Microsomal Aromatase Research Articles

Bisphenol a alternatives suppress human and rat aromatase activity: QSAR structure-activity relationship and in silico docking analysis

The use of alternative substances to replace bisphenol A (BPA) has been encouraged. The objective of this study was to evaluate the effects of BPA and 9 BPA alternatives on human and rat aromatase (CYP19A1) in human and rat placental microsomes. The results revealed that bisphenol A, AP, B, C, E, F, FL, S, and Z, and 4,4′-thiodiphenol (TDP) inhibited human CYP19A1 and bisphenol A, AP, B, C, FL, Z, and TDP inhibited rat CYP19A1. The IC50 values of human CYP19A1 ranged from 3.3 to 172.63 μM and those of rat CYP19A1 ranged from 2.20 to over 100 μM. BPA alternatives were mixed/competitive inhibitors and inhibited estradiol production in BeWo placental cells. Molecular docking analysis showed that BPA alternatives bind to the domain between heme and steroid and form a hydrogen bond with catalytic residue Met374. Pharmacophore analysis showed that there were one hydrogen bond donor, one hydrophobic region, and one ring aromatic hydrophobic region. Bivariate correlation analysis showed that molecular weight, alkyl atom weight, and LogP of BPA alternatives were inversely correlated with their IC50 values. In conclusion, BPA alternatives can inhibit human and rat CYP19A1 and the lipophilicity and the substituted alkyl size determines their inhibitory strength.

Investigation of antiaromatase activity using hepatic microsomes of Nile tilapia (Oreochromis niloticus).

Microsomal aromatase enzymes of humans and rats have been used in antiaromatase assays, but enzyme activity is species-specific. The current study extracted hepatic microsomes of Nile tilapia (Oreochromis niloticus) to investigate and compare the antiaromatase activity of chrysin, quercetin, and quercitrin. This activity was evaluated using a dibenzylfluorescein (DBF) assay. Results revealed that the age and body weight of Nile tilapia affected the yield of extracted microsomes. Extraction of hepatic microsomes of Nile tilapia was most effective when using a reaction medium with a pH of 8.0. A DBF assay using Nile tilapia microsomes revealed significant differences in levels of antiaromatase activity for chrysin, quercetin, and quercitrin. Chrysin was the most potent aromatase inhibitor, with an IC50 of 0.25 mg/mL. In addition, chrysin is an aromatase inhibitor that also inhibits the proliferation of cancer cells. Hepatic microsomes of Nile tilapia can be used to investigate and compare the antiaromatase activity of different compounds.

Post-translational dual regulation of cytochrome P450 aromatase at the catalytic and protein levels by phosphorylation/dephosphorylation.

The post-translational regulation of aromatase has not been well characterized as compared with transcriptional regulation. Several studies of post-translational regulation have focused on decreases in catalytic activity following phosphorylation. We report here dual post-translational regulation of aromatase, at the catalytic activity and protein levels. Microsomal aromatase prepared from JEG-3 cells was rapidly inactivated and subsequently degraded in the presence of a cytosolic fraction with calcium, magnesium, and ATP. In a reconstituted system consisting of microsomal and cytosolic fractions, aromatase was protected from protein degradation by treatment with alkaline phosphatase, whereas degradation was enhanced by treatment with calcineurin inhibitors (FK506 and cyclosporin A). Furthermore, aromatase was protected from degradation by treatment with kinase inhibitors, especially the calcium/calmodulin kinase inhibitors KN62 and KN93. Similarly to the reconstituted system, aromatase in cultured JEG-3 cells was protected from degradation by KN93, whereas FK503 increased degradation in the presence of cycloheximide, although cellular aromatase mRNA levels were unchanged by these reagents. Knockdown of calcineurin and calcium/calmodulin kinase II (CaMKII) with small interfering RNAs resulted in a dose-dependent increase in aromatase degradation and protection from degradation, respectively. The cytosol fraction-dependent phosphorylation of microsomal aromatase was inhibited by calcineurin, KN62, and KN93, and promoted by CaMKII and FK506. These results indicate that aromatase is regulated acutely at the catalytic activity level and subsequently at the enzyme content level by CaMKII/calcineurin-dependent phosphorylation/dephosphorylation.

