Estrogenic Isoflavones Research Articles

Soy Isoflavones Modify Liver Free Radical Scavenger Systems and Liver Parameters in Sprague–Dawley Rats

Soyfoods contain estrogenic isoflavones--namely, genistein (G) and daidzein (D)--that, like estrogens, display physiological effects in humans and animals. Previously we and others have demonstrated antioxidant and cardioprotective effects of orally ingested soy diets and soy isoflavones. The overall objective of this study was to test the effects of injected soy isoflavones, G and D, on liver lipids, liver free radical scavenger systems, and parameters of cardiovascular risk. Forty male rats were injected with G, D, estradiol (E), or a vehicle control (V) for 6 weeks. At the end of the study, body weight, food intake, feed efficiency ratio (FER), plasma glucose and cholesterol, abdominal fat pad weight, reproductive organ weight, liver weight, liver lipids, and liver free radical scavenger systems were compared. Food intake was significantly (P < .04) higher in the D-, G-, and E-treated animals compared with V-treated animals. FER was lower (P < .001) in D-, G-, and E-treated animals compared with the V- and the E-treated animals. Body weight, testis weight, and prostate weight were markedly (P < .001) lower in the E-treated animals compared with D-, G-, and V-treated animals. Intraabdominal fat pad weights were also significantly (P < .001) lower in the E group, although this effect was lost when corrected for body weight. Liver weights were considerably lower in the D-, G-, and E-treated animals versus the V group (P < .001). Total plasma cholesterol was reduced (P < .05) in D- and E-treated animals versus the V group. Liver lipids appeared to be unchanged by the isoflavones and slightly elevated by E treatment (P < .02). Liver catalase levels were numerically higher in the D- and E-treated animals compared with the V group (P < .1). Similarly, Cu/Zn superoxide dismutase (SOD) activity was significantly elevated in the D and E groups (P < .01), while G treatment (P < .03) elevated SOD to a lesser degree, versus the V group. These results suggest that subcutaneous injections of the naturally occurring soy isoflavone D and, to a lesser extent, G exert cardioprotective effects and stimulate antioxidant systems, while minimizing the undesirable effects elicited by E treatment.

Isoflavone, glyphosate, and aminomethylphosphonic acid levels in seeds of glyphosate-treated, glyphosate-resistant soybean.

The estrogenic isoflavones of soybeans and their glycosides are products of the shikimate pathway, the target pathway of glyphosate. This study tested the hypothesis that nonphytotoxic levels of glyphosate and other herbicides known to affect phenolic compound biosynthesis might influence levels of these nutraceutical compounds in glyphosate-resistant soybeans. The effects of glyphosate and other herbicides were determined on estrogenic isoflavones and shikimate in glyphosate-resistant soybeans from identical experiments conducted on different cultivars in Mississippi and Missouri. Four commonly used herbicide treatments were compared to a hand-weeded control. The herbicide treatments were (1) glyphosate at 1260 g/ha at 3 weeks after planting (WAP), followed by glyphosate at 840 g/ha at 6 WAP; (2) sulfentrazone at 168 g/ha plus chlorimuron at 34 g/ha applied preemergence (PRE), followed by glyphosate at 1260 g/ha at 6 WAP; (3) sulfentrazone at 168 g/ha plus chlorimuron at 34 g/ha applied PRE, followed by glyphosate at 1260 g/ha at full bloom; and (4) sulfentrazone at 168 g/ha plus chlorimuron at 34 g/ha applied PRE, followed by acifluorfen at 280 g/ha plus bentazon at 560 g/ha plus clethodim at 140 g/ha at 6 WAP. Soybeans were harvested at maturity, and seeds were analyzed for daidzein, daidzin, genistein, genistin, glycitin, glycitein, shikimate, glyphosate, and the glyphosate degradation product, aminomethylphosphonic acid (AMPA). There were no remarkable effects of any treatment on the contents of any of the biosynthetic compounds in soybean seed from either test site, indicating that early and later season applications of glyphosate have no effects on phytoestrogen levels in glyphosate-resistant soybeans. Glyphosate and AMPA residues were higher in seeds from treatment 3 than from the other two treatments in which glyphosate was used earlier. Intermediate levels were found in treatments 1 and 2. Low levels of glyphosate and AMPA were found in treatment 4 and a hand-weeded control, apparently due to herbicide drift.

