Uterotrophic Activity Research Articles

Comments on "In Vitro and in Vivo Estrogenicity of UV Screens"

LetterOpen AccessComments on "In vitro and in vivo estrogenicity of UV screens". H M Bolt, C Guhe, and G H Degen H M Bolt Search for more papers by this author , C Guhe Search for more papers by this author , and G H Degen Search for more papers by this author Published:1 August 2001https://doi.org/10.1289/ehp.109-a358Cited by:18AboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InReddit FiguresReferencesRelatedDetailsCited by Bora N, Mazumder B and Chattopadhyay P (2017) Prospects of topical protection from ultraviolet radiation exposure: a critical review on the juxtaposition of the benefits and risks involved with the use of chemoprotective agents, Journal of Dermatological Treatment, 10.1080/09546634.2017.1364691, 29:3, (256-268), Online publication date: 3-Apr-2018. Gilbert E, Roussel L, Serre C, Sandouk R, Salmon D, Kirilov P, Haftek M, Falson F and Pirot F (2016) Percutaneous absorption of benzophenone-3 loaded lipid nanoparticles and polymeric nanocapsules: A comparative study, International Journal of Pharmaceutics, 10.1016/j.ijpharm.2016.03.018, 504:1-2, (48-58), Online publication date: 1-May-2016. Gilbert E, Pirot F, Bertholle V, Roussel L, Falson F and Padois K (2013) Commonly used UV filter toxicity on biological functions: review of last decade studies, International Journal of Cosmetic Science, 10.1111/ics.12030, 35:3, (208-219), Online publication date: 1-Jun-2013. Sambandan D and Ratner D (2011) Sunscreens: An overview and update, Journal of the American Academy of Dermatology, 10.1016/j.jaad.2010.01.005, 64:4, (748-758), Online publication date: 1-Apr-2011. Díaz-Cruz M and Barceló D (2009) Chemical analysis and ecotoxicological effects of organic UV-absorbing compounds in aquatic ecosystems, TrAC Trends in Analytical Chemistry, 10.1016/j.trac.2009.03.010, 28:6, (708-717), Online publication date: 1-Jun-2009. Lautenschlager S, Wulf H and Pittelkow M (2007) Photoprotection, The Lancet, 10.1016/S0140-6736(07)60638-2, 370:9586, (528-537), Online publication date: 1-Aug-2007. Morohoshi K, Yamamoto H, Kamata R, Shiraishi F, Koda T and Morita M (2005) Estrogenic activity of 37 components of commercial sunscreen lotions evaluated by in vitro assays, Toxicology in Vitro, 10.1016/j.tiv.2005.01.004, 19:4, (457-469), Online publication date: 1-Jun-2005. Kullavanijaya P and Lim H (2005) Photoprotection, Journal of the American Academy of Dermatology, 10.1016/j.jaad.2004.07.063, 52:6, (937-958), Online publication date: 1-Jun-2005. Pauwels M and Rogiers V (2004) Safety evaluation of cosmetics in the EU, Toxicology Letters, 10.1016/j.toxlet.2004.01.026, 151:1, (7-17), Online publication date: 1-Jun-2004. Ashby J, Tinwell H, Odum J and Lefevre P (2018) Natural variability and the influence of concurrent control values on the detection and interpretation of low-dose or weak endocrine toxicities., Environmental Health Perspectives, 112:8, (847-853), Online publication date: 1-Jun-2004. Mueller S, Kling M, Arifin Firzani P, Mecky A, Duranti E, Shields-Botella J, Delansorne R, Broschard T and Kramer P (2003) Activation of estrogen receptor α and ERβ by 4-methylbenzylidene-camphor in human and rat cells: comparison with phyto- and xenoestrogens, Toxicology Letters, 10.1016/S0378-4274(03)00016-X, 142:1-2, (89-101), Online publication date: 1-Apr-2003. Degen G, Janning P, Wittsiepe J, Upmeier A and Bolt H (2002) Integration of mechanistic data in the toxicological evaluation of endocrine modulators, Toxicology Letters, 10.1016/S0378-4274(01)00504-5, 127:1-3, (225-237), Online publication date: 1-Feb-2002. Degen G, Janning P, Diel P and Bolt H (2002) Estrogenic isoflavones in rodent diets, Toxicology Letters, 10.1016/S0378-4274(02)00009-7, 128:1-3, (145-157), Online publication date: 1-Mar-2002. Tinwell H, Lefevre P, Moffat G, Burns A, Odum J, Spurway T, Orphanides G and Ashby J (2018) Confirmation of uterotrophic activity of 3-(4-methylbenzylidine)camphor in the immature rat., Environmental Health Perspectives, 110:5, (533-536), Online publication date: 1-May-2002.Sudakin D (2018) Comments on "Bioaerosol lung damage in a worker with repeated exposure to fungi in a water-damaged building"., Environmental Health Perspectives, 109:11, (A516-A517), Online publication date: 1-Nov-2001.Trout D (2018) Correspondence: Bioaerosol Lung Damage: Trout's Response, Environmental Health Perspectives, 109:11, (A516-A517), Online publication date: 1-Nov-2001.Zeger S (2018) Correspondence: Correction: Exposure Measurement Error in Time-Series Air Pollution Studies, Environmental Health Perspectives, 109:11, (A517-A517), Online publication date: 1-Nov-2001.Ashby J (2018) Correspondence: Confirmation of Uterotrophic Activity for 4-MBC in the Immature Rat, Environmental Health Perspectives, 109:11, (A517-A517), Online publication date: 1-Nov-2001. Vol. 109, No. 8 August 2001Metrics About Article Metrics Publication History Originally published1 August 2001Published in print1 August 2001 Financial disclosuresPDF download License information EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted. Note to readers with disabilities EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact [email protected]. Our staff will work with you to assess and meet your accessibility needs within 3 working days.