Chemopreventive activity of Cnidii Rhizoma for breast cancer

Chemopreventive potential for human breast cancer was assessed in vitro with Cnidii Rhizoma extract. Cnidii Rhizoma inhibited cell proliferation in estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) human breast carcinoma cell lines. Cytochrome P450 (CYP) 1A1-mediated ethoxyresorufin O-deethylase (EROD) activity was inhibited by Cnidii Rhizoma in a concentration-dependent manner. In addition, Cnidii Rhizoma extract caused inhibition of microsomal aromatase (estrogen synthase) activity. Ornithine decarboxylase (ODC) activity was reduced to 40.3% of the control after 6 h treatment with Cnidii Rhizoma (5 mg/mL) in MCF-7 breast cancer cells. Cnidii Rhizoma extract markedly reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated matrix metalloproteinase (MMP)-9 activity. These results suggest that Cnidii Rhizoma could be of therapeutic value in preventing human breast cancer.

219: Maternal-fetal gestational characteristics and microsomal aromatase activity in human placentas

219: Maternal-fetal gestational characteristics and microsomal aromatase activity in human placentas

A comparison of two human cell lines and two rat gonadal cell primary cultures as in vitro screening tools for aromatase modulation

Environmental toxicants are a serious health concern, and numerous studies have been devoted to studying the effects of environmental Endocrine Disrupting Chemicals (EDCs). The balance between androgens and estrogens controls the function of many EDC-sensitive organs, and the aromatase enzyme plays a key role in maintaining this balance. In vitro studies have suggested that aromatase expression and activity is a promising biomarker for initial screenings of putative hormonal disrupting compounds. To further validate the aromatase biomarker, we tested several EDCs (atrazine, bisphenol A, methoxychlor, methoxychlor metabolite HPTE, vinclozolin, vinclozolin metabolite M2) in four different models (human cell lines H295R and JEG-3, rat primary cultures of granulosa and leydig cells). We evaluated the similarities/differences in the chemical impact on aromatase mRNA levels and enzymatic activity for the different species and cell types. Aromatase gene expression was assessed by q-RT-PCR, and enzymatic activity was assessed via a tritiated water method with either intact cells or isolated microsomes. The aromatase gene mRNA levels and cellular enzymatic activity varied between the four different models tested, which suggests that the EDC effect varies among different cell types. However, regulation of microsomal aromatase activity appeared to be conserved across all the species and cell types tested. These results suggest that several well characterized complementary cellular models are required to fully characterize the effects of putative EDCs and predict the in vivo effects.

Retinol inhibits aromatase activity and expression in vitro

Aromatase converts androgens into estrogens and is thought to supply a local source of estrogen that facilitates the growth of hormone-responsive tumor cells. Inhibition of aromatase is therefore an important chemopreventive strategy. We investigated the effect of retinol and selected retinoids on the activity and expression of aromatase in two human carcinoma cell lines in vitro. Retinol (ROH) and all- trans retinoic acid (ATRA) significantly inhibited aromatase activity in a concentration-dependent manner in microsomes isolated from JEG-3 human placental carcinoma cells, whereas 9- cis and 13- cis retinoic acid had significant inhibitory activity only at the highest concentrations tested. Similar results were observed in an assay of cellular aromatase activity in MCF-7 human breast cancer cells. Enzyme kinetic studies by double-reciprocal plot demonstrated that ROH inhibited microsomal aromatase activity in a mixed manner. In addition, ROH suppressed both the basal and cAMP-induced expression of aromatase mRNA in MCF-7 cells and inhibited transcription controlled by a cAMP-responsive element. These results suggest that aromatase activity and expression are a molecular target of ROH and chemopreventive retinoids, an activity that may underlie, in part, their inhibitory effects on hormone-dependent cancer.

