Indi an Journal of Experimental Biology Vol. 37, May 1999, pp. 439-443

Altered lipid peroxidation and antioxidant potential in human uterine tumors

Subhansu Ray & Parul Chakrabarti*

Department of Chemistry, Bose In stitute, 93/ 1, A.P.C. Road , Calcutta 700009, India

Received 23 July 1998; revised 25 February 1999

A comparative study of lipid peroxidation and an tioxidant potential has been made in human uterus and uterine tumor. Two types of uterine tumor used are : tumor (I), a fibroid which is the commonest benign solid tumor in uterus and tumor (It) . an adenoillyoilla. TUlllor microsomes are less susceptible to lipid peroxidation induced by both enzymic (NADPH­ADP-Fc " and xanthine-xanthine-oxidase) and non-enzymic (ascorbate-Fe2

' ) systems exeept in the ease of tumor (/I ) Illicro­somes when Induced with xanthine-xanthine oxidase. Resistance of tumor mierosoilles to lipid peroxidation is associated wi th the low content of subst rates in the form of polyun saturated fatty ac ids (PUF As), hi gher leve l of a-tocopherol , reduced ~ Iut athion e and protein thi ols and altered enzymi c antioxidant potential (catalase and superoxide dismutase) .

Oxygen free radical-induced cellular damages have b~en impli ca ted in many pathobiological conditions, malignancy, aglllg process and degenerative diseases etc l

. Lipid peroxidation has been identified as one of the basic reactions involved in oxygen free radical induced ce llul ar damages2

. A delicate balance be­tween the availability of the substrates for lipid per­oxidation (name ly polyunsaturated fatty acids) in membrane and ce llular antioxidant defense systems., e.g ., ' superoxide di smutase, catalase, protein thiols, glutathIOne, a-tocopherol etc . is essential to protect the body against oxidative stress3

.4 . a-Tocopherol protects rat uterine tissues from lipid peroxidation5

•

Uterine tumor \Vhich is found in a lmost all age groups of women is a serious problem . Tumor (I) is a fibroid which is the commonest benign solid tumor in uterus. Tumor (II) is an adenomyoma where malignant trans­formation t S possibl e. These two types have been used in the present study . A comparative eva luation of the lipid peroxidation and antioxidant potential in these tumors and control ti ssues is presented in this communication.

Materials and Methods

Collection of samples--Samples were collected from the Obstretrics and Gynaecology Department of the Nil Ratan Sarkar Medical College & Hospital , Calcutta after getti ng clear consent of the patients and

* Corr~spondent author Fax o. (033)-350-6790. E-Illail : paru l@boseinst.ernet.in

the clinicians. The sa mples were col lected in normal saline and quickly stored at - 70°C till the time of analys is. Uterine tissues taken from the subject with dysfunctional uterine bleeding and prolapse were used as control ti ssues. Tumor (I) was fibroid tumor originated from muscle tissue of uterus and tumor (II ) was adenomyoma, a di ffused endometriosis within uterine wall.

Tissue homogenate and microsome prepara­

tion- Tissues were homogenized in phosphate buff­ered saline [(PBS) 10 mM, pH 7.4] in a Sorvall om­nimixer, centrifuged at 2000 g to remove cell debris and nuclei and the supernatant termed as "tissue ho­mogenate" was centri fuged at 10,000 g for 20 min and at 105 ,000 g for 60 min. The 105 ,000 g pellet was used as the microsomal fraction. Protein concen­tration was measured6 in the tissue homogenate, mi­crosomal pellet and the supernatant.