Effect of rodent diets on the sexual development of the rat.

Five rodent diets have been evaluated for their possible effect on the sexual development of the rat. Groups of 12 pregnant Alpk rats were fed one of the following combinations of diets during pregnancy and postnatally: RM3/RM1, AIN-76A/AIN-76A, RM3/AIN-76A, Teklad Global 2016 (Global)/Global and Purina 5001/Purina 5001. AIN-76A is phytoestrogen-free while the other diets contained varying amounts of phytoestrogens. The phytoestrogens genistein and daidzein were determined in the diets studied, and the concentrations found agreed with earlier estimates. RM3/RM1 was selected as the control group, as this has been used routinely in this laboratory for the past decade. Determinations were made in offspring of the times of vaginal opening and first estrus among the females, and of prepuce separation and testes descent among the males. At postnatal day (PND) 26 the females from 6 of the 12 litters were terminated and tissue weights measured. Males from 6 of the 12 litters were similarly studied at PND 68. Animals from the remaining litters were transferred to RM1 diet at PND 70. Termination of the study was at PND 128 (males) and PND 140 (females) when body weights and tissue weights were determined. Marked differences in body weight, sexual development, and reproductive tissue weights were observed for rats maintained on AIN-76A or Purina 5001, with only minimal effects among rats maintained on the Global diet. These comparisons were against RM3/RM1 as the reference diet. However, using Purina 5001 as the reference diet reversed the direction of the differences seen when using RM3/RM1 as the reference diet. The differences observed when using RM3/RM1 as reference diet occurred mainly postnatally. In addition, the fact that similar differences were seen for the phytoestrogen-free diet, AIN-76A, and the phytoestrogen-rich diet, Purina 5001, indicate that these effects are more likely to be caused by nutritional differences between the diets that then have centrally mediated effects on rodent sexual development, rather than individual dietary components affecting peripheral estrogen receptors (ER). This proposal is supported by abolition of the uterotrophic activity of AIN-76A and Purina 5001 (relative to RM3/RM1) in the immature rat by coadministration of the gonadotrophin-releasing hormone (GnRH) antagonist ANTARELIX: The present data indicate that choice of diet may influence the timing of sexual development in the rat, and consequently, that when evaluating the potential endocrine toxicity of chemicals, the components of rodent diets used should be known, and as far as is possible, controlled.

Synthesis and biological evaluation of some novel 4 H-benzopyran-4-one derivatives as nonsteroidal antiestrogens

The preparation and characterization of some novel 2- and 3-substituted-7-methoxy-4 H-1-benzopyran-4-one are presented. The synthesized compounds were evaluated for their uterotrophic, antiuterotrophic and antiimplantation activities in mature female albino rats. 3-Benzyl-7-methoxy-4 H-1-benzopyran-4-one ( 14) showed the highest uterotrophic activity (87%) based on dry uterine weight gain. The antifertility activity, as assessed by the post-coital antiimplantation activity test, was of weak potency for most compounds (14–29%). Among the products, the 2-(4′-methoxyphenyl)-7-methoxy-4 H-1-benzopyran-4-one ( 19) exhibited the highest antiestrogenic activity of 65%. It also elicited 31% of the uterotrophic activity of estradiol.

Dose-dependent bone-sparing effects of dietary isoflavones in the ovariectomised rat.