Moderate intakes of intact soy protein rich in isoflavones compared with ethanol-extracted soy protein increase HDL but do not influence transforming growth factor β1 concentrations and hemostatic risk factors for coronary heart disease in healthy subjects

Soybeans contain estrogenic isoflavones that may influence plasma concentrations of transforming growth factor beta(1) (TGF-beta(1)) and plasma lipid and hemostatic risk factors for coronary heart disease. We compared the effects of moderate intakes of soy protein containing intact phytoestrogens (high-isoflavone diet) and soy protein from which most of the phytoestrogens had been extracted (low-isoflavone diet) on active TGF-beta(1) concentrations and plasma lipid and hemostatic risk factors for coronary heart disease. A randomized crossover trial was conducted in 22 young, healthy, normolipidemic subjects (5 men and 17 women) who consumed diets providing 56 or 2 mg isoflavones/d for 17 d each with a 25-d washout period between treatments. Fasting blood samples were obtained on days 13 and 14 of each treatment to measure plasma isoflavone, lipid, fibrinogen, and active TGF-beta(1) concentrations and factor VII coagulant and plasminogen activator inhibitor type 1 activities. Plasma isoflavone concentrations were 100-999 times greater after the high-isoflavone diet than after the low-isoflavone diet (P < 0.05). Plasma HDL-cholesterol and apolipoprotein A-I concentrations were 4% (95% CI: 1%, 8%) and 6% (95% CI: 3%, 10%) higher, respectively, after the high-isoflavone diet than after the low-isoflavone diet (P < 0.01 for both). Compared with soy protein from which most of the phytoestrogens have been extracted, soy protein with intact phytoestrogens increases HDL-cholesterol and apolipoprotein A-I concentrations but does not influence LDL-cholesterol, TGF-beta(1), or fibrinogen concentrations; factor VII coagulant activity; or plasminogen activator inhibitor type 1 activity in normolipidemic, healthy subjects.

The Phenotype of the Aromatase Knockout Mouse Reveals Dietary Phytoestrogens Impact Significantly on Testis Function

Estrogen is synthesized in the testis, both in Leydig cells and seminiferous epithelium, and its importance in spermatogenesis is highlighted by the phenotype of the aromatase knockout (ArKO) mouse. These mice are unable to synthesize endogenous estrogens. The males develop postmeiotic defects by 18 wk of age. We hypothesized that maintenance of spermatogenesis in younger animals may be mediated by exogenous estrogenic substances. Dietary soy meal, contained in almost all commercial rodent diets, provides a source of estrogenic isoflavones. We thus investigated spermatogenesis in wild-type and ArKO mice raised on a diet containing soy, compared with a soy-free diet, to elucidate the biological action of phytoestrogens on the testis. In ArKO mice, dietary phytoestrogens could partially prevent disruptions to spermatogenesis, in that they prevented the decline in germ cell numbers. They also seemed to maintain Sertoli cell function, and they blocked elevations in FSH. The impairment of spermatogenesis seen in soy-free ArKOs occurred in the absence of a decreased gonadotropic stimulus, suggesting that the effects of dietary phytoestrogens are independent of changes to the pituitary-gonadal axis. Our study highlights the importance of estrogen in spermatogenesis and shows that relatively low levels of dietary phytoestrogens have a biological effect in the testis.

The phenotype of the aromatase knockout mouse reveals dietary phytoestrogens impact significantly on testis function.

