Biotransformation Of Estrogens Research Articles

Seasonal variations of fluorescent dissolved organic matter control estrone biodegradation potential in eutrophic waters affected by allochthonous and autochthonous sources

The spatial–temporal variation of dissolved organic matter (DOM) influences contaminant biodegradation in natural water systems. This study explored seasonal patterns of DOM concentration and composition and the consequent effect on estrone (E1) biodegradation in water columns at the northern bays of Lake Taihu. During bloom seasons, the increased concentration of autochthonous DOM (autoDOM) with higher Fresh/Humic ratios, lower aromatics, molecular weights, and humification degrees exhibited a more substantial promotion on E1 biodegradation than the processed autoDOM after algal bloom. Meanwhile, the inflow rivers imported high-molecular-weight humic-like substances with strong aromaticity in flood seasons, impairing the E1 biodegradation of affected regions. End-member mixing experiments further confirmed that the mixing of algal-derived DOM facilitated the mediation of river water-derived DOM in E1 biodegradation, but microbial ageing reduced the promotive effect of autoDOM and resulted in a weak relationship between DOM source indicators and E1 biodegradation. A significant and positive correlation was further found between E1 biodegradation and DOM bioreactivity (P < 0.001), both of which were well predicted by DOM optical properties as predictor variables in partial least squares regression (PLS-R) modelling (R2 values of 0.78 and 0.90, respectively). The ratio of fluorescence peak T and C was identified as the most effective predictor, and SUVA254 and slope ratio (SR) were moderately influential factors controlling carbon-normalized E1 biodegradation potential. These results suggested that while algal blooms would likely cause enhanced biotransformation of estrogens in freshwater lakes, the import of allochthonous DOM or the microbially aged autoDOM with high-molecular-weight may lead to higher estrogen concentrations in post-bloom seasons.

Influence of breast cancer risk factors on proliferation and DNA damage in human breast glandular tissues: role of intracellular estrogen levels, oxidative stress and estrogen biotransformation

Breast cancer etiology is associated with both proliferation and DNA damage induced by estrogens. Breast cancer risk factors (BCRF) such as body mass index (BMI), smoking, and intake of estrogen-active drugs were recently shown to influence intratissue estrogen levels. Thus, the aim of the present study was to investigate the influence of BCRF on estrogen-induced proliferation and DNA damage in 41 well-characterized breast glandular tissues derived from women without breast cancer. Influence of intramammary estrogen levels and BCRF on estrogen receptor (ESR) activation, ESR-related proliferation (indicated by levels of marker transcripts), oxidative stress (indicated by levels of GCLC transcript and oxidative derivatives of cholesterol), and levels of transcripts encoding enzymes involved in estrogen biotransformation was identified by multiple linear regression models. Metabolic fluxes to adducts of estrogens with DNA (E-DNA) were assessed by a metabolic network model (MNM) which was validated by comparison of calculated fluxes with data on methoxylated and glucuronidated estrogens determined by GC– and UHPLC–MS/MS. Intratissue estrogen levels significantly influenced ESR activation and fluxes to E-DNA within the MNM. Likewise, all BCRF directly and/or indirectly influenced ESR activation, proliferation, and key flux constraints influencing E-DNA (i.e., levels of estrogens, CYP1B1, SULT1A1, SULT1A2, and GSTP1). However, no unambiguous total effect of BCRF on proliferation became apparent. Furthermore, BMI was the only BCRF to indeed influence fluxes to E-DNA (via congruent adverse influence on levels of estrogens, CYP1B1 and SULT1A2).

Development and validation of LC-ESI-MS/MS methods for quantification of 27 free and conjugated estrogen-related metabolites

An imbalance in the estrogen metabolism has been associated with an increased risk of breast cancer development. Evaluation of the estrogen biotransformation capacity requires monitoring of various estrogen metabolites. Up to now, only some estrogen metabolites could be measured in urine. However, in order to offer tailor made nutritional support or therapies, a complete estrogen metabolite profile is required in order to identify specific deficiencies in this pathway for each patient individually. Here, we focused on this need to quantify as many as possible of the estrogen-related metabolites excreted in urine. The method was developed to quantify 27 estrogen-related metabolites in small urine quantities. This entailed sample clean-up with a multi-step solid phase extraction procedure, derivatisation of the metabolites in the less water-soluble fraction through dansylation, and analyses using liquid chromatography–tandem mass spectrometry (LC-MS/MS). The metabolites accurately quantified by the method devised included parent estrogens, hydroxylated and methylated forms, metabolites of the 16α-hydroxyestrogen pathway, sulphate and glucuronide conjugated forms, precursors and a related steroid hormone. This method was validated and enabled quantification in the high picograms and low nanograms per millilitre range. Finally, analyses of urine samples confirmed detection and quantification of each of the metabolites.

