National Toxicology Program

Selected toxicity information from HSDB, one of the National Library of Medicine's databases. ¹

Names (NTP)

• 4H-1-BENZOPYRAN-4-ONE, 5,7-DIHYDROXY-3-(4-HYDROXYPHENYL)- (9CI)
• GENISTEIN (ENDOCRINE DISRUPTOR)
• 4H-1-BENZOPYRAN-4-ONE, 5,7-DIHYDROXY-3-(4-HYDROXYPHENYL)- (9CI)
• Endocrine disruptor (Genistein)
• Genistein

Human Toxicity Excerpts

• EPIDEMIOLOGY STUDIES: ... The Study of Women's Health Across the Nation (SWAN), is a multiethnic, community based, longitudinal study of women aged 42 to 52 years at entry. Dietary isoflavone intakes were estimated from an interviewer-administered food frequency questionnaire at baseline. Intakes of genistein and daidzein were highly correlated (r = 0.98); therefore, analyses were conducted using genistein only. The SWAN includes white, African American, Hispanic, Chinese, and Japanese women; this analysis was performed only in the latter two ethnic groups because the others had minimal genistein intake (median, <4 ug/day). Cognitive function tests were measured at the fourth annual follow-up visit using the East Boston Memory Test (Immediate and Delayed), Symbol Digit Modalities Test, and Digit Span Backward Test. Ethnicity-specific general linear models were used to examine the relationship between each of these cognitive tests and energy-adjusted genistein intake controlling for age, menopause stage, ever-use of any hormones, and current use of any hormones. Among 195 Japanese and women and 185 Chinese women, median intakes of genistein (ug/day) were 6,788 and 3,534, respectively. No associations between genistein intake and measures of cognitive performance were found in either ethnic group. [Huang MH et al; Menopause 13 (4): 621-30 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• ALTERNATIVE and IN VITRO TESTS: HepG2 human hepatoma cells were transfected with various concentrations of receptor plasmids encoding human ERalpha or ERbeta, and complement 3-luciferase and pCMV beta-galactosidase plasmids. Transfected cells were treated with various concentrations of either estradiol, genistein, genistein-GA (isolated from bile of genistein treated rats), genistein-S (chemically synthesized) or dimethylsulfoxide (control vehicle). Following 24-hr incubation, cell lysates were assayed for luciferase activity (receptor activation) and beta-galactosidase activity (transfection control). Estradiol and genistein activated both ER alpha and beta, however, neither genistein-GA nor genistein-S activated either receptor or antagonized the ability of estradiol to activate these receptors. These data demonstrate that conjugation eliminates the estrogenic activity of genistein and that it is important to determine the ratio of free genistein to conjugated forms when estimating estrogenic potency. [Borghoff S et al; Toxicologist 78 (1-S): 119 (2004) ]**PEER REVIEWED**
  
