Hydrogen peroxide formation and lipid peroxidation in rat uterus effect of hormones and vitamin Ε

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1 J. Biosci., Vol. 10, Number 3, September 1986, pp Printed in India. Hydrogen peroxide formation and lipid peroxidation in rat uterus effect of hormones and vitamin Ε SHYAMALI MUKHERJEE, MIHIR NAG, TULTUL NAYYAR, INDRANI MAITRA, PARUL CHAKRABARTI and PRAMOD R. DASGUPTA Department of Chemistry, Bose Institute, 93/1, A.P.C. Road, Calcutta , India MS received 13 November 1985; revised 30 April 1986 Abstract. The effect of estradiol 17ß and progesterone given separately as well as in combination on the rate of hydrogen peroxide formation and lipid peroxidation in the uteri of ovariectomized rats was studied. Estradiol in 3 µg dose per day per animal elicited maximum stimulatory response and progesterone (100 µg), on the other hand, was without any such effect. However, progesterone given along with estradiol completely prevented the effect due to the latter. In the same way, vitamin E, a well known antioxidant was found to be extremelv effective in protecting the uterus from the highly peroxidative action of estradiol 17ß. Keywords. H 2 O 2 -formation; lipid peroxidation; free radicals; malonaldehyde-thiobarbituric reaction; estradiol-17ß; progesterone; vitamin Ε (dl-α-tocopherol). Introduction The presence of the enzyme catalase (EC H 2 O 2 : H 2 O 2 oxidoreductase) and its substrate hydrogen peroxide in the uterine tissue of rat and human has been reported (Dasgupta et al., 1972; Bansal and Dasgupta, 1980; Johri and Dasgupta 1980a, b; Chakrabarti et al., 1982). Formation of many organic hydroperoxides such as unsaturated fatty acid (UFA) hydroperoxides including hydrogen peroxide and finally the thiobarbituric reactive malonaldehyde like substances as a result of free radical chain process in animal tissues, is now well documented (Wills, 1966; Menzel, 1970; Slater, 1972; Oshino and Chance, 1973; Brunori and Rotilio, 1984). Mechanisms of lipid peroxidation have been studied in rat liver, spleen, heart and kidney (Wills, 1966) and in brain (Barber, 1963) both in vitro and in vivo (Slater, 1972). A similar study in animal uterine tissue appeared pertinent from two points of interest: (i) this organ is an active site of protein biosynthesis and of estrogen metabolism, one of the products of which is hydrogen peroxide (Johri, 1978) and (ii) free radicals which set up a chain of biochemical reactions producing numerous highly reactive intermediates (Slater, 1972) may play a role in the process of fertilization and implantation. Abbreviations used: UFA. Unsaturated fatty acid; MA, malonaldehyde; SDS, sodium dodecyl sulphate; TBA, thiobarbituric acid; IUD, intrauterine contraception devices. 311