Qualitative and quantitative differences in estrogen biotransformation in human breast glandular and adipose tissues: implications for studies using mammary biospecimens

Because of its assumed role in breast cancer etiology, estrogen biotransformation (and interaction of compounds therewith) has been investigated in human biospecimens for decades. However, little attention has been paid to the well-known fact that large inter-individual variations exist in the proportion of breast glandular (GLT) and adipose (ADT) tissues and less to adequate tissue characterization. To assess the relevance of this, the present study compares estrogen biotransformation in GLT and ADT. GLT and ADT were isolated from 47 reduction mammoplasty specimens derived from women without breast cancer and were characterized histologically and by their percentages of oil. Levels of 12 unconjugated and five conjugated estrogens were analyzed by GC- and UHPLC-MS/MS, respectively, and levels of 27 transcripts encoding proteins involved in estrogen biotransformation by Taqman® probe-based PCR. Unexpectedly, one-third of specimens provided neat GLT only after cryosection. Whereas 17β-estradiol, estrone, and estrone-3-sulfate were detected in both tissues, estrone-3-glucuronide and 2-methoxy-estrone were detected predominately in GLT and ADT, respectively. Estrogen levels as well as ratios 17β-estradiol/estrone and estrone-3-sulfate/estrone differed significantly between GLT and ADT, yet less than between individuals. Furthermore, estrogen levels in GLT and ADT correlated significantly with each other. In contrast, levels of most transcripts encoding enzymes involved in biotransformation differed more than between individuals and did not correlate between ADT and GLT. Thus, mixed breast tissues (and plasma) will not provide meaningful information on local estrogen biotransformation (and interaction of compounds therewith) whereas relative changes in 17β-estradiol levels may be investigated in the more abundant ADT.

Administration of low dose triclosan to pregnant ewes results in placental uptake and reduced estradiol sulfotransferase activity in fetal liver and placenta

Sulfonation is a major pathway of estrogen biotransformation with a role in regulating estrogen homeostasis in humans and sheep. Previous in vitro studies found that triclosan is an especially potent competitive inhibitor of ovine placental estrogen sulfotransferase, with Kic of <0.1 nM. As the placenta is the main organ responsible for estrogen synthesis in pregnancy in both women and sheep, and the liver is another site of estrogen biotransformation, this study examined the effects of triclosan exposure of pregnant ewes on placental and hepatic sulfotransferase activity. Triclosan, 0.1 mg/kg/day, or saline vehicle was administered to late gestation fetal sheep for two days either by direct infusion into the fetal circulation or infusion into the maternal blood. On the third day, fetal liver and placenta were harvested and analyzed for triclosan and for cytosolic estradiol sulfotransferase activity. Placenta contained higher concentrations of triclosan than liver in each individual sheep in both treatment groups. There was a negative correlation between triclosan tissue concentration (pmol/g tissue) and cytosolic sulfotransferase activity (pmol/min/mg protein) towards estradiol. These findings demonstrated that in the sheep exposed to very low concentrations of triclosan, this substance is taken up into placenta and reduces estrogen sulfonation.

Derivatization-free LC-MS/MS method for estrogen quantification in mouse brain highlights a local metabolic regulation after oral versus subcutaneous administration.