• ALTERNATIVE and IN VITRO TESTS: ... /The authors/ ... studied the effects of genistein, a soy isoflavone with a similar structure to estradiol, on the expression of antioxidant, longevity-related genes. MCF-7 cells (human mammary gland tumor cell line) were incubated for 48 hr with 0.5 uM genistein, a concentration found in the plasma of populations consuming diets rich in soy protein. Peroxide levels were determined by fluorimetry, activation of extracellular-signal regulated kinase (ERK1/2), and nuclear factor kappaB (NFkappaB)-signaling pathways by Western blot analysis and ELISA, respectively, and mRNA expression of antioxidant genes by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Inhibition of basal peroxide levels in MCF-7 cells by genistein was prevented by pretreatment of cells with the estrogen receptor antagonist tamoxifen. Phosphorylation of extracellular regulated kinase (ERK)1/2 led to an activation of NFkappaB, as indicated by increased p50 subunit expression in nuclear extracts, and increased mRNA levels of the antioxidant enzyme manganese-superoxide dismutase (MnSOD). Inhibition of ERK1/2 abrogated genistein-mediated NFkappaB activation and elevated expression of MnSOD. ... [Borras C et al; FASEB J 20 (12): 2136-8 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• ALTERNATIVE and IN VITRO TESTS: ... /The/ influences of soya isoflavones, major soya constituents with endocrine activity, on thyroxine (T4) binding to its distribution proteins /were examined/. Serum binding of iodine-125-labelled L-T4 was analysed in the absence or presence of increasing concentrations of soya isoflavones using non-denaturing PAGE for analysis. Complete displacement of iodine-125-T4 binding to transthyretin (TTR) was observed in human serum incubated with genistein at concentrations >10 uM; interference started at >0.1 uM. Glycitein showed decreased and daidzein the lowest displacement potency. Iodine-125-T4 was displaced to albumin in rat and to T4-binding globulin in human serum. Soya isoflavones also obstruct iodine-125-T4 binding to TTR in human cerebrospinal fluid (CSF). The inhibitory effect was confirmed in direct binding assays using purified TTR with 50% inhibitory concentration values of 0.07 uM for genistein, 0.2 uM for glycitein and 1.8 uM for daidzein. The present study underlined a potent competition of soya isoflavones for T4 binding to TTR in serum and CSF. [Radovic B et al; Br J Nutr 95 (6): 1171-6 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• ALTERNATIVE and IN VITRO TESTS: Neuroendocrine (NE) cells are present in normal prostate and their number appears to be increased in advanced prostate cancer (PCA). /This study examined/ the effect of the phytoestrogen, genistein, on NE differentiation of /human/ LNCaP cells in vitro. ... Six days after continuous genistein treatment, the majority of genistein-surviving cancer cells underwent transdifferentiation into a NE-like phenotype overexpressing the NE markers chromogranin A, synaptophysin, serotonin, and beta-III tubulin. This NE differentiation process was associated with upregulation of the cell cycle modulators p21, p27, and p53, and activation of the MAPK and STAT3 pathways. The data indicate that genistein evokes not only apoptosis but also NE transdifferentiation of PCA cells. [Pinski J et al; 66 (11): 1136-43 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• ALTERNATIVE and IN VITRO TESTS: ... Genistein inhibited cell proliferation in estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) human breast carcinoma cell lines. Cytochrome P450 (CYP) 1A1-mediated ethoxyresorufin O-deethylase (EROD) activity was inhibited by genistein in a concentration-dependent manner. Genistein significantly inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cyclooxygenase-2 activity and protein expression at the concentrations of 10 (p < 0.05), 25 (p < 0.05) and 50 mM (p < 0.01). In addition, ornithine decarboxylase (ODC) activity was reduced to 53.8 % of the control after 6 hr treatment with 50 mM genistein in MCF-7 breast cancer cells. [Shon YH et al; J Biochem Mol Biol 39 (4): 448-51 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• ALTERNATIVE and IN VITRO TESTS: Genistein and parathyroid hormone (PTH) are anabolic agents that stimulate bone formation through their direct actions in osteoblastic cells. /The objective of/ the present study was to determine whether genistein modulates the actions of PTH in human osteoblastic SaOS-2 cells in an estrogen-depleted condition. The present results showed that genistein (10 -8 to 10 -6 m) induced alkaline phosphatase (ALP) activity and osteoprotegrin (OPG) expression in SaOS-2 cells in a dose-dependent manner. These effects could be completely abolished by co-treatment with estrogen antagonist ICI 182780 (7alpha-[9-[(4,4,5,5,5-pentafluoropentyl)sulfonyl]nonyl]-estra-1,3,5(10)-triene-3,17beta-diol). Genistein (at 1 uM) could stimulate the mRNA expression of receptor activator of NF-kappaB ligand (RANKL). As OPG and RANKL are known to modulate osteoclastogenesis, the ability of genistein to modulate OPG and RANKL expression in SaOS-2 cells suggested that it might modulate osteoclastogenesis through its direct actions on osteoblastic cells. PTH (at 10 nM) stimulated ALP activity, induced RANKL mRNA expression and suppressed OPG mRNA expression in SaOS-2 cells, confirming its bi-directional effects on osteoblastic cells. Pre-treatment of SaOS-2 cells with genistein and estrogen not only enhanced PTH-induced ALP activity, but also attenuated PTH up regulation of RANKL mRNA expression and PTH down regulation of OPG mRNA expression. Taken together, the present study provides the first evidence that genistein could modulate the actions of PTH in human osteoblastic SaOS-2 cells in an estrogen-depleted condition. [Chen WF, Wong MS; Br J Nutr 95 (6): 1039-47 (2006) ]**PEER REVIEWED** PubMed Abstract
  

