Induction Of Aromatase Research Articles

EP2 and EP4 Receptors Regulate Aromatase Expression in Human Adipocytes and Breast Cancer Cells: EVIDENCE OF A BRCA1 AND p300 EXCHANGE

Cytochrome P450 aromatase (aromatase), a product of the CYP19 gene, catalyzes the synthesis of estrogens from androgens. Because aromatase-dependent estrogen biosynthesis has been linked to hormone-dependent breast carcinogenesis, it is important to elucidate the mechanisms that regulate CYP19 gene expression. The main objective of this study was to identify the receptors (EP) for prostaglandin E(2) (PGE(2)) that mediate the induction of CYP19 transcription in human adipocytes and breast cancer cells. Treatment with PGE(2) induced aromatase, an effect that was mimicked by either EP(2) or EP(4) agonists. Antagonists of EP(2) or EP(4) or small interference RNA-mediated down-regulation of these receptors suppressed PGE(2)-mediated induction of aromatase. PGE(2) via EP(2) and EP(4) stimulated the cAMP-->protein kinase A pathway resulting in enhanced interaction between P-CREB, p300, and the aromatase promoter I.3/II. Overexpressing a mutant form of p300 that lacks histone acetyltransferase activity suppressed PGE(2)-mediated induction of aromatase promoter activity. PGE(2) via EP(2) and EP(4) also caused a reduction in both the amounts of BRCA1 and the interaction between BRCA1 and the aromatase promoter I.3/II. Activation of the aromatase promoter by PGE(2) was suppressed by overexpressing wild-type BRCA1. Silencing of EP(2) or EP(4) also blocked PGE(2)-mediated induction of the progesterone receptor, a prototypic estrogen-response gene. In a mouse model, overexpressing COX-2 in the mammary gland, a known inducer of PGE(2) synthesis, led to increased aromatase mRNA and activity and reduced amounts of BRCA1; these effects were reversed by knocking out EP(2). Taken together, these results suggest that PGE(2) via EP(2) and EP(4) activates the cAMP-->PKA-->CREB pathway leading to enhanced CYP19 transcription and increased aromatase activity. Reciprocal changes in the interaction between BRCA1, p300, and the aromatase promoter I.3/II contributed to the inductive effects of PGE(2).

Molecular characterization of the injury‐induced aromatase transcript in the adult zebra finch brain

In the zebra finch (Taeniopygia guttata), the aromatase gene is transcribed from one of two promoters resulting in two transcripts constitutively expressed in brain or ovary. These transcripts differ only in Exon 1 which lies in the 5' un-translated region (UTR). An inducible form of aromatase is expressed following brain injury in glia. Towards characterizing this transcript, we (a) examined the up-regulation of amplicons within the aromatase transcript using quantitative PCR (qPCR), (b) performed 5' and 3' rapid amplification of cDNA ends (RACE) on injured brain RNA and (c) sequenced the injury-induced aromatase transcript. qPCR suggested that inducible aromatase may contain a novel 3'UTR. However, neither 3' nor 5' RACE revealed novel UTRs in the injured telencephalon. We then sequenced aromatase from injured entopallium, a region that lacks detectable constitutive aromatase. Inducible aromatase was identical in sequence to the known neural aromatase transcript. These data suggest that injury-induced aromatase differs from ovarian, but is indistinguishable from neuronal aromatase. We suggest that an injury-specific signal in glia may modulate aromatase transcription. Alternatively, injury-induced aromatase transcription may be silenced under constitutive conditions. To the best of our knowledge, this is the first report that documents the sequence of inducible aromatase in any vertebrate.

Effects of Atrazine on Fish, Amphibians, and Aquatic Reptiles: A Critical Review

The herbicide atrazine is widely used in agriculture for the production of corn and other crops. Because of its physical and chemical properties, atrazine is found in small concentrations in surface waters—habitats for some species. A number of reports on the effects of atrazine on aquatic vertebrates, mostly amphibians, have been published, yet there is inconsistency in the effects reported, and inconsistency between studies in different laboratories. We have brought the results and conclusions of all of the relevant laboratory and field studies together in this critical review and assessed causality using procedures for the identification of causative agents of disease and ecoepidemiology derived from Koch's postulates and the Bradford–Hill guidelines. Based on a weight of evidence analysis of all of the data, the central theory that environmentally relevant concentrations of atrazine affect reproduction and/or reproductive development in fish, amphibians, and reptiles is not supported by the vast majority of observations. The same conclusions also hold for the supporting theories such as induction of aromatase, the enzyme that converts testosterone to estradiol. For other responses, such as immune function, stress endocrinology, parasitism, or population-level effects, there are no indications of effects or there is such a paucity of good data that definitive conclusions cannot be made.

