Intracerebral estrogen provision increases cytogenesis and neurogenesis in the injured zebra finch brain

To determine whether or not local, injury‐induced aromatization and/or estrogen provision can affect cyto‐ or neuro‐genesis following mechanical brain damage, two groups of adult male zebra finches sustained bilateral penetrating brain injuries. The first received contralateral injections of vehicle or the aromatase inhibitor fadrozole. The second group received contalateral injections of fadrozole, or fadrozole with 17β‐estradiol. Subsequent to injury, birds were injected with the thymidine analog 5‐bromo‐2′‐deoxyuridine (BrdU). Two weeks following injury, the birds were perfused, and coronal sections were labeled using antibodies against BrdU and the neuronal proteins HuC/HuD. In a double blind fashion, BrdU positive cells and BrdU/Hu double‐labeled cells in the subventricular zone (SVZ) and at the injury site (INJ) were imaged and sampled. The average numbers of cells per image were compared across brain regions and treatments using repeated measures ANOVAs and, where applicable, post‐hoc, pairwise comparisons. Fadrozole administration had no detectable effect on cytogenesis or neurogenesis, however, fadrozole coupled with estradiol significantly increased both measures. The dorsal SVZ had the greatest proportion of new cells that differentiated into neurons, though the highest numbers of BrdU labeled and BrdU, Hu double‐labeled cells were detected at the INJ. In the adult zebra finch brain, local estradiol provision can increase cytogenesis and neurogenesis, however, whether or not endogenous glial aromatization is sufficient to similarly affect these processes remains to be seen. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 71: 170‐181, 2011     

Aromatase expression and cell proliferation following injury of the adult zebra finch hippocampus

Estrogens can be neuroprotective following traumatic brain injury. Immediately after trauma to the zebra finch hippocampus, the estrogen-synthetic enzyme aromatase is rapidly upregulated in astrocytes and radial glia around the lesion site. Brain injury also induces high levels of cell proliferation. Estrogens promote neuronal differentiation, migration, and survival naturally in the avian brain. We suspect that glia are a source of estrogens promoting cell proliferation after neural injury. To explore this hypothesis, we examined the spatial and temporal relationship between glial aromatase expression and cell proliferation after neural injury in adult female zebra finches. Birds were ovariectomized and given a blank implant or one filled with estradiol; some birds were also administered an aromatase inhibitor or vehicle. All birds received penetrating injuries to the right hippocampus. Twenty-four hours after lesioning, birds were injected once with BrdU to label mitotically active cells and euthanized 2 h, 24 h, or 7 days later. The brains were processed for double-label BrdU and aromatase immunocytochemistry. Injury-induced glial aromatase expression was unaffected by survival time and aromatase inhibition. BrdU labeling was significantly reduced at 24 h by ovariectomy and by aromatase inhibition; effects were partially reversed by E2 replacement. Irrespective of ovariectomy, the densities of aromatase immunoreactive astrocytes and BrdU-labeled cells at known distances from the lesion site were highly correlated. These data suggest that injury-induced glial aromatization may influence the reorganization of injured tissue by providing a rich estrogenic environment available to influence cellular incorporation.     

Mechanisms of the actions of aromatase inhibitors 4-hydroxyandrostenedione, fadrozole, and aminoglutethimide on aromatase in JEG-3 cell culture