The dose-dependent bone-sparing effects of dietary isoflavones (IF) were investigated in adult (7-month-old) Wistar rats. Forty animals were ovariectomised, allocated into four groups of ten rats each, and immediately treated orally with IF at 0 (OVX), 20 (IF20), 40 (IF40) or 80 (IF80) microg/g body weight per d for 91 d; ten sham-operated (SH) controls received the same diet without added IF. Animals were killed on day 91. Both femoral failure load and total femoral, diaphyseal or metaphyseal bone mineral densities (BMD) were lower in OVX animals than in SH animals. Urinary deoxypyridinoline (DPD) excretion, a marker of bone resorption, and plasma osteocalcin (OC) levels, a marker of osteoblast activity, were higher in OVX animals than in SH animals. Total femoral and diaphyseal BMD and femoral failure load were similar in IF-treated rats and SH rats. Although metaphyseal BMD in IF40 or IF80 rats was similar to that in SH rats, its value was lower in IF20 rats than in controls. The day 91 urinary DPD excretion in IF40 and IF80 rats, but not in IF20 rats, was similar to that in SH rats. Day 91 plasma OC concentrations in IF-treated rats were similar to day 45 values, but were decreased in OVX and SH rats. Thus, daily IF consumption prevented ovariectomy-induced bone loss, both by depressing bone resorption and stimulating osteoblast activity. Moreover, as only the highest IF level induced a weak uterotrophic activity, the optimal IF dose which preserves both cancellous and cortical bone, but exhibits no oestrogen-like effects on the uterus, was 40 microg/g body weight per d.

The uterotrophic activity of nonylphenol in the rat is not mediated by aromatase enzyme induction

p-Nonylphenol (NP) is weakly estrogenic to rodents and to some species of fish. All evidence to date has indicated that the estrogenic effects of NP are due to the interaction of NP with the estrogen receptor. Recent findings of increased plasma estradiol in fish exposed to NP have, however, led to the proposal of an alternative mechanism for NP-induced estrogenicity in this species, possibly via induction of aromatase enzymes. In the present studies, this hypothesis was investigated in rats using the aromatase inhibitor anastrozole. The results indicated that the uterotrophic action of NP, as with estradiol used as a positive control, is mediated directly by its interaction with uterine ER, rather than an indirect effect via aromatase enzyme induction. Circulating levels of estradiol were unchanged after NP treatment and the aromatase inhibitor anastrozole failed to inhibit NP-induced uterine growth. These results are consistent with previous published data on NP in rodents.

Rutin inhibits ovariectomy-induced osteopenia in rats.

Several studies suggest that polyphenols might exert a protective effect against osteopenia. The present experiment was conducted to observe the effects of rutin (quercetin-3-O-glucose rhamnose) on bone metabolism in ovariectomized (OVX) rats. Thirty 3-month-old Wistar rats were used. Twenty were OVX while the 10 controls were sham-operated (SH). Among the 20 OVX, for 90 days after surgery 10 were fed the same synthetic diet as the SH or OVX ones, but 0. 25% rutin (OVX + R) was added. At necropsy, the decrease in uterine weight was not different in OVX and OVX + R rats. Ovariectomy also induced a significant decrease in both total and distal metaphyseal femoral mineral density, which was prevented by rutin consumption. Moreover, femoral failure load, which was not different in OVX and SH rats, was even higher in OVX + R rats than in OVX or SH rats. In the same way, on day 90, both urinary deoxypyridinoline (DPD) excretion (a marker for bone resorption) and calciuria were higher in OVX rats than in OVX + R or SH rats. Simultaneously, plasma osteocalcin (OC) concentration (a marker for osteoblastic activity) was higher in OVX + R rats than in SH rats. High-performance liquid chromatography (HPLC) profiles of plasma samples from OVX + R rats revealed that mean plasma concentration of active metabolites (quercetin and isorhamnetin) from rutin was 9.46+/-1 microM, whereas it was undetectable in SH and OVX rats. These results indicate that rutin (and/or its metabolites), which appeared devoid of any uterotrophic activity, inhibits ovariectomy-induced trabecular bone loss in rats, both by slowing down resorption and increasing osteoblastic activity.

Effects of estradiol and estradiol sulfamate on the uterus of ovariectomized or ovariectomized and hypophysectomized rats

Estradiol sulfamate (J995), estradiol-17β with a substituted sulfamate group in position 3, has much higher systemic estrogenic activity after oral administration than 17β-estradiol (E2) due to reduced hepatic metabolism of the drug. The lower dose necessary for achievement of adequate systemic estrogenic effects results in a substantial reduction of otherwise commonly observed hepatic side-effects. This makes J995 a strong candidate as an estrogen suitable for oral administration. The present study was performed to examine and compare the effects of J995 and E2 on the uterus after oral or subcutaneous administration to ovariectomized or ovariectomized+hypophysectomized female rats, in particular on the levels of the estrogen receptor (ER) (α+β), ERα mRNA and insulin-like growth factor-I (IGF-I) mRNA. The ER levels were determined using a ligand binding assay and the mRNA levels using solution hybridization. The doses of J995 or E2 were chosen to achieve comparable uterotrophic activity. The rats were treated with hormones for 7 days and the treatment was initiated 14 days after surgery. We conclude that there are no major differences in the uterine response to treatment with J995 or E2 with respect to the effects on ER and ERα mRNA levels. The IGF-I mRNA level though, is more affected by J995 than by E2 after 7 days of treatment, indicating a prolonged effect of J995.