Modulation of in Situ Estrogen Synthesis by Proline-, Glutamic Acid-, and Leucine-Rich Protein-1: Potential Estrogen Receptor Autocrine Signaling Loop in Breast Cancer Cells

In situ estrogen synthesis is implicated in tumor cell proliferation through autocrine or paracrine mechanisms especially in postmenopausal women. Several recent studies demonstrated activity of aromatase, an enzyme that plays a critical role in estrogen synthesis in breast tumors. Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1/MNAR) is an estrogen receptor (ER) coregulator, and its expression is deregulated in breast tumors. In this study, we examined whether PELP1 promotes tumor growth by promoting local estrogen synthesis using breast cancer cells (MCF7) that stably overexpress PELP1. Immunohistochemistry revealed increased aromatase expression in MCF7-PELP1-induced xenograft tumors. Real-time PCR analysis showed enhanced activation of the aromatase promoter in MCF7-PELP1 clones compared with MCF7 cells. Using a tritiated-water release assay, we demonstrated that MCF7-PELP1 clones exhibit increased aromatase activity compared with control MCF-7 cells. PELP1 deregulation uniquely up-regulated aromatase expression via activation of aromatase promoter I.3/II, and growth factor signaling enhanced PELP1 activation of aromatase. PELP1-mediated induction of aromatase requires functional Src and phosphatidylinositol-3-kinase pathways. Mechanistic studies revealed that PELP1 interactions with ER-related receptor-alpha and proline-rich nuclear receptor coregulatory protein 2 lead to activation of aromatase. Immunohistochemistry analysis of breast tumor array showed increased expression of aromatase in ductal carcinoma in situ and node-positive tumors compared with no or weak expression in normal breast tissue. Fifty-four percent (n = 79) of PELP1-overexpressing tumors also overexpressed aromatase compared with 36% (n = 47) in PELP1 low-expressing tumors. Our results suggest that PELP1 regulation of aromatase represents a novel mechanism for in situ estrogen synthesis leading to tumor proliferation by autocrine loop and open a new avenue for ablating local aromatase activity in breast tumors.

Inflammatory Status Influences Aromatase and Steroid Receptor Expression in Endometriosis

Aberrant up-regulation of aromatase in eutopic endometrium and implants from women with endometriosis has been reported. Aromatase induction may be mediated by increased cyclooxygenase-2 (COX-2). Recently, we demonstrated that progesterone receptor (PR)-A and PR-B serve an antiinflammatory role in the uterus by antagonizing nuclear factor kappaB activation and COX-2 expression. PR-C, which antagonizes PR-B, is up-regulated by inflammation. Although estrogen receptor alpha (ERalpha) is implicated in endometriosis, an antiinflammatory role of ERbeta has been suggested. We examined stage-specific expression of aromatase, COX-2, ER, and PR isoform expression in eutopic endometrium, implants, peritoneum, and endometrioma samples from endometriosis patients. Endometrial and peritoneal biopsies were obtained from unaffected women and those with fibroids. Aromatase expression in eutopic endometrium from endometriosis patients was significantly increased compared with controls. Aromatase expression in endometriosis implants was markedly increased compared with eutopic endometrium. Aromatase mRNA levels were increased significantly in red implants relative to black implants and endometrioma cyst capsule. Moreover, COX-2 expression was increased in implants and in eutopic endometrium of women with endometriosis as compared with control endometrium. As observed for aromatase mRNA, the highest levels of COX-2 mRNA were found in red implants. The ratio of ERbeta/ERalpha mRNA was significantly elevated in endometriomas compared with endometriosis implants and eutopic endometrium. Expression of PR-C mRNA relative to PR-A and PR-B mRNA was significantly increased in endometriomas compared with eutopic and control endometrium. PR-A protein was barely detectable in endometriomas. Thus, whereas PR-C may enhance disease progression, up-regulation of ERbeta may play an antiinflammatory and opposing role.