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Non-Human Toxicity Excerpts

• LABORATORY ANIMALS: Acute Exposure: Two acute studies were conducted in male and female 7-week-old Hanlbm Wistar rats and 8-week-old outbred Wistar Crl:(WI)BR rats. The Hanlbm Wistar rats were fed a genistein-free diet and the Wistar Crl:(WI)BR rats were fed standard animal diet. The rats were administered genistein (99.5-99.6% purity) in a single gavage dose of 2000 mg/kg bw and observed for 2 weeks. The rats were then killed and necropsied. Liver and kidney weights were measured in the Hanlmb rats. (The number of rats treated and observed was not stated.) All rats survived, and there were no gross effects at necropsy or changes in organ or body weights. In the Wistar Crl:(WI)BR rats, lethargy was noted in all males and 1 female on day 1 and alopecia was observed on days 14 and 15. The study authors concluded that genistein has low toxicity. [National Toxicology Program, Center For the Evaluation of Risks To Human Reproduction; NTP-CERHR Expert Panel Report on the Reproductive and Developmental Toxicity of Genistein; (NTP-CERHR-GENISTEIN-06) April 2006. Available at http://cerhr.niehs.nih.gov/chemicals/genistein-soy/genistein/Genistein_Report_final.pdf as of August 14, 2007 ]**PEER REVIEWED**
  
• LABORATORY ANIMALS: Subchronic or Prechronic Exposure: ... In the 13-week study that was conducted according to GLP, 15 /outbred Wistar/ rats/sex/group were fed diets containing genistein doses of 0, 5, 50, or 500 mg/kg bw/day. Following treatment, 10 rats/sex/group were killed and 5 rats/sex/group were allowed to recover for 4 weeks to determine reversibility of treatment-related effects. No treatment-related deaths were observed. Body weights were lower in the 500 mg/kg bw/day group compared to the control group (18% lower for males and 10% lower for females). Body weights of males increased during the recovery period but were still lower compared to controls at the end of the study. During the first month of treatment, feed intake was reduced in male rats of the 500 mg/kg bw/day group. Hematology, clinical chemistry, and urinalysis parameters were monitored following 11 weeks of treatment (data were not shown). Red blood cell parameters were reportedly decreased and reticulocyte levels were increased in males and females of the 500 mg/kg bw/day group. Slight changes in clinical chemistry parameters included decreased glucose and increased uric acid, sodium, and chloride in high-dose males and decreased uric acid and increased calcium, total protein, and phospholipid in high-dose females. Uric acid crystals were increased in females of the 500 mg/kg bw/day group. Non-reproductive organ weight changes in high-dose males included slight increases in relative (to body weight) heart, thyroid, kidney, and adrenal weights. Relative to body weight, testis weights was increased (by 19%) in high-dose males [possibly due to decreased body weight]. Relative liver and kidney weights were increased in females of the 500 mg/kg bw/day group. Relative uterine weight of high-dose females was increased (by 41%). (The study authors did not present data for non-reproductive organ weights.) All animals were necropsied, and histopathological evaluations were conducted in tissues from control and high-dose animals. There were no treatment-related gross or histopathological alterations. Ophthalmologic parameters were also unaffected. With the exception of body weight effects in males, none of the treatment-related effects were observed following the 4-week recovery period. (No recovery data were reported by study authors.) [National Toxicology Program, Center For the Evaluation of Risks To Human Reproduction; NTP-CERHR Expert Panel Report on the Reproductive and Developmental Toxicity of Genistein; (NTP-CERHR-GENISTEIN-06) April 2006. Available at http://cerhr.niehs.nih.gov/chemicals/genistein-soy/genistein/Genistein_Report_final.pdf as of August 14, 2007 ]**PEER REVIEWED**
  
• LABORATORY ANIMALS: Subchronic or Prechronic Exposure: The subchronic and chronic safety of genistein were evaluated in the Beagle dog including a 4-week study and a 52-week safety study with a 13 week interim sacrifice and a 4 week recovery period. In both studies at doses of 50, 150 and 500 mg/kg/day, genistein was well tolerated. In the 4 week study, except for an increase in uterine weights in female dogs at 500 mg/kg/day, there were no other treatment related findings. ... In the 4-week study, the no observed adverse effect level (NOAEL) for genistein was considered to be >500 mg/kg/day and the no observed effect level (NOEL) was considered to be 150 mg/kg/day. [McClain RM et al; Food Chem Toxicol 43 (10): 1461-82 (2005) ]**PEER REVIEWED** PubMed Abstract
  