17β-Estradiol (17β-E2) is a steroid with pleiotropic actions. In addition to being a sexual hormone, it is also produced in the brain where it modulates the reproductive axis. It has been shown that 17β-E2 also acts on synaptic plasticity and plays a role in neurological pathways and in neurodegenerative diseases. Assaying this steroid in the brain is thus interesting to improve our knowledge of 17β-E2 effects in the brain. However, 17β-E2 concentration in the central nervous system has been reported to be of a few nanograms per gram wet weight (nanomolar range concentration); therefore, its quantification requires both an efficient extraction process and a sensitive detection method. Herein is presented a derivatization-free procedure based on solid-phase extraction followed by LC-MS/MS analysis, targeted on 17β-E2, its isomer17α-E2, and its metabolites estrone (E1) and estriol (E3). This extraction process allowed reaching 96% 17β-E2 recovery from the mouse brain. Limit of detection (LOD) and limit of quantification (LOQ) values of 0.5 and 2.5pmolmL-1, respectively, were reached for both 17α-E2 and 17β-E2. LOD values for E1 and E3 were 0.01 and 0.025pmolmL-1, respectively. The variation coefficients for intra- and inter-assays were 6 and 14%, respectively, for both estradiol forms. The method was applied to assess estrogen levels in the mouse brain and hippocampus after 17β-E2 acute (subcutaneous injection) and chronic (drinking water) physiological administration. Total estrogen levels were determined after enzymatic deconjugation and compared to free estrogen levels. While 17α-E2 was not detected in biological samples, 17β-E2 and metabolite measurements highlight a local biotransformation of estrogens after physiological administration via drinking water. Graphical abstract Method workflow: After oral or subcutaneous Estradiol administration, mouse brain or hippocampus was removed. Samples were homogenized and prepared according to a liquid-liquid extraction, followed by a solid-phase extraction. Then, LC-MS/MS was optimized to quantify 17ß-E2, its isomer17α-E2, its metabolites estrone (E1) and estriol (E3) and their conjugates.

The endocrine disrupting alkylphenols and 4,4′-DDT interfere with estrogen conversion and clearance by mouse liver cytosol

Endocrine disrupting chemicals (EDCs) are ubiquitous compounds known for negative impacts on reproductive functions and for increasing cancer risk. EDCs are believed to cause the harmful effects in part through their inappropriate low-affinity binding to steroid receptors and other possible non-receptor mediated paradigms, however there is a need to further elucidate other mechanisms involving the direct and indirect impact of EDCs on reproductive functions.We examined the metabolism of 17β-estradiol (E2) and estrone (E1) by cell-free hepatic cytosol in the presence of alkylphenols (nonylphenol/NP and 4-tert-octylphenol/tOP), Dichlorodiphenyltrichloroethane (4,4′-DDT) and other EDCs. Tandem liquid chromatography mass spectrometry was utilized to quantitatively assess the impact of each EDC on estrogen clearance, inter-conversions and downstream metabolism by mouse liver cytosol.The results revealed that NP and tOP (0.1–3μg/mL) significantly reduced the hepatic cytosol clearance and biotransformation of estrogens with inclination for accumulating E2, the stronger estrogen form, than E1. Alkylphenols also caused up to a 34-fold increase in the E2/E1 ratio possibly by suppressing the hepatic E2→E1 conversion by 17β-hydroxysteroid dehydrogenase (17βHSD) types 2, 4 while displaying a weaker inhibition of E1→E2 conversion by type 1, 17βHSD. On the other hand, the pesticide 4,4′-DDT was a weaker inhibitor of clearance of estrogens by the cytosol preparations when compared to alkylphenols, whereas chemicals such as phthalates and atrazine were ineffective.Our data suggest that exposure to NP, tOP and DDT can indirectly increase the estrogenic load by suppressing the hepatic clearance of estrogens and by elevating the E2/1 ratio and could therefore increase the risk of reproductive lesions.

Effects of azocyclotin on gene transcription and steroid metabolome of hypothalamic⿿pituitary⿿gonad axis, and their consequences on reproduction in zebrafish (Danio rerio)

