(CANCER RESEARCH 45, 2425-2428, June 1985]

Effects of Aromatase Inhibitors, Aminoglutethimide, and 4- Hydroxyandrostenedione on Cyclic Rats and Rats with 7,12- Dimethylbenz(a)anthracene-induced Mammary Tumors1

Lih-Yuh Wing, Wesley M. Garrett, and Angela M. H. Brodie2

Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21201

ABSTRACT synthesis by virtue of its effects on cytochrome P-450, a com ponent of the aromatase complex (6, 19). AG, first introduced 4-Hydroxyandrostenedione (4-OHA) is a more potent and spe clinically as an anticonvulsant, has been shown to be beneficial cific inhibitor of aromatase (estrogen synthetase) than amino- to postmenopausal (15) but not premenopausal breast cancer glutethimide (AG). The two inhibitors were compared in rats with patients (14). 7,12-dimethylbenz(a)anthracene-induced, hormone-dependent In this paper, we compared the effectiveness of 4-OHA and tumors and in normal cyclic rats treated for 4 and 2 weeks, AG in the rat with carcinogen (DMBA)-induced mammary tumors respectively. Ovaríanestradici levels and aromatase activities and investigated whether the 2 compounds might act synergis- were not consistently reduced, and tumors regressed in only tically. Since the DMBA-induced tumors are dependent on ovar two of eight rats treated with AG. In animals treated with 4-OHA ian steroids, this model resembles the premenopausal rather or 4-OHA:AG, the total tumor volume, estradici levels, and than postmenopausal breast cancer patient. Although we have aromatase activity decreased by >70%. Ovaríanweights and demonstrated inhibition of ovarian estrogen secretion and aro plasma luteinizing hormone (LH) levels were also reduced by 4- matase activity by AG in acute studies (1), we have now deter OHA but increased by AG. Uterine weights were not altered by mined the long-term effect of this compound on estrogen and AG treatment but were increased by 4-OHA. Similar but more gonadotropin levels as well as on mammary tumor growth. consistent results were obtained with these treatments in normal, cyclic rats. In ovariectomized rats, AG had no effect, whereas 4- OHA decreased LH levels and increased uterine weights. The MATERIALS AND METHODS results suggest that, although AG reduces ovarian estrogen 4-OHA was prepared as described previously (4). AG was kindly secretion by aromatase inhibition, this may lead to an increase donated by Dr. C. A. Browntey, Ciba Pharmaceutical Co., Summit, in LH secretion. Increased LH may promote ovarian growth and NJ. [1,2-3H]Androstenedione was obtained from New England Nuclear aromatase synthesis, counteracting the inhibitory action of AG to some extent. 4-OHA which inactivates aromatase may also Corp. and checked for purity on thin-layer chromatography prior to use. [1-3H]Androstened¡one was prepared from the latter by overnight treat prevent new enzyme synthesis by directly inhibiting gonadotro- ment in sodium hydroxide in 8 ml of methanol to remove 3H at C-2. [1- pins. This would result in more effective reduction in ovarian 3H]Androstenedione was then extracted with méthylènedichloride and estrogen production by 4-OHA than AG during long-term treat purified by thin-layer chromatography on silica gel-coated plates ment. (etherhexane, 3;1).

