Aromatase in the Central Nervous System1

[CANCER RESEARCH (SUPPL.) 42, 3274s-3276s, August 1982] 0008-5472/82/0042-OOOOS02.00 Aromatase in the Central Nervous System1

F. Naftolin and Neil J. MacLusky'

Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut Oo'570

Abstract ies on placental aromatase. Evidence of further metabolism of the formed estrogen by the brain was also adduced in these Central (central nervous system and pituitary) aromatization early experiments, since the addition of large amounts of appears to be a fundamental process for endocrine control and unlabeled estrogen to the incubation (estrogen trap) resulted development. Metabolism of androgens to estrogens and the in the retention of more of the newly formed estrogen from the subsequent metabolism of estrogens have been proven in androgen substrate (21). Subsequent studies have shown this many species, including humans, and linked to estrogen action. metabolism of estrogens to include hydroxylation at positions Thus, aromatization appears to initiate or to be involved in 2, 4, or 16 and indicate possible sources of varying estrogen activities of importance to endocrine function at the central agonist-antagonist, neurotransmitter-effective, mutagenic com level and their effects peripherally. pounds (16). In the context of breast cancer, central aromatization relates These in vitro methods have been applied by us and others to the control of gonadotrophins and other pituitary-brain hor to confirm and localize central estrogen formation. Among the mones which may effect metabolism at the level of the breast. animals studied, adult and perinatal rats were shown to have For example, follicle-stimulating hormone can increase aro the ability to form estrogen, this evidence confirming the initial matization and may be a factor in the control of such metabo hypothesis regarding the basis of rodent sexual differentiation. lism in breast tissue. Direct in vivo evidence of this activity was furnished in rhesus monkeys during collaborative experiments with Professor R. Aromatization by Central Tissues White (21). Perfusion of the isolated monkey brain-pituitary Aromatization of androgens by CMS3 tissues (Chart 1) has with radiolabeled androgens resulted in the formation and localized retention of estrogen. Studies showing the selective been proven to exist in all species studied thus far (4, 13, 21 ). uptake of newly formed estrogen following administration of The levels of activity are relatively modest compared to tissues radiolabeled androgen to whole animals are also consistent classically known as estrogen-producing sources, such as with central aromatization in vivo (14, 27). placenta and ovary. However, the evidence for in vitro and in vivo CNS aromatization is firm and includes localization to Control of Central Aromatization areas of neuroendocrine activity, particularly the hypothalamus and limbic system. While the anterior pituitary gland has oc Experiments, largely in the adult rabbit, revealed that central casionally been shown to form measurable estrogen in vitro, aromatization is responsive to endocrine and, perhaps, to this is less consistent. It is now established that CNS aromatase constitutional factors. Activity is increased by castration and activity is found in the microsomal compartment, occurs in exposure to androgens and estrogen. Progestins diminish aro areas known to contain estrogen receptors, and responds to matization by CNS tissues. Central tissues from males regularly sex steroids and other endocrine changes. Coupled with evi have greater aromatization activity than do identical tissues dence of other CNS steroidal and nonsteroidal hormone pro from females (16). Callard ef al. (4) showed an increase in in duction, these findings contribute to our acceptance of the vitro aromatization by turtle CNS with time of incubation. Canick hypothalamus and its linked limbic structures as endocrine et al. (5) and Vaccaro ef al. (26) showed diminution of aroma tissues. The proof of CNS aromatization rests upon 3 types of tase activity by cultured hypothalamic neurons under the influ evidence: direct in vitro studies; direct in vivo studies; and ence of norepinephrine and isoproterenol. The progestational indirect in vivo studies. These data have been reviewed previ antiandrogen cyproterone acetate when given prior to dissec ously, and we will only touch upon details used to tie the tion curtails in vitro CNS aromatization (14). No evidence of biochemical findings to some of the effects of central (CNS and product inhibition of CNS aromatization by estrogens has been pituitary) aromatization. reported. Several groups have shown a change in aromatiza Early animal studies led us to the conclusion that one well- tion during the pre-postnatal period (8, 21 ); however, the cause known "androgen action," sexual differentiation of the rodent of these age-related changes is unknown. The anatomic loca brain (10, 17), required local estrogen formation (22). This tion of CNS aromatase has been beautifully mapped by mi- "aromatization hypothesis" led to direct in vitro testing of crodissection techniques (24). human fetal CNS tissues for their ability to aromatize andro gens. Positive results were obtained using the method that Kenneth Ryan's laboratory had developed previously for stud- Implications of Central Aromatization