The widely used organotins have the potential to disrupt the endocrine system, but little is known of underlying mechanisms of azocyclotin toxicity in fish. The objective of the present study was to investigate the impact of azocyclotin on reproduction in zebrafish. Adult zebrafish were exposed to 0.09 and 0.45μg/L azocyclotin for 21days, and effects on steroid hormones and mRNA expression of the genes belonging to the hypothalamic⿿pituitary⿿gonad (HPG) axis were investigated. Mass spectrometry methodology was developed to profile steroids within the metabolome of the gonads. They were disrupted as a result of azocyclotin exposure. Alterations in the expression of key genes associated with reproductive endocrine pathways in the pituitary (lhβ), gonad (cyp19a1a, cyp17a1 and 17β-hsd3), and liver (vtg1, vtg2, cyp1a1, comt, ugt1a and gstp1) were correlated with significant reductions in estrogen in both sexes and increased testosterone in females. Azocyclotin-induced down-regulation of cyp19a1a in males suggested a reduction in the rate of estrogen biosynthesis, while up-regulation of hepatic cyp1a1 and comt in both sexes suggested an increase in estrogen biotransformation and clearance. Azocyclotin also induced change in the expression of 17β-hsd3, suggesting increased bioavailability of 11-ketotestosterone (11-KT) in the blood. Furthermore, the down-regulation of lhβ expression in the brains of azocyclotin-exposed fish was associated with inhibition of oocyte maturation in females and retarded spermatogenesis in males. As a histological finding, retarded development of the ovaries was found to be an important cause for decreased fecundity, with down-regulation of vtg suspected to be a likely underlying mechanism. Additionally, relatively high concentrations of azocyclotin in the gonads may have directly caused toxicity, thereby impairing gametogenesis and reproduction. Embryonic or larval abnormalities occurred in the F1 generation along with accumulated burdens of azocyclotin in F1 eggs, following parental exposure. Overall, our results indicate that exposure to azocyclotin can impair reproduction in fish, and induce toxicity related abnormalities in non-exposed offspring.

Correlation between prostate volume and single nucleotide polymorphisms implicated in the steroid pathway.

A few preliminary studies have suggested a link between some genetics variants and benign prostatic hyperplasia (BPH). Our goal was to study the link between a set of single nucleotide polymorphisms (SNPs) implicated in the steroid pathway and accurate measurement of prostate volume in a cohort of men who underwent radical prostatectomy. Clinical and pathological data including prostate weight were obtained from 611 Caucasian patients with small volume, localized prostate cancer treated by radical prostatectomy. Patients were genotyped for 90 SNPs located inside or nearby genes implicated in the steroid pathway (Sequenom iPLEX). Correlation between prostate weight and genotypes from each SNP was studied by analysis of covariance, adjusted on age and tumor stage. A Bonferroni correction was applied, and the SNPs implicated were then incorporated in a multivariable model. Seven SNPs located in or nearby genes implicated in steroid hormone metabolism were significantly associated with prostate volume: HSD17B2 (rs1119933), ESR2 (rs8006145), SULT2B1 (rs279451), NQO1 (rs2917670), ESR1 (rs1569788), GSTP1 (rs1138272), and CYP19A1 (rs17523880). Significant association was maintained after multivariate analysis for four SNPs, indicating their independent association with prostate volume. The power of the association of each SNP with prostate volume was comparable to the effect of age. The strongest associations were found with variants in ESR1, ESR2, HSD17B2, and CYP19A1 genes, indicating a potential role of the estrogen signaling pathway in genesis of BPH. Our results are in favor of an implication of estrogen biotransformation and signaling pathways in the pathophysiology of BPH.

Hypoxia disrupts gene modulation along the brain–pituitary–gonad (BPG)–liver axis

Hypoxia alters sex hormone concentrations leading to reproductive impairment in fish; however the mechanisms underlying these effects remain largely unknown. Using zebrafish (Danio rerio), this study is the first to demonstrate that hypoxia causes endocrine disruption by simultaneously acting on multiple targets along the brain–pituitary–gonadal (BPG)–liver axis in fish. Alterations in the expression of key genes associated with reproductive endocrine pathways in the brain (sGnRH), pituitary (FSHβ and LHβ), gonads (FSH-R, LH-R, HMGR, StAR, CYP19A, CYP11A, CYP11β and 20β-HSD), and liver were correlated with significant reductions of estradiol in females and testosterone in males. Hypoxia also induced sex-specific and tissue-specific changes in the expression of estrogen, androgen, and membrane progestin receptors along the BPG axis, suggesting disruption of the feedback and synchronization of hormone signals. Furthermore, the hypoxia-induced upregulation of hepatic sex hormone-binding globulin suggests an increase in hormone transport and reduced bioavailability in blood, while upregulation of hepatic CYP3A65 and CYP1A in females suggests an increase in estrogen biotransformation and clearance. Given that the regulation of reproductive hormones and the BPG–liver axis are highly conserved, this study provides new insights into the hypoxia-induced endocrine disrupting mechanisms and reproductive impairment in other vertebrates.

