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Estrogen effects on NADH oxidase and superoxide dismutase in prepubertal female rats
651
3032
ESTROGEN EFFECTS ON NADH OXIDASE AND S~EROXID~ DIS~TASE IN PREPUBERT~ ABBE
RATS
R. Rajan, M.J. Daly and V.V.R. Reddy Department of Obstetrics and Gynecology; Temple University School of Medicine; Philadelphia, Pa. 19140 Received 9-16-82 ABSTRACT
Thirty-four day old, ovariectomisedrats were treated with increasing doses of estradiol, 2-hydroxyestradiol2,3-dimethyl ether (23E2), 4-hydroxyestradiol3,4-dimethyl ether (34E2) and 4-methoxyestradiol (4ME2) for five days by subcutaneous injection. Superoxide dismutase, phenol activated NADH oxidase and uterine dry weights were determined. Only estradiol was found to be uterotrophic and increased NADH oxidase activity in these experiments. Both 23E2 and 34E2 treatment reduced the enzyme activity significantly. Though 4ME2 showed a decrease in NADH oxidase at O.O5~g/lOOgm body weight there was no further decrease at higher dose {5~g/lOO~). The superoxide dismutase (SOD) in uterus and liver was unaffected by estradiol, while and 4ME significantly reduced SOD in both liver and uterus. 23E 34E The& res%ts indiczte that 23E 34E and 4ME , in spite of their nonuterotrophic property, affect u8' erine2metaboligm. Furthermore, in view of the reports indicating the importance of SOD levels in various tumors and since catecholestrogensare observed to reduce SOD levels in liver and uterus, it is suggestive that catecholestrogensmay play an important role in the pathophysiologyof certain tumors.
INTRODUCTION
Estrogens are known to induce various enzymes in uterus and affect uterine metabolism. The phenol-activatedNADH oxidase, which catalyses reduced NAD oxidation in the presence of an added phenol in in vitro system is one of the enzymes that is induced by estradiol -treatment in immature rats (1). Catecholestrogenshave been suggested to be peripherally inactive but active at central level (2). Though catecholestrogensare weak uterotrophic agents, they are reported to bind estrogen receptors in uterus (3). It has been reported recently
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(4) that 2-hydroxyestradiol can induce peroxidase in uterus at high doses, but inhibitory and antiestrogenic when given with estradiol. However, the effect of catecholestrogens on NADH oxidase is not known. Superoxide dismutase (SOD) is one of the important enzymes associated with the regulation of superoxide anions and formation of hydrogen peroxide in various tissues.
Various tumors have been re-
ported to possess altered levels of SOD and this was suggested to be important in planning chemotherapeutic regimens in cancer treatment (5).
Since estrogens are known to influence uterine tumor development
and we have observed a decrease in catecholestrogen synthesis in human leiomyoma (22), we attempted to determine the effects of these estrogens on SOD activity in uterus.
In this report, we present evidence
that treatment with catecholestrogen methyl ethers inhibit both NADH oxidase and SOD activity in immature rat uterus.
MATERIALS AND METHOD Estradiol-178, superoxide dismutase and reduced NADH were purchased from Sigma Chemical Company. 2-Hydroxyestradiol 2,3-dimethylether (23E2), 4-hydroxyestradiol 3,4-dimethyl ether (343 > and 4methoxyestradiol (4ME2) were prepared earlier (6). All $he steroids were purified by repeated crystallizations. Twenty-five day old Sprague Dawley rats were obtained from Charles River and ovariectomised under light ether anesthesia. The animals were divided into several groups of five animals. Steroids were dissolved in sesame oil and dilutions were adjusted in order to inject O.lml of the solutions per each injection. All the groups received daily subcutaneous injections for five days starting on 34th day of age. Groups 1 and 2 received estradiol O.O5~g/lOOgm and 0.5ug/lOOgm body weight respectively. Groups 3,4 and 5 received 23E 0.05, 0.5 and 5ug/lOOgm body weight respectively. The treatmen 8 schedule for 34E2 and 4ME2 was the same as for 23E . Oil injected group served as controls. The animals were sacrificed by decapitation within 18 hr after the last injection. Uteri were excised from the animals after decapitation. The tissues were trimmed of the adhering adipose and connective tissues