' Presented at the Conference "Aromatase: New Perspectives for Breast Central aromatization forms a critical link between biochem Cancer." December 6 to 9, 1981, Key Biscayne, Fla. This work was supported ical and anatomic aspects of endocrine regulation. The effects by NIH Grant HD13587. National Institute of Child Health and Human Develop of this in situ steroidogenesis are local, but they are amplified ment. 2 Fellow of the Alfred P. Sloan Foundation. by subsequent changes in control of the target organs of the 3 The abbreviations used are: CNS. central nervous system; ERN, cell nuclear pituitary and of the brain itself. Estrogens (and androgens, estrogen receptor: PRC. cylosol progestin receptor; LH. luteinizing hormone. probably acting via central aromatization) are important in the

3274s CANCER RESEARCH VOL. 42

Proven Hypolhalamic MetabolismRing A of Steroids one can uncouple the feedback effect of testosterone on gonadotrophins by treatment with the estrogen antagonist clomi-

Reduction Aromatizaron Hydroxylation phene (20). Changes in available estrogen affect gonadotro phin release, and diminished estrogen, whether due to failure C21 CorticoÃds Progesims - of direct gonadal secretion or of available androgen for periph eral conversion, will result in elevated circulating LH and folli cle-stimulating hormone. One of the effects of unopposed