Uterotrophic Activity of Bisphenol A in the Immature Mouse

Bisphenol A (BPA) has been evaluated in eight independent immature mouse uterotrophic assays using the subcutaneous route of administration, and in a single study employing oral gavage. The dose range covered was from 0.02 μg to 300 mg/kg BPA and some experiments were supplemented by assessments of uterine hypertrophy and hyperplasia. Pooling of the test data indicates no uterotrophic activity for the chemical. However, in a subset of the subcutaneous injection studies, where control uterine weights were relatively low, significant, but weak, uterotrophic activity was observed over a range of dose levels, but in the complete absence of a dose relationship. In the oral gavage study, no increases in uterine weight were seen, but there were increases in uterine labeling with bromodeoxyuridine at 200–300 mg/kg BPA. The present study illustrated that when a large number of observations are made, a certain level of chance observations may be made, and that surrogates for an increase in uterine weight do not necessarily enhance assay sensitivity, albeit such data may complement uterine weight data. The data indicate that reducing control uterine weights may enhance assay sensitivity, but that animal body weight is an imperfect indicator of control uterine weight. The data also show that it is possible for individual investigators to be unable to confirm their own observations. It is concluded that BPA may be weakly uterotrophic to the mouse under specific conditions of test, and in the complete absence of a dose–response relationship to this activity. However, overall, we have failed to define BPA as reproducibly active in the immature mouse uterotrophic assay, and in that sense, our data are broadly consistent with those reported earlier by Coldham et al. (Environ. Health Perspect. 105, 734–742, 1997) in 1997 using a similar assay.

Estradiol-Type Activity of Coumestrol in Mature and Immature Ovariectomized Rat Uterotrophic Assays

Makaverich et al. [Environ Health Perspect 103:574-581 (1995)] reported that the uterotrophic activity of the phytoestrogen coumestrol in the immature ovariectomized rat was atypical in that it was not associated with increased uterine hyperplasia and DNA content. We previously reported that coumestrol gave a typical estradiol-type uterotrophic response in the immature intact rat, yielding increases in uterine epithelial cell height, glandular formation, cell labeling, and DNA content. These papers did not answer the question of whether there is a basic difference between the ovariectomized and the intact rat uterotrophic assays. In this paper, we report that coumestrol gives a typical estradiol-type uterotrophic response in uterotrophic assays using immature intact, immature ovariectomized, and mature ovariectomized rats. We concluded that the uterotrophic activity of coumestrol is typical of the natural estrogen estradiol.

Estradiol-type activity of coumestrol in mature and immature ovariectomized rat uterotrophic assays.

Makaverich et al. [Environ Health Perspect 103:574-581 (1995)] reported that the uterotrophic activity of the phytoestrogen coumestrol in the immature ovariectomized rat was atypical in that it was not associated with increased uterine hyperplasia and DNA content. We previously reported that coumestrol gave a typical estradiol-type uterotrophic response in the immature intact rat, yielding increases in uterine epithelial cell height, glandular formation, cell labeling, and DNA content. These papers did not answer the question of whether there is a basic difference between the ovariectomized and the intact rat uterotrophic assays. In this paper, we report that coumestrol gives a typical estradiol-type uterotrophic response in uterotrophic assays using immature intact, immature ovariectomized, and mature ovariectomized rats. We concluded that the uterotrophic activity of coumestrol is typical of the natural estrogen estradiol.

Immature Rat Uterotrophic Assay of Diethylstilbestrol, Ethynyl Estradiol and Atrazine.

