Genotoxic effects of estrogens

Estrogens are associated with several cancers in humans and are known to induce tumors in rodents. In this review a mechanism of carcinogenesis by estrogens is discussed which features the following key events: (1) Steroid estrogens are metabolized by estrogen 2- and 4-hydroxylases to catecholestrogens. Target organs of estrogen-induced carcinogenesis, hamster kidney or mouse uterus, contain high levels of estrogen 4-hydroxylase activity. Since the methylation of 4-hydroxyestradiol by catechol-O-methyltransferase is inhibited by 2-hydroxyestradiol, it is proposed that a build up of 4-hydroxyestrogens precedes estrogen-induced cancer. (2) The catecholestrogen or diethylstilbestrol (DES) are oxidized to semiquinones and quinones by the peroxidatic activity of cytochrome P-450. The quinones are proposed to be (the) reactive intermediates of estrogen metabolism. (3) The quinones may be reduced to catecholestrogens and DES and redox cycling may ensue. Redox cycling of estrogens has been shown to generate free radicals which may react to form the organic hydroperoxides needed as cofactors for oxidation to quinones. (4) The quinone metabolites of catechol estrogens and of DES bind covalently to DNA in vitro whereas DNA binding in vivo has only been examined for DES. When DES is administered to hamsters, the resulting DES-DNA adduct profile in liver, kidney, or other organs closely matches that of DES quinone-DNA adducts in vitro. In vitro, DES-DNA adducts are chemically unstable and are generated in incubations with organic hydroperoxide as cofactor. It is proposed that the instability of adducts and the lower sensitivity of previous assay methods contributed to the reported failures to detect adducts. Steroid estrogen-DNA adducts in vivo are currently under investigation. (5) Tumors are postulated to arise in cells rapidly proliferating due to the growth stimulus provided by the estrogenic activity of the primary estrogen or of hormonally potent metabolites such as 4-hydroxyestradiol. The covalent modification of DNA in these cells is temporary because of the chemical instability of adducts and will result in altered genetic messages in daughter cells, whereas in non-proliferating cells there may be no lasting genetic damage. The sequence of events described above is a plausible mechanism for tumor initiation by estrogens and is partially substantiated by experimental evidence obtained in vitro and/or in vivo.     

Urinary steroid evaluations to monitor ovarian function in exotic ungulates: IV. Estrogen metabolism in the okapi (Okapia johnstoni)

In this investigation, an attempt was made to identify the major urinary estrogen metabolites in the okapi by radioisotope infusion and by chromatographic separation procedures. The results suggest that the urinary estrogens are excreted in concentrations below the limit of sensitivity of conventional assay systems. In addition, a variety of contaminants with estrogenic immunoreactivity are present in okapi urine that are not necessarily correlated with follicular activity. A great proportion of circulating estrogen is eliminated in fecal material. However, the rate of elimination is slow, and the extraction procedures necessary to detect the metabolites are tedious, which would prevent the practical application of this procedure for monitoring follicular activity.     

Biochemical and computational insights into the anti-aromatase activity of natural catechol estrogens

High levels of endogenous estrogens are associated with increased risks of breast cancer. Estrogen levels are mainly increased by the activity of the aromatase enzyme and reduced by oxidative/conjugative metabolic pathways. In this paper, we demonstrate for the first time that catechol estrogen metabolites are potent aromatase inhibitors, thus establishing a link between aromatase activity and the processes involved in estrogen metabolism. In particular, the anti-aromatase activity of a set of natural hydroxyl and methoxyl estrogen metabolites was investigated using biochemical methods and subsequently compared with the anti-aromatase potency of estradiol and two reference aromatase inhibitors. Catechol estrogens proved to be strong inhibitors with an anti-aromatase potency two orders of magnitude higher than estradiol. A competitive inhibition mechanism was found for the most potent molecule, 2-hydroxyestradiol (2-OHE(2)) and a rational model identifying the interaction determinants of the metabolites with the enzyme is proposed based on ab initio quantum-mechanical calculations. A strong relationship between activity and electrostatic properties was found for catechol estrogens. Moreover, our results suggest that natural catechol estrogens may be involved in the control mechanisms of estrogen production.     