INTRODUCTION In Vitro Assay of Aromatase Activity

It is now evident that breast cancer patients whose tumors Microsomes were prepared as described previously from human term contain significant concentrations of estrogen receptors are likely placenta (16) and ovaries of rats pretreated for 12 days with 100 IU of to respond to hormonal therapy (12). The development of strat pregnant mares' serum gonadotropin (Sigma) injected on alternate days egies that block the action of estrogens or limit their production (4). The microsomes, equivalent to 50 mg (wet weight) of tissue, were should be useful in treating this type of patient. Our approach is incubated with [1,2-3H]androstenedione (200,000 dpm/1 MM) in 2 ml of to develop potent and safe compounds which selectively inhibit 0.1 M phosphate buffer containing an oxygen-generating system [NADP estrogen production. We have identified a number of steroids (4, (5 mg):glucose 6-phosphate (3 mg):1 ID glucose-6-phosphate dehydro- 16) which are specific inhibitors of aromatase, and several appear genase] at 37°C under an oxygen atmosphere (5). Various concentra to inactivate the enzyme in an irreversible manner (2, 11). 4- tions of inhibitors were added in ethanol to the dry flask together with OHA3 is the most potent of these, and its in vivo inhibition of one drop of propylene glycol. The solvent was evaporated prior to adding ovarian aromatase and estrogen production in rats (5) as well as the microsomal mixture. After 30-min incubation, chloroform was added inhibition of peripheral aromatization in monkeys (3) have been to the vials. An aliquot of the aqueous phase was removed and mixed reported previously by us. In the first clinical use of this com with an equal volume of 2.5% charcoal, and after centrifugation, the pound, we reported recently that estradici levels were reduced, radioactivity in the supernatant was measured. The amount of estrogen measureable tumors were significantly decreased, and soft tis produced was calculated from the tritium released during aromatization sue and bone metastatic lesions were healed in postmenopausal of [1,2-3H]androstenedione and converted to 3H2O (18). breast cancer patients (7). AG is also an inhibitor of estrogen Animal Experiments 1This work was supported by CA-27440. 2 To whom requests for reprints should be addressed, at Pharmacology and Tumor-bearing Rats. All animals were female Sprague-Dawley rats Experimental Therapeutics, School of Medicine, University of Maryland, Room 407, from Charles River Breeding Laboratories, and they were housed under BRB, Baltimore, MD 21201. 'The abbreviations used are: 4-OHA, 4-hydroxyandrostene-3,17-dione; AG, controlled conditions of temperature, humidity, and light (14 h) and fed aminoglutethimide; DMBA, 7.12-dimethylbenz(a)anthracene: LH, luteinizing hor ad libitum. The rats were gavaged at 50 to 55 days of age with 20 mg mone; FSH, follicle-stimulating hormone. of DMBA (Sigma) in 2 ml of sesame oil as described (10). Animals which Received 9/10/84; revised 1/24/85; accepted 2/13/85. had been exposed to the carcinogen less than 6 months beforehand