The purpose of this study was to decide the appropriate administration period in uterotrophic assay and to evaluate the usefulness of uterotrophic assay using two typical estrogen agonists and one antagonist, when chemicals were given orally. Diethylstilbestrol (DES), ethynyl estradiol (EE), or Atrazine (ATZ) was administered to immature Crj: CD (SD) IGS rats orally at doses of 0.01-1 μg/kg/day, 0.06-6 μg/kg/day, or 0.5-50 mg/kg/day from postnatal day (PND) 21 for 3 or 7 days, respectively. Additional groups of rats given ATZ orally at the above doses were also treated with 17β-estradiol (E2) subcutaneously at a dose of 0.3 μg/rat/day. A vehicle control group given only olive oil was established in each study, and a group given only E2 was included in the study of ATZ also. No abnormal clinical signs were detected in any of the groups. There were no significant differences in body weight between the vehicle control and each of the groups given DES, EE or ATZ, or the group given only E2 and each of the groups given ATZ plus E2. Premature vaginal opening was observed from 6 days in all rats given 6 μg/kg EE or ATZ plus E2. Uterine fluid was also present in all rats given 6 μg/kg EE or ATZ plus E2 for 3 or 7 days. Uterine weight was significantly higher in the rats given 1 μg/kg DES for 3 days and 6 μg/kg EE for 3 or 7 days, but no increases were detected in any of the rats given DES for 7 days. Although uterotrophic activity was not detected in any of the rats given only ATZ, co-treatment with 50 mg/kg ATZ and E2 for 3 days reduced the E2-induced increases in uterine weight. The present results demonstrated that the appropriate period of oral administration for detecting the estrogenic properties of DES and EE or anti-estrogenic properties of ATZ using the immature rat uterotrophic assay was 3 days and ATZ exerted weak anti-estrogenic effects on the uterus.

Uterotrophic Activity of a "Phytoestrogen-Free" Rat Diet

LetterOpen AccessUterotrophic activity of a "phytoestrogen-free" rat diet. J Ashby, H Tinwell, and J Odum J Ashby Search for more papers by this author , H Tinwell Search for more papers by this author , and J Odum Search for more papers by this author Published:1 January 2000https://doi.org/10.1289/ehp.108-a12cCited by:12AboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InReddit "Uterotrophic activity of a "phytoestrogen-free" rat diet.." Environmental Health Perspectives, 108(1), pp. A12–A13FiguresReferencesRelatedDetailsCited by Prongay K, Lewis A, Hurn P and Murphy S (2010) Dietary soy may not confound acute experimental stroke infarct volume outcomes in ovariectomized female rats, Laboratory Animals, 10.1258/la.2009.009031, 44:3, (238-246), Online publication date: 1-Jul-2010. Charles G, Gennings C, Tornesi B, Kan H, Zacharewski T, Bhaskar Gollapudi B and Carney E (2007) Analysis of the interaction of phytoestrogens and synthetic chemicals: An in vitro/in vivo comparison, Toxicology and Applied Pharmacology, 10.1016/j.taap.2006.11.029, 218:3, (280-288), Online publication date: 1-Feb-2007. Thigpen J, Setchell K, Padilla-Banks E, Haseman J, Saunders H, Caviness G, Kissling G, Grant M and Forsythe D (2007) Variations in Phytoestrogen Content between Different Mill Dates of the Same Diet Produces Significant Differences in the Time of Vaginal Opening in CD-1 Mice and F344 Rats but Not in CD Sprague-Dawley Rats, Environmental Health Perspectives, 115:12, (1717-1726), Online publication date: 1-Dec-2007. Masutomi N, Shibutani M, Takagi H, Uneyama C, Takahashi N and Hirose M (2003) Impact of dietary exposure to methoxychlor, genistein, or diisononyl phthalate during the perinatal period on the development of the rat endocrine/reproductive systems in later life, Toxicology, 10.1016/S0300-483X(03)00269-5, 192:2-3, (149-170), Online publication date: 1-Nov-2003. Wade M, Lee A, McMahon A, Cooke G and Curran I (2003) The influence of dietary isoflavone on the uterotrophic response in juvenile rats, Food and Chemical Toxicology, 10.1016/S0278-6915(03)00168-6, 41:11, (1517-1525), Online publication date: 1-Nov-2003. Owens W, Ashby J, Odum J and Onyon L (2003) The OECD program to validate the rat uterotrophic bioassay. Phase 2: dietary phytoestrogen analyses., Environmental Health Perspectives, 111:12, (1559-1567), Online publication date: 1-Sep-2003.Ashby J, Owens W, Odum J and Tinwell H (2003) The intact immature rodent uterotrophic bioassay: possible effects on assay sensitivity of vomeronasal signals from male rodents and strain differences., Environmental Health Perspectives, 111:12, (1568-1570), Online publication date: 1-Sep-2003. Degen G, Janning P, Diel P and Bolt H (2002) Estrogenic isoflavones in rodent diets, Toxicology Letters, 10.1016/S0378-4274(02)00009-7, 128:1-3, (145-157), Online publication date: 1-Mar-2002. Barlow S, Greig J, Bridges J, Carere A, Carpy A, Galli C, Kleiner J, Knudsen I, Koëter H, Levy L, Madsen C, Mayer S, Narbonne J, Pfannkuch F, Prodanchuk M, Smith M and Steinberg P (2002) Hazard identification by methods of animal-based toxicology, Food and Chemical Toxicology, 10.1016/S0278-6915(01)00117-X, 40:2-3, (145-191), Online publication date: 1-Feb-2002. Thigpen J, Haseman J, Saunders H, Locklear J, Caviness G, Grant M and Forsythe D (2002) Dietary factors affecting uterine weights of immature CD-1 mice used in uterotrophic bioassays, Cancer Detection and Prevention, 10.1016/S0361-090X(02)00122-8, 26:5, (381-393), Online publication date: 1-Nov-2002. Ashby J, Tinwell H and Odum J (2001) DNA adducts, estrogenicity and rodent diets, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 10.1016/S0027-5107(01)00224-X, 483:1-2, (105-106), Online publication date: 1-Nov-2001. Ashby J, Tinwell H, Odum J, Kimber I, Brooks A, Pate I and Boyle C (2000) Diet and the aetiology of temporal advances in human and rodent sexual development, Journal of Applied Toxicology, 10.1002/1099-1263(200009/10)20:5<343::AID-JAT719>3.0.CO;2-O, 20:5, (343-347), Online publication date: 1-Sep-2000. Vol. 108, No. 1 January 2000Metrics About Article Metrics Publication History Originally published1 January 2000Published in print1 January 2000 Financial disclosuresPDF download License information EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted. Note to readers with disabilities EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact [email protected]. Our staff will work with you to assess and meet your accessibility needs within 3 working days.