Estrogen carcinogenesis in Syrian hamster tissues: role of metabolism

Evidence for a role of estrogen metabolism in hormonal carcinogenesis was obtained with the Syrian hamster as an in vivo model system. Both natural and synthetic estrogens are capable of inducing a high incidence of renal carcinomas in this species. A high incidence of hepatocellular carcinomas can also be induced in the hamster with synthetic estrogens such as ethinyl estradiol or diethylstilbestrol, provided alpha-naphthoflavone (ANF) is present in the diet. Although steroid receptor-mediated hormonal events appear to be intimately involved in the process of in vivo cell transformation of both tissues, certain observations strongly suggest that nonhormonal events are also important. Despite their potent estrogenic activity at the doses used, ethinyl estradiol and alpha-zearalanol induce relatively low renal tumor incidences after 9.0 and 10.0 months of continuous treatment, respectively. A role for the metabolism of estrogens to reactive intermediates is also suggested by studies showing estrogen-induced renal tumorigenesis can be partially inhibited by concomitant administration of ANF or ascorbic acid. Consistent with this is the general correlation between the amount of catechol estrogen formed by a compound, as mediated by estrogen 2-/4-hydroxylase, and renal carcinogenicity data. Recently, additional supporting evidence has been obtained from studies involving the irreversible binding of reactive metabolites of steroidal or stilbene estrogens to hamster liver microsomal proteins.     

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.     

Removal of natural and xeno-estrogens during conventional wastewater treatment

The ecological impacts of natural estrogens and xenoestrogens in treated wastewater include altered sexual development and sex ratios among continuously exposed organisms. The primary sources of estrogenic activity in wastewater are natural estrogens such as estrone, 17β-estradiol and estriol and synthetic compounds like 17α-ethinylestradiol, alkylphenols and alklphenol ethoxylates. Precursors in raw wastewater can yield estrogenic intermediates during wastewater treatment. All these compounds can be destroyed by biochemical processes, albeit at significantly different rates or under different conditions. That is, estrogenic compounds can be, but are not always, destroyed by conventional wastewater treatment processes, suggesting that conventional processes can be optimized for removal of estrogenic activity from wastewater. Sorption to sludges derived from wastewater treatment affects the fates of hydrophobic xenoestrogens such as nonylphenol, in part because the biodegradability of sorbed contaminants is limited. It may also be possible to tailor sludge stabilization processes to remove trace contaminants, including estrogens. For example, there are significant differences in the efficiencies of aerobic and anaerobic digestion for destruction of alkylphenols and probably other estrogenic compounds with aromatic moieties. Because advanced wastewater treatment is not economically feasible for most communities, there is ample incentive to develop accurate relationships between operational parameters and removal of estrogenic compounds during secondary wastewater treatment.     

Effects of 2-fluoroestradiol on lipid metabolism in the ovariectomized rat

Biological activities of estrogen molecules are altered by fluorination of ring A, and the resulting impairment to form catechols. 2-fluoroestradiol (2-F-E2) has been found to be devoid of carcinogenic action despite its high estrogenic potency; its metabolic effects are so far unknown. This study was designed to investigate the effects of 2-F-E2 on lipid metabolism, as compared to those of estradiol-17 beta(E2). Ovariectomized rats received E2 or 2-F-E2 by s.c. injection at a dose of 60 micrograms for three consecutive days. Parameters measured were weights of parametrial fat depots, fat cell volumes, levels of triacylglycerol and acylcholesterol in plasma, and enzymatic responses to the estrogens in isolated parametrial fat cells as evaluated in terms of lipoprotein lipase (LPL) and hormone-sensitive lipase (HSL) activities. 2-F-E2 and E2 were found to produce comparable decreases in fat depots, cell volumes and plasma levels of acylcholesterol whereas plasma triacylglycerol was unchanged. Both estrogens decreased LPL, and increased HSL activities to the same extent. Thus, 2-F-E and E2 exhibited comparable effects on lipid metabolism. These effects appeared to depend mainly on the estrogenic potency of these molecules, and to be distinct from their carcinogenic action. Despite its high estrogenic potency, 2-F-E2 was found to be slightly less estrogenic than E2.     

