Influence of alpha tocopherol during carcinogenesis in uterine cervix of mice

Nutrition Research, Vol. 20, No. 2, pp. 261-272. 2000 Copyright 0 2000 Elsevier Science Inc. Printed in the USA. All rights resewed 0271.5317/00/&see front matter ELSEVIER

INFLUENCE

PII: SO271-5317(99)00158-X

OF ALPHA TOCOPHEROL DURING CARCINOGENESIS CERVIX OF MICE

IN UTERINE

Sarmishtha De, M.Sc., Archana Sengupta, Ph.D., R.N. Chakraborty, M.D and Sukta Das, Ph.D. Departments of Cancer Chemoprevention and Preventive Oncology, Chittararjan National Cancer Institute. Calcutta, India.

ABSTRACT The effect of supplemental alpha tocopherol and its mode of action were evaluated during carcinogenesis in uterine cervix of mice by chronic exposure to 20-methylcholanthrene (MC). Oral administration of alpha tocopherol restricted the progression of dysplastic changes in cervix uteri which lead to carcinoma in situ during chronic exposure to MC. Enhanced mitogenic stimulation of peripheral blood lymphocytes (PBL) indicating improved immune response was also noted following treatment. A decrease in micronucleus frequency in the vaginal exfoliated cells suggested reduced genotoxic effect of the carcinogen after alpha tocopherol administration. A decrease in lipid peroxides following ingestion of alpha tocopherol imply its protective role on DNA damage by oxidative free radicals. Similarly, elevated glutathione level and increased activities of glutathione+transferase, glutathione peroxidase, catalase and superoxide dismutase indicated involvement of alpha tocopherol in host’s detoxification system . c,2lMWl Elrewr Sc*ence Inc. KEY WORDS: Alpha tocopherol, Chemoprevention, 20-Methylcholanthrene, Micronucleus, Antioxidant enzymes, Mitogenic response.

INTRODUCTION Alpha tocopherol is a major lipid soluble antioxidant vitamin and free radical scavenger, present as an integral component of cellular membranes and having important biological functions (1). This vitamin is known to protect cellular membranes from oxidative damage and lipid peroxidation (2,3). Supplementation of this antioxidant is also reported to decrease oxidative DNA damage (4). In view of these properties, alpha tocopherol has been considered to be one of the potential chemopreventive agents for cancer. Experimental studies have revealed the protective effects of this vitamin against development of sarcomas and carcinomas at different *Corresponding Author: Sukta Das, Department of Cancer Chemoprevention, Chittaranjan National Cancer Institute, 37. S.P. Mukherjee Road, Calcutta 700 026, India. Tel.: 91 033 4765101/02/04, ext. 316.

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sites, although reports to the contrary also exist (5). The protective effect of alpha tocopherol on carcinogenesis of uterine cervix haa not yet been welI documented, although some experimental studies and human trials, including our own, are underway. The present report furnishes observation on the effect of supplemental alpha tocopherol during carcinogenesis of the cervical epithelium and its possible mode of action.

MATEFUALS AND METHODS Exnerimental

animals:

Swiss albino virgin female mice, in the age group of 5-6 weeks, weighing 20-23 g were obtained from the animal colony of our Institute. The mice were caged in groups of 5 per cage and kept in alternating periods of light and dark conditions of 12 h each. Standard animal food pellets (Lipton India Ltd.) and water were provided ad libitum. Chronic exposure to 20 methylcholanthrene (MC, Sigma Chemicals) was effected by pipetting the carcinogen dissolved in acetone gently into the cervix through the vaginal opening at a dose of 1OmgKg body weight daily for 30 days. Alpha toWDhero1 treatment: Oral administration of alpha tocopherol by gavage (Evion drops, E. Merck India) was started from the same day as carcinogen application. Different doses were tried initially and the minimum effective dose was ascertained as 100 mgKg body weight/day. ExDerimental arouus : Group I- normal mice, Group II- normal mice treated with alpha tocopherol, Group IIImice receiving carcinogen, Group IV- mice receiving carcinogen and alpha tocopherol, Group IVmice receiving only vehicle without carcinogen. Vaginal exfoliated cells were collected at weekly intervals and smear preparations made for cytological observation, which was confiied by histopathological study (6) . Micronucleus frequency (MN) was also examined in exfoliated cells (7,8) . All biochemical estimations were made on day 31 of the experiment. Glutathione (GSH) was estimated in the liver cytosolic fraction by using dithiobis(2&ro)-benzoic acid (DTNB) by the method described by Sedlack and Lindsay (9). Glutathione-S-transferase (GST) activity was measured in the liver cytosol as expressed by formation of 1-chloro-2-4-dinitrobenzene (CDNB)GSH conjugate following the method of Habig et al (10). Lipid peroxidation was estimated in liver microsomal fraction by measuring the thiobarbituric acid reactive substances (TBARS) formed in the tissue using the method of Okahawa et al (11). Glutathione peroxidase (GPx) activity was determined in the postmitochondrial fraction of liver by the method of Paglia and Valentine (12). Activity of catalase (CAT) was estimated by the method of Luck (13) in the liver post nuclear homogenate. Superoxide dismutase (SOD) activity was determined by quantification of pyrogallol auto oxidation inhibition by the method of Markhmd and Marklund (14). Protein was estimated by Lowry’s method (15). Blastogenesis of peripheral blood lymphocytes (PBL) was detetmined in vitro by the method of Moorhead et al (16). Mononuclear cells, from blood of experimental mice, were separated on Ficoll gradient and cultured in medium RPMI (Gibco) with

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10% foetal calf serum (Gibco) and phytohaemagglutinin (PHA,Sigma Chemicals) at a concentration of 20 ug /lo6 cells for 72 h (17). Tritiated thymidine incorporation by the PHA stimulated cells was determined and blastogenesis expressed as cpm / lo6 cells. Statistical analvsis of data : The data were statistically evaluated using Student’s t-test.

RESULTS Cyto-Histological

Observations:

The epithelial cells of the uterine cervix are exfoliated into the vagina. The normal five days oestrus cycle in female mice , consisting of proestrus , estrus , metaesttus and diestrus can be distinguished in vaginal smears by their characteristic cytological features. This normal cyclic variation in the cervical epithelium was disturbed following application of the carcinogen. After seven days of MC exposure mild dysplasia was noted (65%) which progressed to carcinoma-insitu after 30 days (96%) through stages that can be identified as moderate and severe dysplasia. Alpha tocopherol supplementation during MC administration restricted the cyto-histological changes in the cervical epithelium in 72Oh cases upto day 14, though the estrous cycle was disturbed. Mild dysplasia appeared around day 21(50%) and 15% moderate dysplasia was noted after 30 days (Tablel) and none developed carcinoma in situ during this period. No marked and consistent change was found in the vehicle treated group, so this group was discontinued after histo-cytological study. TABLE

1

Effect of Alpha Tocopherol on Development

Groups

MC treated MC + alpha tocopherol treated

Micronucleus

of Cervical Neoplasia in Mice.

Carcinoma in situ Day 30

31150 O/50

Test:

Observation of vaginal exfoliated epithelial cells revealed a significant increase in MN frequency (P
S. DE et al.

264 Biochemical Observations

:

GSH level and GST activiQ : In the carcinogen treated group GSH level and GST activity in liver were found to be decreased (P
60

10

FIG 1. Effect of alpha tocopherol on micronucleus frequency. Micronucleus frequency (MN) was counted per 1000 exfoliated cells of uterine cervix in normal (N), normal + alpha tocopherol treated (N+T), carcinogen treated (MC) and carcinogen + alpha tocopherol treated (MC+T) mice. Data represents mean f SE (n=8).

tocopherol treatment significantly enhanced the level of GSH almost two fold and activity of GST about four times fkom the normal values noted in the present investigation (P
K3N EN+1

HMC

BMC+T

P

x II

FIG 2. Effect of alpha tocopherol on GSH level and GST activity. Data represents mean f SE (n=8). N- normal, N+T- normal + alpha tocopherol treated, MC- carcinogen treated and MC+T- carcinogen + alpha tocopherol treated.

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Effect More than ten fold elevation value was noted following MC administration (P
265

of lipid peroxides over the notmal Oral intake of alpha tocopherol and carcinogen treated groups, the 3).