AHTN and HHCB show weak estrogenic — but no uterotrophic activity

The ubiquitous presence of the polycyclic musks AHTN (6-acetyl-1,1,2,4,4,7-hexamethyltetraline) and HHCB (1,2,4,6,7,8-hexahydro-4,6,6,7,8-hexamethylcyclopenta-γ-2-benzopyreen) in surface waters and their identification in human milk fat together with their polycyclic nature, which makes them potential candidates for interference with estrogen receptors, prompted us to assess these compounds for their potential estrogenic effects. We therefore investigated the effects of AHTN and HHCB in ERα- and ERβ-dependent gene transcription assays with Human Embryonal Kidney 293 (HEK293) cells, which have proven to be very suitable to estimate the estrogenic activity of compounds with low binding activity (Kuiper, G.G., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., Van der Saag, P.T., Van der Burg, B., Gustafsson, J.Å., 1998. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor β. Endocrinology 139, 4252–4264). Both AHTN and HHCB were found to induce a slight but dose-dependent stimulation of transcriptional activity in the transiently ERα transfected HEK293 cells. This weak estrogenic response was not observed in the ERβ transiently transfected cells. However, these cells were less responsive to estradiol than the ERα transfected HEK293 cells. Also, no significant increase in transcriptional activity was observed in HEK293 cell lines, permanently expressing the same estrogen-responsive reporter gene construct and either ERα or ERβ. In the classical uterine weight assay performed in juvenile Balb/c mice, no uterotrophic activity of AHTN and HHCB was noted at relatively high dietary exposure levels up to 50 and 300 ppm, respectively, at which levels an increase in liver weight was evident. Also the vitellogenin production by carp hepatocytes, a sensitive marker of estrogenic activity, was not affected by these two fragrance materials (Smeets, J.M.W., Rouhani Rankouhi, T., Nichols, K.M., Komen, H., Kaminsky, N.E., Giesy, J.P., Van den Berg, M., 1999. In vitro vitellogenin production by carp ( Cyprimus carpio) hepatocytes as a screening method for determining (anti-) estrogenic activity of xenobiotics. Toxicol. Appl. Pharmacol., 157, 68–76). Therefore it is concluded that these compounds have very weak estrogenic potency, too weak to induce estrogenic effects in wildlife species or humans at the current levels of exposure. These results give further support to the promiscuity of estrogen receptors.

Induction of hyperplasia and increased DNA content in the uterus of immature rats exposed to coumestrol.