Selective inhibition of estrogen production with aromatase inhibitors has been found to be an effective strategy for breast cancer treatment. Most studies have focused on inhibitor screening and in vitro kinetic analysis of aromatase inhibition using placental microsomes. In order to determine the effects of different inhibitors on aromatase in the whole cell, we have utilized the human choriocarcinoma cell line, JEG-3 in culture to compare and study three classes of aromatase inhibitors, 4-hydroxyandrostenedione, fadrozole (CGS 16949A), and aminoglutethimide. Fadrozole is the most potent competitive inhibitor and aminoglutethimide is the least potent among the three. However, stimulation of aromatase activity was found to occur when JEG-3 cells were preincubated with aminoglutethimide. In contrast, 4-OHA and fadrozole caused sustained inhibition of aromatase activity in both JEG-3 cells and placental microsomes, which was not reversed even after the removal of the inhibitors. 4-OHA bound irreversibly to the active site of aromatase and caused inactivation of the enzyme which followed pseudo-first order kinetics. However, 4-OHA appears to be metabolized rapidly in JEG-3 cells. Sustained inhibition of aromatase induced by fadrozole occurs by a different mechanism. Although fadrozole bound tightly to aromatase at a site distinct from the steroid binding site, the inhibition of aromatase activity by fadrozole does not involve a reactive process. None of the inhibitors stimulated aromatase mRNA synthesis in JEG-3 cells during 8 h treatment. The stimulation of aromatase activity by AG appeared to be due to stabilization of aromatase protein. According to these results, 4-OHA and fadrozole would be expected to be more beneficial in the treatment of breast cancer patients than AG. The increase in aromatase activity by AG may counteract its therapeutic effect and might be partially responsible for relapse of breast cancer patients from this treatment.     

Brain aromatase is neuroprotective

The expression of aromatase, the enzyme that catalyzes the biosynthesis of estrogens from precursor androgens, is increased in the brain after injury, suggesting that aromatase may be involved in neuroprotection. In the present study, the effect of inactivating aromatase has been assessed in a model of neurodegeneration induced by the systemic administration of neurotoxins. Domoic acid, at a dose that is not neurotoxic in intact male mice, induced significant neuronal loss in the hilus of the hippocampal formation of mice with reduced levels of aromatase substrates as a result of gonadectomy. Furthermore, the aromatase substrate testosterone, as well as its metabolite estradiol, the product of aromatase, were able to protect hilar neurons from domoic acid. In contrast, dihydrotestosterone, the 5 alpha-reduced metabolite of testosterone and a nonaromatizable androgen, was not. These findings suggest that aromatization of testosterone to estradiol may be involved in the neuroprotective action of testosterone in this experimental model. In addition, aromatase knock-out mice showed significant neuronal loss after injection of a low dose of domoic acid, while control littermates did not, indicating that aromatase deficiency increases the vulnerability of hilar neurons to neurotoxic degeneration. The effect of aromatase on neuroprotection was also tested in male rats treated systemically with the specific aromatase inhibitor fadrozole and injected with kainic acid, a well characterized neurotoxin for hilar neurons in the rat. Fadrozole enhanced the neurodegenerative effect of kainic acid in intact male rats and this effect was counterbalanced by the administration of estradiol. Furthermore, the neuroprotective effect of testosterone against kainic acid in castrated male rats was blocked by fadrozole. These findings suggest that neuroprotection by aromatase is due to the formation of estradiol from its precursor testosterone. Finally, a role for local cerebral aromatase in neuroprotection is indicated by the fact that intracerebral administration of fadrozole enhanced kainic acid induced neurodegeneration in the hippocampus of intact male rats. These findings indicate that aromatase deficiency decreases the threshold for neurodegeneration and that local cerebral aromatase is neuroprotective. Brain aromatase may therefore represent a new target for therapeutic approaches to neurodegenerative diseases.     

Aromatase-independent testosterone conversion into estrogenic steroids is inhibited by a 5 alpha-reductase inhibitor

Estrogens are generated mainly by the action of aromatase, which converts testosterone to estradiol and androstenedione to estrone. However, in addition to estradiol and estrone, a variety of other steroids, whose synthesis is not dependent on aromatase, can stimulate the estrogen receptor. Here we show that testosterone is converted into such estrogenic steroids by aromatase-negative HeLa cells. This aromatase-independent generation of estrogenic steroids is seen in aromatase-positive MCF-7 cells as well. In both cell lines, the synthesis of estrogenic steroids was blocked by inhibition of testosterone conversion into dihydrotestosterone using a 5 alpha-reductase inhibitor finasteride, suggesting that they are generated downstream of dihydrotestosterone. This finding raises the possibility that the combination of a 5 alpha-reductase inhibitor and an aromatase inhibitor may reduce estrogenic steroids in vivo more completely than an aromatase inhibitor alone.     