Estrogen is synthesized in the testis, both in Leydig cells and seminiferous epithelium, and its importance in spermatogenesis is highlighted by the phenotype of the aromatase knockout (ArKO) mouse. These mice are unable to synthesize endogenous estrogens. The males develop postmeiotic defects by 18 wk of age. We hypothesized that maintenance of spermatogenesis in younger animals may be mediated by exogenous estrogenic substances. Dietary soy meal, contained in almost all commercial rodent diets, provides a source of estrogenic isoflavones. We thus investigated spermatogenesis in wild-type and ArKO mice raised on a diet containing soy, compared with a soy-free diet, to elucidate the biological action of phytoestrogens on the testis. In ArKO mice, dietary phytoestrogens could partially prevent disruptions to spermatogenesis, in that they prevented the decline in germ cell numbers. They also seemed to maintain Sertoli cell function, and they blocked elevations in FSH. The impairment of spermatogenesis seen in soy-free ArKOs occurred in the absence of a decreased gonadotropic stimulus, suggesting that the effects of dietary phytoestrogens are independent of changes to the pituitary-gonadal axis. Our study highlights the importance of estrogen in spermatogenesis and shows that relatively low levels of dietary phytoestrogens have a biological effect in the testis.

Goitrogenic and estrogenic activity of soy isoflavones.

Soy is known to produce estrogenic isoflavones. Here, we briefly review the evidence for binding of isoflavones to the estrogen receptor, in vivo estrogenicity and developmental toxicity, and estrogen developmental carcinogenesis in rats. Genistein, the major soy isoflavone, also has a frank estrogenic effect in women. We then focus on evidence from animal and human studies suggesting a link between soy consumption and goiter, an activity independent of estrogenicity. Iodine deficiency greatly increases soy antithyroid effects, whereas iodine supplementation is protective. Thus, soy effects on the thyroid involve the critical relationship between iodine status and thyroid function. In rats consuming genistein-fortified diets, genistein was measured in the thyroid at levels that produced dose-dependent and significant inactivation of rat and human thyroid peroxidase (TPO) in vitro. Furthermore, rat TPO activity was dose-dependently reduced by up to 80%. Although these effects are clear and reproducible, other measures of thyroid function in vivo (serum levels of triiodothyronine, thyroxine, and thyroid-stimulating hormone; thyroid weight; and thyroid histopathology) were all normal. Additional factors appear necessary for soy to cause overt thyroid toxicity. These clearly include iodine deficiency but may also include additional soy components, other defects of hormone synthesis, or additional goitrogenic dietary factors. Although safety testing of natural products, including soy products, is not required, the possibility that widely consumed soy products may cause harm in the human population via either or both estrogenic and goitrogenic activities is of concern. Rigorous, high-quality experimental and human research into soy toxicity is the best way to address these concerns. Similar studies in wildlife populations are also appropriate.

Transplacental transfer of the phytoestrogen daidzein in DA/Han rats.

Disposition and transplacental transfer of the phytoestrogen daidzein was studied in pregnant DA/Han rats on day 18 of gestation. Daidzein concentrations were determined by HPLC in maternal blood, maternal organs (liver, kidney, uterus), placenta and fetuses (liver and residual tissues) at specific times (5, 10, 20, 40 and 120 min) after intravenous administration of 10 mg/kg body weight. Early after injection, the majority of circulating daidzein was still in the aglycone form; at later time points the majority consisted of conjugates. The initially high isoflavone concentration in maternal plasma (about 25 microg/ml at 5 min) decreased rapidly within the first hour, and after 2 h total daidzein was below 1 microg/ml. Despite its efficient conjugation, daidzein was rapidly distributed in the organism: peak concentrations were attained 10 min after intravenous administration in all tissues analysed, with mean values of about 31 microg/g in maternal liver, 13 microg/g in kidneys and 5 microg/g in the uterus. Placenta contained about one-tenth the hepatic daidzein concentration, and fetal liver about 1/30 the peak concentration of maternal liver (i.e. 1.3 microg/g, which is one-third the placental concentration). Daidzein levels in tissues then declined in parallel with those in maternal blood. The data show that daidzein is transferred across the placenta of DA/Han rats to fetuses. This is indicative of a rapid transfer from the mother to the fetus, but also that efficient hepatic extraction of daidzein from the maternal blood occurs. Since dietary phytoestrogens account for a significant proportion of human exposure to potential endocrine modulators, and since the placenta does not represent a barrier to daidzein or related estrogenic isoflavones, the consequences of these exposures early in life should be examined and monitored carefully.