2 312 Shyamali Mukherjee et al. In the present investigation an attempt has been made to determine the effect of estrogen and progesterone on the rate of hydrogen peroxide formation and the degree of lipid peroxidation in the rat uterine tissue. Materials and methods Adult virgin regularly cycling female albino rats (Wistar strain) ( g) supplied by a local dealer were maintained under standard husbandry conditions at the Institute on a complete diet (developed here) and water ad lib, for a fortnight before any treatment. Bilateral overiectomy was performed aseptically under ether anaesthesia. After a rest period of 10 days following surgery the animals were injected intramuscularly with estradiol-17ß and progesterone dissolved in ground nut oil once daily for 4 consecutive days and sacrificed 24 h after the last injection. Both the uteri of each animal were carefully dissected out, trimmed free from adhering fat and extraneous tissues, and freed from intraluminal fluid by opening out the uteri. After some preliminary mincing with a scalpel it was then homogenized in glass homogenizer with 0 05 Μ phosphate buffer (ph 7 4) solution such that 1 ml of the final homogenate corresponded to 100 mg of tissue (wet weight). The homogenates were centrifuged at 2700 g for 10 min and the supernatants were used for various analyses. All the steps commencing from dissection of the tissue till the preparation of the homogenates were performed in cold (4 C). The animals were divided into the following groups: (i) Intact normal, having regular estrous cycle; (ii) ovariectomized control, receiving treatment with groundnut oil only; (iii) ovariectomized receiving estradiol only; (iv) ovariectomized, treated with progesterone only; (v) ovariectomized, treated simultaneously with both estradiol andprogesterone; and (vi) ovariectomized, treated with vitamin Ε at the rate of 10 mg per 100 g of body weight from day 1 of post-operation till the end of estradiol administration. The dose of hormones used is given in tables 1 and 2. Six animals were taken in each group. Estimation of protein Protein concentration of the homogenates of uteri was estimated according to the method of Lowry et al. (1951). Estimation of lipid peroxidation in terms of malonaldehyde (MA): Thiobarbituric acid reaction (Ohkawa et al., 1979). The reaction mixture contained 0 1 ml of tissue homogenate, 0 2 ml of 8 1% sodium dodecyl sulphate (SDS), 1 5 ml of 20% acetic acid and 1 5 ml of 0 8% aqueous solution of thiobarbituric acid (TBA). The ph of 20% acetic acid was adjusted with 1 Ν NaOH to 3 5. The mixture was finally made up to 4 0 ml with distilled water and heated at 95 C for 60 min on an oil bath. After cooling under tap water, 1 0 ml of distilled water and 5 0 ml of a mixture of n-butanol and pyridine (15:1 by vol) was added and the mixture was shaken vigorously on a vortex mixer. After centrifugation at 2200 g for 5 min the absorbance of the organic layer (upper layer) was measured immediately at 532 nm using appropriate controls in a Carry 17D model spectrophotometer.

3 Hydrogen peroxide and free radicals in rat uterus 313 Table 1. Effect of graded doses of estradiol-17ß and progesterone on the rate of H2O2 formation and lipid peroxidation in the uterine tissue of ovariectomized rat. Results are expressed as mean ± standard error of the mean. Figures in parentheses indicate the number of rats. Hormones were administered at the dose indicated once daily for 4 consecutive days. Control rats received vehicle only. Table 2. Effect of combined treatment with estradiol (E 2 ) and progesterone (P) on the rate of H 2 O 2 formation and lipid peroxidation in the uterine tissue of ovariectomized rat. Results are expressed as mean ± standard error of the mean. Figures in parentheses indicate the number of rats. Estrogen and progesterone were administered once daily at the dose indicated for 4 consecutive days. * The control group received groundnut oil only.

4 314 Shyamali Mukherjee et al. Estimation of H 2 O 2 (Lichtenberg and Wellner, 1968) The reaction mixture, in a final volume of 1 ml, contained 40 µmol of Tris-HCl (ph 7 5), 1 nmol of scopoletin, and 10 µg of peroxidase both in buffer. The solution was preincubated for 5 min at 37 C; then 0 1 ml of tissue homogenate was added and incubated for 15 min at 37 C with mild shaking in a Dubnoff shaker and the reaction was stopped by the addition of 4 ml of 0 1 Μ sodium borate (ph 10) and the mixture was then centrifuged at 2200 g for 5 min. The fluorescence of the clear supernatant was measured at 25 C with excitation monochromator set at 395 nm and the emission monochromator set at 470 nm in a Perkin Elmer Spectrofluorimeter (Model MDF 44B). The control contained all the reagents except peroxidase. Student's 't' test was applied for the calculation of significance. Results Table 1 illustrates that estradiol-17ß does, indeed, stimulate the formation of H 2 O 2 and of free radicals production, as indicated by the production of MA, in the ovariectomized rat uterus and that the dose of 3 µg per day is optimal (P< 0 001). However, the maximal effects obtained does not reach the levels of these two parameters observed in the intact normal group. Progesterone even at a dose as high as 100 µg per day is without any such effect on the uterus, although a massive dose ( µg/day) appears to show some positive effect (P <0 05). Table 2 shows the results of the effect of treatment with an optimal dose of 3 µg of estradiol-17ß along with progesterone in two different doses of 100 µg and 500 µg. It is found that the stimulatory effect of estradiol-17ß on H 2 O 2 production and lipid peroxidation has been substantially neutralized by 100 µg of progesterone However, when a pharmacological dose of progesterone (500 µg) is used along with 3 µg of estradiol, a massive increase in the H 2 O 2 formation is noted; however, no such rise in lipid peroxidation is found. The results of administration of vitamin Ε on the increased formation of H 2 O 2 and lipid peroxidation in ovariectomized rat uterus under the influence of estradiol-17ß are presented in table 3. As expected, vitamin Ε prevented the Table 3. Effect of vitamin Ε on H 2 O 2 formation and lipid peroxidation in ovariectomized rat uterus. * Mean ± SEM. Figures in parentheses indicate total number of animals. Estradiol dose 3 µg/day/animal for 4 days from day 10 post-operation; vitamin Ε 30 mg/day/ 100 g body weight of rat for 14 days post-operation.