Induction and estimation of lipid peroxida­

tion--Lipid peroxidation was estimated in terms of thiobarbituric acid-reactive substances (TBARS) and malondialdehyde (MDA) was taken to represent the TBARS. Three systems were used to induce lipid peroxidation in the microsomal preparation : (i) ascorbate - Fe2

+, (ii) NADPH - ADP - Fe3+ and (iii)

xanthine .. xanthine oxidase7.8

. Optimal concentrations of different inducers and optimal time of induction were determined. Final assays were carried out with optimal concentrations at optimal time as follows : Microsomal preparation in PBS (I mg protein/ml) was incubated with (i) ascorbate (500. flm) and Fe2

+ (8

440 INDIAN J. EXP mOL., MAY 1999

)lm) at 37°C for 30 min or (ii) NADPH (0.3 mM), ADP (0.2 mM) and Fe3

+ (10 )lm) at 37°C for 30 min or (iii) xanthine (0.6 mM) and xanthine oxidase (3 x 10-2 unit) at 37°C for 30 min . The reaction was stopped by the addition of EDT A (5.8 mM) and after addition of 0.8% thiobarbituric acid (TBA) the mix­ture was incubated at 95°C for 15 min. The absorb­ance was read at 532 nm. A calibration cunre was prepared by generating MDA from freshly prepared 1,1,3,3/-tetramethoxy propane (TMP) by acid hy­drolysi s9 . Control experiments were carried out with microsome alone without any inducer.

F(1tty acid analyses oj microsomal phospholip­ids-Lipids were extracted from microsomes as fol­lows 10 : Microsomes were thoroughly homogenised using the following solvent systems in succession: (i) ch lorofornl : methanol (2 : I , v/v), (ii) chloroform : methanol (I :2, v/v), (iii) chloroform: methanol : wa­ter ( I :2:0.8, v/v/v) for 10 min with each solvent sys­tem. The phospholipid fraction was separated from the total lipid by preparative thin-l ayer chromatogra­phy (TLC) on si lica gel G (20 x 20 cm, thickness I mm) using petroleum ether: ether (4 : I , v/v) as devel­oping solvent, molybdenum blue and iodine vapour for identification and hydrolyzed with 0.2 M metha-110lic KOHli to isolate free fatty acids. The fatty acid methyl esters were prepared with etheral di­azomethane l2, analysed by gas-liquid chromatography and identified by comparing the retention times with those of the reference methyl esters .

Catalase and superoxide dismutase (SOD) acti­vity----Catalase activity was measured spectropho­tometrically by decomposition of hydrogen peroxide (HZ0 2) 13 and expressed as unit per mg protein . The SOD activity was measured using xanthine oxidase­nitroblue tetrazolium (NBT) method J4

. One unit of thi s enzyme activity is the amount of protein which gives half-max imal inhibition ofNBT.

Measurement oj cellular reduced glutathione (GSH) and microsomal protein thiol content--The tissue homogenate (I mg protein/ml) was treated with 25% trichloroacetic acid (TCA), centrifuged at 3,000 g for 5 min, the supernatant (0.1 to 0.2 ml) was treated with 5,5/ -dithiobis (2-nitrobenzoic acid) (DTNB, 0.4 mM) in 0 .2 M phosphate buffer (PH 8) for measuring GSH I5 and absorbance was read at 412 nm. The microsomal protein thiol content was meas­ured l6 by suspending the microsomes (1.5 mg pro­tein/ml) in phosphate buffer (50 mM, pH 8) contain-

ing 1 % SDS (0.1 ml); protein precipitated with 6.5% TCA and dissolved in the same buffer. The solution was treated with DTNB (100 )lM) and the absorbance was read at 412 nm using glutathione as external standard.

Quantitalion oj a-tocopherol--a-Tocopherol content in the lipid extract of microsome was esti­mated by reverse phase HPLC (Waters, Inc) using a C 18 column (Nova Pack, 150 x 3.9 mm, 6 )lm) with a variable wavelength UV detector (Hewlett Packard) and standard a-tocopherol 17.

Statistical methods-Mean va ues and standard deviation (S.D.) were calculated and analysed statis­tically by unpaired Student 's t test.