Sexually dimorphic basal water absorption at the isolated pelvic patch of Japanese tree frog, Hyla japonica

Frogs ingest little water orally, but absorb the majority of the water needed for normal physiological performance through a specific region of the ventral skin, the pelvic patch. We observed non-stimulated (basal water absorption, BWA) water flux through the isolated pelvic patch in vitro in Japanese tree frog (Hyla japonica). We found that water flux through non-stimulated skin from the pelvic patch was greater in males than females. This water flux was confirmed as BWA by observing no effect following the in vitro administration of propranolol and [adamantaneacetyl(1), O-Et-D-Tyr(2), Val(1), aminobutyryl(6), Arg(8, 9)] vasopressin, which are a beta-adrenergic receptor antagonist and a vasopressin V2 receptor antagonist, respectively. We further examined this phenomenon following gonadectomy, treatment with sex hormones (E2, 17beta-estradiol; TP, testosterone propionate), estrogenic chemicals (BPA, bisphenol A; MTX, methoxychlor) or prolactin (PRL, a hormone regulated by sex hormones that has osmoregulatory activity). Ovariectomy increased BWA in females. Injection (in vivo treatment) of E2 or PRL reduced BWA in males, whereas TP injection increased BWA in females. However, the in vitro addition of E2, TP, or PRL to the Ringer's solution on the serosal side of the ventral skin patch did not alter BWA. Subsequently, we injected (in vivo treatment) BPA or MTX, environmental chemical contaminants with known hormonal actions in mammals. Injection of BPA or MTX reduced BWA in males as observed following treatment with E2. These results provide the first evidence of sexual dimorphism in BWA through the isolated pelvic patch. The gonad appears essential for observed sexual dimorphism in BWA, and we hypothesize that sex hormones regulate the release of PRL, that in turn influences BWA indirectly. E2 is known to exert a specific stimulatory effect on PRL secretion. In addition, we have observed that some endocrine disrupting contaminants also eliminate the sexual dimorphism in BWA observed in the Japanese tree frog.     

The pharmacognosy of Humulus lupulus L. (hops) with an emphasis on estrogenic properties

As the population ages, there is an ever-increasing need for therapeutic agents that can be used safely and efficaciously to manage symptoms related to postmenopausal estrogen deficiency. Endogenous estrogens, e.g., 17beta-estradiol, of exogenous mammalian origin, e.g., horses, have long been used to manage such symptoms. There are more than 20 different classes of phytochemicals that have demonstrated affinity for human estrogen receptors in vitro. Some studies on exogenous estrogenic substances of botanical origin (phytoestrogens), such as standardized formulations of plant extracts with in vitro and in vivo estrogenic activity from soy (Glycine max Merill.) and red clover (Trifolium pratense L.), suggest clinical efficacy. Few clinical data for phytoestrogens other than isoflavonoids are available. In an exhaustive review of the literature through 2003, only two clinical trials were identified that were designed to evaluate the effect of hops (Humulus lupulus L.) on symptoms related to menopause. Folkloric, chemical, and biological literature relating primarily to the use of hops for their estrogenic activity, and two human clinical trials, are reviewed.     

Correlation of substance P-induced desensitization with substance P amino terminal metabolites in the mouse spinal cord.

Intrathecal injection of mice with substance P (SP) or its C-terminal fragments results in a behavioral syndrome characterized by reciprocal caudally directed biting and scratching. Repeated injection of SP, but not SP C-terminal fragments, results in a decrease in the intensity of, or desensitization to, these SP-induced behaviors. Peptidase inhibitors, phosphoramidon (PH), bacitracin (BAC), diprotin A (DPA) and angiotensin converting enzyme inhibitor (ACEI OR SQ20881), together with [3H]SP, were used to investigate the possible accumulation of tritiated N-terminal metabolites in the mouse spinal cord in vivo during the development of desensitization to SP. SP N-terminal metabolites in the spinal cord were quantified by reverse-phase HPLC. The magnitude of SP-induced desensitization correlated well (r = .95) with total SP N-terminal metabolites recovered from the spinal cords of the same mice studied in vivo. The magnitude of SP-induced desensitization was also found to be negatively correlated (r = .95) with total recovered intact [3H]SP. The rank order of potency of the peptidase inhibitors in decreasing the magnitude of SP-induced desensitization was BAC = PH much greater than ACEI greater than DPA. The order of potency for in vitro inhibition of SP metabolism using synaptic membrane-derived peptidases was BAC greater than PH much greater than ACEI. These results support the hypothesis that desensitization to SP-induced behaviors depends, at least in part, on the concentration of SP N-terminal metabolites in the spinal cord.     