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.

Optimization of a Yeast Estrogen Screen and Its Applicability to Study the Release of Estrogenic Isoflavones from a Soygerm Powder

Here we describe a redesigned protocol of the yeast estrogen screen developed by Routledge and Sumpter. The redesigned test comprises two steps. First, a large amount of yeast with estrogenic compounds is incubated for 24 hr. Subsequently, a mixture of cycloheximide and the chromogenic substrate chlorophenol red-beta-d-galactopyranoside (CPRG) is added. The cycloheximide stops protein synthesis and allows for an end-point measurement of beta-galactosidase activity generated during the first 24 hr. CPRG is converted to chlorophenol red and reflects beta-galactosidase activity, which is indicative of the estrogenic activity. The modifications shorten the duration of the assay at least 1 day and avoid interference of the estrogenic CPRG or chlorophenol red. The redesigned and the original protocol were used to study the estrogenic activity of bisphenol A, methoxychlor, p,p'-DDT, and isoflavones (genistein, daidzein, and glycitein). Bisphenol A, methoxychlor, and genistein triggered higher levels of beta-galactosidase activity in the redesigned protocol. Estrogenic activity of p,p'-DDT could only be demonstrated with the redesigned protocol. Glycitein and daidzein failed to give a response with both protocols. We also studied deconjugation of beta-glycosidic isoflavones present in soygerm powder. Treatment of the soygerm powder with beta-glycosidase released isoflavones. The estrogenic response of the samples was confirmed with the redesigned protocol and correlated with the amount of genistein present. The release of isoflavones under conditions prevailing in the intestines was studied. Bacterial beta-glycosidase present in the large intestine released isoflavones, and moderate estrogenic activity could be demonstrated.

Optimization of a yeast estrogen screen and its applicability to study the release of estrogenic isoflavones from a soygerm powder.

Here we describe a redesigned protocol of the yeast estrogen screen developed by Routledge and Sumpter. The redesigned test comprises two steps. First, a large amount of yeast with estrogenic compounds is incubated for 24 hr. Subsequently, a mixture of cycloheximide and the chromogenic substrate chlorophenol red-beta-d-galactopyranoside (CPRG) is added. The cycloheximide stops protein synthesis and allows for an end-point measurement of beta-galactosidase activity generated during the first 24 hr. CPRG is converted to chlorophenol red and reflects beta-galactosidase activity, which is indicative of the estrogenic activity. The modifications shorten the duration of the assay at least 1 day and avoid interference of the estrogenic CPRG or chlorophenol red. The redesigned and the original protocol were used to study the estrogenic activity of bisphenol A, methoxychlor, p,p'-DDT, and isoflavones (genistein, daidzein, and glycitein). Bisphenol A, methoxychlor, and genistein triggered higher levels of beta-galactosidase activity in the redesigned protocol. Estrogenic activity of p,p'-DDT could only be demonstrated with the redesigned protocol. Glycitein and daidzein failed to give a response with both protocols. We also studied deconjugation of beta-glycosidic isoflavones present in soygerm powder. Treatment of the soygerm powder with beta-glycosidase released isoflavones. The estrogenic response of the samples was confirmed with the redesigned protocol and correlated with the amount of genistein present. The release of isoflavones under conditions prevailing in the intestines was studied. Bacterial beta-glycosidase present in the large intestine released isoflavones, and moderate estrogenic activity could be demonstrated.