Insulin-Like Growth Factor I Enhances the Expression of Aromatase P450 by Inhibiting Autophagy

Aromatase, a key enzyme of estrogen biosynthesis, is transcriptionally regulated by many growth factors. IGF-I enhances aromatase activity in a variety of cells, but the mechanism of action has not been determined. We herein report our finding of a novel mechanism of action for IGF-I. IGF-I enhanced the dexamethasone (DEX)-induced aromatase activity by 30% in serum-starved THP-1 cells. The increase was associated with a corresponding increase in the level of aromatase protein but not with any change in the mRNA level. Metabolic labeling experiments revealed that IGF-I inhibited the degradation of aromatase. We identified pepstatin A as the most effective inhibitor of aromatase degradation by in vitro assay. Using a nontoxic concentration of pepstatin A, we examined IGF-I's action on aromatase distribution in microsomes and lysosomes. In the presence of pepstatin A, DEX caused an increase in the amount of aromatase in both microsomes and lysosomes, and IGF-I attenuated the DEX-induced accumulation of aromatase in lysosomes and, conversely, enhanced its accumulation in the microsomes. The addition of serum abolished the IGF-I-induced changes. The transport from microsome to lysosome was fluorescently traced in cells using a recombinant aromatase. IGF-I selectively reduced the aromatase signal in the lysosomes. Finally, we observed that IGF-I enhanced the aromatase activity by 50% as early as 1 h after treatment; furthermore, rapamycin, an enhancer of autophagy, completely negated the effect of IGF-I on the enzyme. These results indicate that IGF-I enhances aromatase by the inhibition of autophagy.

Inhibition of aromatase (P450Arom) by some 1-(benzofuran-2-ylmethyl)imidazoles.

Studies of a series of 1-(benzofuran-2-ylmethyl)imidazoles, 1-5, previously proposed as potential agents for prostatic cancer by their inhibition of 17beta-hydroxylase:17,20-lyase (P450 17), have been extended to their selectivity against placental microsomal aromatase (P450(Arom)) in man. The compounds were 3-7-fold more potent than aminoglutethimide and had some selectivity for P450 17 as expressed by the ratio (IC50 P450(Arom))/(IC50 P450) 17)/17.0 (2), 10.3 (3), 34.6 (4) and 42.0 (5), where IC50 is the concentration resulting in 50% inhibition. The lower potency of 1-5 towards P450(Arom) compared with the racemic alpha-phenyl-substituted compounds (6, 80-1000 x aminoglutethimide) and some racemic alpha-methyl (8.5 and 12.2 x aminoglutethimide) and alpha-ethyl (12.1 and 32.9 x aminoglutethimide) analogues has been rationalized. This work selectively extends studies of the P450 17 inhibitor 5, a potential prostatic cancer agent, towards other cytochrome P450 enzymes in the steroidogenic pathway and provides a general method for determining the relative influence of chemical manipulation of a parent inhibitor towards two enzymes in the pathway using additional literature data.

Pomegranate Ellagitannin–Derived Compounds Exhibit Antiproliferative and Antiaromatase Activity in Breast Cancer Cells In vitro

Estrogen stimulates the proliferation of breast cancer cells and the growth of estrogen-responsive tumors. The aromatase enzyme, which converts androgen to estrogen, plays a key role in breast carcinogenesis. The pomegranate fruit, a rich source of ellagitannins (ET), has attracted recent attention due to its anticancer and antiatherosclerotic properties. On consumption, pomegranate ETs hydrolyze, releasing ellagic acid, which is then converted to 3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one ("urolithin") derivatives by gut microflora. The purpose of this study was to investigate the antiaromatase activity and inhibition of testosterone-induced breast cancer cell proliferation by ET-derived compounds isolated from pomegranates. A panel of 10 ET-derived compounds including ellagic acid, gallagic acid, and urolithins A and B (and their acetylated, methylated, and sulfated analogues prepared in our laboratory) were examined for their ability to inhibit aromatase activity and testosterone-induced breast cancer cell proliferation. Using a microsomal aromatase assay, we screened the panel of ET-derived compounds and identified six with antiaromatase activity. Among these, urolithin B (UB) was shown to most effectively inhibit aromatase activity in a live cell assay. Kinetic analysis of UB showed mixed inhibition, suggesting more than one inhibitory mechanism. Proliferation assays also determined that UB significantly inhibited testosterone-induced MCF-7aro cell proliferation. The remaining test compounds also exhibited antiproliferative activity, but to a lesser degree than UB. These studies suggest that pomegranate ET-derived compounds have potential for the prevention of estrogen-responsive breast cancers.