Results Lipid peroxidation- Lipid peroxidation has been

measured (Table 1) in the microsoma l membrane of the reference and the tumor tissues using different prooxidant systems, vi z., (i) asco bate - Fe2

+ (non­enzymic system), (ii) NADPH-ADP-Fe3

+ (enzymic system) and (ii i) xanthine-xanthine oxidase (an en­zym ic system to generate superoxide O~2 and H20 z). Lipid peroxidation is almost four-fold and three-fold more in the control microsomes in comparison with that obtained from tumor (1) and rumor (II) respec­tively, when treated with NADPH - ADP - Fe3

+. This almost doubled in the control microsomes in com­parison with the tumor (1) microsome when treated with xanthine - xanthine oxidase. But the value is quite reverse in the case of tumor (II) , where it is two times higher than the control microsome. The differ­ence is not so significant in ascorbate-Fe2

+ system. Lipid peroxidation is almost similar in all the systems without any inducer.

Fatty acid projile--Analyses of the fatty acid composition of phospholipid of the tumor and refer­ence microsomes (Table 2) reveal that there is a sig­nificant reduction (P < 0.001) in the levels of Czo5 and C22:6 fatty acids with a concomitant increase in the level of C I80 fatty acid in both the tumor types. C204

fatty acid level is higher in the tumor (II) microsomes and lower in the tumor (I) microsomes.

Catalase and SOD a ctivities--Superoxide dismu­tase (SOD) activity in both the tumor microsomes is significantly lower (P < 0.001) than that in the control microsomes whereas catalase activity is less altered (P < 0.05) (Table 3) .

RA Y & CHAKRABARTI : LIPID PEROXIDATION & ANTIOXIDANT POTENTIAL IN UTERINE TUMORS 441

Table I- Lipid peroxidation (measured as nM MDA formedimg protein/min) in microsome derived from tumor and control ti ssues. [Values are mean ± S.D. Number of determination for control tissues is 5 and for tumor is 8]

System

Without an y inducer Induced with : Ascorbate - Fc1

'

NADPH-ADP-Fc'" Xanthine-xanthine oxidase Xanthine-xanthine ox idase + catalase Xanthine-xanthine oxidase + superoxide di smutase Xanthine-xanthine oxidase + catalase + supcrox ide dismutase

P va lues: " < () I; /, < 0.05; r < 0.0 I; " < 0.00 I.

Control

1.17 ± 0.21

1.98 ± 0.24 9.5 ± 0.89 2.8 ± 0.53

1.44 ± 0.65 1.32 ± 0.24

1.05±034

a-Tocopherol content-a-Tocopherol is p:-esent in very high quantity in tumor (1) microsomal lipid and in very low amount in tumor (II) lipid (Table 3) .

Cellular GSH and microsomal protein thiol--Cellular GSH concentration (Table 3) is three and half times more (P < 0 .001) in tumor (I) and not so high in tumor (II). The level of protein thiol is al­most one and hal f times more in the tumor micro­some (I) in comparison with the control microsomes . The leve l observed in tumor (II) microsomes is al­most similar to the control microsomes. Incubation of microsomes wi th xanthine-xanthine oxidase appre­ciably reduces the value in all the cases. Pre­incubation of the microsomes with superoxide dis­mutase followed by treatment with xanthine-xanthine oxidase gives increased thiol value which is almost close to those found in the untreated systems (Table 3).

Discussion The present study reports lipid peroxidation in mi­

crosomes of normal and human uterine tumor tissues under same proxidant conditions. Lipid peroxidation with~ut any proox idant or inducer is almost similar in the normal and tumor microsomes (Table 1). In NADPH - ADP - Fe' + catalysed system, stimulation in lipid peroxidation is very high in the normal mi­crosome. In both the tumor microsomes, this enzyme­catalysed (NADPH - ADP - Fe3+) lipid peroxidation is significantly lower in comparison with the normal microsomes. Human uterine microsomes are more sensitive to NADPH-induced lipid peroxidation like rat uterine microsomes I g . The present results are also in agreement with the report on normal rat liver and

Tumor (I) Tumor (II )

1.01 ± 0.11 108 ± 0.17

1.57 ± 0.34" 2.59 ± 0.61 " 2.45 ± 0.98" 3.54 ± 0.77"

1.5 ± 0.7 1r 5.6±1 .2" 1.12 ± 0. 14 2.07 ± 0.62 0.88±0.14r \.92 ± 0.44"

0.8 ± 0.26 \.9 ± 0.3Y

Table 2-Fatty acid composition (ex pressed as a percentage of total fatty acid) of phospholipids of microsome.