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and weighed. About one half of each tissue was then dried at 6070°C until a constant weight was obtained. Total dry weights were calculated from the fraction of the initial weight used for drying. Estimation of Phenol Activated NADH Oxidase The procedure followed for determination of NADH oxidase was essentially the same as the method of Temple -et al (7). Briefly, uterine homogenates were prepared in 0.25M sucrose (pH.7.4) to yield a 101% solution. Homogenates were centrifuged at 1OOOg for 15 minutes and the supernatant used for estimations of enzymes. The incubation mixture contained 0.015M phosphate buffer (pH.7.7), 1.5umoles of maganese chloride, 0.4umoles of NADH, 34 nanomoles of estradiol dissolved in 0.05ml propylene @ye01 and O.lml of homogenate. The mixture was incubated at 30 C for 30 minutes and the reduction in absorbency at 34Ornl.l was recorded against the control incubations containing 0.05ml propylene glycol. The enzyme activity was found to -be linear up to 30 to 45 minutes and activity is expressed as )Jmoles of NADH oxidized per minute mg protein. The incubation mixture for estimation of 2,4 dichlorphenol (2,4-DCP) activated NADH oxidase contained same components as in estradiol-activated oxidase determination, except that 0.015M phosphate buffer (pH.7.0) was used and estradiol was replaced with 0.751.imolesof 2,4-DCP. Determination of Superoxide Dismutase in Uterus and Liver Superoxide dismutase was estimated by the method reported recently (8), using nitroblue tetrazolium. The tissues were homogenized at (0-4OC) in 0.154M NaCl (1:50). A 5ml aliquot was shaken vigorously with 5ml of ethanol: chloroform mixture for 3 minutes and centrifuged for 1 hr at 1500g at 0-4'C. The supernatant (0.25ml) was used for assay. The incubation mixture contained cacodylic acid buffer (pH8.2) 0.5m1, triton X-100 (16% solution) O.lml, nitroblue tetrazolium (1M) 0.4ml and tissue extract 0.25ml. The reaction was started by adding 2Ovl of pyrogallol (0.1% solution). The mixture was incubated at 37'C for 5 minutes and the reaction stopped with 2M formic acid buffer (pH 3.5). The blue formazan color was determined at 54Oml.l. SOD was inactivated by heating the enzyme extracts in a boiling water bath for 2 minutes and the inactivated enzyme extracts served as controls. The rate of superoxide reaction inhibited by SOD was calculated according to the definition of McCord and Fridovich (9). A fifty percent inhibition of the reaction was considered to be equal to one unit of the enzyme. The standard SOD obtained from Sigma Chemical Co. with 3.3 units/pgm protein was used as reference standard. Protein was estimated by the method of Lowry -et al (lb).
654
RESULTS
The results presented are an average of two independent experiments with a total of 8-10
It is observed that
4ME2 has a weak uterotrophic activity, while 23E2 and 34E2 are nonuterotrophic, even at high doses. TABLE I EFFECT OF CATECHOLESTROGENMETHYL ETHERS ON UTERINE DRY WEIGHT IN OVARIECTOMISEDHATS Treatment Dosage Oil Estradiol 23E2 3432 4ME2
Uterine Dry Weights (mg) 0.0 12.2 f.3.7
0.05vg 35.0+10.5 17.7+ 5.7 14.77 2.6 18.8+ - 3.7
0.51.18 40.4+13.0 18.17 7.6 15.5, 2*7 18.6+ - 6.3
5Fig 19.8+11.3 16,6T 1.9 28.3t 5.6
Animals were treated with steroid doses of 0.051.18, O.Sug and S.Opg/ 1OOgm body weight by daily subcutaneous injections for five days. Each group contained 8-10 animals. Results are mean + S.D. The results presented in fig. 1 and fig. 2 indicate that estradiol increased the phenol-activatedNADH oxidase activity in the uteri of these rats. The increase of 2,4-DCP-activatedNADH oxidase is about 4 times that of control, while estradiol-activatedoxidase increase is only about 50% over the controls. There is no significant effect observed at 0.05ugm dose of estradiol. A dose dependent decrease in NADH oxidase is observed for both 23E2 and 34E2. The decrease in
estradiol-activated and 2,4-DCP-activated NADH oxidase was comparable in both treatments.
The enzyme activity is significantly reduced by
subphysiological levels of 4ME2.
However, a further decrease in es-
tradiol-activated oxidase by higher doses of 4ME2 is not significant (Fig.l), whereas an increase to the normal levels of oxidase is seen in 2,4-DCP-activated enzyme activity (Fig.2).