Prostaglandin E2 Induces Breast Cancer–Related Aromatase Promoters via Activation of p38 and c-Jun NH2-Terminal Kinase in Adipose Fibroblasts

Aromatase is the key enzyme for estrogen biosynthesis. A distal promoter, PI.4, maintains baseline levels of aromatase in normal breast adipose tissue. In contrast, malignant breast epithelial cells secrete prostaglandin E(2) (PGE(2)), which stimulates aromatase expression via proximal promoters PI.3/PII in a cyclic AMP (cAMP)- and protein kinase C (PKC)-dependent manner in adjacent breast adipose fibroblasts (BAF), leading to increased local concentrations of estrogen. Although an effective treatment for breast cancer, aromatase inhibitors indiscriminately abolish estrogen synthesis in all tissues, causing major side effects. To identify drug targets to selectively block aromatase and estrogen production in breast cancer, we investigated PGE(2)-stimulated signaling pathways essential for aromatase induction downstream of cAMP and PKC in human BAFs. Here, we show that PGE(2) or its surrogate hormonal mixture dibutyryl cAMP (Bt(2)cAMP) + phorbol diacetate (PDA) stimulated the p38, c-jun NH(2)-terminal kinase (JNK)-1, and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathways. Inhibition or small interfering RNA-mediated knockdown of p38 or JNK1, but not ERK, inhibited PGE(2)- or Bt(2)cAMP + PDA-induced aromatase activity and expression via PI.3/PII. Conversely, overexpression of wild-type p38alpha or JNK1 enhanced PGE(2)-stimulated aromatase expression via PII. PGE(2) or Bt(2)cAMP + PDA stimulated c-Jun and activating transcription factor-2 (ATF2) phosphorylation and binding to the PI.3/PII region. Specific activation of protein kinase A (PKA) or EPAC with cAMP analogues stimulated p38 and JNK1; however, only PKA-activating cAMP analogues induced aromatase expression. The PKC activator PDA effectively stimulated p38 and JNK1 phosphorylation but not aromatase expression. Taken together, PGE(2) activation of p38 and JNK1 via PKA and PKC is necessary for aromatase induction in BAFs, and p38 and JNK1 are potential new drug targets for tissue-specific ablation of aromatase expression in breast cancer.

Aromatase excess in cancers of breast, endometrium and ovary

Pathogenesis and growth of three common women's cancers (breast, endometrium and ovary) are linked to estrogen. A single gene encodes the key enzyme for estrogen biosynthesis named aromatase, inhibition of which effectively eliminates estrogen production in the entire body. Aromatase inhibitors successfully treat breast cancer, whereas their roles in endometrial and ovarian cancers are less clear. Ovary, testis, adipose tissue, skin, hypothalamus and placenta express aromatase normally, whereas breast, endometrial and ovarian cancers overexpress aromatase and produce local estrogen exerting paracrine and intracrine effects. Tissue-specific promoters distributed over a 93-kb regulatory region upstream of a common coding region alternatively control aromatase expression. A distinct set of transcription factors regulates each promoter in a signaling pathway- and tissue-specific manner. In cancers of breast, endometrium and ovary, aromatase expression is primarly regulated by increased activity of the proximally located promoter I.3/II region. Promoters I.3 and II lie 215 bp from each other and are coordinately stimulated by PGE 2 via a cAMP-PKA-dependent pathway. In breast adipose fibroblasts exposed to PGE 2 secreted by malignant epithelial cells, PKC is also activated, and this potentiates cAMP-PKA-dependent induction of aromatase. Thus, inflammatory substances such as PGE 2 may play important roles in inducing local production of estrogen that promotes tumor growth.

Foxl2 Up-Regulates Aromatase Gene Transcription in a Female-Specific Manner by Binding to the Promoter as Well as Interacting with Ad4 Binding Protein/Steroidogenic Factor 1

Increasing evidence suggests the crucial role of estrogen in ovarian differentiation of nonmammalian vertebrates including fish. The present study has investigated the plausible role of Foxl2 in ovarian differentiation through transcriptional regulation of aromatase gene, using monosex fry of tilapia. Foxl2 expression is sexually dimorphic, like Cyp19a1, colocalizing with Cyp19a1 and Ad4BP/SF-1 in the stromal cells and interstitial cells in gonads of normal XX and sex-reversed XY fish, before the occurrence of morphological sex differentiation. Under in vitro conditions, Foxl2 binds to the sequence ACAAATA in the promoter region of the Cyp19a1 gene directly through its forkhead domain and activates the transcription of Cyp19a1 with its C terminus. Foxl2 can also interact through the forkhead domain with the ligand-binding domain of Ad4BP/SF-1 to form a heterodimer and enhance the Ad4BP/SF-1 mediated Cyp19a1 transcription. Disruption of endogenous Foxl2 in XX tilapia by overexpression of its dominant negative mutant (M3) induces varying degrees of testicular development with occasional sex reversal from ovary to testis. Such fish display reduced expression of Cyp19a1 as well as a drop in the serum levels of 17beta-estradiol and 11-ketotestosterone. Although the XY fish with wild-type tilapia Foxl2 (tFoxl2) overexpression never exhibited a complete sex reversal, there were significant structural changes, such as tissue degeneration, somatic cell proliferation, and induction of aromatase, with increased serum levels of 17beta-estradiol and 11-ketotestosterone. Altogether, these results suggest that Foxl2 plays a decisive role in the ovarian differentiation of the Nile tilapia by regulating aromatase expression and possibly the entire steroidogenic pathway.