• LABORATORY ANIMALS: Chronic Exposure or Carcinogenicity: ... The authors compared the effects of daily sc injections of 1mg/kg bw of genistein and vehicle (2% DMSO in peanut oil) for 20 weeks on N-nitroso-N-methylurea (NMU)-induced tumorigenesis in adult female rats. Genistein significantly increased tumor cross-sectional area and tumor multiplicity but not the tumor incidence and latency period when compared with the vehicle treated group. The serum E(2) levels of the genistein treated group were significantly higher than those of the vehicle treated group at 1 and 2 months after treatment which is the time when most of the rats developed tumors. There were no significant differences in the length of the estrous cycle, food consumption and weights of body, livers, uteri and ovaries between the two groups. The data shows that supplementation of genistein at a dosage comparable to the isoflavone consumption in humans did not affect the reproductive system but resulted in enhancement of NMU-induced tumorigenesis in adult female rats. ... [Kijkuokool P et al; Cancer Lett 242 (1): 53-9 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• LABORATORY ANIMALS: Chronic Exposure or Carcinogenicity: In the 52-week study that was conducted according to GLP, 30 /outbred WIstar/ rats/sex/group were fed diets providing genistein doses of 0, 5, 50, or 500 mg/kg bw/day. Five rats/sex/group were killed following 26 weeks of treatment and 20 rats/sex/group were killed following 52 weeks of treatment. Five rats/sex/group were allowed to recover for 8 weeks during which time they received no treatment. There were no treatment-related deaths during the study. A higher rate of alopecia in male and female rats of the high-dose group was the only clinical sign of toxicity reported. No effects were noted for ophthalmologic parameters. ... A number of statistically significant effects on hematology and clinical chemistry parameters were observed. ... Some of the hematological effects persisted through the recovery period, but all clinical chemistry effects were resolved during recovery. Organ weights were measured at weeks 26 and 52. The only significant organ weight effects that the authors considered to be treatment-related at 52 weeks were increased relative weights of adrenal and spleen (males and females), prostate (7%), testis (52%), ovary (394%), and uterus (275%) in the 500 mg/kg bw/day group. Increases in adrenal, spleen, and uterus weights were also observed following 26 weeks of treatment. Increased ovary weight was the only organ weight effect that persisted through the recovery period. Other significant organ weight effects occurred, but the study authors concluded that those effects resulted from reduced body weight gain. At the 52-week necropsy, uterine horn dilation was observed in 7 females of the 500 mg/kg bw/day group and watery cysts in ovaries were noted in 4, 3, and 12 females of the low-, mid-, and high-dose group. (It is assumed that about 20 females/dose group were examined.) ... In male rats, epididymal vacuolation was observed at 500 mg/kg bw/day and prostate inflammation was observed at >/=50 mg/kg bw/day. In female rats, the study authors reported histopathology alterations in ovaries and uterus/cervix at >/=50 mg/kg bw/day. ...Histopathological changes in vagina and mammary gland were observed at 500 mg/kg bw/day. ... (The study authors reported an increase in osteopetrosis in males and females at >/=50 mg/kg bw/day; however it appears that the increase at 50 mg/kg bw/day was observed only at 26 weeks in females (2/5 females of the 50 mg/kg bw/day group and 5/5 females of the 500 mg/kg bw/day group affected versus 0/5 controls affected).) Extramedullary hemopoiesis [incidence and severity not indicated] was reported to occur in the spleen at all doses and was stated to be a compensatory response to decreased bone marrow resulting from bone thickening. Liver histopathology was observed in males and females at 500 mg/kg bw/day. Many of the histopathology observations observed at 52 weeks (i.e., effects in liver, bone, epididymides, prostate, ovaries, uterus, and vagina) were also observed at 26 weeks. Following the 8-week recovery period, osteopetrosis in females and epididymal vacuolation were the only persistent histopathological effects observed at the high dose. [National Toxicology Program, Center For the Evaluation of Risks To Human Reproduction; NTP-CERHR Expert Panel Report on the Reproductive and Developmental Toxicity of Genistein; (NTP-CERHR-GENISTEIN-06) April 2006. Available at http://cerhr.niehs.nih.gov/chemicals/genistein-soy/genistein/Genistein_Report_final.pdf as of August 14, 2007 ]**PEER REVIEWED**
  