Time-resolved fluoroimmunoassay of plasma daidzein and genistein

We present a method for the determination of the phytoestrogens daidzein and genistein in plasma (serum). These weakly estrogenic isoflavones occur in soybeans and in smaller amounts in some other beans and plants. It has been suggested that they may afford protection against prostate and breast cancer. The method is based on time-resolved fluoroimmunoassay (TR-FIA) using a europium chelate as a label. After synthesis of 4′- O-carboxymethyl-daidzein and 4′- O-carboxymethyl-genistein the compounds are coupled to bovine serum albumin (BSA), then used as antigens to immunize rabbits. The tracers with the europium chelate are synthesized using the same 4′- O-derivative of the isoflavones. After enzymatic hydrolysis and ether extraction the immunoassay is carried out using the VICTOR 1420 multilabel counter (Wallac Oy, Turku, Finland). The antisera cross-reacted to some extent with some isoflavonoids but not with flavonoids. The cross-reactivity seems not to influence the results, which were highly specific for both compounds. The correlation coefficients between the TR-FIA methods and the reference method based on isotope dilution gas chromatography-mass spectrometry were high; r-values were about 0.95–0.99 depending on concentration. The intra-assay coefficients of variation (CV%) for daidzein and genistein at three different concentrations vary 3.2–4.5 and 3.2–4.1, respectively. The inter-assay CVs vary 5.0–6.3 and 4.5–5.3, respectively. The working ranges of the daidzein and genistein assays are 1.0–216 and 1.7–370 nmol/l, respectively. The plasma values ( n = 80) of daidzein and genistein are very low in Finnish subjects (mean for daidzein, 3.8 ± 6.8 and for genistein, 3.2 ± 7.6 nmol/l; median value for daidzein 1.5 and for genistein 1.4 nmol/l).

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.

Developmental effects of dietary phytoestrogens in Sprague-Dawley rats and interactions of genistein and daidzein with rat estrogen receptors alpha and beta in vitro.

Estrogenic isoflavones, such as genistein and daidzein, are present in virtually all natural-ingredient rodent diets that use soy as a source of protein. Since these compounds are endocrine-active, it is important to determine whether the amounts present in rodent diets are sufficient to affect sexual development. The present study consisted of in vitro and in vivo parts. In the in vitro portion, human hepatoma cells were transfected with either rat estrogen receptor (ER) alpha or beta plus an estrogen-responsive luciferase reporter gene. Genistein and daidzein were complete agonists at both ERs, genistein being more potent than daidzein, and both compounds were more potent at ER beta than ER alpha. In combined studies with estradiol, genistein exerted additive effects with estradiol in vitro. In the in vivo portion of the study, groups of six pregnant Sprague-Dawley females were fed one of the following four diets, and the pups were maintained on the same diets until puberty: (1) a natural-ingredient, open-formula rodent diet (NIH-07) containing 16 mg genistein and 14 mg daidzein per 100 g of feed; (2) a soy- and alfalfa-free diet (SAFD) in which casein and corn oil were substituted for soy and alfalfa meal and soy oil, respectively, that contained no detectable isoflavones; (3) SAFD containing 0.02% genistein (GE.02); or (4) SAFD containing 0.1% genistein (GE.1). In the GE.1 group, effects of dietary genistein included a decreased rate of body-weight gain, a markedly increased (2.3-fold) uterine/body weight (U/BW) ratio on postnatal day (pnd) 21, a significant acceleration of puberty among females, and a marginal decrease in the ventral prostate weight on postnatal day (pnd) 56. However, developmental differences among the groups fed SAFD, GE.02, or NIH-07 were small and suggested minimal effects of phytoestrogens at normal dietary levels. In particular, on pnd 21, the U/BW ratio of the GE.02 and NIH-07 groups did not differ significantly from that of the SAFD group. Only one statistically significant difference was detected between groups fed SAFD and NIH-07: the anogenital distance (AGD) of female neonates on pnd 1 whose dams were fed NIH-07 was 12% larger than that of neonates whose dams were fed SAFD. The results suggest that normal amounts of phytoestrogens in natural-ingredient rodent diets may affect one developmental parameter, the female AGD, and that higher doses can affect several other parameters in both males and females. Based on these findings, we do not suggest replacing soy- and alfalfa-based rodent diets with phytoestrogen-free diets in most developmental toxicology studies. However, phytoestrogen-free diets are recommended for endocrine toxicology studies at low doses, to determine whether interactive effects may occur between dietary phytoestrogens and man-made chemicals.