The importance of local synthesis of estrogen within the breast

Tumor aromatase has been correlated with clinical response to treatment with aminoglutethimide in patients with estrogen receptor-positive advanced breast cancer. There was a significant positive relationship between aromatase status and likelihood of response to therapy, none of five patients with aromatase-negative tumors responding compared with 11 of 18 having aromatase-positive cancers. Measurement of aromatase in sequential biopsies of large primary tumors before and during treatment with aminoglutethimide-hydrocortisone showed a marked but paradoxical rise in activity following therapy. Assays of aromatase in adipose tissue from the different quadrants of mastectomy specimens from patients with breast cancer indicate that activity was always higher in quadrants associated with tumor as compared with non-involved quadrants. These results emphasize the importance of local estrogen synthesis within the breast in terms of both the natural history and behavior of breast cancers.     

Role of astroglia in estrogen regulation of synaptic plasticity and brain repair.

Astroglia are targets for estrogen and testosterone and are apparently involved in the action of sex steroids on the brain. Sex hormones induce changes in the expression of glial fibrillary acidic protein, the growth of astrocytic processes, and the degree of apposition of astroglial processes to neuronal membranes in the rat hypothalamus. These changes are linked to modifications in the number of synaptic inputs to hypothalamic neurons. These findings suggest that astrocytes may participate in the genesis of androgen-induced sex differences in synaptic connectivity and in estrogen-induced synaptic plasticity in the adult brain. Astrocytes and tanycytes may also participate in the cellular effects of sex steroids by releasing neuroactive substances and by regulating the local accumulation of specific growth factors, such as insulin-like growth factor-I, that are involved in estrogen-induced synaptic plasticity and estrogen-mediated neuroendocrine control. Astroglia may also be involved in regenerative and neuroprotective effects of sex steroids, since astroglia formation after brain injury or after peripheral nerve axotomy is regulated by sex hormones. Furthermore, the expression of aromatase, the enzyme that produces estrogen, is induced de novo in astrocytes in lesioned brain areas of adult male and female rodents. Since astroglia do not express aromatase under normal circumstances, the induction of this enzyme may be part of the program of glial activation to cope with the new conditions of the neural tissue after injury. Given the neuroprotective and growth-promoting effects of estrogen after injury, the local production of this steroid may be a relevant component of the reparative process.     

Acute and chronic effects of an aromatase inhibitor on territorial aggression in breeding and nonbreeding male song sparrows

Many studies have demonstrated that male aggression is regulated by testosterone. The conversion of testosterone to estradiol by brain aromatase is also known to regulate male aggression in the breeding season. Male song sparrows (Melospiza melodia morphna) are territorial not only in the breeding season, but also in the nonbreeding season, when plasma testosterone and estradiol levels are basal. Castration has no effect on nonbreeding aggression. In contrast, chronic (10 day) aromatase inhibitor (fadrozole) treatment decreases nonbreeding aggression, indicating a role for estrogens. Here, we show that acute (1 day) fadrozole treatment decreases nonbreeding territoriality, suggesting relatively rapid estrogen effects. In spring, fadrozole decreases brain aromatase activity, but acute and chronic fadrozole treatments do not significantly decrease aggression, although trends for some behaviors approach significance. In gonadally intact birds, fadrozole may be less effective at reducing aggression in the spring. This might occur because fadrozole causes a large increase in plasma testosterone in intact breeding males. Alternatively, estradiol may be more important for territoriality in winter than spring. We hypothesize that sex steroids regulate male aggression in spring and winter, but the endocrine mechanisms vary seasonally.     