• LABORATORY ANIMALS: Chronic Exposure or Carcinogenicity: ... The chronic safety of genistein were evaluated in the Beagle dog including a ... a 52-week safety study with a 13 week interim sacrifice and a 4 week recovery period. ... Doses of 50, 150 and 500 mg/kg/day /were well tolerated/. ... . In the 52-week study, the primary effects of genistein were observed on the reproductive tract, which included for male dogs: reduced size and/or weight of the testes, epididymis and prostate of 2/2 dogs after 13 weeks of treatment and in 1/4 dogs after 52 weeks of treatment at 500 mg/kg/day. The histological changes observed in the affected dogs at 500 mg/kg/day indicated atrophy of the testes and prostate gland and absent spermatozoa in the epididymus. At the mid-dose of 150 mg/kg/day, although there was a reduction to a lesser extent in testes weight after 13, but not 52 weeks, there were no histopathological changes. In female dogs, the reproductive tract effects included increased uterine weight at 500 mg/kg/day after 13 weeks of treatment, but not after 52 weeks of treatment. There was also a small decrease in ovarian weights at 150 and 500 mg/kg/day after 13 weeks and at 500 mg/kg/day after 52 weeks of treatment. There were no histopathological correlates to the changes in organ weights in female dogs. In the 4-week recovery group dogs, no changes were observed in dogs previously treated for 52 weeks with 500 mg/kg/day of genistein. ... For the 52-week study, the NOAEL is considered to be >500 mg/kg/day and the NOEL is considered to be 50 mg/kg/day. [McClain RM et al; Food Chem Toxicol 43 (10): 1461-82 (2005) ]**PEER REVIEWED** PubMed Abstract
  
• LABORATORY ANIMALS: Chronic Exposure or Carcinogenicity: ... Pregnant rat dams were treated daily with sc injections of 20, 100 or 300 ug genistein, 20 ug zearalenone, or vehicle between days 15 and 20 of gestation. The offspring were given 7, 12-dimethylbenz(a)anthracene (DMBA) at the age of 2 months to induce mammary tumors. The results indicate that in utero exposure to genistein, but not to zearalenone, dose-dependently increased the incidence of DMBA-induced mammary tumors, when compared with the controls. Tumor growth characteristics were not altered. Prior to the carcinogen administration, the number of estrogen receptor binding sites, determined using a ligand binding assay, were significantly elevated in the mammary glands of genistein offspring. In contrast, the mammary protein kinase C (PKC) activity was significantly reduced in the genistein offspring. ... /The/ results suggest that a maternal exposure to sc administration of genistein can increase mammary tumorigenesis in the offspring, mimicking the effects of in utero estrogenic exposures. Further, increased estrogen receptor protein levels and reduced PKC activity in the mammary gland may be involved in increasing susceptibility to carcinogen-induced mammary tumorigenesis in rats exposed to genistein in utero. [Hilakivi-Clarke L et al; Oncol Rep 6 (5): 1089-95 (1999) ]**PEER REVIEWED** PubMed Abstract
  
• LABORATORY ANIMALS: Chronic Exposure or Carcinogenicity: ... The carcinogenic potential of genistein ... /was investigated/ in an experimental animal model previously reported to result in a high incidence of uterine adenocarcinoma after neonatal diethylstilbestrol (DES) exposure. Outbred female CD-1 mice were treated on days 1-5 with equivalent estrogenic doses of DES (0.001 mg/kg/day) or genistein (50 mg/kg/day). At 18 months, the incidence of uterine adenocarcinoma was 35% for genistein and 31% for DES. These data suggest that genistein is carcinogenic if exposure occurs during critical periods of differentiation. ... [Newbold RR et al; Cancer Res 61 (11): 4325-8 (2001) ]**PEER REVIEWED** PubMed Abstract
  
• LABORATORY ANIMALS: Chronic Exposure or Carcinogenicity: ... A large multigenerational experiment involved feeding 0, 5, 100, or 500 ppm genistein in the diet to intact male and female rats from conception until either weaning, postnatal day 140, or continuously for 2 years. Vertebrae (lumbar and caudal) were collected from these animals at necropsy at 2 years of age and subjected to dual-energy x-ray absorptiometry (DXA) scanning to measure bone mineral density (BMD), bone mineral content (BMC), and bone area. Femurs were collected, and length, cross-sectional area, and cortical bone area were measured directly. Serum was collected for measurement of pyridinoline (PYD) and alkaline phosphatase (ALP). BMD was not affected by genistein in any phase of the experiment. In female rats treated continuously with genistein, BMC and bone area were reduced in the 500 ppm group compared to the 5 ppm group in the lumbar vertebrae, and in all treatment groups compared to control in the caudal vertebrae. In both males and females treated continuously, the cross-sectional area of the femur was reduced in rats treated with 500 ppm compared to those treated with 5 ppm. In female rats treated continuously, PYD was higher in the 100 and 500 ppm groups than in the 0 and 5 ppm groups. In conclusion, the effects of genistein on reproductively-intact rats were not dramatic. High dose of genistein throughout the lifespan resulted in decreased bone size, which may reduce the force required to break the bone. [Hotchkiss CE et al; Bone 37 (5): 720-7 (2005) ]**PEER REVIEWED** PubMed Abstract
  