Dietary Estrogenic Isoflavones Are Potent Inhibitors of β-Hydroxysteroid Dehydrogenase of P testosteronii

The isoflavones daidzein, genistein, biochanin A and formononetin selectively inhibit the γ-isozymes of mammalian alcohol dehydrogenase (ADH). Since γ-ADH is the only ADH isoform that catalyzes 3β-hydroxysteroid oxidation, it was conjectured that these isoflavones might also inhibit other enzymes involved in 3β-hydroxysteroid metabolism. P. testosteronii β-hydroxysteroid dehydrogenase (β-HSD) was used to evaluate this hypothesis. Indeed, all isoflavones that inhibit γ-ADH were found to be potent inhibitors of β-HSD. Both the 3β- and 17β-HSD activities of the enzyme are inhibited. Kinetic analyses with pregnenolone (3-beta-OH) and testosterone (17-beta-OH) as substrates reveal that daidzein and genistein inhibit β-HSD competitively with respect to the sterol substrates. Their K i values are very similar and range from 0.013 to 0.02 μM. These results suggest that isoflavones may exert some of their biological effects by modulating activities of enzymes that metabolize steroids critical to hormonal and/or neuronal functions.

Genetic damage and the inhibition of 7,12-dimethylbenz[a]anthracene-induced genetic damage by the phytoestrogens, genistein and daidzein, in female ICR mice.

Populations consuming soybeans have reduced rates of breast, colon and prostate cancer possibly due, in part, to the presence in soybeans of two estrogenic isoflavones, genistein and daidzein. This study investigated the genotoxicity of these soya isoflavones and their interactions with 7,12-dimethylbenz[a]anthracene (DMBA)-induced sister chromatid exchanges (SCE) in bone marrow cells and DNA adduct formations in liver and mammary glands of mice. Groups of female ICR mice were pretreated i.p. with daidzein and/or genistein (10-20 mg/kg per day for 6 days or 50 mg/kg per 12 h for 3 days) or with the solvent, dimethylsulfoxide (DMSO). The mice were implanted with bromodeoxyuridine (BrdU) tablets s.c., and treated with DMBA (50 mg/kg) i.p. and colchicine (4 mg/kg) i.p. 24, 23, and 2 h before sacrifice, respectively. In bone marrow cells. DMBA alone induced 11.73 +/- 1.42 SCE/cell compared to 4.35 +/- 0.83 SCE/cell in the DMSO treated controls (P = 0.001). DMBA induced 20% fewer SCE (P < 0.05) in mice pretreated with daidzein, genistein or a combination of genistein and daidzein (6 x 20 mg/kg per day for 6 days) when compared to mice that received no pretreatments. Genistein at 50 mg/kg per 12 h for 3 days also inhibited DMBA-induced SCE by 20%. However, treatment for 3 days with 50 mg/kg per 12 h of genistein or daidzein alone, or a combination of daidzein plus genistein (without DMBA treatment) also induced more SCE than treatment with only the solvent (DMSO, P < 0.05). Pretreatment with both the low and the high doses of daidzein plus genistein or the high dose of genistein reduced the replication index of bone marrow cells when compared to pretreatment with DMSO (P < 0.05). Pretreatment with genistein reduced DMBA-induced DNA adduct formation by 34%, but this was only marginally significant (P = 0.08) due to the large inter-individual variability in adduct levels. These results show that genistein and daidzein suppress SCE and possibly DNA adduct formation induced by the known carcinogen, DMBA. This response to a low dose isoflavone exposure may be partly responsible for the protective effect against endocrine cancers of soya consumption.

Phytoestrogen Content and Estrogenic Effect of Legume Fodder

This study is a summary of Finnish investigations of the phytoestrogen content of legume plants, red clover, white clover, alfalfa, and goat's rue. In addition to the chemical analyses, biological studies were performed. Uterine weight of immature rats was used as an indicator of the estrogenic effect of the fodder used. All red clover varieties studied contained estrogenic isoflavones, especially formononetin and biochanin-A. The phytoestrogen content varied from 1.0% to 2.5% of dry matter. The biological study of white clover showed a clear estrogenic effect not visible through chemical analysis. Alfalfa contains small quantities of formononetin and biochanin-A, but 25-65 ppm coumestrol in dry matter. The estrogenic effect of alfalfa was obvious in the biological study. Goat's rue did not contain any known phytoestrogens, and the biological study was completely negative.