A Delayed Gonadotropin-Dependent and Growth Factor-Mediated Activation of the Extracellular Signal-Regulated Kinase 1/2 Cascade Negatively Regulates Aromatase Expression in Granulosa Cells

Human chorionic gonadotropin and human FSH (hFSH) elicit a transient increase in ERK1/2 phosphorylation lasting less than 60 min in immature granulosa cells expressing a low density of gonadotropin receptors. In cells expressing a high density of receptors, human chorionic gonadotropin and human FSH elicit this fast transient increase in ERK1/2 phosphorylation and also a delayed and more sustained increase that is detectable after 6-9 h. Both the early and delayed increases in ERK1/2 phosphorylation can be blocked with inhibitors of protein kinase A, the epidermal growth factor receptor kinase, metalloproteases, and MAPK kinase. The delayed effect, but not the early effect, can also be blocked with an inhibitor of protein kinase C. Because the delayed increase in ERK1/2 phosphorylation correlates with low aromatase expression in response to gonadotropins, we tested the effects of these inhibitors on aromatase expression. These inhibitors had little or no effect on gonadotropin-induced aromatase expression in cells expressing a low density of receptors, but they enhanced gonadotropin-induced aromatase expression in cells expressing a high density of receptors. Phorbol esters also induced a prolonged increase in ERK1/2 phosphorylation and, when added together with hFSH, blocked the induction of aromatase expression by hFSH in cells expressing a low density of hFSH receptor. A MAPK kinase inhibitor reversed the inhibitory effect of the phorbol ester on aromatase induction. We conclude that the effects of gonadotropins on ERK1/2 phosphorylation are mediated by epidermal growth factor-like growth factors and that the delayed effect is partially mediated by protein kinase C and acts as a negative regulator of aromatase expression.

Effects of In Ovo Exposure to Imazalil and Atrazine on Sexual Differentiation in Chick Gonads

We examined the effects of atrazine and imazalil, 2 commonly used pesticides, on sexual differentiation in chickens. Atrazine and imazalil were injected into fertile eggs on d 0. At hatching, sex genotype and phenotype were determined. Gonads were stereomicroscopically and histologically observed. In ovo exposure of atrazine (0.01 to 3 mg/egg) did not influence hatchability, whereas imazalil exposure (2 mg/egg) inhibited hatch-ability. The sex genotype matched the sex phenotype in controls, atrazine, and imazalil-exposed groups. In control females, the right gonad was regressed at hatching. Regression of the right gonad, however, was inhibited following atrazine and imazalil exposure. In atrazine-exposed female chicks, the left gonads had normal ovary structures, and the remaining right gonads had ovary medulla-like structures. In imazalil-exposed females, some left gonads had an ovary medulla-like structure without the cortex as well as tubules, and the right gonad had testis-like structures. There was no change in male gonads at hatching following atrazine and imazalil exposure. Aromatase activity of the left gonad from female chicks was not changed by any concentration of atrazine exposure. These results suggest that atrazine and imazalil inhibit regression of the right gonad in female chicks, although it is not clear whether the remaining right gonad has aromatase activity. In ovo exposure to atrazine influences sexual differentiation of the ovary by different mechanisms from imazalil, possibly by the induction of aromatase in the right gonad, whereas it is confirmed that imazalil inhibits in vitro aromatase activity in the chick ovary. The results indicated that in ovo exposure to imazalil inhibits sexual differentiation of the ovary by inhibiting aromatase activity.

O-74: Inflammatory status influences aromatase and steroid hormone receptor expression in endometriosis

Enhanced aromatase expression has been detected both in eutopic endometrium (EE) and endometriotic lesions of women with endometriosis. Induction of aromatase may be mediated by an inflammatory cascade associated with the pathogenesis of this disease. Moreover, persistent inflammatory stimuli and increased aromatase activity may trigger events leading to tissue-specific alterations in estrogen (ER) and progesterone (PR) receptor expression, which may also impact the disease. The objectives of this study were to examine the effects of tissue inflammation on expression of aromatase, ER and PR isoforms in specimen sets collected from patients with and without endometriosis.