Fig.1: Effect of estrogens on estradiol activated NADH oxidase in rat uterus plotted on a semi log scale. Estradiol 178(0-A); 34E2 (o-o); 23E2 (0-n); 4ME2 (o--r));control (4). Treatment schedule and experimental conditions are as described in the materials and methods section.
Fig.2: Effect of estrogens on 2,4-DCP-activated NADH activity in rat uterus plotted on a semi log scale. Estradiol 17B(P-A); 34E2 (o-o); 23E (o--a); 4ME Treatmen 8 (0-o); control f*). schedule and experimental conditions are as described in the materials and methods section.
It is observed that superoxide dismutase is present in the uterine tissue of rats (Table 2). present in liver.
The levels in uterus were about 50% of that
Estradiol treatment did not affect SOD activity in
uterus, while a 30% reduction in liver enzyme was observed.
Both u-
terine and liver SOD were found to be significantly reduced by 23E2, 34E2 and 4ME2.
However, 34E2 affected uterine enzyme at 5ngm/lOOgm
s
656
TIFIIICOXDDB
only. There is an apparent dose-related decrease in SOD activity in both tissues by catecholestrogenmethyl ethers. TABLE 2 EFFECTS OF CATECHOLESTROGENSON SUPEROXIDE DISMUTASE IN RAT LIVER AND UTERUS Treatment
Superoxide Dismutase (Units/~ Protein) Liver
Oil Estradiol.0.05ug Estradiol.0.51-18 23E2 0.05j.lg 23R2 0.5i.rg 34E2 0.05ug 34E2 0.5118 0.05118 4ME2 0.5ng
4.5 + 1.0 3.0 7 1.3 2.8 5 0.5* 2.0 -I0.2* 2.3 + 1.5 0.5 SF: 0.2* 1.5 ?: 0.7* 0.8 5 O.l*
Uterus 3.6 + 0.8 3.3 T 1.3 3.4 T 1.8 0.9 + 0.56: 1.5 T 0.9* 3.4 T 2.9 2.9 ?: 1.2 2.7 T 1.8* 1.0 z o.s*
Results presented are mean + S-D,: See materials and methods section for-details of treatme% schedule, determination of SOD and calculation of the activity. *P < 0.05 compared to control.group.
DISCUSSION
Though various studies have utilized free catecholestrogensand demonstrated biological activity of these compounds, the physiological significance of eatecholestrogens -per se is questioned because of their high MCR (11). Since the majority of circulating catecholestrogensare methylated (2) and -~ in situ demethylation of estrogen methyl ethers has been reported (12), we have used methylated catecholestrogensin our studies. The present results demonstrating that catecholestrogenmethyl. ethers affect the uterine enzyme activity support the theory that these compunds may be physiologically important. Furthermore, methylated catecholestrogens-per se may be kmportant in regulating some aspects
of estrogen induced changes in target tissues. However, the mechanism of action of these compounds is not clear. The competition for estrogen receptor binding in uterus for fsee catecholestrogenshas been reported (3). Since we have not determined the estrogen receptors in the present experiments, it is not known if the methylated catecholestrogens induced changes in uterinemet,abolismare receptor mediated. Uterine peroxidase and phenol-activatedNAUH oxidase have been shown to be induced by estrogens in immature rats (13). It was also reported
(4)
that peroxidase activity is correlated with the activity
of farmation of water soluble products of estrone by uterine extracts and nuclear estrogen receptor content (LQ.
The relationship between
phenol-activatedNADH oxidase and peroxidase is not clear. However, it was suggested that peroxldase may be involved in NAUH oxidation(l5). The induction of NADH oxidase in these animals by estradiol.supports the earlier reports (1,7) that this enzyme is inducible in uterine tissues of immature rats by estrogens. Recent reports by Tsibris et al (16) and Jellinck and Newcombe (17) indicating that 2-hydroxy-_estradiol inhibited -_I in vitro peroxidase activity are in accordance with the present observations on NADH oxidase activity. The results obtained in the present study indicating that catecholestrogenmethyl ethers inhibit the NADH oxidase activity (Fig. 1,2) suggest that these compounds may play an important role in uterine metabolism. Though catecholestrogenshave bean reported to inhibit estradiol induced peroxidase (4), we do not know if catecholestrogenmethyl ethers can reduce the estradiol induced NADH oxidase in these aniIELLS.