The variable metabolic response to dietary isoflavones in humans.

The aim of this study was to better understand the metabolic fate of dietary estrogenic isoflavones in humans. Twelve volunteers were challenged with soya flour and urinary levels of the isoflavones daidzein (Da), genistein (Gen), and glycitein (Gly), and the isoflavonoid metabolites equol (Eq) and O-desmethylangolensin (O-Dma) determined by GC and GC-MS. Prior to challenge, Da, Gen, and Gly were present in the urine of all participants at low levels and Eq and O-Dma were present in 9/12 and 10/12 participants, respectively. Urinary levels of all five diphenols were increased in each individual on the day following challenge, returning to approximately prechallenge levels on the second or third day post-challenge. Mean post-challenge peak levels of the five diphenols compared with pre-challenge levels were Da (4x), Gen (8x), Gly (5x), Eq (45x), and O-Dma (66x). However, there was considerable individual variation in this metabolic response with peak levels of Eq showing the highest variation (1527x). An inverse relationship between Eq and O-Dma excretion was also found post-challenge suggesting individual variability in the preferred metabolic pathways of dietary isoflavones.

Estrogenic soybean isoflavones and chronic disease: Risks and benefits

Many edible plants contain natural estrogens called phytoestrogens. These compounds possess mixed estrogen agonist-antagonist properties that are organ-specific in vivo. We have focused on estrogenic soybean isoflavones because of their potential extensive dietary availability. In this article, we review the clinical and experimental evidence for the possible benefits and risks of ingestion of estrogenic isoflavones throughout the life span, and highlight areas needing further elucidation.

Study of the phytoestrogen content of goat’s rue (Galega orientalis), alfalfa (Medicago sutiva) and white clover (Trifolium repens)

Studies were conducted to determine the phytoestrogen content of goat’s rue (Galega orientalis Lam.), alfalfa (Medicago saliva L.) and white clover (Trifolium repens L.), all belonging to the Fabaceae family subjected to test cultivation at research stations of the Agricultural Research Centre of Finland. Apart from some insignificant quantities, goat’s rue did not contain any known phytoestrogens. Even in biological studies it had no estrogenic effect. The estrogenic effect of alfalfa was apparently due to coumestrol, which was discovered in the samples in quantities of 34-65 ppm. All white clover varieties contained very small quantities of estrogenic isoflavones and coumestrol, and they did not explain the increased weight of the immature rat uterus observed in the biological studies.

Persistent infertility in ewes after prolonged exposure to oestradiol-17 beta.

Merino ewes were treated with implants which released 300 micrograms oestradiol-17 beta per day or 5 mg progesterone per day, or both, for 9 months (Months 1-9), and after an 11-month intermission were treated again for 6 months (Months 20-26). Ewes were run with rams at Months 16, 28 and 40. Fertility was not affected by the first exposure period, but the second exposure to oestradiol reduced the fertility of ewes at both subsequent mating periods. Affected ewes returned to service more frequently (P less than 0.01) and were less likely to conceive (P less than 0.05). After mating, a normal population of spermatozoa was established in the caudal cervix, but transport through the cervix was impaired in affected ewes and there were fewer spermatozoa (P less than 0.01) in the cranial cervix. In affected ewes, the spinnbarkeit of cervical mucus was reduced (P less than 0.05), and the histological appearance of the cervix changed, looking like that of the uterus. Treatment with progesterone did not affect fertility, cervical mucus or sperm transport, but diminished the histological abnormalities produced by oestradiol (P less than 0.05). These results show that oestradiol-17 beta given after puberty can cause the same kind of permanent sexual transdifferentiation that is produced by the oestrogenic isoflavones in ewes with clover disease. The results suggest that this change may require more than a single exposure to oestrogen.