Administration of the phytoestrogen coumestrol to ovariectomized rats leads to increases in both wet and dry uterine weights in the absence of an increase in uterine DNA content, as reported by Markaverich et al. [Effects of Coumestrol on Estrogen Receptor Function and Uterine Growth in Ovariectomized Rats. Environ Health Perspect 103:574-581 (1995)]. It was not possible to know if the observed atypical uterotrophic response of coumestrol was associated uniquely with the ovariectomized uterotrophic assay protocol. This question is answered in the present paper. Two experiments are described in which three daily oral gavage administrations of 60 mg/kg/day coumestrol to immature AP rats were followed by assessment of the reproductive tract on the fourth day. In both experiments coumestrol increased uterine fluid content and increased the weights of the uterus, cervix, and vagina. In addition, bromodeoxyuridine staining of uterine sections enabled confirmation of uterine hyperplasia for the coumestrol-treated animals. In the second experiment, total uterine DNA was determined; it doubled in the coumestrol-treated animals. Estradiol benzoate acted as the positive control agent for both of these experiments, and in each case it gave similar responses to those seen for coumestrol. We conclude that the uterotrophic activity of the phytoestrogen coumestrol in the immature intact rat is typical of the activity of the natural estrogen estradiol.

Short-term tests of estrogenic potential of plant stanols and plant stanol esters.

To test for potential estrogenic activity of plant stanols and plant stanol esters, two short-term tests were performed. These were the E-screen test, which measures a substance's ability to induce proliferation of estrogen-responsive human breast adenocarcinoma (MCF-7) cells in culture, and an in vivo test, which measures uterotrophic activity in immature female rats fed the test substance. Four samples of vegetable oil-derived stanols (containing 88-99% stanols) were tested in the E-screen test, and one sample of wood-derived and one of vegetable oil-derived stanol fatty acid esters were tested in the in vivo test. In the E-screen test, the positive control substance, 17beta-estradiol, at 100 pM, produced a statistically significant, 11.6-fold increase in cell proliferation, as measured by sulforhodamine B staining. None of the stanol preparations produced any increase in cell proliferation when tested at 1, 10, and 100 microM. The highest dose of each stanol sample was associated with microscopic evidence of cytotoxicity and crystalline precipitation in the culture dishes. In the in vivo test, the positive control compound, diethylstilbestrol, produced a significant, dose-related increase in absolute and relative uterus weight in young female rats (17 days old at the start of treatment) fed the compound at 5, 10, and 20 ppb in the diet for 4 days. Neither of the two stanol ester preparations caused any significant change in absolute or relative uterus weight when fed at a concentration of 8.3% in the diet for 4 days. Thus, under the conditions of testing used, neither the free stanols nor the stanol fatty acid ester preparations showed evidence of estrogenic or uterotrophic activity.

The pharmacological profile of a novel norpregnane progestin (trimegestone)

Trimegestone is a novel norpregnane progestin that is being developed, in combination with estradiol, for the treatment of menopausal symptoms. The pharmacological characteristics of trimegestone have been evaluated in both in vitro and in vivo test systems, and compared with reference progestins. Interaction with hormonal steroid receptors from animal tissues and with human recombinant receptors in vitro has demonstrated that trimegestone has a high specificity and potency for the progesterone receptor, no affinity for the estrogen receptor, and weak affinity for androgen, glucocorticoid and mineralocorticoid receptors. With respect to progestomimetic activity in vivo, trimegestone was more potent than reference progestins in the endometrial transformation test in the rabbit, preventing the uterotrophic effect of estradiol in the immature mouse bioassay, and had more effect on traumatic deciduoma formation and greater oral antiovulatory activity in the rat. In vivo, trimegestone effectively maintained pregnancy in the rat, but was devoid of any uterotrophic activity. Trimegestone showed no androgenic, glucocorticoid, antiglucocorticoid or mineralocorticoid activity, but did show some antiandrogenic and antimineralocorticoid activity at higher doses. Administration of trimegestone to ovariectomized rats, in combination with estradiol, inhibited the uterotrophic effects of estradiol. At doses up to 1 mg/kg intravenously and 30 mg/kg orally, trimegestone was not associated with any unwanted pharmacological effects. Overall, the results show trimegestone to have a favorable pharmacological profile with potent progestomimetic activity.

Idoxifene: a novel selective estrogen receptor modulator prevents bone loss and lowers cholesterol levels in ovariectomized rats and decreases uterine weight in intact rats.