Design,synthesis, and biological evaluation of novel estradiol–bisphosphonate conjugates as bone-specific estrogens

Bone deficiency causes osteoporosis and often decreases quality of life in patients with rheumatoid arthritis. Estrogens are known to protect elderly women from bone loss. Synthesis of new estradiol–bisphosphonate conjugates (E₂–BPs) was accomplished and their in vivo activity as bone-specific estrogens were examined. Among them, MCC-565 showed selective estrogenic activity in bones; but it showed little estrogenic activity in the uterus. We also found that the linker moiety in E₂–BPs was essential for the absorption and specificity of the conjugates.     

Drug metabolism by cultured human hepatocytes: how far are we from the in vivo reality?

The investigation of metabolism is an important milestone in the course of drug development. Drug metabolism is a determinant of drug pharmacokinetics variability in human beings. Fundamental to this are phenotypic differences, as well as genotypic differences, in the expression of the enzymes involved in drug metabolism. Genotypic variability is easy to identify by means of polymerase chain reaction-based or DNA chip-based methods, whereas phenotypic variability requires direct measurement of enzyme activities in liver, or, indirectly, measurement of the rate of metabolism of a given compound in vivo. There is a great deal of phenotypic variability in human beings, only a minor part being attributable to gene polymorphisms. Thus, enzyme activity measurements in a series of human livers, as well as in vivo studies with human volunteers, show that phenotypic variability is, by far, much greater than genotypic variability. In vitro models are currently used to investigate the hepatic metabolism of new compounds. Cultured human hepatocytes are considered to be the closest model to the human liver. However, the fact that hepatocytes are placed in a microenvironment that differs from that of the cells in the liver raises the question of to what extent drug metabolism variability observed in vitro actually reflects that in the liver in vivo. This issue has been examined by investigating the metabolism of the model compound, aceclofenac (an approved analgesic/anti-inflammatory drug), both in vitro and in vivo. Hepatocytes isolated from programmed liver biopsies were incubated with aceclofenac, and the metabolites formed were investigated by HPLC. The patients were given the drug during the course of clinical recovery, and the metabolites, largely present in urine, were analysed. In vitro and in vivo data from the same individual were compared. There was a good correlation between the in vitro and in vivo relative abundance of oxidised metabolites (4'-OH-aceclofenac + 4'-OH-diclofenac; Spearman's rho = 0.855), and the hydrolysis of aceclofenac (diclofenac + 4'-OH-aceclofenac + 4'-OH-diclofenac; rho = 0.691), while the conjugation of the drug in vitro was somewhat lower than in vivo. Globally, the metabolism of aceclofenac in vitro correlated with the amount of metabolites excreted in urine after 16 hours (rho = 0.95). Overall, although differing among assays, the in vitro/in vivo metabolism data for each patient were surprisingly similar. Thus, the variability observed in vitro appears to reflect genuine phenotypic variability among the donors.     

In vivo estrogenic and antiestrogenic activity of phenolphthalein and derivative compounds

The estrogenic activity of phenolphthalein and other related triphenylmethane dyes was evaluated in vivo in the immature rat uterus. Phenolphthalein behaved as a partial agonist of estradiol in stimulating the growth of rat uterus. Other specific estrogenic effects of the dye included an increase of the uterine DNA content, histological changes and induction of estrogen-modulated secretory proteins. The progressive introduction of side chains in the triphenylmethane skeleton concomitantly decreased the estrogenic activity. Triphenylmethanes competed with [3H]estradiol for the binding to the estrogen receptor in vitro, the relative binding affinity being correlated with the estrogenic potency observed in vivo. Phenolphthalein also showed antiestrogenic activity that could be overcome by increasing the dose of estradiol.     

Synthesis of a specific protein induced by zearalenone and its derivatives in rat uterus

Zearalenone and its derivatives (alpha-zearalenol and alpha-zearalanol), estrogenic mycotoxins produced by Fusarium species, when added in vivo and in vitro to immature rat uteri, induced the incorporation of labeled amino acids into a specific uterine protein (induced protein). When immature rat uteri were incubated with alpha-zearalenol in vitro, the maximum induction of the induced protein synthesis was obtained with 1 x 10(-6) M and the induction was detected 15 min after the start of the incubation. Moreover, this induction was strongly inhibited by prior addition of inhibitors of RNA synthesis such as alpha-amanitin and actinomycin D. The molecular weight of the induced protein obtained by the in vivo and in vitro treatments with zearalenone and alpha-zearalenol was estimated to be about 52,000 by means of SDS-polyacrylamide gel electrophoresis. These findings clearly indicate that these estrogenic mycotoxins, despite their non-steroidal structures, exhibit an estrogenic activity toward target tissues in a similar manner to that of natural estrogens.     