Effect on GPx. CAT and SOD actititv : Chronic exposure to MC produced a significant decrease in GPx activity (PcO.05) as compared to notmal animals. There was an increase of the GPx activity by five fold following concomittant treatment with alpha tocopherol (P
3.E d h

2.5 -

F

2-

n 8

l.S-

,: ? P

l-

:: 2 I-

0.5 -

FIG 3. Effect of alpha tocopherol on lipid peroxidation.Estimation was ma& in liver microsomal fix&on. Data represents mean f SE (n=@. N- normal, N+T- normal + alpha tocopherol treated, MC- carcinogen treated and MC+T- carcinogen + alpha tocopherol treated.

Mitonenic stimulation

of PBL

Response of PBL to PHA is a good indicator of lymphocyte activity which has a major role in tumour immunology. Mitogenic response was found to be depressed after 30 days of carcinogenic exposure; alpha tocopherol was found to activate the same (P
S. DE et al.

DISCUSSIONS In view of the antioxidant property of alpha tocopherol the present study was an attempt to evaluate the effect of this vitamin during chemical carcinogenesis of cervical epithelium in mice Experimental studies have shown that alpha tocopherol inhibited carcinogenesis and tumour growth in skin breast and oral cavity (5,18-19) but our study is perhaps the first

?? N ?? N+T .MC

GPx

GItK+T

CAT

SOD

FIG 4. Effect of alpha tocopherol on antioxidant defence enzymes. GPx, CAT and SOD activity were measured in livers. Data represents mean f SE (n=Q N- normal, N+T- normal + alpha tocopherol treated, MC- carcinogen treated and MC+T- carcinogen + alpha tocopherol treated.

report to demonstrate the influence of alpha tocopherol on uterine cervix. Moreover most studies withdraw the carcinogen after initiation, but we studied the effect during chronic carcinogenic exposure and therefore is of more signiI?cance with respect to assessment of the protective influence of alpha tocopherol. In this connection it may be mentioned that our prehminary results of human intervention trials with supplemental antioxidant vitamins have shown that vitamin E at a dose of 400 n&lay given continuously for 6-12 months was effective not only in preventing progression of pre-neoplastic lesions but also produced regression in some cases (20). In the present study alpha tocopherol was administered at a very high dose (1OOmgKgday for 30 days), though the notmal physiological requirement is much lower, because this was found to be the minimal effective dose in our experimental model receiving a chronic exposure to the carcinogen. Appearance of mild dysplasia apparent from day 7 of MC administration with the establishment of carcinoma-m-situ around day 30 could be delayed following treatment with alpha tocopherol upto the second week after which mild to moderate dysplasia was evident which persisted and did not progress to carcinoma in situ within the experimental period i.e., 30 days, inspite of continuous exposure to the carcinogen.

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TABLE 2

Effect of Alpha Tocopherol on Response of Peripheral Blood Lymphocytes towards Mitogenic Stimulation

Groups

(n=6)

3H Thymidine uptake CPM / lo6 cells (mean + SE)

Normal

1687.0 + 76.02

MC treated

1339.7 + 52.32

MC+ alpha tocopherol treated

1967.3 +_59.19

Several studies have established that development of cancer involves genetic damage, which may be either induced or spontaneous due to exogenous or endogenous factors (21-23). The increased frequency of micronucleus, both in terms of number of MN per cell and number of cells with MN, reflect the extent of DNA damage (24). Konopacka et al (25) reported that pretreatment with vitamin E (100-200 mg&$day) could protect mice from clastogenic activity of gamma rays, as measured by MN assay. We noted that alpha tocopherol (100 mgKgiday) administered along with carcinogen could reduce the MN munber as well as number of epithelial cells with MN which had increased significantly following chronic exposure to MC. MN frequency may increase even in apparently normal subjects due to age related spontaneous DNA damage or endogenous factors (26), thereby increasing the risk of cancer . Our observation on reduction of MN frequency in normal animals also following treatment with alpha tocopherol suggest that it may, by virtue of its protective effect on DNA damage, be useful for protecting cells from development of neoplasia. Fenech et al (27) however is of opinion that supplemental vitamin E is unlikely to affect chromosome damage occuring spontaneously in humans. Glutathione, the major free thiol present in all animal cells, participate in diverse biological processes, including detoxification of xenobiotics (28), and glutathione-S-transferases catalyze the conjugation of GSH to the electrophilic foreign compounds (10) for their elimination from the system. Observations on the association of GSH and GST during carcinogenesis and in cancer is often contradictory and conflicting. A decreased level of GSH was reported in cervical neoplasia and invasive carcinoma (29-30). Low plasma GSH was also observed in malignancies of breast, lung, liver, prostate and in lymphoma (31). GSH content in the liver was found to be increased following carcinogen administration which was in proportion to the carcinogenic potency (32), and there are reports to show an increased GST activity during malignancies (33-36). We noted a reduced liver GSH level and GST activity after 30 days of MC exposure, although at the initial stages a sharp elevation was observed ( unpublished data ). It is possible that initially the host’s defense system provides cellular protection by increasing the