5 Hydrogen peroxide and free radicals in rat uterus 315 increase in the formation of H 2 O 2 as well as of lipid peroxidation which occurs under the influence of estradiol-17ß. Furthermore, it is noteworthy that the protective action of vitamin Ε against free radical mediated lipid peroxidation is highly pronounced; the production of MA is reduced to half of that observed in the ovariectomized control uterus (P < 0 001). Discussion Dasgupta and coworkers (Bansal and Dasgupta, 1980; Johri and Dasgupta, 1980a, b; Chakrabarti et al., 1982; Nayyar et al., 1983) demonstrated an increase in the production of H 2 O 2 in the uteri of rat and of human under the influence of intrauterine contraceptive devices (IUD ) in utero and that such rise in the rat uterus was also dependent on the blood level of estrogen. It was suggested that H 2 O 2 being a highly toxic metabolic intermediate could explain the contraceptive action of IUD's. Meanwhile, evidence accumulated suggesting that H 2 O 2 is an active intermediate in the redox process initiating the production of free radicals in various tissues of the body, with a potential to cause tissue injury (Menzel, 1970; Slater, 1972; Janzen, 1980; Brunori and Rotilio, 1984). Experiments in vivo and in vitro have demonstrated the production of free radicals under the influence of poisonous substances like CCl 4 on the basis of the production of MA which produces a coloured substance with TBA, in various tissues, mainly in liver (Slater, 1972). However, no published report is known of similar studies in the uterine tissue which is a target for estradiol, and metabolism of which is reported to produce hydrogen peroxide (H 2 O 2 ) (McNabb and Jellinck, 1976; Johri, 1978). The results presented in table 1 clearly show that both H 2 O 2 formation and lipid peroxidation occur in the rat uterus under normal physiological condition and both these parameters are drastically lowered (P<0 01) following ovariectomy. However, treatment of spayed animals with estradiol-17ß, but not with progesterone, restores substantially (P<0 01) the preoperative levels in the uterus. Thus it is apparent that H 2 O 2 formation and lipid peroxidation in the rat uterus are normal physiological events and are partly, but not entirely, estrogen dependent; possibly in intact normal animals other pathways besides metabolism of estrogen (McNabb and Jellinck, 1976; Johri, 1978) are operative in the uterus for the production of free radicals (Slater, 1972). The effects of combined treatment with estradiol-17ß and progesterone (table 2) are as expected to some extent. The well documented antiestrogen effect of progesterone has been clearly manifested and the stimulatory influence of estradiol on free radical formation has been practically neutralized (P<0.01). However, it was interesting to note that a massive dose of 500 µg of progesterone along with estradiol-17ß records a phenomenal increase in the level of H 2 O 2 without affecting the extent of lipid peroxidation. Questions arise about the fate and disposal of such high quantities of H 2 O 2. No explanation can be offered on the basis of the present data. However, it seems possible that the excess amount of progesterone may undergo degradation or is disposed of by a mechanism involving H 2 O 2. In vitro studies may be useful in solving these questions. Many antioxidants, synthetic as well as natural, are known to effectively prevent lipid peroxidation in canned food materials (Slater, 1972). Vitamin Ε (α-tocopherol-dl) is of particular interest so far