Inhibition of placental aromatase activity in a cell free assay by ovarian protein.

Previously we identified a fraction of follicular fluid (follicle regulatory protein: FRP) which inhibits granulosa cell aromatase activity. During the course of these studies the question of FRP acting via autocrine as well as paracrine mechanisms arose in addition to the need for a more efficient method of screening for aromatase inhibitory activity during the purification of FRP. Accordingly, we assessed the effects of FRP on aromatase activity in a microsomal assay. Placental microsome preparations were preincubated for 20 minutes with or without FRP prior to a 20 minute incubation with testosterone. Significantly less conversion of testosterone to estrogen occurred with FRP compared to control preincubation. When follicular protein was added without pre-incubation, there was no apparent change in microsomal aromatase activity, whereas after a 20 minute pre-incubation with the follicular protein fraction, significantly less testosterone was converted into estrogen. When various concentrations of FRP were assayed in the placental aromatase assay, a dose-response curve demonstrated a 50% inhibitory dose (ID50) of approximately 400 micrograms/ml. To further purify the aromatase inhibitory activity, 5 mg of the crude follicular fluid preparation was eluted through an anion exchange column via HPLC using a sodium acetate gradient. The fractions in the central elution peak contained aromatase inhibitor activity with ID50 values of 25-160 micrograms/ml. Thus Fractions were further purified by elution through a gel exclusion column via HPLC which demonstrated inhibition of cell free placental aromatase activity in the 15,000-18,000 molecular weight range with an ID50 of 5 micrograms/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Aromatase inactivation by 2-substituted derivatives of the suicide substrate androsta-1,4-diene-3,17-dione

To gain the structure–activity relationship of Δ 1-androstenediones (Δ 1-ADs) as mechanism-based inactivator of aromatase, series of 2-alkyl- and 2-alkoxy-substitiuted Δ 1-ADs ( 6 and 9) as well as 2-bromo-Δ 1-AD ( 14) were synthesized and tested. All of the inhibitors examined blocked aromatase in human placental microsomes in a competitive manner. In a series of 2-alkyl-Δ 1-ADs ( 6), n-hexyl compound 6f was the most powerful inhibitor with an apparent K i value of 31 nM. The inhibitory activities of 2-alkoxy steroids 9 decreased in relation to length of the alkyl chain up to n-hexyloxy group ( K i: 95 nM for methoxy 9a). All of the alkyl steroids 6 along with the alkoxy steroid 9, except for the ethyl and n-propyl compounds 6b and 6c, caused a time-dependent inactivation of aromatase. The inactivation rates ( k inact: 0.020–0.084 min −1) were comparable to that of the parent compound Δ 1-AD. The inactivation was prevented by the substrate AD, and no significant effect of l-cysteine on the inactivation was observed in each case. The results indicate that the 2-hexyl compound 6f act as the most powerful mechanism-based inactivator of aromatase among Δ 1-AD analogs and may be submitted to the preclinical study in estrogen-dependent breast cancer.

Synthesis and biochemical studies of 17-substituted androst-3-enes and 3,4-epoxyandrostanes as aromatase inhibitors

A series of 5α-androst-3-enes and 3α,4α-epoxy-5α-androstanes were synthesized and tested for their abilities to inhibit aromatase in human placental microsomes. In these series the original C-17 carbonyl group was replaced by hydroxyl, acetyl and hydroxyimine groups. Inhibition kinetic analysis on the most potent steroid of these series revealed that it inhibits the enzyme in a competitive manner (IC 50 = 6.5 μM). The achieved data pointed out the importance of the C-17 carbonyl group in the D-ring of the studied steroids as a structural feature required to reach maximum aromatase inhibitory activity. Further, at least one carbonyl group (C-3 or C-17) seems to be essential to effective aromatase inhibition.