Intracellular aromatase and its relevance to the pharmacological efficacy of aromatase inhibitors

An important feature of the pharmacological profile of aromatase inhibitors is the ability of the various inhibitors to inhibit intracellular aromatase. It is now well documented that a large proportion of breast tumors express their own aromatase. This intratumoral aromatase produces estrogen in situ and therefore may contribute significantly to the amount of estrogen to which the cell is exposed. Thus it is not only important that aromatase inhibitors potently inhibit the peripheral production of estrogen and eliminate the external supply of estrogen to the tumor cell, but that they in addition potently inhibit intratumoral aromatase and prevent the tumor cell from making its own estrogen within the cell. To study the inhibition of intracellular aromatase we have compared the aromatase-inhibiting potency of the non-steroidal aromatase inhibitors, letrozole, anastrozole and fadrozole in a variety of model cellular endocrine and tumor systems which contain aromatase. We have used hamsters ovarian tissue fragments, adipose tissue fibroblasts from normal human breast, the MCF-7Ca human breast cancer cell line transfected with the human aromatase gene and the JEG-3 human choriocarcinoma cell line. Although letrozole and anastrozole are approximately equipotent in a cell-free aromatase system (human placental microsomes), letrozole is consistently 10–30 times more potent than anastrozole in inhibiting intracellular aromatase in intact rodent cells, normal human adipose fibroblasts and human cancer cell lines. Whether these differences between letrozole and anastrozole are seen in the clinical setting will have to await the results of clinical trials which are currently in progress.     

Prognostic significance of aromatase and estrone sulfatase enzymes in human breast cancer

The aromatase and estrone sulfatase enzymes are important sources of local synthesis of biologically active estrogens in human breast cancer. Significant intratumoral aromatase activity was detected in 91/145 (63%) of tumors and estrone sulfatase was detected in 93/104 (89%) of tumors. There was no relationship between aromatase activity and tumor size, site, nodal status, menopousal status or estrogen receptor status. There was a significant correlation between the aromatase activity and histological grade, with an excess of aromatase-positive in the high grade tumors (P = 0.03). There was a marginally inverse correlation between the aromatase activity and time to relapse (P < 0.1), a significant correlation between aromatase activity and survival after relapse (P < 0.05) but not with overall survival (P < 0.1). Intratumoral estrone sulfatase activity was not significantly correlated to any putative prognostic factors, nor with time to relapse nor overall survival time.     

Aromatase inhibitor and 17alpha-methyltestosterone cause sex-reversal from genetical females to phenotypic males and suppression of P450 aromatase gene expression in Japanese flounder (Paralichthys olivaceus)

The sex of Japanese flounder (Paralichthys olivaceus) is easily altered by water temperature or sex steroid hormone treatment during the period of sex determination. We have previously shown that rearing the genetically female larvae at high water temperature caused the suppression of P450 aromatase (P450arom) gene expression in the gonad and phenotypic sex-reversal of the individuals to males (Kitano et al. 1999. J Mol Endocrinol 23:167-176). In the present study, we show that treatment of genetically female larvae with fadrozole (aromatase inhibitor) or 17alpha-methyltestosterone induces sex-reversal as well as suppression of P450arom gene expression. The effect of fadrozole was counteracted by co-administration of estradiol-17beta. Effective periods for fadrozole treatment to induce sex-reversal were similar to those for high water temperature treatment. RT-PCR did not detect P450arom mRNA in gonad of the sex-reversed, phenotypic males. These results indicate that sex-reversal of the genetically female larvae by aromatase inhibitor (or 17alpha-methyltestosterone) may be due to the suppression of P450arom gene expression and the resultant decrease in the amount of estrogen.     