Modulation of aromatase activity and mRNA by various selected pesticides in the human choriocarcinoma JEG-3 cell line

Aromatase enzyme plays a central role in steroidogenesis by converting androgens to estrogens and has been proposed as an important molecular target for many environmental endocrine disrupters chemicals. In this study, we have screened 30 selected pesticides with known, unknown or supposed effects on aromatase activity, for their ability to modulate aromatase activity in the human choriocarcinoma JEG-3 cell line after both short (2 h) and long exposure (24 h). All pesticides were tested at concentrations up to 10 μM that did not cause cytotoxicity after 24 h of exposure, as verified by the MTT viability assay. Four pesticides inhibited aromatase activity after 2 h of exposure: prochloraz (IC 50 < 1 μM), fenbuconazole (IC 50 = 1.1 μM), propiconazole (IC 50 = 1.5 μM) and fenarimol (IC 50 = 3.3 μM). Among them, prochloraz and fenbuconazole also exerted inhibitory effects after 24 h. Toxaphen (10 μM) and heptachlor (10 μM) inhibited aromatase activity after 24 h exposure only. Nine pesticides induced aromatase activity: aldrin, chlordane, cypermethrin, parathion-methyl, endosulfan, methoxychlor, oxadiazon, metolachlor and atrazine after 24 h of exposure, while tributyltin induced aromatase activity at 1 nM and 3 nM after both 2 h and 24 h of exposure, respectively. To further investigate the mechanisms of aromatase induction we measured CYP19 mRNA expression and showed that methoxychlor, aldrin, chlordane and tributyltin induced the transcription of the cyp19 gene. In addition, none of the aromatase inducers transactivated the retinoic acid receptor (RAR) in JEG-3 stably transfected with a RARE-luciferase plasmid while the RAR agonist TTNPB induced both aromatase and luciferase expression in these cells. Our results, which provide new data for fenbuconazole, as an inhibitor of human aromatase, and for eight pesticides as aromatase inducers, are discussed with regards to the regulation of aromatase expression in the JEG-3 cellular context.

Endogenous testicular D-aspartic acid regulates gonadal aromatase activity in boar

D-aspartic acid (D-Asp), aromatase enzyme activity and the putative D-Asp involvement on aromatase induction have been studied in the testis of mature boars. The peroxidase-antiperoxidase and the indirect immunofluorescence methods, applied to cryostat and paraffin sections, were used to evaluate D-Asp and aromatase distributions. D-Asp level was dosed by an enzymatic method performed on boar testis extracts. Biochemical aromatase activity was determined by in vitro experiments carried out on testis extracts. D-Asp immunoreactivity was found in Leydig cells, and, to a lesser extent, in germ cells. Analogously, aromatase immunoreactivity was present in Leydig cells, but absent from seminiferous tubule elements. In vitro experiments showed that the addition of D-Asp to testicular tissue acetone powder induced a significant increase of aromatase activity, as assessed by testosterone conversion to 17beta-estradiol. Enzyme Km was not affected by D-Asp (about 25 nM in control and D-Asp added tests). These findings suggest that D-Asp could be involved in the local regulation of aromatase in boar Leydig cells and intervenes in this organ's production of estrogens.

Inhibition and Induction of Aromatase (CYP19) Activity by Brominated Flame Retardants in H295R Human Adrenocortical Carcinoma Cells

Brominated flame retardants (BFRs) are persistent and ubiquitous chemicals in the environment, and they are found at increasing levels in tissues of wildlife and humans. Previous in vitro studies with the BFR class of polybrominated diphenyl ethers (BDEs) have shown endocrine-disrupting properties. Our study assessed the potential effects of nineteen BDEs, five hydroxylated BDEs (OH-BDEs), one methoxylated BDE (CH(3)O-BDE), tetrabromobisphenol-A (TBBPA), its dibromopropane ether derivative (TBBPA-DBPE), and the brominated phenols/anisols 2,4,6-tribromophenol (TBP), 4-bromophenol (4BP) and 2,4,6-tribromoanisole (TBA) on the catalytic activity of the steroidogenic enzyme aromatase (CYP19) in H295R human adrenocortical carcinoma cells. Effects were studied in the concentration range from 0.5 to 7.5 microM; exposures were for 24 h. Both 6-OH-BDE47 and 6-OH-BDE99 showed an inhibitory effect on aromatase activity at concentrations >2.5 microM and >5 microM, respectively. However, 6-OH-BDE47 also caused a statistically significant increase in cytotoxicity (based on mitochondrial MTT reduction and lactate dehydrogenase-leakage [LDH]) at concentrations >2.5 microM that could explain in part the apparent inhibitory effect on aromatase activity. Compared to 6-OH-BDE47, the methoxy analog (6-CH(3)O-BDE47) did not elicit a cytotoxic effect, whereas significant inhibition of aromatase remained. TBP caused a concentration-dependent induction of aromatase activity between 0.5 and 7.5 microM (with a maximum of 3.8-fold induction at 7.5 microM). This induction was not observed when a OH- group replaced the CH(3)O- group or when bromine atoms adjacent to this OH- group were absent. These in vitro results provide a basis for studies of more detailed structure-activity relationships between these brominated compounds and the modulation of aromatase activity.