Xanthones from the botanical dietary supplement mangosteen (Garcinia mangostana) with aromatase inhibitory activity.

Twelve xanthone constituents of the botanical dietary supplement mangosteen (the pericarp of Garcinia mangostana) were screened using a noncellular, enzyme-based microsomal aromatase inhibition assay. Of these compounds, garcinone D (3), garcinone E (5), alpha-mangostin (8), and gamma-mangostin (9) exhibited dose-dependent inhibitory activity. In a follow-up cell-based assay using SK-BR-3 breast cancer cells that express high levels of aromatase, the most potent of these four xanthones was gamma-mangostin (9). Because xanthones may be consumed in substantial amounts from commercially available mangosteen products, the consequences of frequent intake of mangosteen botanical dietary supplements require further investigation to determine their possible role in breast cancer chemoprevention.

해양심층수의 cytochrome P450 1A1, aromatase 및 MMP-9 활성 억제 효과

동해 해양심층수의 유방암예방 효능과 전이에 미치는 영향을 알아보기 위해 cytochrome P450 1A1 활성과 aromatase 활성 및 유방암세포의 침윤성, 이와 관련된 MMP-9 의 활성과 그 단백질 발현에 미치는 영향을 조사하였다. 해양심층수는 체내외의 여러 화학물질을 체내에서 활성화시켜 발암이나 돌연변이 등을 유발시키는 것으로 알려진 cytochrome P450 1A1을 경도의존적으로 저해시켰다. 또한 호르몬 의존성 유방암의 진행에 관여하는 aromatase의 활성도 경도의존적으로 저해시켰다(<TEX>$5.6{\sim}51.9%$</TEX>). 해양심층수 처리에 의해 사람유방암세포인 MDA-MB-231 세포의 침윤성은 <TEX>$73.7{\sim}29.4%$</TEX>로 감소하였으며, 세포의 침윤시 작용하는 단백질 분해 효소인 MMP-9의 활성과 단백질 발현도 경도의존적으로 억제되었다. 따라서 해양심층수는 유방암 예방과 전이관련의 더 많은 연구에 의해 유방암 예방과 전이 억제작용을 증명할 수 있을 것으로 보인다. Deep sea water from the East sea was tested for breast cancer chemoprevention and metastasis by measuring the activities of cytochrome P450 1A1 and aromatase, invasiveness, and activity and expression of matrix metalloproteinase (MMP)-9 in breast MDA-MB-231 cancer cell. The in vitro incubation of rat liver microsome with deep sea water (a hardness range of <TEX>$100{\sim}1,000$</TEX>) showed a hardness-dependent inhibition of 7,12-dimethylbenz[a]anthracene (DMBA)-induced cytochrome P450 1A1 activity. Deep sea water showed 27.1, 45.4 and 51.9% inhibition of microsomal aromatase activity at the hardness of 600, 800 and 1,000, respectively. In addition deep sea water inhibited not only the invasiveness of 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated MDA-MB-231 cells through matrigel-coated membrane in a hardness-dependent manner but also the activity and expression of MMP-9 in MDA-MB-231 cell.

Ovarian P450 aromatase activity in the catfish Heteropneustes fossilis: Seasonal changes and effects of catecholestrogens