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Downloaded from cancerres.aacrjournals.org on October 6, 2021. © 1985 American Association for Cancer Research. AROMATASE INHIBITORS IN CYCLIC AND TUMOR-BEARING RATS were selected for experiments when at least one tumor per rat had Table1 reached a diameter of 2 cm. Tumors were measured with calipers once Comparison of effectiveness of 4-OHA and AG as inhibitors of aromatization a week, and the tumor volume was calculated [Va ir r-?r-¿(9)].Groups of The standard assay is described ¡nthe text using 1 pM androstenedione. rats were given injections s.c. twice daily with 4-OHA, AG, or 4-OHA: AG in steroid suspending vehicle (Klucil: 0.3% hydroxypropylcellulose). The 4-OHA daily dose of inhibitor was 50 mg/kg, which was the dose found effective Tissue source A3 previously for 4-OHA (4). Controls were given injections of the vehicle. Human placenta 0.3 17.5 All AG-treated animals were given 0.9% NaCI solution (saline) to drink. Rat ovary 1.65 24.5 Vaginal smears were taken daily to determine stages of the estrous , 50% inhibitory concentration. cycle. On the last day of the experiment, animals were given injections at 9:00 a.m. Three h later, Mood (1 ml) was collected from the ovarian 100, vein. Control and AG-treated rats were bled on diestrus, since animals treated with 4-OHA were consistently diestrus. The ovaries and uterus were then removed and weighed. The ovaries were frozen until assayed. Each pair of ovaries was homogenized, and aromatase activity was determined from the 3H2O formed after incubating the homogenate with 70 FJ-'HJandrostenedione (200,000 dpm/35 nm) in 2 ml of 0.1 M phosphate buffer containing the NADPH-generating systems for 30 min at 37°C under an oxygen atmosphere. 2 50 Estradici antibody was kindly donated by Dr. D. Collins, Emory School c of Medicine, GA. Cross-reaction of the antibody with estrone was 1.4%. o There was no cross-reactivity with up to 100 ng of AG or with 1 to 10 ing of 4-OHA, although this compound contributed a background of approximately 2 pg/tube. Charcoal-treated, steroid-free rat plasma (0.05 ml) was extracted with ether and added to all standard curve tubes. The 20 same volume of plasma from the treated rats was extracted and assayed. For the controls, a 0.025-ml sample and 0.025 ml of steroid-free rat plasma were extracted. All samples were measured in duplicate in one assay. The coefficient of variation of the assay is 5.8% (n = 7). The 0.25 0.5 1.0 10 100 sensitivity of the assay is 1 pg/tube. inhibitor Healthy Cycling Rats. Normal female Sprague-Dawley rats (220 to Chart 1. Inhibition of aromatization of androstenedione by 4-OHA and AG. 250 g), without tumors, were treated identically to the above for 2 weeks. Aromatization was determined from tritium released (3H20) from |1.2-JH|andro- Blood was collected, and assays were performed as already described. stenedione (200,000 dpm/*iM) incubated for 30 min with various concentrations of Inhibitor, an oxygen-generating system, and microsomes from human placenta (•) Ovariectomized Rats. Normal rats were ovariectomized 2 weeks or pregnant mares' serum gonadotropin-primed rat ovaries (O). prior to treatment as described above. After 2 weeks of treatment, heart blood was collected, and LH concentrations were measured. Uteri were weighed. +100

Statistics

Data were analyzed by one-way analysis of variance, followed by !+50 Newman-Keuis multiple-range analyses. As indicated in the tables, anal yses were performed on logs-transformed data if Cochran's test of the raw data revealed heterogeneous variances.

RESULTS

In Chart 1 is shown the dose response data for inhibition of aromatization of 1 ¡Mandrostenedione by 4-OHA and AG. It is -50 interesting to tiote that the activity of 4-OHA is about 5-fold greater in microsomes from human placenta than from rat ova ries, whereas AG has similar potency in both tissues. The 50%- 4-OHA -100 inhibltory concentrations of the compounds are shown in Table 1. 4-OHA is approximately 60 times more potent than AG in 1234 human placenta! microsomes. weeks Chart 2. Effect of 4-OHA and AG on DMBA-induced, hormone-dependent mam Chart 2 shows the percentage of change in total tumor volume with 4-OHA, AG, or 4-OHA:AG versus controls. In the control mary tumors in the rat. All animals were given injections s.c. twice daily for 4 weeks. Tumor volumes were measured weekly. •8 animals given injections of group, 2 of 12 tumors regressed, but there were 14 new tumors vehicle; •,10 animals given injections of 4-OHA (50 mg/kg/day). O, 8 animals after 4 weeks, resulting in an increase in total tumor volume. 4- given injections of AG (50 mg/kg/day); A, 6 animals given injections of 4-OHA and OHA causes marked tumor regression as reported previously AG (each 50 mg/kg/day). CONT, control. (5). After 4 weeks, although one tumor did not regress, the total tumor volume of 18 tumors on 10 rats had decreased by 75%. the experiment, there were 11 new tumors, so that there was Four new tumors occurred during the first week of treatment, no decrease in the total tumor volume. The combined treatment but all regressed completely by the fourth week. Treatment with of 6 rats with 4-OHA and AG caused tumor regression to a AG decreased the volume of 10 of 12 tumors, but at the end of similar extent as with 4-OHA alone. Five of the 6 original tumors