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658
TLEIROIDS
The ratio between estradiol-activated and 2,4-DCP-activated NADH oxidase activities in control animals is 0.8-1.2.
However, this
ratio increased to 2.5-3.5 in estradiol or catecholestrogen treated rats.
A ratio of about 10 for these enzyme activities was reported
in earlier reports (7). the discrepancies
At present, it is not possible to ascertain
in this ratio.
It is interesting to note that the
inhibition of estradiol-activated NADH oxidase does not increase with increasing the 4ME2 dose.
Furthermore, initial decrease and an in-
crease to the control levels in 2,4-DCP-activated enzyme activity by increasing the 4ME2 dose is observed.
These results may suggest
that as 4ME2 increases in circulation, the net concentrations of free 4-hydroxyestradiol
increases at tissue level, thus eliciting
a positive estrogenic effect.
The free catecholestrogen may counter-
act some of the inhibitory activity of 4ME2.
This is further evi-
denced by the uterine dry weight increase at high dose of 4ME2 in these rats (Table 1). 4-hydroxyestradiol (18).
It has been reported by Fishman -et al that
is a more potent estrogen than its methyl ether
At equimolar concentrations of estrogens, the induction
and inhibition of estradiol-activated oxidase are comparable, while 2,4-DCP-dependent enzyme activity is induced by estradiol to a much greater extent than its inhibition by catecholestrogen methyl ethers.
It is not known if this differential effect is due
to the presence of two isomeric enzymes or to differential activation of separate active sites on the same enzyme.
However, the
physiological significance of this enzyme in uterine metabolism is unknown at present.
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659
S'IIBOIDm
Our results demonstrating the presence of superoxide dismutase (SOD) in the rat uterine tissues is in support of an earlier report (19) demonstrating this enzyme in human uterus.
The levels of SOD
in neoplastic tissues is suggested to be important in designing certain chemotherapeutic regimens (5).
Furthermore, SOD is shown
to reduce the rytotoxicity of various chemical agents (20). Though there is great variation in SOD levels in tumors of different origin, there is a general trend towards reduced SOD levels in malignant tissues (21). The present results demonstrate for the first time that estrogens affect SOD activity in liver and uterus.
Fur-
thermore, these experiments indicating that SOD levels are decreased by catecholestrogen methyl ethers may suggest an important role for these compounds in pathophysiology of tumors of uterus and liver. However, it is still not known if catecholestrogens affect tumor metabolism. ABBREVIATIONS: E := estradiol = 1,3,5(10)-estratriene-3,178-diol. 25E2 = 2-hydroxyestradiol 2,3-dimethyl ether = 1,3,5(10)-estratriene2,3,178-trio1 2,3 dimethyl ether. 34E12 = 4-hydroxyestradiol 3,4-dimethyl ether = 1,3,5(10)-estratriene3,4,17&triol 3,4 dimethyl ether. 4ME., = 4-methoxyestradiol = 1,3,5(10)-estratriene-3,4,178-trio1 4 methyl ether. REFERENCES 1. 2. 3. 4. 5. 6. 7.
Hollander, V.P., Stephens, M.L. and Adamson, T.E., Endocrinol, 66, 39, (1960). Ball, P. and Knuppen, R., Acta Endocrinol. Suppl., 232, l(1980). Merriam, G.R., Maclusky, N.J., Picard, M.K. and Nafzin, F., Steroids, 36, 1 (1980). Jellinck, P.K. and Newcombe, A., J. Steroid Biochem., !,1193, (1977). Oberley, L.W. and Buettner, G.R., Can. Res., 2, 1141, (1979). Reddy, V.V.R., Rajan, R. and Daly, M.J., Acta Endocrinol., 96, 7, (1981). Tempie, S., Hollander, V.P., Hollander, N. and Stephens, M.L.,
660
8. 9. 10. 11. 12. 13. 14. 15. 16.
17. 18. 19. 20. 21. 22.