Prog.llmsl gonadotrophin secretion might be an increase in peripheral aromatization (6, 9). Breast tissue can aromatize androgens (7, Androgens-Â». 23), and might respond to this stimulation. The impact of local formation of estrogen by breast tissue has not been assessed; however, estrogens induce local peroxidase formation in uterus (12) and the catechol estrogen-metabolizing enzyme catechol O-methyltransferase is found in high concentration in the breast Estrogens (11). As well, estrogens and their metabolic products, the catechol estrogens, modulate prolactin secretion (16). While no direct relationship between prolactin and breast neoplasia has been shown, the effects of estrogen and prolactin in Chart 1. A scheme of In vivo and in vitro metabolism of steroid substrates ( far left) by neuroendocrine tissue. Reproduced with permission from Naftolin (18). stimulating breast growth and functions are undisputed and cannot be overlooked in any such considerations. growth of neuntes and synaptic circuitry and in the develop ment of neuroendocrine controls. These latter include the Summary timing of sexual maturation, control of sexual behavior, and gonadotrophin regulation (2, 19). Estrogen exposure may also Central aromatization has been proven in many species and play a role in aging phenomena in the rodent brain (3). Gen is present throughout life. It appears to be a fundamental erally, these effects result from the administration of aromatiz- process, the importance of which in determining growth and able androgens or estrogens, and, while a role for androgens development of central and peripheral tissues outweighs the per se has not been ruled out, there is clear evidence that relatively small quantitative capacity of this system for steroid aromatase blockers, antisera to estrogens, and nonsteroidal metabolism. Central aromatization also furnishes precursor estrogen antagonists can block these activities, while antian- estrogen for subsequent formation of catechol estrogens, some drogens have minimal or no effect (1, 15, 20). Moreover, of which are active biological substances. The result of this in nonandrogens such as exogenously administered nonsteroidal situ metabolism of androgens includes intracellular changes in estrogen agonists and catechol estrogens can produce such estrogen and progestin receptor status and may be the basis effects as sexual differentiation of the rodent brain (17). for such endocrine actions as gonadotrophin and prolactin Recently, Krey ef al. (14, 15) have explored the effects of regulation. Thus, the linkage between central aromatization testosterone on gonadotrophin control and sex behavior in the and the biology of the breast may be important for events rat by injecting testosterone and following changes in the related to abnormal as well as normal breast function. hypothalamic content of cell nuclear estradiol, cell ERN, and PRC. When this was done in castrated androgen-insensitive Acknowledgments male rats, it was seen that testosterone administration in creased hypothalamic nuclear estradiol and both ERN and We appreciate the assistance of Jacki Fitzpatrick in the preparation of this PRC levels while inducing lordosis behavior and cyclic LH manuscript. release. After injection of [3H]testosterone, approximately 70% of recoverable nuclear radiolabeled steroid was newly formed References estradiol. Krey ef al. have also used the aromatase inhibitor 1. Beyer. C., Morali, G., Naftolin, F., Larsson. K., and Perez-Palacios. G. Effect androstatrienedione and the nonsteroidal androgen antagonist of some antiestrogens and aromatase inhibitors on androgen induced sexual flutamide in assessing the mechanism of administered testos behavior in castrated male rats. Horm. Behav., 7: 353-363, 1976. terone in causing lordosis and cyclic LH release in castrated 2. Brawer, J. R., Naftolin, F.. Martin, J., and Sonnenschein, C. Effects of a single injection of estradiol valerate on the hypothalamic arcuate nucleus normal female rats. They found that the actions of testosterone and on reproductive function in the female rat. Endocrinology, 103: 501- could be blocked by androstatrienedione but not by flutamide 512, 1978. 3. Brawer, J. R., Schnipper, H., and Naftolin, F. Ovary-dependent degeneration and that induced lordosis behavior and cyclic LH release were in the hypothalamic arcuate nucleus. Endocrinology, 707. 274-279, 1980. accompanied by increased ERN and PRC. This work in cas 4. Callard, G. V., Petro, Z., and Ryan, K. J. Conversion of androgen to estrogen trated rats has been mentioned to show some of the in vivo and other steroids in the vertebrate brain. Am. Zool., )8: 511-523, 1978. aspects of extragonadal aromatization. Failure of all investiga 5. Canick, J. A., Vaccaro, D. E., Ryan, K. J., and Leeman, S. E. The aromati zation of androgens by primary monolayer cultures of fetal rat hypothalamus. tors thus far to prove the existence of measurable aromatization Endocrinology, 700. 250-253, 1977. by rat fat plus nuclear localization of estradiol in the hypothal 6. Frieden, E. H., Patkin, J. K., and Mills, M. Effects of follicle stimulating amus of Krey's experimental animals indicates that this model hormone (FSH) upon steroid aromatization in vitro. Proc. Soc. Exp. Biol. Med., 729. 606-609, 1968. may bear close relevance to events occurring as a result of 7. Gallegos, A. J., and Canales, E. S. In vitro estrogen formation by human central aromatization. mammary gland. In: Endocrine Society, 51st annual meeting, abstract 99, 1969. Estrogens formed in the brain or elsewhere are important in 8. George, F. W., Tobleman, W. T., Milewich, L.. and Wilson, J. D. Aromatase gonadotrophin homeostasis. Evidence in humans indicates that activity in the developing rabbit brain. Endocrinology, i 02. 86-91, 1978.