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Downloaded from cancerres.aacrjournals.org on October 6, 2021. © 1985 American Association for Cancer Research. AROMATASE INHIBITORS IN CYCLIC AND TUMOR-BEARING RATS were less than half their original size after 4 weeks. Although 4 weights were unaffected by AG. They were also not decreased new tumors developed, 3 had regressed completely by the end by 4-OHA. As in the experiment with tumor-bearing rats shown of the treatment period. in Table 2, LH values were decreased by 4-OHA and increased Rats treated with AG alone continued to cycle, whereas 4- (3-fold) by AG relative to the controls. OHA-treated animals showed only diestrus smears. All animals To determine the mechanism of the effects of these com were therefore autopsied on diestrus for blood collections. Mean pounds on LH secretion and uterine weight, rats ovariectomized estradici concentrations in ovaríanvein samples and ovarian 2 weeks previously were treated for 2 weeks. In these animals, aromatase activity were decreased in AG-treated rats (Table 2), there was no significant difference in the LH values or uterine but the difference was not significant compared to controls. weights between controls and AG-treated rats (Table 4). How There was considerable variability among the AG-treated rats, ever, LH values were markedly suppressed, and uterine weights but in 4 of 6 rats, values were similar to the controls with only 2 were increased more than 2-fold by 4-OHA. animals having significantly decreased estradiol concentration. However, there was no con-elation between the response of DISCUSSION tumors and estrogen concentrations. There was a consistent and significant decrease in estradiol secretion and aromatase We have reported previously that 4-OHA is a potent inhibitor activity with 4-OHA and also with 4-OHA:AG treatment. LH levels of aromatase and causes time-dependent inactivation of the measured in peripheral plasma were significantly reduced by 4- enzyme (2, 4). These observations have been confirmed by OHA and by 4-OHA:AG in comparison with control values in others (8), and the evidence suggests that 4-OHA may be a ko« samples collected from rats in diestrous (Table 2). In contrast, or active site-directed inhibitor. AG appears to be a competitive LH values were increased approximately 2-fold by AG in com inhibitor of cytochrome P-450 and is a weaker inhibitor of aro parison to controls. Prolactin concentrations were very variable, matase than 4-OHA. It is not known whether the differences in and there was no significant difference between treated and activity of the 4-OHA in the rat ovarian microsomes versus human control groups (data not shown). The mean ovarian weight was placental microsomes are due to species differences or to tissue also increased by AG but decreased by 4-OHA and 4-OHA:AG. difference. If the former were true, it implies that 4-OHA may be Uterine weight was unaffected by AG. Despite reduced estrogen more active in the human in vivo. In contrast, no difference was levels, uterine weight was not decreased by 4-OHA or by 4- noted in the activity of AG in the 2 systems. OHA:AG treatment. In the tumor-bearing rats, there was considerable variability in Because of the variability in the concentrations of estradiol the response of tumors, estradiol secretion, and aromatase during AG treatment and because the cycle pattern, in the control activity to AG treatment. Estradiol secretion was inhibited in 2 animals as well as the AG-treated rats, was irregular, healthy rats but not in other animals. Although many of the original cycling rats were treated with AG and 4-OHA for 2 weeks. As tumors decreased in volume, 11 new tumors appeared and shown in Table 3, the results were similar to those after 4 weeks increased during treatment, but the overall effect of AG on the of treatment in the tumor-bearing rats. Although there was much tumors was not significantly different from controls (Chart 2). In less variation in the estradiol values, there was no significant one animal, there was a marked decrease in both tumor volume reduction by AG treatment compared to control values. Uterine and estradiol secretion (<25 pg/ml, value not included in the