The Differential Effects of the Gonadotropin Receptors on Aromatase Expression in Primary Cultures of Immature Rat Granulosa Cells Are Highly Dependent on the Density of Receptors Expressed and the Activation of the Inositol Phosphate Cascade

Signaling pathways mediating the divergent effects of FSH and LH on aromatase in immature rat granulosa cells were studied by infecting cells with increasing amounts of adenoviral vectors for the human LH receptor (hLHR) or FSH receptor (hFSHR). Increasing amounts of Ad-hLHR, used at a multiplicity of infection (MOI) of 20 or 200 viable viral particles/cell, increased human chorionic gonadotropin (hCG) binding and hCG-induced cAMP and Akt phosphorylation, but inositol phosphates only increased in response to hCG in cells infected with 200 MOI Ad-hLHR. In contrast, hCG increased aromatase expression in cells infected with 20, but not in cells infected with 200, MOI Ad-hLHR. Cells infected with 20 or 200 MOI Ad-hFSHR showed increased hFSH binding and hFSH-induced Akt phosphorylation, but the hFSH-induced cAMP response was unchanged relative to control cells. However, hFSH was able to stimulate the inositol phosphate cascade in the Ad-hFSHR-infected cells, and the hFSH induction of aromatase was abolished. We also found that activation of C kinase or expression of a constitutively active form of Galphaq inhibited the induction of aromatase by hFSH or 8Br-cAMP. We conclude that the differential effects of FSH and LH on aromatase in immature granulosa cells are highly dependent on gonadotropin receptor density and on the signaling pathways activated. We propose that aromatase is induced by common signals generated by activation of the FSHR and LHR (possibly cAMP and Akt) and that the activation of the inositol phosphate cascade in cells expressing a high density of LHR or FSHR antagonizes this induction.

Welcome to the Revolution: Integrative Biology and Assessing the Impact of Endocrine Disruptors on Environmental and Public Health

Concern continues to grow over the negative impact of endocrine disrupting chemicals on environmental and public health. The number of identified endocrine disrupting chemicals is increasing, but biological endpoints, experimental design, and approaches for examining and assessing the impact of these chemicals are still debated. Although some workers consider endocrine disruption an "emerging science," I argue here that it is equally, a "merging science" developing in the tradition of integrative biology. Understanding the impact of endocrine disruptors on humans and wildlife is an examination of "context dependent development" and one that Scott Gilbert predicted would require a "new synthesis" or a "revolution" in the biological sciences. Here, I use atrazine as an example to demonstrate the importance of an integrative approach in understanding endocrine disruptors.Atrazine is a potent endocrine disruptor that chemically castrates and feminizes amphibians and other wildlife. These effects are the result of the induction of aromatase, the enzyme that converts androgens to estrogens, and this mechanism has been confirmed in all vertebrate classes examined (fish, amphibians, reptiles, birds, and mammals, including humans). To truly assess the impact of atrazine on amphibians in the wild, diverse fields of study including endocrinology, developmental biology, molecular biology, cellular biology, ecology, and evolutionary biology need to be invoked. To understand fully the long-term impacts on the environment, meteorology, geology, hydrology, chemistry, statistics, mathematics and other disciplines well outside of the biological sciences are required.

Induction of Cyclin D2 in Rat Granulosa Cells Requires FSH-dependent Relief from FOXO1 Repression Coupled with Positive Signals from Smad