Estrogen mediation of injury-induced cell birth in neuroproliferative regions of the adult zebra finch brain

Estrogens influence neuronal differentiation, migration, and survival in intact brains. In injured brains, estrogens can also be neuroprotective. In Experiment 1, following a unilateral penetrating injury to the hippocampus (HP), adult female zebra finches were injected once with BrdU to label mitotic cells then sacrificed 2 h, 1 day, or 7 days postinjection. Cell proliferation was dramatically enhanced in the ipsilateral HP, as well as in neuroproliferative areas including the subventricular zone (SVZ) proximal to the injury. This increase was seen at all time points investigated. Ovariectomy (OVX) substantially suppressed proliferation bilaterally especially in the SVZ indicating that gonadal hormones influenced cell proliferation in both the intact and injured hemisphere. To determine if estrogens were directly involved, estrogen was depleted in Experiment 2 through either OVX or administration of the aromatase inhibitor fadrozole (FAD). Birds were implanted with estradiol or blank followed 2 weeks later by a unilateral penetrating lesion to the HP. Injury-induced substantial proliferation, which was again significantly suppressed bilaterally in both OVX and FAD birds. Estrogen replacement reversed this effect in FAD but not OVX birds therefore the suppression following OVX may be due in part to nonestrogenic influences. Suppression of cell birth in FAD birds was indeed due to the removal of endogenous sources of estrogen. Results therefore indicate that estrogens are directly involved in the brain's response to injury and may be acting to provide a rich environment for the production and perhaps protection of new cells.     

Effect of androstenedione on growth of untransfected and aromatase-transfected MCF-7 cells in culture

Aromatase is present in human breast tumors and in breast cancer cell lines suggesting the possibility of in-situ estrogen production via the androstenedione to estrone and estradiol pathway. However, proof of the biologic relevance of aromatase in breast cancer tissue requires the demonstration that this enzyme mediates biologic effects on cell proliferation. Accordingly, we studied the effects of the aromatase substrate, androstenedione, on the rate of proliferation of wild-type and aromatase-transfected MCF-7 breast cancer cells. Androstenedione did not increase cell growth in wild-type MCF-7 cells which contained relatively low aromatase activity and produced 4-fold more estrone than estradiol. In contrast, aromatase-transfected cell contained higher amounts of aromatase, produced predominantly estradiol, and responded to androstenedione with enhanced growth. An aromatase inhibitor fadrozole hydrochloride, blocked the proliferative effects of androstenedione providing evidence for the role of aromatase in this process. As further evidence of the requirement for aromatase, cells transfected with the neomycin resistance expression plasmid but lacking the aromatase cDNA did not respond to androstenedione. These studies provide evidence that aromatase may have a biologic role for in-situ synthesis of estrogens of breast cancer tissue.     

Altered gene expression in the brain and liver of female fathead minnows Pimephales promelas Rafinesque exposed to fadrozole

The fathead minnow Pimephales promelas is a small fish species widely used for ecotoxicology research and regulatory testing in North America. This study used a 2000 gene oligonucleotide microarray to evaluate the effects of the aromatase inhibitor, fadrozole, on gene expression in the liver and brain tissue of exposed females. Reproductive measures, plasma vitellogenin and gene expression data for the brain isoform of aromatase (cytP19B), vitellogenin precursors and transferrin provided evidence supporting the efficacy of the fadrozole exposure. Unsupervised analysis of the microarray results identified 20 genes in brain and 41 in liver as significantly up-regulated and seven genes in brain and around 45 in liver as significantly down-regulated. Differentially expressed genes were associated with a broad spectrum of biological functions, many with no obvious relationship to aromatase inhibition. However, in brain, fadrozole exposure elicited significant up-regulation of several genes involved in the cholesterol synthesis, suggesting it as a potentially affected pathway. Gene ontology-based analysis of expression changes in liver suggested overall down-regulation of protein biosynthesis. While real-time polymerase chain reaction analyses supported some of the microarray responses, others could not be verified. Overall, results of this study provide a foundation for developing novel hypotheses regarding the system-wide effects of fadrozole, and other chemical stressors with similar modes of action, on fish biology.     