Idoxifene, a novel selective estrogen receptor modulator, was tested for its effects on bone loss, serum cholesterol, and uterine wet weight and histology in the ovariectomized (Ovx) rat. Idoxifene (0.5 mg/kg x day) completely prevented loss of both lumbar and proximal tibial bone mineral density (BMD). In an intervention study, idoxifene (0.5 and 2.5 mg/kg x day) completely prevented further loss of both lumbar and proximal tibial BMD during a 2-month treatment period commencing 1 month after surgery, when significant loss of BMD had occurred in the Ovx control group. Idoxifene reduced total serum cholesterol, which was maximal at 0.5 mg/kg x day. Idoxifene alone displayed minimal uterotrophic activity in Ovx rats and inhibited the agonist activity of estrogen in intact rats. Histologically, myometrial and endometrial atrophy were observed in both idoxifene and vehicle-treated Ovx rats. In this report, we also provide molecular-based evidence to support the observations in vivo of a novel selective estrogen receptor modulator (SERM) mechanism of action in bone and endometrial cells. Idoxifene is an agonist through the estrogen response element (ERE) and exhibits similar postreceptor effects to estrogen in bone-forming osteoblasts. Idoxifene also stimulates osteoclast apoptosis, and these pleiotropic effects ultimately could contribute to the maintenance of bone homeostasis. However, idoxifene differs from estrogen in a tissue-specific manner. In human endometrial cells, where estrogen is a potent agonist through the ERE, idoxifene has negligible agonist activity. Moreover, idoxifene was able to block estrogen induced gene expression in endometrial cells, which is in agreement with the observation in the intact rat study. In the uterus, idoxifene has a pharmacologically favorable profile, lacking agonist and therefore growth-promoting activity. Together with its cholesterol lowering effect and lack of uterotrophic activity, these data suggest that idoxifene may be effective in the prevention of osteoporosis and other postmenopausal diseases without producing unwanted estrogenic effects on the endometrium.

Uterotrophic activity of bisphenol A in the immature rat.

Bisphenol A (BPA) is active in immature AP rat uterotrophic assays when evaluated using either the oral or the subcutaneous (sc) injection routes of exposure (three daily administrations of 400-800 mg/kg BPA). Premature vaginal opening was seen for 8 of 14 animals exposed to 600 and 800 mg/kg BPA by sc injection. Vaginal opening was not produced by BPA in the gavage studies. These results are consistent with those of Dodds and Lawson [Nature 137:96 (1936)] who found that BPA induces persistent vaginal cornification in ovariectomized rats exposed to three twice-daily injections of 85 mg/kg BPA (total daily dose 170 mg/kg), but they conflict with the reported inactivity of BPA in the immature mouse uterotrophic assay. The uterotrophic activity of diethylstilbestrol in the rat is also established (0.04 mg/kg/day for three days).

Uterotrophic Activity of Bisphenol A in the Immature Rat

Bisphenol A (BPA) is active in immature AP rat uterotrophic assays when evaluated using either the oral or the subcutaneous (sc) injection routes of exposure (three daily administrations of 400-800 mg/kg BPA). Premature vaginal opening was seen for 8 of 14 animals exposed to 600 and 800 mg/kg BPA by sc injection. Vaginal opening was not produced by BPA in the gavage studies. These results are consistent with those of Dodds and Lawson [Nature 137:96 (1936)] who found that BPA induces persistent vaginal cornification in ovariectomized rats exposed to three twice-daily injections of 85 mg/kg BPA (total daily dose 170 mg/kg), but they conflict with the reported inactivity of BPA in the immature mouse uterotrophic assay. The uterotrophic activity of diethylstilbestrol in the rat is also established (0.04 mg/kg/day for three days).

Estrogen Response Elements Can Mediate Agonist Activity of Anti-estrogens in Human Endometrial Ishikawa Cells

Anti-estrogens like hydroxytamoxifen (OHT) have mixed agonist/antagonist activities, leading to tissue-specific stimulation of cellular proliferation. Partial agonist activity of OHT can be observed in vitro in endometrial carcinoma cells like Ishikawa. Here, we have compared several anti-estrogens (including extensively characterized OHT and pure anti-estrogens such as ICI164, 384 and RU58,668, which are devoid of uterotrophic activity) for their capacity to stimulate promoters containing estrogen response elements (EREs) or AP1-binding sites (12-O-tetradecanoylphorbol-13-acetate response elements, TREs), the two types of DNA motifs known to mediate transcriptional stimulation by estrogen receptors. Assays were performed in Ishikawa cells either by transient transfection or by using cell lines with stably propagated reporter vectors. In transient transfection experiments, none of the anti-estrogens displayed agonist activity on the promoters tested. In contrast, significant transcriptional stimulation was observed with low concentrations of OHT and RU39,411 in Ishikawa cells stably propagating reporter constructs containing a minimal ERE3-TATA promoter. In addition, micromolar concentrations of OHT, but not of RU39,411, stimulated stably propagated AP1-responsive reporter constructs. No transcriptional stimulation of ERE- or TRE-containing promoters was observed with the pure anti-estrogens ICI164,384 and RU58,668. These results indicate that the presence of estrogen response elements in promoters is sufficient to mediate cell-specific agonism of anti-estrogens at the transcriptional level, and that stimulation of AP1 activity may be restricted to a subset of anti-estrogens possessing agonist activity on EREs. In addition, our results suggest that transient transfections do not fully recapitulate in vivo conditions required to observe agonist activity of anti-estrogens.