• LABORATORY ANIMALS: Chronic Exposure or Carcinogenicity: ... 7, 12-dimethylbenz(a)anthracene (DMBA) solution (0.5% in mineral oil) was applied topically to the left cheek pouch of male Syrian golden hamsters three times a week for 6 weeks. Two days after the last treatment of DMBA, genistein suspended in distilled water (10 mg/kg body wt/day) or same volume of distilled water was administered into the animals by gavage daily for 12 weeks. The genistein treatment decreased the visible oral tumor incidence to 40.7 (11/27) from 53.6% (15/28) of the positive control, but the difference was not statistically significant (P = 0.34). /There was also/ no significant difference in the average number of tumors/tumor-bearing hamster, the average tumor volume, or latency ... observed between the control and the genistein-treated group. Vascular density in oral squamous cell carcinoma (OSCC) of the genistein-treated group and that of the control group was similar and there was no significant difference. Importantly, three animals in the genistein-treated group produced poorly-differenciated fibrosarcomas in the DMBA-painted cheek pouches. ... [Yang Y et al; Carcinogenesis 27 (3): 578-83 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• LABORATORY ANIMALS: Developmental or Reproductive Toxicity: The present studies report the effects on neonatal rats of oral exposure to genistein during the period from birth to postnatal day (PND) 21. ... Failure to demonstrate significant exposure of the newborn pups via the mothers milk led /the authors/ to subcutaneously inject genistein into the pups over the period PND 1-7, followed by daily gavage dosing to PND 21. The targeted doses throughout were 4 mg/kg/day genistein (equivalent to the average exposure of infants to total isoflavones in soy milk) and a dose 10 times higher than this (40 mg/kg genistein). The dose used during the injection phase of the experiment was based on plasma determinations of genistein and its major metabolites. Diethylstilbestrol at 10 ug/kg was used as a positive control agent for assessment of changes in the sexually dimorphic nucleus of the preoptic area (SDN-POA). Administration of 40 mg/kg genistein increased uterus weights at day 22, advanced the mean day of vaginal opening, and induced permanent estrus in the developing female pups. Progesterone concentrations were also decreased in the mature females. There were no effects in females dosed with 4 mg/kg genistein, the predicted exposure level for infants drinking soy-based infant formulas. There were no consistent effects on male offspring at either dose level of genistein. Although genistein is estrogenic at 40 mg/kg/day, as illustrated by the effects described above, this dose does not have the same repercussions as diethylstilbestrol in terms of the organizational effects on the SDN-POA. [Lewis RW et al; Toxicol Sci 71 (1): 74-83 (2003) ]**PEER REVIEWED** PubMed Abstract
  