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T=XSOIDB
J. Biol. Chem., 235, 1504, (1960). Minamui, M. and Yoshikawa, H., Clin. Chem. Acta., 92, 337, (1979). McCord, J.M. and Fridovich, I., J. Biol. Chem., 244, 6049, (1969). Lowry, O.H.,Rosenbough, N.J., Farr, A.L. and Randall, R.J., J. Biol. Chem., 193, 265, (1951). Kno, S., Brandon, D., Merriam, G.R., Loriaux, D.L. and Lipsett, M.B., Steroids, 36, 463, (1980). Reddy, V.V.R., Raan, R. and Lam, P., Trans. Am. Sot. Neurothem., 12, 267, (1981). Jellinck, P.H. and Lyttle, C.R., Adv. Enzy. Reg., 2, 17, (1973). McNabb, T. and Jellinck, P.H., Steroids, 2, 681, (1976). Akazawa, T. and Conn, E.E., J. Biol. Chem., 232, 403, (1958). Tsibris, J.C.M., Trujillo, Y.P., Fernandez, B.B., Bardawil, W.A., Kunigk, A. and Spellacy, W.N., J. Clin. Endo. Metab., 2, (1982), in press. Jellinck, P.H. and Newcombe, A., J. Steroid Biochem., 2, 681, (1980). Martucci, C.P. and Fishman, J., Endocrinol., 105, 1288,(1979). Westman, N.G. and Marklund, S.L., Cancer Res., fi,2962,(1981). Galvan, L., Huang, C.H., Prestayke, A.W., Stont, J.T., Evans, J.E. and Crooke, S.T., Can. Res., 41, 5103, (1981). Sykes, J.A., McCormick, F.X. and G'Brien, T.J., Can. Res., 2, 2759, (1978). Reddy, V.V.R., Hanjani, P. and Rajan, R., Steroids, 2, 195, (1981).
3032
ESTROGEN EFFECTS ON NADH OXIDASE AND S~EROXID~ DIS~TASE IN PREPUBERT~ ABBE
RATS
R. Rajan, M.J. Daly and V.V.R. Reddy Department of Obstetrics and Gynecology; Temple University School of Medicine; Philadelphia, Pa. 19140 Received 9-16-82 ABSTRACT
Thirty-four day old, ovariectomisedrats were treated with increasing doses of estradiol, 2-hydroxyestradiol2,3-dimethyl ether (23E2), 4-hydroxyestradiol3,4-dimethyl ether (34E2) and 4-methoxyestradiol (4ME2) for five days by subcutaneous injection. Superoxide dismutase, phenol activated NADH oxidase and uterine dry weights were determined. Only estradiol was found to be uterotrophic and increased NADH oxidase activity in these experiments. Both 23E2 and 34E2 treatment reduced the enzyme activity significantly. Though 4ME2 showed a decrease in NADH oxidase at O.O5~g/lOOgm body weight there was no further decrease at higher dose {5~g/lOO~). The superoxide dismutase (SOD) in uterus and liver was unaffected by estradiol, while and 4ME significantly reduced SOD in both liver and uterus. 23E 34E The& res%ts indiczte that 23E 34E and 4ME , in spite of their nonuterotrophic property, affect u8' erine2metaboligm. Furthermore, in view of the reports indicating the importance of SOD levels in various tumors and since catecholestrogensare observed to reduce SOD levels in liver and uterus, it is suggestive that catecholestrogensmay play an important role in the pathophysiologyof certain tumors.
INTRODUCTION
Estrogens are known to induce various enzymes in uterus and affect uterine metabolism. The phenol-activatedNADH oxidase, which catalyses reduced NAD oxidation in the presence of an added phenol in in vitro system is one of the enzymes that is induced by estradiol -treatment in immature rats (1). Catecholestrogenshave been suggested to be peripherally inactive but active at central level (2). Though catecholestrogensare weak uterotrophic agents, they are reported to bind estrogen receptors in uterus (3). It has been reported recently
VoZume 40, Number 6
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Deoember, 1982
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(4) that 2-hydroxyestradiol can induce peroxidase in uterus at high doses, but inhibitory and antiestrogenic when given with estradiol. However, the effect of catecholestrogens on NADH oxidase is not known. Superoxide dismutase (SOD) is one of the important enzymes associated with the regulation of superoxide anions and formation of hydrogen peroxide in various tissues.
Various tumors have been re-
ported to possess altered levels of SOD and this was suggested to be important in planning chemotherapeutic regimens in cancer treatment (5).
Since estrogens are known to influence uterine tumor development
and we have observed a decrease in catecholestrogen synthesis in human leiomyoma (22), we attempted to determine the effects of these estrogens on SOD activity in uterus.
In this report, we present evidence
that treatment with catecholestrogen methyl ethers inhibit both NADH oxidase and SOD activity in immature rat uterus.