Characterization of a cis-acting element involved in cell-specific expression of the zebrafish brain aromatase gene

The cytochrome P450 Aromatase is the key enzyme catalyzing the conversion of androgens into estrogens. In zebrafish, the brain aromatase is encoded by cyp19b. Expression of cyp19b is restricted to radial glial cells bordering forebrain ventricles and is strongly stimulated by estrogens during development. At the promoter level, we have previously shown that an estrogen responsive element (ERE) is required for induction by estrogens. Here, we investigated the role of ERE flanking regions in the control of cell-specific expression. First, we show that a 20 bp length motif, named G x RE (glial x responsive element), acts in synergy with the ERE to mediate the estrogenic induction specifically in glial cells. Second, we demonstrate that, in vitro, this sequence binds factors exclusively present in glial or neuro-glial cells and is able to confer a glial specificity to an artificial estrogen-dependent gene. Taken together, these results contribute to the understanding of the molecular mechanisms allowing cyp19b regulation by estrogens and allowed to identify a promoter sequence involved in the strong estrogen inducibility of cyp19b which is specific for glial cells. The exceptional aromatase activity measured in the brain of teleost fish could rely on such mechanisms.     

Extracellular matrix protein SC1/hevin in the hippocampus following pilocarpine-induced status epilepticus

Pilocarpine-induced status epilepticus (SE) mimics many features of temporal lobe epilepsy and is a useful model to study neural changes that result from prolonged seizure activity. In this study, distribution of the anti-adhesive extracellular matrix protein SC1 was examined in the rat hippocampus following SE. Western blotting showed decreased levels of SC1 protein in the week following SE. Immunohistochemistry demonstrated that the decrease in overall SC1 protein levels was reflected by a reduction of SC1 signal in granule cells of the dentate gyrus. Interestingly, levels of SC1 protein in neurons of the seizure-resistant CA2 sector of the hippocampus did not change throughout the seizure time course. However, at 1 day post-SE, a subset of neurons of the hippocampal CA1, CA3, and hilar regions, which are noted for extensive neuronal degeneration after SE, exhibited a transient increase in SC1 signal. Neurons exhibiting enhanced SC1 signal were not detected at 7 days post-SE. The cellular stress response was also examined. A prominent induction of heat-shock protein (Hsp70) and Hsp27 was detected following SE, while levels of constitutively expressed Hsp40, Hsp90, Hsp110, and Hsc70 showed little change at the time points examined. The subset of neurons that demonstrated a transient increase in SC1 colocalized with the cellular stress marker Hsp70, the degeneration marker Fluoro-Jade B, and the neuron activity marker activity-regulated cytoskeleton-associated protein (Arc). Taken together, these findings suggest that SC1 may be a component of the 'matrix response' involved in remodeling events associated with neuronal degeneration following neural injury.     

Ependyma as a Possible Morphological Basis of Ischemic Preconditioning Tolerance in Rat Spinal Cord Ischemia Model: Nestin and Fluoro-Jade B Observations

1. To test our hypothesis that a transient nonlethal ischemic insult benefits the lumbosacral spinal cord ischemic injury, nestin, the marker of proliferating cells, and Fluoro-Jade B, the marker of degenerating cells, were used in rats. Morphological outcome was evaluated after 12-min ischemia versus 12-min ischemia preconditioned by 3-min ischemic period and 30-min recirculation (IPC), in each group followed by 2, 3, and 4 days of posttreatment survival. 2. Twelve-minute ischemia, inducing nestin-positivity in ependyma and reactive astrocytes at the L(1-3) spinal cord segments, shows this region as the viable region of spinal cord in all postischemic survival periods. On the other hand, abundance of Fluoro-Jade B-positive cells, distributed throughout the dorsal horn and intermediate zone of L4-S2 segments, points out the most injured spinal cord region by ischemia. 3. After the same ischemic insult in IPC rats only a few nestin-positive ependymal cell and reactive astrocytes appeared beside the nestin-positive vessels in the lower lumbar and sacral spinal cord segments of all survival periods. The appearance of nestin-positive cells in the spinal cord segments, which "should have been affected" by ischemia indicates protection of this region by the IPC treatment. 4. The number and density evaluation of Fluoro-Jade B fluorescent cells of L4-S2 segments after ischemia and IPC confirmed that degenerating cells were significantly reduced in the IPC rats in all survival periods. 5. Our results showing the immunohistochemical response of epemdyma, committed to the presence of viable tissue, indicate that the ependymal cells may contribute to the ischemic resistance in the IPC rats.     

