,
ANOVA). The mean age for the control group was
34.5 years, however for GIN and invasive cancer
groups, the mean age was found to be 38.5 and
43.0 years, respectively. Mean plasma GSH content
for the various groups are depicted in Table 1. The
data show that mean plasma GSH content was fo.und
to be significantly different among the various groups
using one-way ANOVA (P = 0.0001, ANOVA). The

2.3. Biochemical kuzlysis l‘ahle I

Peripheral blood samples were coltected in hepar- Plasma total gtutathione in women with warying grades of CIN
lnized tubes and analyzed within 2-3 h. Each speci- and invasive cancer
men- was centrifuged at 809 X R for 15 min at 4°C. -
1’lasm.a was pipetted out and used for total GSH esti- Subiects N GSH* @molJml)
mation. Total plasma GSH was estimated according
COIItrOlS 60 I.082 f 0.334
to the method of Tietze [21]. Briefly, an aliquot of
CIN I and II 50 0.983 + 0.5 I.7
plasma was mixed with precipitating solution (1.67% C-IN III so 0.724 + 0 236
glacial metaphosphoric acid, 0.20% EDTA, 30% Invasive cancer 30 0.62 + 0. I7 I
NaCl) and the mixture was left on ice for 10 min.
After centrifugation at 800 x g for 10 min, the su- f I: 0.0001. 4NOVA
 A. Kumar et al. 1 Cancer Letters 94 (1995) 107-l 11 109

Table 2 Table 3 shows the odds ratio values and their cor-
Multiple comparison of plasma GSH content between the pairs of responding 95% CI for the various groups in all
various cervical morphologies quartiles of plasma GSH. For CIN III, a significant
OR value of 0.27 (CI = 0.084l.86) was found in
Type of pairs Statistical findings
fourth quartiles of plasma GSH. However for inva-
Controls versus sive cancer, significant OR values of 0.36 (CI =
CIN 1 and II NS 0.09-1.42) and 0.19 (0.044.77), respectively were
CIN III s found in the third and fourth quartiles of plasma
Invasive cancer S GSH. No significant OR values was found in CIN I
CIN I and I1 versus
and II groups (P > 0.05).
CIN III NS
Invasive cancer S
CIN III versus 4. Discussion
Invasive cancer S
The main finding of this preliminary study is a
Note: S, significant difference (mean difference > CD values).
significant decrease for plasma GSH in high grade
NS, non-significant difference (mean difference < CD values).
CIN (CIN III) and invasive cancer. Also the high
content of plasma GSH was found to be protective
controls were found to have the highest plasma GSH against the development of cervical cancer, based on
content (1.082,~mollml of plasma), however the in- the odds ratio values (Table 3). These findings sug-
vasive cancer group was found to have the lowest gest a plausible association of plasma GSH with
plasma GSH content (0.622 pmollml of plasma). The uterine cervical carcinogenesis.
multiple comparison for the plasma GSH between the The level of GSH inside the cell depends firstly on
pairs of various cervical morphologies is illustrated the rate of synthesis and therefore, on the pool of
in Table 2. It has been observed that mean plasma cystein, glycine, glutamate and their assembling en-
GSH content was found to be significantly different zymes. Secondly, it depends on the rate of its utiliza-
between the pairs of controls and CIN III (1.082 ver- tion via glutathione S-transferase (GST) and glu-
sus 0.724,~mollml of plasma) and controls and inva- tathione peroxidase (GPX) system [ 131. The impor-
sive cancer (1.082 versus 0.622 pmollml of plasma). tance of red cell GSH and its maintenance in the re-
Other pairs which were found to be significantly dif- duced form has been recognized. Red cells are found
ferent were (i) CIN I and II and invasive cancer and to have very low GSSG content, since GSSG is
(ii) CIN III and invasive cancer (Table 2). Further the known to be continually transported outward from
gradual decrease in the plasma GSH was found as the the red cells into the plasma [20]. The existence of
severity of disease increased. plasma GSH has been appreciated only recently. The

Table 3
The odds ratios and 95% confidence interval for the various quartiles of plasma GSH in various grades of CIN and invasive cancer

Plasma GSH CIN I and II CIN III Invasive cancer

quartiles
(umol/ml) % OR(CI) % OR(C1) % OR(C1)

0.150-0.650 18 1 26 1 34
0.651-1.150 26 0.74 38 0.75 30 0.45
(0.23-2.48) (0.26-2.19) (0.13-1.62)
1.1.5-1.800 18 0.60 18 0.42 20 0.36*
(0.16-2.19) (0.13-1.41) (0.09-l .42)
1.801-2.450 38 0.82 18 0.27* 16 0.19*
(0.27-2.52) (0.08-0.86) (0.04-0.77)

* Significant P values (P < 0.05).

amount of GSH is thought to be very low compared ing cervical car&o-genesis. However, it will be in-
with its red cell content, eO.001 of red cell contents teresting to investigate the GSH in the exfoliated
f]. cells of cervical precancer/cancer women. Further
A number of investigators have measured the studies on the followup of the patients are underway
plasma GSH content of human and rats. In some in- to find out any correlation between the GSH content
stances, separate estimation of reduced and oxidized and progression of disease.
GSH have been made. In human, however, the level
of GSH is so low as to make this distinction imprac- Acknowledgment
tical between the reduced and oxidized forms. There-
fore, only total GSH (CSH + GSSG) has been esti- The authors wish to thank Mrs Kavita Magon for
mated. Several researchers have found altered GSH her help in collection of blood samples.
~oncrntration in a number of human cancers [5,6].
The GSH content has been found to be increased in References
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