MATERIALS AND METHOD Estradiol-178, superoxide dismutase and reduced NADH were purchased from Sigma Chemical Company. 2-Hydroxyestradiol 2,3-dimethylether (23E2), 4-hydroxyestradiol 3,4-dimethyl ether (343 > and 4methoxyestradiol (4ME2) were prepared earlier (6). All $he steroids were purified by repeated crystallizations. Twenty-five day old Sprague Dawley rats were obtained from Charles River and ovariectomised under light ether anesthesia. The animals were divided into several groups of five animals. Steroids were dissolved in sesame oil and dilutions were adjusted in order to inject O.lml of the solutions per each injection. All the groups received daily subcutaneous injections for five days starting on 34th day of age. Groups 1 and 2 received estradiol O.O5~g/lOOgm and 0.5ug/lOOgm body weight respectively. Groups 3,4 and 5 received 23E 0.05, 0.5 and 5ug/lOOgm body weight respectively. The treatmen 8 schedule for 34E2 and 4ME2 was the same as for 23E . Oil injected group served as controls. The animals were sacrificed by decapitation within 18 hr after the last injection. Uteri were excised from the animals after decapitation. The tissues were trimmed of the adhering adipose and connective tissues
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653
and weighed. About one half of each tissue was then dried at 6070°C until a constant weight was obtained. Total dry weights were calculated from the fraction of the initial weight used for drying. Estimation of Phenol Activated NADH Oxidase The procedure followed for determination of NADH oxidase was essentially the same as the method of Temple -et al (7). Briefly, uterine homogenates were prepared in 0.25M sucrose (pH.7.4) to yield a 101% solution. Homogenates were centrifuged at 1OOOg for 15 minutes and the supernatant used for estimations of enzymes. The incubation mixture contained 0.015M phosphate buffer (pH.7.7), 1.5umoles of maganese chloride, 0.4umoles of NADH, 34 nanomoles of estradiol dissolved in 0.05ml propylene @ye01 and O.lml of homogenate. The mixture was incubated at 30 C for 30 minutes and the reduction in absorbency at 34Ornl.l was recorded against the control incubations containing 0.05ml propylene glycol. The enzyme activity was found to -be linear up to 30 to 45 minutes and activity is expressed as )Jmoles of NADH oxidized per minute mg protein. The incubation mixture for estimation of 2,4 dichlorphenol (2,4-DCP) activated NADH oxidase contained same components as in estradiol-activated oxidase determination, except that 0.015M phosphate buffer (pH.7.0) was used and estradiol was replaced with 0.751.imolesof 2,4-DCP. Determination of Superoxide Dismutase in Uterus and Liver Superoxide dismutase was estimated by the method reported recently (8), using nitroblue tetrazolium. The tissues were homogenized at (0-4OC) in 0.154M NaCl (1:50). A 5ml aliquot was shaken vigorously with 5ml of ethanol: chloroform mixture for 3 minutes and centrifuged for 1 hr at 1500g at 0-4'C. The supernatant (0.25ml) was used for assay. The incubation mixture contained cacodylic acid buffer (pH8.2) 0.5m1, triton X-100 (16% solution) O.lml, nitroblue tetrazolium (1M) 0.4ml and tissue extract 0.25ml. The reaction was started by adding 2Ovl of pyrogallol (0.1% solution). The mixture was incubated at 37'C for 5 minutes and the reaction stopped with 2M formic acid buffer (pH 3.5). The blue formazan color was determined at 54Oml.l. SOD was inactivated by heating the enzyme extracts in a boiling water bath for 2 minutes and the inactivated enzyme extracts served as controls. The rate of superoxide reaction inhibited by SOD was calculated according to the definition of McCord and Fridovich (9). A fifty percent inhibition of the reaction was considered to be equal to one unit of the enzyme. The standard SOD obtained from Sigma Chemical Co. with 3.3 units/pgm protein was used as reference standard. Protein was estimated by the method of Lowry -et al (lb).