Ovarian follicles undergo exponential growth in response to follicle-stimulating hormone (FSH), largely as a result of the proliferation of granulosa cells (GCs). In vitro under serum-free conditions, rat GCs differentiate in response to FSH but do not proliferate unless activin is also present. In the presence of FSH plus activin, GCs exhibit enhanced expression of cyclin D2 as well as inhibin-alpha, aromatase, steroidogenic factor-1 (SF-1), cholesterol side chain (SCC), and epiregulin. In this report we sought to identify the signaling pathways by which FSH and activin promote GC proliferation and differentiation. Our results show that these responses are associated with prolonged Akt phosphorylation relative to time-matched controls and are dependent on phosphatidylinositol 3-kinase (PI 3-kinase) and Smad2/3 signaling, based on the ability of the PI 3-kinase inhibitor LY294002 or infection with adenoviral dominant negative Smad3 (DN-Smad3) mutant to attenuate induction of cyclin D2, inhibin-alpha, aromatase, SCC, SF-1, and epiregulin. The DN-Smad3 mutant also abolished prolonged Akt phosphorylation stimulated by FSH plus activin 24 h post-treatment. Infection with the adenoviral constitutively active forkhead box-containing protein, O subfamily (FOXO)1 mutant suppressed induction of cyclin D2, aromatase, inhibin-alpha, SF-1, and epiregulin. Transient transfections of GCs with constitutively active FOXO1 mutant also suppressed cyclin D2, inhibin-alpha, and epiregulin promoter-reporter activities. Chromatin immunoprecipitation results demonstrate in vivo the association of FOXO1 with the cyclin D2 promoter in untreated GCs and release of FOXO1 from the cyclin D2 promoter upon addition of FSH plus activin. These results suggest that proliferation and differentiation of GCs in response to FSH plus activin requires both removal of FOXO1-dependent repression and positive signaling from Smad2/3.

CpG Island methylation patterns in various histotypes of endometrial cancer

Our laboratory is interested in defining molecular pathways that help distinguish various histotypes of endometrial cancer. Type 1 endometrial cancer (low grade endometrioid adenocarcinoma) is characterized by superficial myometrial invasion, low clinical stage, and excellent prognosis. Type 2 endometrial cancer (grade 3 endometrioid adenocarcinoma, uterine papillary serous carcinoma, and malignant mixed mullerian tumor) is associated with deep myometrial invasion, metastasis, advanced clinical stage, and poor prognosis. One approach that we have used to define the molecular characteristics of these endometrial tumors is to analyze CpG island methylation patterns. Gene methylation is an important mechanism of tumor suppressor gene inactivation in a wide variety of neoplasms. We have modified existing techniques for methylation-specific PCR for effective use in formalin-fixed, paraffin-embedded tissues. To date, we have analyzed a large series (greater than 100) different endometrial tumors for gene methylation patterns, using a panel of 12 different genes. A subset of the data for endometrioid tumors and uterine MMMT will be presented at the symposium. The endometrioid and MMMT groups have similar percentages of tumors with a methylator phenotype (tumor with two or more genes methylated). However, the pattern of gene methylation was distinct for several genes. Similar to previous studies, we found methylation of MLH1 and resulting microsatellite instability to be extremely uncommon in uterine MMMT compared to endometrioid adenocarcinoma. Methylation of MINT 31, a common event in endometrioid tumors, is also significantly less common in MMMT. Several important genes showed similar methylation frequencies in the endometrioid and MMMT groups. These include apoptosis-promoting genes (DAP-K and RASSF1) and cell cycle regulatory genes (p14 and p16). RASSF1, which encodes a pro-apoptotic protein downstream from RAS, is methylated in a majority of the endometrioid tumors and MMMT. These results are intriguing, especially considering that the established frequency of RAS mutation in endometrial cancer is relatively low (12–25% of cases). It has been proposed by several different groups that RAS mutation and methylation of RASSF1 may be mutually exclusive events. Our preliminary findings for endometrial cancer certainly support this assertion. Finally, analysis of COX-2 methylation has yielded exciting results. Previously, it had been proposed that induction of COX-2 leads to PGE2 synthesis, which causes induction of aromatase. Increased aromatase levels contribute to increased local production of estrogens, which contributes to tumor cell growth. Therefore, we were surprised to find that nearly half of the endometrioid tumors and MMMT demonstrated methylation of COX-2. Since gene methylation usually implies decreased gene expression, these findings did not support the “COX-2/aromatase/estrogen” pathway in endometrial cancer. Quantitative PCR and immunohistochemistry supported these findings. Collectively, these results suggest that elevated COX-2 is not an important mechanism in at least half of endometrial cancers.

A risk assessment of atrazine use in California: human health and ecological aspects