[Detai ls are same as in Table I]

Fatty ac ids Control Tumor (I) Tumor (I I)

14 : 0 49 ± 0.21 10.03 ± 0.8" 2.4 ± 0.2" 15 : 0 3.26 ± 0.95 6.15 ± 0.88 5.1 ± 0.3 16 :0 13.26 ± 0.87 14.1 ± 0.64 19.1 ± 1.2 18 :0 11 83 ± 0.89 21.42 ± 2.45" 18.7 ± \.69" 18 : 24.29 ± 2.44 17.1±0.78" 25.41 ± 1.33 18 : 2 12.04 ± 2.87 II . I± 2.3 5.3 ± 0.86" 18 : 3 6.12 ± 1.5 7.9 ± 2.4 6.4 ± 0.91 20 : 4 10.72 ± 1.1 7.98 ± 1.35" 14.2 ± 0.48" 20 : 5 6.33 ± 0.44 1.55 ± 0.29" 2.1 ± 0.28" 22 : 2 2.85 ± 0.41 22 : 6 4.29 ± 0.56 2.6 ± 0.4" 1.2 ± 0.25"

P values:" < 0.001 ;" < 0.01

rapidly proliferating Novikoff hepatoma and the Yo­shida ascites hepatoma cells and microsome prepared from them 19 . Lipid peroxidation does not occur to a large extent even when tumor (I) microsomes are ex­posed to superoxide radical generating system (xan­thine - xanthine oxidase) (Table I) .

Superoxide di smutase activity and catalase activity in tumor tissue homogenates and microsomes re flect the altered enzymatic antioxidant potential of the systems (Table 3). This is in agreement with an earlier report on endometrial cancer in some Japanese and Finnish women20

. The result is very significant in view o f the proposed signalling role of superoxide radical (0 -2) in proliferation of fibroblast and other systems21

.

NADPH-dependent (cytochrome P-4S0) lipid peroxi­dation is parallel to 0 '2 generation by xanthine -xanthine oxidase. Recovery of lipid ---peroxidation

442 INDIAN 1. EX P SIOL., MA Y 1999

Table }--Levels of catalase, superoxide dismutase, a-tocopherol and protein thiol in microsomes of tumor and corresponding con­trol tissue and so lubl e reduced g lutathione in the respective ti ssue

homogenates. [Detail s are same as in Table I]

Parameter Contro l Tumor (I ) Tumor (II )

Catalase 2 ± 1 I ± 0.5" I ± OS' (unitlmg protein) Superox ide 3± 1 0 .4 ± 0.2" 0 .3 ± 0 . 1 h

di smutase (unitlmg prote in ) a -Tocopherol 2. 18 ± 0. 15 4.41 ± 0.2 1h 0 .65± 0.006h

(nM /mg total lip id) Protein thiol 166 ± 0 .55 2.5 ± 0.42" 1.84 ± 032 (IlM/mg protein) Induced with : a) Xanthine-xanthine 0 .42 ± 0 .12 069 ± 0 .17" 0.46 ± 0.09 oxidase

b) Xanthine-xanthine 1.55 ± 0 .24 2.48 ± 0 .3 Ic 1.76 ± 0.41 ox idase + superoxide dismutase Reduced glutathione 1.0 ± 0 .09 3.62 ± 0.14b 1.8 ± O.4c

(Ilglmg soluble pro-te in )

P values: " < 0.05 ; h < 0.001 ; < < 0.01 ; d < 0. 1.

level almost to the endogenous level after addition of SOD (Table 1) is in agreement with the observation that SOD inhibits the xanthine - xanthine oxidase de­pendent lipid peroxidation22

. Catalase which converts HzOz to H20 , has been shown to be of primary im­portance in erythrocyte defense against H20 2 than the glutathione-dependent action of glutathione peroxi­dase which converts also H20 2 to H20 .