Table 2 Effect of 4-OHA and AG treatment on rats with DMBA-induced mammary tumor Rats were given injections s.c. twice daily for 4 weeks of 4-OHA and/or AG (50 mg/kg/day). Blood was collected from the ovaríanvein 3 h after the last injection, and then ovaries were removed, stored frozen, and homogenized, and aromatase activity was determined. Heart blood was collected at sacrifice, and LH concentrations were determined in the plasma. Values with same superscripts (c, e, and I ) were significantly different. Aromatase (% of activity/ Ovarian wt (mg)d LH (ng/ml)s Treatment mg of protein) Estradiol (pg/ml) Uterine wt (mg) ±1.03"(6) ±100.3°(5) ±29.8* (7) Control4-OHAAG ±34.1e (7) 45.8 ±5.2e (8) 0.43 ±0.06e(8) 140.0 ±18.7°(8) 14.7 ±6.4e (10) 544.3 ±21.0e (10) 117.3 + 7.3' (8) 1.58±0.25 (6) 326.8 ±153.4 (5) 297.3 + 50.8 (7) 424.5 ±36.3 (8) AG:4-OHA79.6±5.56'e(7)°'-e69.8 ±4.3 (6)3.10 0.67 ±0.21e(5)474.0 140.5 ±24.2 (4)167.7 10.8 + 3.1e (6)432.3 473.2 ±14.9e (6) " Statistical analyses of differences were performed on iog.-transformed data, since Cochran's test of raw values revealed heterogeneous variances. " Mean ±SE. c Significance (P < 0.01). d Numbers in parentheses, number of animals. " Significance (P< 0.005).

Tables Effect of 4-OHA and AG on normal cyclic rats Rats were given injections s.c. twice daily for 2 weeks of 4-OHA or AG (50 mg/kg/day). Procedures were the same as for Table 1. (ng/ml)"107.3 (pg/ml)e433.3 TreatmentControl (mg)300.5wt wt(mg)e77.2 ±19.5t>'c(12)*e ±74.8e (6) ±12.3e (12) ±1.8e 47.1 ±12.3e (5) 154.0 ±14.5e (5) 419.2 ±31.6e (6) 4-OHA 68.4 ±5.6 322.8 ±27.1e (7)Estradiol AGLH 284.0 + 55.0 (5)Uterine367.7 ±32.4 (6)Ovarian101 .9 + 8.0e (7) * Statistical analyses of differences were performed on lock-transformed data, since Cochran's test of raw values revealed heterogeneous variances. * Mean ±SE. c Significantly different (P < 0.05). Numbers in parentheses, number of animals. e Significantly different (P < 0.005).