A risk assessment of the triazine herbicide atrazine has been conducted by first analyzing the toxicity database and subsequently estimating exposure. Margins of safety (MOS) were then calculated. Toxicity was assessed in animal studies and exposure was estimated from occupational and dietary sources. In acute toxicity studies, atrazine caused developmental toxicity in the rabbit [no observed effect level (NOEL) 5 mg kg(-1) day(-1)] and cardiotoxicity in a dog chronic study (NOEL 0.5 mg kg(-1) day(-1)); cancer (mammary glands) resulted from lifetime exposure. The mammary tumors, which occurred specifically in female Sprague-Dawley rats, were malignant, increased in a dose-dependent manner and were also observed with other, related triazines. Evidence for a genotoxic basis for these tumors was either equivocal or negative. Triazines have been shown to be clastogenic in Chinese hamster ovary cells, in vitro, but without showing a convincing dose/response relationship. Atrazine can be converted into genotoxic N-nitrosoatrazine in the environment or the digestive system, suggesting that N-nitrosamines derived from triazines could be oncogenic. However, it was concluded that N-nitrosotriazines are unlikely to play a significant role in triazine-induced rat mammary gland tumors. An endocrine basis for the mammary tumors, involving premature aging of the female SD rat reproductive system, has been proposed. A suppression of the luteinizing hormone surge during the estrus cycle by atrazine leads to the maintenance of elevated blood levels of 17beta-estradiol (E2) and prolactin. The mechanism for tumor development may include one or more of the following: the induction of aromatase (CYP19) and/or other P450 oxygenases, an antagonist action at the estrogen feedback receptor in the hypothalamus, an agonist action at the mammary gland estrogen receptor or an effect on adrenergic neurons in the hypothalamic-pituitary pathway. None of these has been excluded as a target because there has been a lack of a rigorous attempt to address the mechanism of action for mammary tumors at the molecular level. The potential occupational exposure to atrazine was assessed during mixing, loading and application. Absorbed daily dosage values were 1.8-6.1 microg kg(-1) day(-1). The MOS values (animal NOEL/human exposure) for short-term (acute) exposure were 820-2800. Longer-term occupational exposure and risk were also calculated. Detectable crop residues are generally absent at harvest. Theoretical calculations of acute dietary exposure used tolerance levels, along with secondary residues, and water, for which there is a maximum contamination level; atrazine plus the three main chlorotriazine metabolites were combined. MOS values were above 2000 for all population subgroups. Dietary exposure to atrazine is therefore extremely unlikely to result in human health hazard. Recent publications have reported a possible feminization of frogs, measured in laboratory and field studies. This is assumed to be due to the induction of aromatase, but no measurements of enzyme activity have been reported. In field studies, the water bodies with the greatest numbers of deformed frogs sometimes had the lowest concentrations of atrazine. Other studies have also cast doubt on the feminization theory, except perhaps at very high levels of atrazine. Epidemiology studies have investigated the possibility that atrazine may result in adverse effects in humans. Although some studies have claimed that atrazine exposure results in an elevated risk of prostate cancer, the published literature is inconclusive with respect to cancer incidence.

Co-culture of Primary Human Mammary Fibroblasts and MCF-7 Cells as an In Vitro Breast Cancer Model

Approximately 60% of all breast tumors are estrogen-responsive and chemicals that show estrogenic or anti-estrogenic properties are able to interact with breast tumor growth. In a breast tumor, adipose stromal cells (fibroblasts) surrounding the epithelial tumor contain the aromatase enzyme, which converts androgens into estrogens. Exposure to aromatase inducers can therefore lead to increased estrogen levels and possibly to accelerated breast tumor growth. Subsequently, breast tumor cells synthesize and secrete elevated levels of factors such as prostaglandin E2 (PGE2), interleukin-6 (IL-6), and IL-6 soluble receptor (IL-6sR), which in turn have the ability to stimulate aromatase gene transcription in fibroblasts, establishing a positive feedback loop. In this study, a technique that allows for culturing MCF-7 epithelial breast tumor cells and healthy primary human mammary fibroblasts together in one compartment was developed. To establish the positive feedback loop, the co-culture was exposed to estrogenic compounds. RNA was isolated and reverse-transcriptase polymerase chain reaction (RT-PCR) was performed on the aromatase and pS2 genes. Exposure of the co-culture to estradiol (E2), diethylstilbestrol (DES), and bisphenol-A (BPA), resulted in a three- to seven-fold increase of pS2 transcription levels. Furthermore, pS2 transcription levels increased even more when the aromatase substrate testosterone (20 nM) was present in the co-culture medium. Exposure of the co-culture to the aromatase inducer dexamethasone (DEX) resulted in increased pS2 transcription levels, as well as increased aromatase transcription levels. Simultaneous exposure to DEX and the synthetic anti-estrogen ICI 182,780 almost completely blocked the pS2 response. The aromatase induction response was not altered by ICI 182,780 treatment. Simultaneous exposure to DEX and the non-steroidal aromatase inhibitor fadrozole, abolished the effect of the presence of testosterone in the co-culture medium, but did not result in pS2 gene transcription levels as low as seen after exposure to ICI 182,780. These observations indicate the presence of a positive feedback loop in our co-culture system. This co-culture provides a more sophisticated and sensitive system to detect direct and indirect estrogenic effects of compounds and their possible effects on breast tumor promotion.