6 316 Shyamali Mukherjee et al. as in vivo protection of the living tissue is concerned (Lucy, 1978; Fukuzawa et al., 1981). For example, Hochstein and Ernster (1963) demonstrated the antioxidant action of vitamin Ε in many diverse conditions in vitro. Draper and Csallany (1969) have also reviewed these effects of vitamin E. In the present study, vitamin Ε was administered before the commencement of hormone (estradiol) treatment of the spayed animals. It was interesting to observe that the vitamin offers protection against the peroxidative influence of estradio- 17ß in the uterus (table 3). Whereas the level of H 2 O 2 production was significantly kept under control (P<0 01) the level of lipid peroxidation was markedly diminished compared to the spayed control level (P<0 001). Such remarkable diminution of lipid peroxidation reaching half the level of spayed control may mean that the vitamin Ε defends not only against H 2 O 2 mediated free radical formation, but also against those produced by other pathways. Thus on the basis of present study, it appears that (i) lipid peroxidation is a normal physiological phenomenon of the rat uterus; (ii) it is dependent on estrogen, (iii) progesterone can neutralise the stimulatory effect of estradiol on lipid peroxidation, (iv) a non-hormonal antioxidant like vitamin Ε is extremely effective in protecting the tissue against lipid peroxidation and (ν) estradiol metabolism and H 2 O 2 formation constitute the major pathway for the production of free radicals in the uterus. Acknowledgements Thanks are due to Prof. B. B. Biswas for his kind interest and support, the Regional Sophisticated Instrument Centre for the use of the instruments, and the Department of Science and Technology, New Delhi for the financial aid. A grant from the World Health Organization, Geneva, in the form of essential supplies is thankfully acknowledged. References Bansal, R. and Dasgupta, P. R. (1980) Indian J. Med. Res., 72, 66. Barber, A. A. (1963) Implication of Organic Peroxides in Radiobiology Radiat. Res. Suppl., No. 3, p. 33. Brunori, M. and Rotilio, G. (1984) Methods Enzymol., 105, 22. Chakraborti, A. S., Chatterjee, P., Dutta, S. and Dasgupta, P. R. (1982) Int. J. Fertil., 27, 132. Dasgupta, P. R., Pande, J. Κ., Garg, R., Srivastava, Κ. and Kar, Α. Β. (1972) Contraception, 6, 459. Draper, Η. Η. and Csallany, Α. S. (1969) Fed. Proc. Fed. Am.Soc. Exp. Biol., 28, Fukuzawa, Κ., Chida, H.,Tokumura, Α. and Tsukatani, Η. (1981) Arch. Biochem. Biophys., 206, 173. Hochstein, P. and Ernster, L. (1963) Biochem. Biophys. Res. Commun., 12, 388. Janzen, E. G. (1980) in Free Radical in Biology (ed. W. A. Pryor) (New York: Academy Press) Vol. 4, p Johri, R. K. (1978) Mechanism of Action of Copper and Copper-bearing Intrauterine Devices, Ph.D. thesis, Kanpur University, Kanpur. Johri, R. K. and Dasgupta, P. R. (1980a) J. Endocrinol., 86, 477. Johri, R. K. and Dasgupta, P. R. (1980b) J. Reprod. Fertil., 59, 495. Klebanoff, S. J. (1965) Endocrinology, 76, 301. Lichtenberg, L. A. and Wellner, D. (1968) Anal. Biochem., 26, 313. Lowry, O. H., Rosenbrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem.,

7 Hydrogen peroxide and free radicals in rat uterus 317 Lucy, J. A. (1978) in Tocopherol, Oxygen and Biomembranes, (eds C. deduve and O. Hayaishi) (Amsterdam: Elsevier). McNabb, Τ. and Jellinck, P. H. (1976) Steroids, 27, 681. Menzel, D. Β. (1970) Annu. Rev. Pharmacol., 10, 379. Nayyar, T., Mukherjee, S., Nag, M. K., Chakrabarti, P. and Dasgupta, P. R. (1983) Indian J. Physiol. Allied Sci., 37, 64. Ohkawa, Η., Ohishi, Ν. and Yagi, K. (1979) Anal. Biochem., 95, 351. Oshino, N. P. and Chance, Β. (1973) Biochim. Biophys. Acta, 154, 117. Slater, T. F. (1972) in Free Radical Mechanism in Tissue Injury, (London: Pion Ltd., Brondesbury Park) p. 13. Wills, E. D. (1966) Biochem. J., 99, 667.