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Table 4 tageous, since peripheral aromatase appears not to be regulated Effect of 4-OHAand AG on ovariectomized rats by LH and FSH (13). Rats were ovariectomized 2 weeks previously, and 7 animals per group were given injections s.c. of 4-OHA or AG (50 mg/kg/day) twice daily for 2 weeks. Procedureswere the same as for Table 1. ACKNOWLEDGMENTS LH (ng/ml)a Uterinewt (mg)" Treatment The assistance of John Nolan, Dr. Cheng Hong, and Dr. Qiam-ChunYu in the 1092.9 ±68.56'c 102.7±4.9C Control animal studies and of Marsha Morgan for preparation of the manuscript is greatly 4-OHA 71.3±14.0C 269.4 ±18.2e appreciated. AG 1148.4 ±123.1 110.3 ±5.8 * Statistical analyses of differences were performed on log.-transformed data, since Cochran's test of raw values revealed heterogeneousvariances. REFERENCES 6 Mean ±SE. c Significantlydifferent (P < 0.005). 1. Brodie, A. M. H., Garrett, W. M., Hendrickson, J. R., and Tsai-Morris, C-H. Effects of aromatase inhibitor 4-hydroxyandrostenedione and other com pounds in the 7,12-dimethylbenz(a)anthracene-inducedbreast carcinoma model. Cancer Res., 42; 3360-3364,1982. 2. Brodie, A. M. H., Garrett, W. M., Hendrickson, J. R., Tsai-Morris, C. H., mean value for Table 2), but 5 animals secreted more than 190 Marcotte, P. A., and Robinson, C. H. Inactivation of aromatase in vitro by 4- pg of estradici per ml and were all within the range of the hydroxyandrostenedioneand 4-acetoxyandrostenedioneand sustainedeffects controls. The total tumor volume for these animals increased in vivo. Steroids, 38; 693-702,1981. approximately 4-fold. Blood could not be collected from 2 other 3. Brodie, A. M. H., and Longcope, C. Inhibition of peripheral aromatization by aromatase inhibitors 4-hydroxy and 4-acetoxy androstenedione. Endocrinol animals. Estrous cycles also continued during AG treatment. The ogy, 106:19-21,1980. estradici secretion by control animals was quite variable, and the 4. Brodie,A. M. H., Schwarzel,W. C., and Brodie,H.J. Studieson the mechanism animals cycled rather erratically. In 4-OHA-treated and in 4- of estrogen biosynthesis in the rat ovary-l. J. Steroid Btochem.,7:7877-7893, OHA:AG-treated rats, estradici levels were reduced compared 1976. 5. Brodie, A. M. H., Schwarzel. W. C., Shaikh, A. A., and Brodie, H. J. The effect to controls, and daily vaginal smears were leukocytic, consistent of an aromatase inhibitor, 4-hydroxy-4-androstene-3,17-dione,an estrogen- with low estradiol production. dependent processes in reproduction and breast cancer. Endocrinology, 700: We have observed previously that 4-OHA and AG decreased 1684-1695,1977. ovarian aromatase activity and estrogen secretion to a similar 6. Chakraborty, J., Hopkins, R., and Parke, D. V. Inhibition studies on the aromatization of androstenedione by human placental microsomal prepara extent in acute experiments in which rats were given injections tions. Biochem. J., 730:19-20,1972. on the morning of proestrus, and tissues and blood were col 7. Coombes, R. C., Goss, P., Dowsett, M., Gazet, J., and Brodie, A. 4-Hydrox- lected 3 h later (1). However, in long-term experiments of 2 and yandrostenedionetreatment of postmenopausalpatients with advancedbreast 4 weeks, it is evident that estradiol suppression is not maintained cancer. Lancet, 2:1237-1239,1984. 8. Covey, D. F., and Hood, W. F. Aromatase enzyme catalysis involved in the by AG to the same degree. The initial 90% inhibition of ovarian potent inhibitionof estrogen biosynthesiscaused by 4-acetoxy and 4-hydroxy- estradiol synthesis by AG may lead to increased LH levels via 4-androstene-3,17-dtone.Mol. Pharmacd., 27:173-180,1982. feedback regulatory mechanisms in the intact rat. Reflex in 9. DeSombre, E. R., and Arbogast, L. Y. Effect of the antiestrogen C1628 in the creases in LH and FSH were observed in premenopausal patients growth of rat mammary tumors. Cancer Res., 34:1971-1976,1974. 