654
RESULTS
The results presented are an average of two independent experiments with a total of 8-10
It is observed that
4ME2 has a weak uterotrophic activity, while 23E2 and 34E2 are nonuterotrophic, even at high doses. TABLE I EFFECT OF CATECHOLESTROGENMETHYL ETHERS ON UTERINE DRY WEIGHT IN OVARIECTOMISEDHATS Treatment Dosage Oil Estradiol 23E2 3432 4ME2
Uterine Dry Weights (mg) 0.0 12.2 f.3.7
0.05vg 35.0+10.5 17.7+ 5.7 14.77 2.6 18.8+ - 3.7
0.51.18 40.4+13.0 18.17 7.6 15.5, 2*7 18.6+ - 6.3
5Fig 19.8+11.3 16,6T 1.9 28.3t 5.6
Animals were treated with steroid doses of 0.051.18, O.Sug and S.Opg/ 1OOgm body weight by daily subcutaneous injections for five days. Each group contained 8-10 animals. Results are mean + S.D. The results presented in fig. 1 and fig. 2 indicate that estradiol increased the phenol-activatedNADH oxidase activity in the uteri of these rats. The increase of 2,4-DCP-activatedNADH oxidase is about 4 times that of control, while estradiol-activatedoxidase increase is only about 50% over the controls. There is no significant effect observed at 0.05ugm dose of estradiol. A dose dependent decrease in NADH oxidase is observed for both 23E2 and 34E2. The decrease in
estradiol-activated and 2,4-DCP-activated NADH oxidase was comparable in both treatments.
The enzyme activity is significantly reduced by
subphysiological levels of 4ME2.
However, a further decrease in es-
tradiol-activated oxidase by higher doses of 4ME2 is not significant (Fig.l), whereas an increase to the normal levels of oxidase is seen in 2,4-DCP-activated enzyme activity (Fig.2).
Fig.1: Effect of estrogens on estradiol activated NADH oxidase in rat uterus plotted on a semi log scale. Estradiol 178(0-A); 34E2 (o-o); 23E2 (0-n); 4ME2 (o--r));control (4). Treatment schedule and experimental conditions are as described in the materials and methods section.
Fig.2: Effect of estrogens on 2,4-DCP-activated NADH activity in rat uterus plotted on a semi log scale. Estradiol 17B(P-A); 34E2 (o-o); 23E (o--a); 4ME Treatmen 8 (0-o); control f*). schedule and experimental conditions are as described in the materials and methods section.
It is observed that superoxide dismutase is present in the uterine tissue of rats (Table 2). present in liver.
The levels in uterus were about 50% of that
Estradiol treatment did not affect SOD activity in
uterus, while a 30% reduction in liver enzyme was observed.
Both u-
terine and liver SOD were found to be significantly reduced by 23E2, 34E2 and 4ME2.
However, 34E2 affected uterine enzyme at 5ngm/lOOgm
s
656
TIFIIICOXDDB
only. There is an apparent dose-related decrease in SOD activity in both tissues by catecholestrogenmethyl ethers. TABLE 2 EFFECTS OF CATECHOLESTROGENSON SUPEROXIDE DISMUTASE IN RAT LIVER AND UTERUS Treatment
Superoxide Dismutase (Units/~ Protein) Liver
Oil Estradiol.0.05ug Estradiol.0.51-18 23E2 0.05j.lg 23R2 0.5i.rg 34E2 0.05ug 34E2 0.5118 0.05118 4ME2 0.5ng
4.5 + 1.0 3.0 7 1.3 2.8 5 0.5* 2.0 -I0.2* 2.3 + 1.5 0.5 SF: 0.2* 1.5 ?: 0.7* 0.8 5 O.l*
Uterus 3.6 + 0.8 3.3 T 1.3 3.4 T 1.8 0.9 + 0.56: 1.5 T 0.9* 3.4 T 2.9 2.9 ?: 1.2 2.7 T 1.8* 1.0 z o.s*
Results presented are mean + S-D,: See materials and methods section for-details of treatme% schedule, determination of SOD and calculation of the activity. *P < 0.05 compared to control.group.
DISCUSSION
Though various studies have utilized free catecholestrogensand demonstrated biological activity of these compounds, the physiological significance of eatecholestrogens -per se is questioned because of their high MCR (11). Since the majority of circulating catecholestrogensare methylated (2) and -~ in situ demethylation of estrogen methyl ethers has been reported (12), we have used methylated catecholestrogensin our studies. The present results demonstrating that catecholestrogenmethyl. ethers affect the uterine enzyme activity support the theory that these compunds may be physiologically important. Furthermore, methylated catecholestrogens-per se may be kmportant in regulating some aspects
of estrogen induced changes in target tissues. However, the mechanism of action of these compounds is not clear. The competition for estrogen receptor binding in uterus for fsee catecholestrogenshas been reported (3). Since we have not determined the estrogen receptors in the present experiments, it is not known if the methylated catecholestrogens induced changes in uterinemet,abolismare receptor mediated. Uterine peroxidase and phenol-activatedNAUH oxidase have been shown to be induced by estrogens in immature rats (13). It was also reported
(4)
that peroxidase activity is correlated with the activity
of farmation of water soluble products of estrone by uterine extracts and nuclear estrogen receptor content (LQ.