Ovarian microsomal aromatase (P450arom) activity was studied in relation to season and incubation of follicles with catecholestrogens [(2-hydroxyestradiol-17β (2-OHE2) and 2-methoxyestradiol-17β (2-methoxyE2)] using a product (estradiol-17β) assay. Peak P450arom activity was noticed in late preparatory phase (April) and it decreased significantly in pre-spawning, spawning and post-spawning phases to give the lowest value in resting phase. Apparent Km and Vmax of the enzyme varied significantly and the values were high in the preparatory (vitellogenic) phase (Km 74.62±1.73nM, Vmax 0.81±0.01pmol/mgprotein/min) and low in the spawning (post-vitellogenic) phase (Km 62.01±1.68nM, Vmax 0.69±0.002pmol/mgprotein/min). The incubation of the ovarian microsomes with 2-OHE2 elicited significant biphasic effects on enzyme activity. In the vitellogenic phase, concentrations of the steroid up to 1μM inhibited enzyme activity significantly with the highest inhibition at 10nM. However, in the post-vitellogenic ovary, the highest inhibition was registered at 100nM. The higher concentrations (10μM or 100μM) did not elicit any significant change compared to the control groups. A comparison of the aromatase inhibition index (AI50, indicates 50% inhibition of aromatase activity) of fadrozole, a known aromatase inhibitor and 2-OHE2 shows that the AI50 was 4.4nM for fadrozole and 0.864nM (vitellogenic phase) and 1.31nM (post-vitellogenic phase) for 2-OHE2 indicating higher potency of the latter. The incubation of the ovarian microsomes with 2-methoxyE2 increased enzyme activity only at the higher concentrations (1–100μM). The results show seasonality in the potential of the ovary to synthesize E2 and the potent enzyme inhibiting activity of 2-OHE2, which is reported for the first time.

4-Hydroxyphenylretinamide (4HPR) derivatives regulate aromatase activity and expression in breast cancer cells

Recent studies exhibit that 4-hydroxyphenylretinamide (4HPR) decreases aromatase activity in breast and placental cells. The effect of synthetic 4HPR analogs on aromatase and expression was examined in three breast cancer cell lines. Most derivatives did not decrease cellular aromatase activity. Two of the analogs even stimulated aromatase activity at the transcriptional level. Only one derivative significantly decreased aromatase in all three breast cancer cell lines and also suppressed CYP19 gene expression in one of the cell line. Placental microsomal aromatase assay rule out the possibility that this compound directly inhibits the aromatase enzyme. A non-genomic mechanism in suppression of cellular aromatase activity of this compound is proposed.

Novel trends in endocrine disruptor testing: the H295R Steroidogenesis Assay for identification of inducers and inhibitors of hormone production

IntroductionOver the past two decades, there has been increasingconcern about the possible impacts of exposure to chem-icals in the environment on endocrine and reproductivesystems in humans and wildlife [1]. To address theseconcerns, national and international programs have beeninitiated to develop new guidelines for the screening andtesting of potential endocrine-disrupting chemicals (EDCs)in vertebrates [2, 3]. The focus of these multi-tieredprograms was to develop in vitro and in vivo assays toidentify and classify chemicals relative to their potentialinteraction with endocrine systems (Tier 1), and then todevelop concentration–response relationships in animalmodels (Tier 2) [2]. In this article we will focus on a newin vitro bioassay to test for the potential of chemicals tointerfere with steroid hormone production. This assay iscurrently being developed as part of Tier 1 of the USEPAEndocrine Disruptor Screening Program and an Organiza-tion for Economic Cooperation and Development (OECD)test method validation program [4].Most of the efforts to develop screening assays forendocrine disruptor testing have been focused on thedevelopment and validation of in vitro hormone receptor-binding assays including the estrogen receptor (ER) and theandrogen receptor (AR) [5–7]. However, it should beacknowledged that there are a number of non-receptor-mediated processes that may also alter endocrine function[8]. These include chemical-induced modulation of theenzymes involved in the production, transformation, orelimination of steroid hormones, which can result inalterations of the absolute and relative concentrations ofhormones in blood and tissues [9]. Often, these non-receptor-mediated effects are caused indirectly via alter-ations of common signal-transduction pathways [10], orthrough direct competitive or non-competitive inhibition ofthe enzyme.Steroid sex hormones are key factors involved in theregulation of reproduction in vertebrates and are alsoinvolved in numerous other processes that are related todevelopment and growth [11]. Thus, chemicals that candisrupt the production of sex steroids may be directlylinked to adverse outcomes for these processes. To addressthe potential issues of exposure to chemicals that may resultin the disturbance of sex steroid homeostasis, therefore,several in vivo test systems have been evaluated. Thesetests include the pubertal male and female rat assays, thefish reproductive screen, and the frog thyroid assay.Furthermore, several in vitro screening methods have beendeveloped including the microsomal aromatase assay and arodent minced-testis assay [2]. Some of these assays suchas the rodent minced-testis have shown some promiseregarding their potential to identify effects on androgenproduction. However, a number of questions have beenraised about the utility of rodent–based explant assays inthat they were found to yield high rates of false positive or