• LABORATORY ANIMALS: Developmental or Reproductive Toxicity: Prolonged estrous cyclicity was observed following neonatal genistein treatment (0.5-50 mg/kg) on Days 1-5 with dose- and age-related increase in severity. Fertility, determined at 2, 4, and 6 months, showed decreased numbers of genistein-treated females (0.5 or 5 mg/kg) delivering live pups and reduced numbers of pups. At 6 months, 60% of 0.5 mg/kg and 40% of 5 mg/kg groups delivered live pups compared to 100% of controls. At 2 months, half the mice treated with 25 mg/kg of genistein and none treated with 50 mg/kg delivered live pups, although half of the latter group showed signs of pregnancy with few small implantation sites. Ovarian function was disrupted in the low genistein-dosed mice with increased numbers of corpora lutea (CLs) compared to controls and increased ovulated oocytes following exogenous gonadotropins treatment. In contrast, mice treated with high genistein doses had decreased numbers of CLs; ovulation could be restored with exogenous gonadotropins. ... [Jefferson WN et al; J AOAC Int 89 (4): 1189-96 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• LABORATORY ANIMALS: Developmental or Reproductive Toxicity: ... Maternal rats were fed diets containing genistein at levels of 0 and 0.5 g/kg diet from pregnancy day 5 to postnatal day 13. No effects of genistein on the delivery, anogenital distance, reproductive organ weight, and body weight of the infants at birth were observed. There were no consistent effects on suckling pups after continuous genistein exposure during their fetal and suckling stages through their mothers, and there was no difference in effects according to the periods of exposure during pregnancy and lactation. ... No significant effect on the growth of offspring after weaning /was observed/. Moreover, while ... the serum concentration of triiodothyronine (T3) in dams decreased, the result was a tendency, not a significant decrease. [Tousen Y et al; J Nutr Sci Vitaminol (Tokyo) 52 (3): 174-82 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• LABORATORY ANIMALS: Developmental or Reproductive Toxicity: Developmental effects of genistein (Gen) on the mammary gland were investigated using outbred female CD-1 mice treated neonatally on day 1-5 by sc injections at doses of 0.5, 5, or 50 mg/kg/day. Examination of mammary gland whole mounts (no. 4) before puberty (4 wk) revealed no morphological differences in development after Gen treatment. However, mice treated with Gen-50 had stunted development characterized by less branching at 5 wk and decreased numbers of terminal end buds at 5 and 6 wk. Conversely, at 6 wk, Gen-0.5-treated mice exhibited advanced development with increased ductal elongation compared with controls. Measurements of hormone receptor levels showed increased levels of progesterone receptor protein and estrogen receptor-beta mRNA in Gen-0.5-treated mice compared with controls; ERalpha expression was decreased after all doses of Gen treatment. Lactation ability, measured by pup weight gain and survival, was not affected after neonatal Gen-0.5 and Gen-5. Mice treated with Gen-50 did not deliver live pups; therefore, lactation ability could not be determined. Evaluation of mammary glands in aged mice (9 months) showed no differences between Gen-0.5-treated mice and controls but mice treated with Gen-5 and Gen-50 exhibited altered morphology including reduced lobular alveolar development, dilated ducts, and focal areas of beaded ducts lined with hyperplastic ductal epithelium. In summary, neonatal Gen exposure altered mammary gland growth and development as well as hormone receptor levels at all doses examined; higher doses of Gen led to permanent long-lasting morphological changes. [Padilla-Banks E et al; Endocrinology 147 (10): 4871-82 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• LABORATORY ANIMALS: Developmental or Reproductive Toxicity: Female rats were fed a phytoestrogen-free diet supplemented with no genistein (free), a low genistein dose (low) or a high genistein dose (high) throughout gestation and lactation. Anogenital distance of male offspring was measured weekly from postnatal days 2 to 21. At puberty (postnatal day 40 to 45) preputial separation, and testis length and width of male offspring were measured. At age 70 days reproductive organ masses, plasma testosterone concentration, sperm counts and sexual behavior were assessed in male offspring. Exposure to genistein resulted in temporary, prepubertal urogenital abnormalities at postnatal days 21 and 40. Males exposed to genistein had smaller anogenital distance and testis size, and delayed preputial separation. Perinatal exposure to genistein also caused long-term dysfunction in reproductive behavior, in which adult males exposed to genistein were less likely to mount, intromit and ejaculate during mating tests. Males exposed to genistein also had lower testosterone concentrations in adulthood. Perinatal genistein exposure results in transient and lasting alterations in masculinization of the reproductive system. [Wisniewski AB et al; J Urol 169 (4): 1582-6 (2003) ]**PEER REVIEWED** PubMed Abstract
  
• LABORATORY ANIMALS: Developmental or Reproductive Toxicity: Sprague-Dawley rats were administered genistein orally at doses of 12.5, 25, 50, or 100 mg/kg on postnatal days 1 through 5 to examine its effects on reproductive function after puberty. In addition, preputial separation and vaginal opening as endpoints of sexual maturation, estrous cycling, sperm count, serum testosterone concentration, and histopathologic changes of reproductive organs of male and female rats were examined. Body weights of male and female rats exposed to genistein at any dose level examined were lower than those of controls. Timing of preputial separation in males and timing of vaginal opening were not affected by genistein treatment. The number of females showing estrous cycle irregularities was increased by genistein treatment. The fertility of female rats exposed neonatally to genistein at 100 mg/kg was disrupted, while neonatal exposure to genistein did not affect male fertility. Neither sperm counts nor serum testosterone concentration were changed by neonatal exposure to genistein. Female rats exposed neonatally to genistein at 100 mg/kg showed histopathologic changes in the ovaries and uterus, while male rats showed no histopathologic alterations in the gonads. The results of this study indicate that early neonatal exposure to genistein caused dysfunction of postpubertal reproductive performance as well as abnormal development of gonads in female but not in male rats. [Nagao T et al; Reprod Toxicol 15 (4): 399-411 (2001) ]**PEER REVIEWED** PubMed Abstract
  