Susceptibility of Human Embryonic Stem Cell-Derived Neural Cells to Japanese Encephalitis Virus Infection

Pluripotent human embryonic stem cells (hESCs) can be efficiently directed to become immature neuroepithelial precursor cells (NPCs) and functional mature neural cells, including neurotransmitter-secreting neurons and glial cells. Investigating the susceptibility of these hESCs-derived neural cells to neurotrophic viruses, such as Japanese encephalitis virus (JEV), provides insight into the viral cell tropism in the infected human brain. We demonstrate that hESC-derived NPCs are highly vulnerable to JEV infection at a low multiplicity of infection (MOI). In addition, glial fibrillary acid protein (GFAP)-expressing glial cells are also susceptible to JEV infection. In contrast, only a few mature neurons were infected at MOI 10 or higher on the third day post-infection. In addition, functional neurotransmitter-secreting neurons are also resistant to JEV infection at high MOI. Moreover, we discover that vimentin intermediate filament, reported as a putative neurovirulent JEV receptor, is highly expressed in NPCs and glial cells, but not mature neurons. These results indicate that the expression of vimentin in neural cells correlates to the cell tropism of JEV. Finally, we further demonstrate that membranous vimentin is necessary for the susceptibility of hESC-derived NPCs to JEV infection.     

17alpha-methyl testosterone is a competitive inhibitor of aromatase activity in Jar choriocarcinoma cells and macrophage-like THP-1 cells in culture

17alpha-methyl testosterone is a synthetic androgen with affinity for the androgen receptor. 17alpha-methyl testosterone is used widely as a component of hormone replacement therapy. Previous reports have indicated that contrary to testosterone, 17alpha-methyl testosterone is not aromatized. However, 17alpha-methyl testosterone still could affect local estrogen formation by regulating aromatase expression or by inhibiting aromatase action. Both possibilities have important clinical implications. To evaluate the effect of 17alpha-methyl testosterone on the expression and activity of aromatase, we tested the choriocarcinoma Jar cell line, a cell line that express high levels of P450 aromatase, and the macrophage-like THP-1 cells, which express aromatase only after undergoing differentiation. We found that in both cell lines, 17alpha-methyl testosterone inhibits aromatase activity in a dose-related manner. The curve of inhibition parallels that of letrozole and gives complete inhibition at 10(-4) M 17alpha-methyl testosterone, determined by the tritium release assay. 17alpha-methyl testosterone does not have detectable effects on aromatase RNA and protein expression by Jar cells. Undifferentiated THP-1 cells had no aromatase activity and showed no effect of 17alpha-methyl testosterone, but differentiated THP-1 (macrophage-like) cells had a similar inhibition of aromatase activity by 17alpha-methyl testosterone to that seen in Jar cells. The Lineweaver-Burke plot shows 17alpha-methyl testosterone to be a competitive aromatase inhibitor. Our results show for the first time that 17alpha-methyl testosterone acts as an aromatase inhibitor. These findings are relevant for understanding the effects of 17alpha-methyl testosterone as a component of hormone replacement therapy. 17alpha-methyl testosterone may, as a functional androgen and orally active steroidal inhibitor of endogenous estrogen production, also offer special possibilities for the prevention/treatment of hormone-sensitive cancers.