Fatty ac id analyses of the microsomal phospho­lipid fractions (Table 2) show that the level of poly­unsaturated fatty acids (PUFAs) is appreciably lower in comparison with that of the saturated fatty acids (SFAs) in tumor (1) microsome. This is consistent with the data obtained with CHF negative chicken embroys and their RSV-virus transfonned counter­parts23

. The PUF A containing three or more double bonds are more vulnerable to peroxidative attack and arachidonic acid (C204 ) and docosahexaenoic acid (C226) are approximately three and five times more readily oxidized than linoleic acid (C I8Z)24. Unsatu­rated fatty acids have been reported to play role in promotion and inhibition of tumor growth . Long chaill fatty acids (C I83 , C20S , C22S ) inhibit tumor growth, and metastasti s is promoted by fatty acids (C I8:2, C I8:3) 2S. PUFAs (C I8 :3, CZO:4 , C20:S and C22:6) have been shown to inhibit the growth of human pancreatic cell lines in vitro26 . Significant reduction (P < 0.001)

in the level of C20S and C226 may play role in tumor promoti on in both the present cases. The major mem­brane bound radica l trap, a-tocopherol is particularly concentrated in these regions. Thi s protects cells from oxidative damage via a free radica l scavanging mechani sm or as a structural component of cell membranes27

.

The hi gh a-tocopherol level (Table 3) in the tumor (I) microsomes argues well wi th the notion that per­oxyl radica l produced in the membrane can be trapped by a -tocopherol, which may explain, partly why the peroxidation index in tumor (I) microsome is low in comparison with the nonnal microsome. Free thiol groups are vi tal in cellul ar defen se against en­dogenous and exogenous oxidant s tress28

. DTNB­reactive protein thio l concentration (Table 3) is also high in tumor (1) microsome. Glutathione level is sig­nificant ly higher (P < 0.00 I) in the tumor (1) ti ssue homogenate as compared to the nonnal one . Increase in the level of gl utathione is not s significant in tu­mor (II) tissue homogena te Cfable 3). Protection by glutathione against the loss of protei n thiols during microsomal lip id peroxidation has been reported in rat liver9. It is suggested that a maintenance of pro­te in thiols may not only protect important me tabolic func tions, but may also afford an antioxidant capacity to membranes and account for one facet of the GSH­dependent inhibition of lip id peroxldation. In tumor (II) microsome, a-tocopherol content is low and PUF A content is hi gh. The other antioxidant poten­tials are not so much altered as tumor (1 ) . Involve­ment of a -tocopherol and prote in thiols in the inhib i­tiOti of microsomal lipid peroxidation by glutathione has been demonstrated in rat liver microsomes3o

.

In summary the present study has identified some of the possible reasons for altered lip id peroxidation in the human uterine tumor microsomes. The lower level of lipid peroxidation in the tumor microsomes is associated with the low content of polyunsaturated fatty acids (PUF A) and higher leve l of a-tocopherol , glutathione and protein thiol in the tumor (type J) mi­crosomes. Tumor microsomes are less sensitive to enzyme-catalysed NADPH-dependent lipid peroxi­dation .

Lipid peroxidation is controlled by a delicate bal­ance between3

.4 the availability of substrates (i.e. polyunsaturated fatty acids) and the . inhibitors. Low level of substrates and high level of inh ibitors (a­tocopherol, protein thio l and glutathione) may ac-

RA Y & CHAKRABARTI : LIPID PEROXIDATION & ANTIOXIDANT POTENTIAL IN UTERINE TUMORS 443

count for low susceptibilities of the human uterine tumor tissues to lipid peroxidation.

The present results further strengthen the general hypothesis that a decreased rate of lipid peroxidation may be related to an increased 'rate of cell division in the highly undifferentiated rapidly proliferating sys­tem l9

.

Acknowledgement The work was carried out with financial support

fronrthe Indian Council of Medical Research (Project No.8406500). The authors would like to thank Dr. Pampa Sarkar, fonnerly associated with the Depart­ment of GynaecoJogy and Obstretics, Nil Ratan Sarkar Medical College, Calcutta, for providing sam­ples and Late Prof. P.R. Dasgupta, Emeritus Scientist, Bose Institute for helpful discussion.

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