Biotransformation of Estrogens in Nitrifying Activated Sludge Under Aerobic and Alternating Anoxic/Aerobic Conditions

Natural and synthetic estrogens present in municipal wastewater can be biodegraded during treatment, particularly in activated sludge. The objective was to assess the extent of transformation of 17-beta-estradiol (E2) and 17-alpha-ethinylestradiol (EE2) by nitrifying activated sludge and evaluate potential relationships between availability of oxygen, nitrification rate, and estrogen removal. For each batch experiment, two reactors were set up--aerobic and alternating anoxic/aerobic-which were then amended with E2 and EE2 from methanolic stock solutions. The EE2 was persistent under anoxic conditions; under aerobic conditions, the observed level of its removal was 22%. The E2 was readily converted to estrone (El)--faster under aerobic (nitrifying) than anoxic (denitrifying) conditions. During the initial anoxic conditions, a metabolite consistent with 17-alpha-estradiol transiently accumulated and was subsequently removed when the reactor was aerated. Higher removal rates of estrogens were associated with higher nitrification rates, which supports the contention that the nitrifying biomass was responsible for their removal.

Impact of Serum Bilirubin on Human Diseases

Bilirubin, a major product of heme catabolism, belongs to compounds with pleiotropic biologic effects. Although for decades it was considered as a metabolite dangerous for human health, recent data indicate that bilirubin exhibits potent antioxidant properties with substantial positive clinical consequences. In a recent issue of Pediatrics , Sedlak and Snyder1 comprehensively reviewed available data on the beneficial role of bilirubin. Although the article covers current knowledge of all the aspects of this topic almost completely, I would like to mention more data that further support a salutary role of bilirubin in the human body. In addition to studies cited in the Sedlak and Snyder article, total serum antioxidant capacity was found to correlate with serum bilirubin levels in our study on adult Gilbert syndrome (GS) patients, and this relation was confirmed in an in vitro study.2 In regard to the cited bilirubin oxidation metabolite study in septic patients, recent studies (mainly from Japan) demonstrated close correlation between these markers (called biopyrrins) and many other pathologic conditions including ischemic heart disease,3,4 congestive heart failure,5 atopic dermatitis,6 and surgical7,8 or even psychological stress.9 The same relationship was also found … Address correspondence to Libor Vitek, MD, PhD, 4th Department of Internal Medicine and Institute of Clinical Biochemistry and Laboratory Diagnostics, 1st Medical Faculty, Charles University of Prague, U Nemocnice 2, Praha 2, 128 08, Czech Republic. E-mail: vitek{at}cesnet.cz

Human Liver Estrogen Sulfotransferase: Identification by cDNA Cloning and Expression

Sulfation is an important pathway in the biotransformation of steroid hormones such as estrogens. Human liver contains three well-characterized sulfotransferase (ST) enzymes, dehydroepiandrosterone (DHEA) ST and two forms of phenol sulfotransferase (PST). Although two of these enzymes, DHEA ST and one form of PST, can catalyze the sulfate conjugation of estrogens, our goal was to test the hypothesis that human liver might also contain a distinct estrogen ST (EST). We used the PCR to clone a human liver EST cDNA by utilizing degenerate oligonucleotide primers designed on the basis of highly conserved EST sequences in non-human species to amplify a 512 nucleotide portion of the open reading frame (ORF) with single-stranded human liver cDNA as a template. Rapid amplification of cDNA ends (RACE) was then used to obtain the 5′- and 3′-ends of the EST cDNA. The ORF of this cDNA consisted of 882 nucleotides and encoded 294 amino acids. The protein encoded by the human liver EST cDNA was 81%, 73%, and 72% identical to the amino acid sequences of guinea pig adrenocortical, bovine placental and rat liver ESTs, respectively. Human liver EST transiently expressed in COS-1 cells was capable of catalyzing the sulfation of estrone, DHEA and 4-nitrophenol, but not that of dopamine. The expressed human liver EST was also characterized with regard to thermal stability, inhibition by 2,6-dichloro-4-nitrophenol (DCNP) and response to NaCl. Identification of human liver EST by cloning and expression of its cDNA should enhance understanding of the enzymology and regulation of the biotransformation of estrogens and other steroids in humans.