10. Huggins, C., Briziarelli, G., and Button. H. Rapid induction of mammary treated with AG (14). Increased gonadotropins may tend to carcinoma in the rat and the influence of hormones on tumors. J. Exp. Med , stimulate aromatase synthesis by the ovaries and counteract the 709: 25-42, 1959. inhibitory effects of AG to some extent. After 2 weeks in the 11. Marsh, D. A., Brodie, H. J., Garrett, W., Tsai-Morris, C-H., and Brodie, A. M. normal cycling animals, there was a 50% reduction in the mean H. Aromatase inhibitors. Synthesis and biological activity of androstenedione value of estradiol which, due to variation, was not significantly derivatives. J. Med. Chem., in press, 1985. 12. McGuire. W. L. An update on estrogen and progesterone receptors in prog different from the control value. However, after 4 weeks of nosis for primary and advanced breast cancer. In: S. lacobelli et al. (éd.), treatment, estradiol production in 5 of 6 tumor-bearing animals Hormones and Cancer, pp. 337-343. New York: Raven Press, 1980. was within the range of values for control animals. This amount 13. Mendelson,C. R., Cleland,W. H., Smith, M. E., and Simpson, E. R. Regulation of aromatase activity of stromal cells devised from human adipose tissue. of estradiol was sufficient to maintain the uterine weight com Endocrinology, 777: 1077-1085,1982. parable to intact control rats. AG appeared to have no direct 14. Samojlik, E., Santen, R. J., and Wells, S. A. Resistance of the ovary to effect on either the uterus or pituitary in ovariectomized rats, blockade of aromatization with aminoglutethimide.J. Clin. Endocrino!.Metab., whereas marked reduction in LH levels by 4-OHA suggests a 5Õ;473-477,1980. direct action of this compound independent of aromatase inhibi 15. Santen, R. J., Santner. S., Davis, B., Veldhuis,J., Samojlik, E., and Ruby, E. Aminoglutethimide inhibits extraglandular estrogen production in postmeno tion. The effect on LH secretion4 as well as on the uterus appears pausal women with breast carcinoma. J. Clin. Endocrino!.Metab., 47; 1257- to be due to weak androgenic activity (<1% testosterone) of 4- 1265, 1978. OHA (20) which may contribute to its efficacy in causing regres 16. Schwarzel, W. C., Kruggel, W., and Brodie, H. J. Studies of the mechanismof sion of DMBA-induced mammary tumors. Thus, 4-OHA by more estrogen biosynthesis. VIII. The development of inhibitors of the enzyme system in human placenta. Endocrinology,92: 866-880,1973. potent aromatase inhibition and gonadotropin suppression may 17. Segaloff, A., Weeth, J. B., Meyer, K. K., Rongone, E. L., and Cunningham,M. prevent new enzyme synthesis and follicular development by the E. G. Hormonaltherapy in cancer of the breast. XIX. Effect of oral administra ovary, resulting in a greater and sustained reduction in estradiol tion of A'-testololactone on the clinicalcourse and hormonalexcretion. Cancer (Phila.),75:633,1962. production than AG. This activity may be of importance in 18. Thompson, E. A., Jr., and Siiteli, P. K. Utilization of oxygen and reduced considering 4-OHA as treatment for patients with functional nicotinamideadeninedinucleotide phosphate by human placentalmicrosomes ovaries. However, in postmenopausal breast cancer patients, during aromatization of androstenedione. J. Bid. Chem., 249: 5364-5372, inhibition of gonadotropins would not be expected to be advan- 1974. 19. Thompson, E. A., Jr., and Siiteri, P. K. The involvement of human placental microsomal cytochrome P-450 in aromatization. J. Biol. Chem., 249: 5373- 5378, 1974. 20. Wing, L-Y., Tsai-Morris, C., and Brodie, A. M. H. The effect of aromatase 4L-Y. Wing, C. H. Tsai-Morris, M. Ghosh, and A. M. H. Brodie. The direct effect inhibitor 4-hydroxyandrostenedioneon sex steroid target tissue (abstract). In: of 4-hydroxyandrostenedione,an aromatase inhibitor, on gonadotropins in the rat, Quebec, Canada: Proceedings of the Seventh International Congress of En submitted for publication. docrinology, 1984.

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Downloaded from cancerres.aacrjournals.org on October 6, 2021. © 1985 American Association for Cancer Research. Effects of Aromatase Inhibitors, Aminoglutethimide, and 4-Hydroxyandrostenedione on Cyclic Rats and Rats with 7,12-Dimethylbenz(a)anthracene-induced Mammary Tumors

Lih-Yuh Wing, Wesley M. Garrett and Angela M. H. Brodie

Cancer Res 1985;45:2425-2428.

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