Carcinogenicity of catechol estrogens in Syrian hamsters

Estradiol and other estrogens induce renal carcinoma in male Syrian hamsters. The mechanism of carcinogenesis still remains unclear. Activation of estrogens to catechol metabolites has in the past been postulated to play a role in estrogen-induced carcinogenesis. Therefore, the carcinogenic activity of catechol estrogens was investigated. After 175 days of treatment, 4-hydroxyestradiol was found to be as carcinogenic as estradiol in male Syrian hamsters (4/5 and 4/5 animals with kidney tumors, respectively). Animals treated with 2-hydroxyestradiol (0/5) or 2-methoxyestradiol (0/6) did not develop renal carcinoma. The catechol estrogens failed to be mutagenic in the Ames test (reversions of his- S. typhimurium to histidine prototrophy in the TA 100 strain). The lack of carcinogenic activity of 2-hydroxyestradiol was not due to a failure to stimulate estrogen-dependent tumor growth. Growth of H-301 cells, an estrogen-dependent hamster kidney tumor cell line, was supported in vivo by estrogens in the following order: estradiol greater than 4-hydroxyestradiol greater than 2-hydroxyestradiol. Stimulation of tumor growth by 2-methoxyestradiol was not detected. It was concluded that the carcinogenic activity of 4-hydroxyestradiol was consistent with a role of catechol metabolites in estrogen-induced carcinogenesis. However, the intrinsic carcinogenic or hormonal activity of 2-hydroxyestradiol probably can not be assessed accurately in vivo because of its rapid methylation and metabolic clearance.     

Metabolism of estrogens in breast cancer.

This extensive literature compilation reviews major studies on estrogen metabolism in cancer, studies which have led to proposed possible etiological roles of estrogens in human breast cancer. Urinary and plasma estrogen excretion patterns and profiles in women with breast cancer are the topics of part 1. Studies of estrogen profiles in women who are at high-risk for breast cancer are critiqued. The estriol hypothesis is presented and criticised in a chapter. The effects of endocrine ablation on urinary estrogen profiles in breast cancer patients are compiled. Production and metabolism of estrogens in women with breast cancer are rendered, including in vivo biotransformation rates and in vitro transformation data. And the search for estrogen metabolites in women with breast cancer is reviewed. In conclusion it is obvious that the question of whether breast cancer patients have an abnormal metabolism of estrogen has not been answered, but further investigations of estrogen metabolism in breast cancer should be continued because: 1) the possibility that estrogens are carcinogenic has not been ruled out; 2) receptors have been discovered which do correlate with hormone dependency of tumors; 3) present evidence suggests that neoplasm may induce abnormal estrogen metabolism; 4) directional changes of estrogen metabolism that occur in pregnancy may also occur in women with target tissue neoplasia; 5) hepatic tissue's relationship to breast cancer has not received attention; and 6) the role of peripheral aromatization in the pathogenesis of mammary cancer is not yet understood.     

Plasma uptake and in vivo metabolism of [Leu]enkephalin following its intraperitoneal administration to rats

To understand better how [Leu]enkephalin (LE) acts to modulate learning and memory in rats, the plasma uptake, disappearance, and metabolism of LE were investigated following its intraperitoneal administration. Concentrations of [3H]-LE and its radioactive metabolites were determined by thin layer chromatography in plasma samples withdrawn from rats at various times after injection of peptide. As measured in rats receiving an IP injection of a dose of LE (3 micrograms/kg) that impairs active avoidance conditioning, the LE was very rapidly metabolized, with greater than 95% of plasma [3H] in the form of metabolites by 1 min after injection. Despite this rapid metabolism, low but measurable quantities of intact LE were detectable in plasma at all sampling times. Consistent with a greater potency of D-Ala2-[D-Leu5]enkephalin (DADLE) than of LE in modulating avoidance conditioning, DADLE was less rapidly metabolized than was LE following its IP administration. The metabolism of DADLE and LE in vivo was more rapid than it was in plasma in vitro, suggesting a role for membrane bound enzymes in the metabolism of IP-administered enkephalins. The data demonstrate that, despite a rapid hydrolysis of LE in vivo, sufficient LE is present in plasma following IP administration of a behaviorally active dose to support a role of circulating intact LE in the modulation of avoidance conditioning.