Temporary decrease in renal quinone reductase activity induced by chronic administration of estradiol to male Syrian hamsters. Increased superoxide formation by redox cycling of estrogen.

Cytochrome P-450-mediated redox cycling between the synthetic estrogen diethylstilbestrol (DES) and diethylstilbestrol-4',4"-quinone (DES Q) has previously been demonstrated. Cytochrome P-450 reductase catalyzes the reduction of DES Q presumably via a semiquinone formed by one-electron reduction. A reducing action of NAD(P)H quinone reductase (EC 1.6.99.2) mediating two-electron reduction of DES Q has been investigated in the present work. Quinone reductase catalyzed the conversion in the presence of NADH or NADPH of DES Q to 53-65% Z-DES, a marker product of reduction. Dicumarol (15 microM), a known specific inhibitor of quinone reductase, inhibited this reduction almost completely. Using microsomes from Syrian hamster kidney, a target organ of estrogen-induced carcinogenesis, the reduction of DES Q was only partially inhibited by dicumarol. Apparent Km values of quinone reductase and cytochrome P-450 reductase were 17.25 and 11.9 microM, respectively. These data demonstrate that in hamster kidney, quinone reductase and cytochrome P-450 reductase compete for the reduction of DES Q. Microsomal 02-. radical generation was stimulated 10-fold over base levels by the addition of 100 microM DES Q. The formation of 02-. radicals was inhibited by addition of superoxide dismutase (0.2 mg/ml) or by 2'-AMP or NADP, known inhibitors of cytochrome P-450 reductase. In contrast, dicumarol enhanced microsome-mediated 02-. formation. It is concluded that cytochrome P-450 reductase in hamster kidney microsomes mediates one-electron reduction of estrogen quinones to free radicals (semiquinones), which may subsequently enter redox cycling with molecular oxygen to form 02-.. Moreover, quinone reductase reduces DES Q directly to E- and Z-DES, and thus may prevent the formation of toxic intermediates during redox cycling of estrogens. Measurements of quinone reductase activity in liver and kidney of hamsters treated with estrogen for various lengths of time revealed a temporary decrease in activity by 80% specifically in the kidney after 1 month of chronic treatment with estradiol. Thus, a temporary decrease in quinone reductase activity, which occurred specifically in estrogen-exposed hamster kidney, may enhance the formation of free radical intermediates generated during biotransformation of estrogens.

Aromatic hydroxylation of estrogens.

The metabolism of an endogenous steroid hormone has traditionally been viewed as a means of terminating its activity and facilitating its excretion. In recent years, however, examples have accumulated (18,50,76) in which such biotransformations have been shown to have activating rather than inactivating functions, placing the role of metabolism in the expression of hormonal action into a new context. The mammalian metabolism of the female sex hormone is almost exclusively oxidative in nature and is domi­ nated by three pathways, of which the largest and fastest is the oxidation of the 17 ,B-hydroxy function to the 17 ketone (23). This process in revers­ ible, but the equilibrium greatly favors the oxidized estrone form, which is the principal substrate for the other two main oxidative transformations, hydroxylation at the C-2 or C-16a positions of the molecule. The 16a-hydroxylative pathway, whose most noted product is estriol, was rec­ ognized early (59), but the hydroxylation at the C-2 position of the aromatic ring has been detected only relatively recently (52). This biotransformation of estrogens and the even more recently discovered hydroxylation at the isomeric C-4 position (83) have attracted much interest and have generated a substantial literature. The nature of these transformations and the biologi­ cal properties of their polyphenolic products, the catechol estrogens, are reviewed in this chapter. This review is selective. The reader seeking more detailed information is directed to an exhaustive survey of the field (6), which covers the available information through 1979.

Estradiol transformation in men with breast cancer.

The biotransformation of radioactive estradiol and estrone was examined in 10 studies of 6 men with breast cancer. Five normal healthy men of comparable age, 9 men with various cancers unrelated to either breast or endocrine system, 1 patient with heart disease and 1 patient with gynecomastia were similarly examined for the pattern of estradiol metabolism. Compared with the well or ill control subjects, the men with breast cancer exhibited sharp diminution in the formation of estrone, 2-hydroxyestrone and 2-methoxyestrone, accompanied by pronounced increase in the production of estriol. The results reported imply a relationship between estrogen biotransformation and an unusual, steroid hormone-related malignancy.