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level of GSH and GST activity which help in neutralizing the action of the xenobiotic. However chronic exposure to MC eventually deranged the defense mechanism by significantly depleting the GSH level and reducing GST activity. Interestingly, oral administration of alpha tocopherol elevated the GSH level and GST activity both in normal and carcinogen treated animals which resulted in delayed action of the carcinogen on the epithelium. Here GSH and GST possibly played a significant role in elimination of the carcinogen. Hu et al (37) had demonstrated a dose dependant increase in plasma GSH after short term supplementation of alpha tocopherol and attributed the effect to the influence of the vitamin on regulation of enzymes involved in the synthesis and metabolism of GSH. The present observations suggest alpha tocopherol-induced increase in the level of GSH and GST activity helps to eliminate the carcinogen and defers the onset of cervical neoplasia and its progression. Besides its involvement in the detoxification process GSH is also considered to play an important role in lymphocyte function (38) and changes in GSH was found to be associated with impaired immune response and increased risk of malignancy . In the present study we noted a reduced response of PBL towards mitogenic stimulation suggesting deficient PBL response during cervical carcinogenesis. Oral administration of alpha tocopherol could increase the lymphocyte response which may be a possible mechanism of vitamin E protection against cancer (3% Generation of reactive species following increased lipid peroxidation and consequent tissue injury and cellular damage increases the risk for cancer (40-41). A number of chemical carcinogens is known to increase formation of lipid peroxides. We observed an elevation of lipid peroxides during MC induced carcinogenesis in the uterine cervix. Decrease in lipid peroxides by alpha tocopherol in this study implies that its chemopreventive action is mediated also by preventing or reducing DNA damage by oxidative free radicals. That alpha tocopherol can decrease lipid peroxidation has been already reported (42-45). Several anti oxidant enzymes like CAT, SOD and GPx can protect cell and cehular DNA from oxidative damage. Cellular damages from radical and non-radical reactive oxygen species including peroxides and superoxides are inactivated enzymatically by CAT, SOD and GPx (46). We observed a significant decrease in GPx and SOD activity in MC treated group compared to normal whereas the CAT activity was increased. Decreased cellular activities of GPx and SOD causes accumulation of reactive species in the body which leads to oxidative damage and progression of carcinogenesis. Intake of alpha tocopherol significantly increased the GPx and SOD activity in both MC treated and normal group of animals which facilitates removal of the peroxides and superoxides produced both in normal conditions and in large amount during carcinogen metabolism, thereby preventing the oxidative damage and malignant transformation. From these studies it can be concluded that alpha tocopherol has the potential to decrease DNA damage and inhibit carcinogen induced cervical neoplasia in mice through its antioxidant function. Additionally, this fat-soluble vitamin can stimulate the host’s immune response. Such actions of alpha tocopherol suggest a protective role of this micronutrient against carcinogenesis and point towards the possibility of its use in future as an effective chemopreventive agent in humans also. Many intervention trials in human preneoplastic conditions with anti-oxidant vitamins including vitamin E are already in progress.

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ACKNOWLEDGEMENT The authors are grateful to Prof..M.Siddiqi, Director CNCI and Dr. R.K.Bhattacharya, Emeritus critical suggestions during preparation of the manuscript. Medical Scientist, ICMR for their Thanks are also due to Ms..Jamuna Chakrabarty and Mr.S.RDatta for their excellent technical assistance.

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