Studies directed towards a mechanistic evaluation of inactivation of aromatase by the suicide substrates androsta-1,4-diene-3,17-diones and its 6-ene derivatives: Aromatase inactivation by the 19-substituted derivatives and their enzymic aromatization

To gain insight into the mechanistic features for aromatase inactivation by the typical suicide substrates, androsta-1,4-diene-3,17-dione (ADD, 1) and its 6-ene derivative 2, we synthesized 19-substituted (methyl and halogeno) ADD and 1,4,6-triene derivatives 8 and 10 along with 4,6-diene derivatives 9 and tested for their ability to inhibit aromatase in human placental microsomes as well as their ability to serve as a substrate for the enzyme. 19-Methyl-substituted steroids were the most powerful competitive inhibitors of aromatase ( K i: 8.2–40 nM) in each series. Among the 19-substituted inhibitors examined, 19-chloro-ADD and its 6-ene derivatives ( 7b and 9b) inactivated aromatase in a time-dependent manner in the presence of NADPH in air while the other ones did not. The time-dependent inactivation was blocked by the substrate AD and required NADPH. Only the time-dependent inactivators 7b and 9b in series of 1,4-diene and 1,4,6-triene steroids as well as all of 4,6-diene steroids 9, except for the methyl compound 9a, served as a substrate for aromatase to yield estradiol and/or its 6-ene estradiol with lower conversion rates compared to the corresponding parent steroids 1,4-diene, 1,4,6-triene and 4,6-diene derivatives. The present findings strongly suggest that the aromatase reaction, 19-oxygenation, at least in part, would be involved in the time-dependent inactivation of aromatase by the suicide substrates 1 and 2, where the 19-substitutent would play a critical role in the aromatase reaction probably though steric and electronic reasons.

Cytotoxic effects and aromatase inhibition by xenobiotic endocrine disrupters alone and in combination

Xenobiotics may cause long-term adverse effects in humans, especially at the embryonic level, raising questions about their levels of exposure, combined effects, and crucial endpoints. We are interested in the possible interactions between xenobiotic endocrine disrupters, cellular viability and androgen metabolism. Accordingly, we tested aroclor 1254 (A1254), atrazine (AZ), o, p′-DDT, vinclozolin (VZ), p, p′-DDE, bisphenol A (BPA), chlordecone (CD), nonylphenol (NP), tributylin oxide (TBTO), and diethylstilbestrol (DES) for cellular toxicity against human embryonic 293 cells, and activity against cellular aromatase, but also on placental microsomes and on the purified equine enzyme. Cellular viability was affected in 24 h by all the xenobiotics with a threshold at 50 μM (except for TBTO and DES, 10 μM threshold), and aromatase was inhibited at non-toxic doses. In combination synergism was observed reducing the threshold values of toxicity to 4–10 μM, and aromatase activity by 50% in some cases. In placental microsomes the most active xenobiotics rapidly inhibited microsomal aromatase in a manner independent of NADPH metabolism. Prolonged exposures to low doses in cells generally amplified by 50 times aromatase inhibition. These xenobiotics may act by inhibition of the active site or by allosteric effects on the enzyme. Bioaccumulation is a feature of some xenobiotics, especially chlordecone, DDT and DDE, and low level chronic exposures can also affect cell signaling mechanisms. This new information about the mechanism of action of these xenobiotics will assist in improved molecular design with a view to providing safer compounds for use in the (human) environment.