The relationship between
phenol-activatedNADH oxidase and peroxidase is not clear. However, it was suggested that peroxldase may be involved in NAUH oxidation(l5). The induction of NADH oxidase in these animals by estradiol.supports the earlier reports (1,7) that this enzyme is inducible in uterine tissues of immature rats by estrogens. Recent reports by Tsibris et al (16) and Jellinck and Newcombe (17) indicating that 2-hydroxy-_estradiol inhibited -_I in vitro peroxidase activity are in accordance with the present observations on NADH oxidase activity. The results obtained in the present study indicating that catecholestrogenmethyl ethers inhibit the NADH oxidase activity (Fig. 1,2) suggest that these compounds may play an important role in uterine metabolism. Though catecholestrogenshave bean reported to inhibit estradiol induced peroxidase (4), we do not know if catecholestrogenmethyl ethers can reduce the estradiol induced NADH oxidase in these aniIELLS.
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TLEIROIDS
The ratio between estradiol-activated and 2,4-DCP-activated NADH oxidase activities in control animals is 0.8-1.2.
However, this
ratio increased to 2.5-3.5 in estradiol or catecholestrogen treated rats.
A ratio of about 10 for these enzyme activities was reported
in earlier reports (7). the discrepancies
At present, it is not possible to ascertain
in this ratio.
It is interesting to note that the
inhibition of estradiol-activated NADH oxidase does not increase with increasing the 4ME2 dose.
Furthermore, initial decrease and an in-
crease to the control levels in 2,4-DCP-activated enzyme activity by increasing the 4ME2 dose is observed.
These results may suggest
that as 4ME2 increases in circulation, the net concentrations of free 4-hydroxyestradiol
increases at tissue level, thus eliciting
a positive estrogenic effect.
The free catecholestrogen may counter-
act some of the inhibitory activity of 4ME2.
This is further evi-
denced by the uterine dry weight increase at high dose of 4ME2 in these rats (Table 1). 4-hydroxyestradiol (18).
It has been reported by Fishman -et al that
is a more potent estrogen than its methyl ether
At equimolar concentrations of estrogens, the induction
and inhibition of estradiol-activated oxidase are comparable, while 2,4-DCP-dependent enzyme activity is induced by estradiol to a much greater extent than its inhibition by catecholestrogen methyl ethers.
It is not known if this differential effect is due
to the presence of two isomeric enzymes or to differential activation of separate active sites on the same enzyme.
However, the
physiological significance of this enzyme in uterine metabolism is unknown at present.
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S'IIBOIDm
Our results demonstrating the presence of superoxide dismutase (SOD) in the rat uterine tissues is in support of an earlier report (19) demonstrating this enzyme in human uterus.
The levels of SOD
in neoplastic tissues is suggested to be important in designing certain chemotherapeutic regimens (5).
Furthermore, SOD is shown
to reduce the rytotoxicity of various chemical agents (20). Though there is great variation in SOD levels in tumors of different origin, there is a general trend towards reduced SOD levels in malignant tissues (21). The present results demonstrate for the first time that estrogens affect SOD activity in liver and uterus.
Fur-
thermore, these experiments indicating that SOD levels are decreased by catecholestrogen methyl ethers may suggest an important role for these compounds in pathophysiology of tumors of uterus and liver. However, it is still not known if catecholestrogens affect tumor metabolism. ABBREVIATIONS: E := estradiol = 1,3,5(10)-estratriene-3,178-diol. 25E2 = 2-hydroxyestradiol 2,3-dimethyl ether = 1,3,5(10)-estratriene2,3,178-trio1 2,3 dimethyl ether. 34E12 = 4-hydroxyestradiol 3,4-dimethyl ether = 1,3,5(10)-estratriene3,4,17&triol 3,4 dimethyl ether. 4ME., = 4-methoxyestradiol = 1,3,5(10)-estratriene-3,4,178-trio1 4 methyl ether. REFERENCES 1. 2. 3. 4. 5. 6. 7.
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