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9. Grodin, J. M.. Siiteri, P. K., and MacDonald. P. C. Source of estrogen 13 Naftolin, F. Metabolism of steroids in the brain. In: V. H. T. James (ed.). production in post-menopausal women. J. Clin. Endocrinol. Metab . 36: Proceedings of the Fifth International Congress of Endocrinology; pp. 29- 207, 1973. 33. Amsterdam: Excerpta Medica, 1976. 10. Harris, G. W., and Naftolin. F. The hypothalamus and control of ovulation. 19 Naftolin, F., and Brawer, J. R. The effect of estrogens on hypothalamic Br Med. Bull. 26. 3-9, 1970. structure and function. Am. J. Obstet. Gynecol., 132: 758-765, 1978. 11. Inoue, K., Tice. L. W., and CrevelÃ¬ng.C. R. Immunocytochemical localization 20 Naftolin, F., Judd, H. L., and Yen, S. S. C. Pulsatile patterns of gonadotro- of catechol-o-methyltransferase. In: E. Usdin, N. Weiner, and M. B. Youdin phins and testosterone in man: the effects of clomiphene with and without (eds.). Structure and Function of Monoamine Enzymes. Modern Pharmacol testosterone. J. Clin. Endocrinol. Metab., 36. 285-288, 1973. ogy-Toxicology. Vol. 10. pp. 835-893. New York: Marcel Dekker. Inc. 21. Naftolin, F., Ryan, K. J., Davies. I. J., Reddy. V. V.. Flores, F.. Petro. Z., 12. Jellinck, P. H., and Newcombe, A. M. Effect of catechol estrogens and KÃ¼hn,M., White, R. J., Takaoka, Y., and Wolin, L. The formation of estrogens estriol on the induction of uterine peroxidase. J. Steroid Biochem., 13: 681 - by central neuroendocrine tissues. Recent Prog. Horm. Res.. 31: 295-319, 683. 1980. 1975. 13. Knapstein, P., David, A., Wu, C-H.. Archer, D., Flickinger, G. F., and 22 Naftolin, F., Ryan. K. J., and Petro, Z. Aromatizaron of androstenedione by Touchstone, J. C. Metabolism of free and sulfoconjugated DHEA in brain the diencephalon. J. Clin. Endocrinol. Metab., 33. 368-370. 1971. tissue in vivo and in vitro. Steroids, 11: 885-896, 1968. 23 Nimrod. A., and Ryan. K. J. Aromatization of androgens by human abdominal 14. Krey, L. C., Ueberberg. I.. MacLusky, N. J., Davis, P. G., and Robbins, R. and breast fat tissue. J. Clin. Endocrinol. Metab., 40: 367-372, 1975. Testosterone increases cell nuclear estrogen receptor levels in the brain of 24 Selmanoff, M. K., Brodkin, L. D., Weiner, R. I., and Siiteri, P. K. Aromatization the Stanley-Gumbreck pseudohermaphrodite male rat: implication for tes and Sa-reduction of androgens in discrete hypothalamic and limbic regions tosterone modulation of neuroendocrine activity. Endocrinology, 110: in of the male and female rat. Endocrinology. 101: 841-848, 1977. press, 1982. 25. Steimer, T., and Hutchison, J. B. Aromatization of testosterone within a 15. Krey, L. C., MacLusky, N. J., Davis, P. G.. Lieberberg. I., and Roy, E. J. discrete hypothalamic area associated with the behavioral action of andro- Different intracellular mechanisms underlie testosterone's suppression of gen in the male dove. Brain Res.. )92: 586-591, 1980. basal and stimulation of cyclic luteinizing hormone release in male and 26 Vaccaro. D. E., Canick, J. A., Livingston, E. G.. Fox, T. O., Ryan, K. J., and female rats. Endocrinology. 110: in press, 1982. Leeman, S. E. Possible effectors of aromatizaron and 5a-reduction in 16. MacLusky. N. J., Clark, C. R.. Paden, C. M., and Naftolin, F. End-organ hypothalamic cell cultures. In: Endocrine Society, 62nd Annual Meeting, metabolism of oestrogens. In: G. P. Lewis and M. Ginsburg (eds.). Mecha Abstract 127, 1980. nisms of Steroid Action, pp. 115-132. Macmillan, London, 1981. 27 Weisz, J., and Gibbs. C. Conversion of testosterone and androstenedione to 17. MacLusky, N. J.. and Naftolin, F. Sexual differentiation of the central nervous estrogens in vitro by the brain of female rats. Endocrinology, 94: 616-620, system. Science (Wash. D. C.), 211: 1294-1303, 1981. 1974.

3276s CANCER RESEARCH VOL. 42

F. Naftolin and Neil J. MacLusky

Cancer Res 1982;42:3274s-3276s.

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