• LABORATORY ANIMALS: Developmental or Reproductive Toxicity: Male Sprague-Dawley rats were fed 25 and 250 mg genistein/kg diet from conception until day 70 postpartum, or 250 and 1000 mg genistein/kg diet from day 56 to 70 postpartum. Exposure to genistein in the diet, starting at conception, resulted in down-regulated androgen receptor (AR), and estrogen receptors (ER)-alpha and -beta mRNA expression in the dorsolateral prostate in a dose-dependent manner. Also, genistein fed to adult rats for 2 weeks reduced mRNA expression of AR, ER-alpha and ER-beta in the dorsolateral prostate. ER-alpha protein levels were significantly reduced in animals fed 1000 mg genistein/kg diet compared to control animals. There were no significant alterations to male reproductive tract histomorphology or weights. ... /The authors/ conclude that dietary genistein down-regulated expression of the AR and ER-alpha and -beta in the rat prostate at concentrations comparable to those found in humans on a soy diet. [Fritz WA et al; Mol Cell Endocrinol 186 (1): 89-99 (2002) ]**PEER REVIEWED** PubMed Abstract
  
• LABORATORY ANIMALS: Developmental or Reproductive Toxicity: Outbred CD-1 mice were treated neonatally on Days 1-5 with the phytoestrogen, genistein (1, 10, or 100 ug per pup per day), and ovaries were collected on Days 5, 12, and 19. Ribonuclease protection assay analysis of ovarian mRNA showed that estrogen receptor beta (ERbeta) predominated over ERalpha in controls and increased with age. Genistein treatment did not alter ERbeta expression, however, ERalpha expression was higher on Days 5 and 12. ERbeta was immunolocalized in granulosa cells, whereas ERalpha was immunolocalized in interstitial and thecal cells. Genistein treatment caused a dramatic increase in ERalpha in granulosa cells. Genistein-treated ERbeta knockout mice showed a similar induction of ERalpha, which is seen in CD-1 mice, suggesting that ERbeta does not mediate this effect. Similar ERalpha induction in granulosa cells was seen in CD-1 mice treated with lavendustin A, a tyrosine kinase inhibitor that has no known estrogenic actions, which suggests that this property of genistein may be responsible. As a functional analysis, genistein-treated mice were superovulated and the number of oocytes was counted. A statistically significant increase in the number of ovulated oocytes was observed with the lowest dose, whereas a decrease was observed with the two higher doses. This increase in ovulatory capacity with the low dose coincided with higher ERalpha expression. Histological evaluations on Day 19 revealed a dose-related increase in multioocyte follicles (MOFs) in genistein-treated mice. Tyrosine kinase inhibition was apparently not responsible for MOFs because they were not present in mice that had been treated with lavendustin; however, ERbeta must play a role, because mice lacking ERbeta showed no MOFs. These data taken together demonstrate alterations in the ovary following neonatal exposure to genistein. [Jefferson WN et al; Biol Reprod 67 (4): 1285-96 (2002) ]**PEER REVIEWED** PubMed Abstract
  
• LABORATORY ANIMALS: Developmental or Reproductive Toxicity: The myelotoxicity of five endocrine active chemicals was evaluated in F1 generation of Sprague-Dawley rats following developmental and adult exposures at three concentration levels. Rats were exposed to genistein (GEN: 25, 250 and 1250 ppm), nonylphenol (NPH: 25, 500 and 2000 ppm), methoxychlor (MXC: 10, 100 and 1000 ppm), vinclozolin (VCZ: 10, 150 and 750 ppm) and ethinyl estradiol (EE2: 5, 25 and 200 ppb) gestationally and lactationally through dams from day 7 of gestation and through feed after weaning on postnatal day (PND) 22 to PND 64. The parameters examined included the number of recovered bone marrow cells, DNA synthesis, and colony forming units (CFU) in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and erythropoietin. Except for the EE2, the concentrations of other individual chemicals in the diet were in an approximate range that allowed for a comparison to be made in terms of myelotoxic potency. Decreases in the DNA synthesis, CFU-GM and CFU-M seemed to be the common findings among the alterations induced by these compounds. Using the numbers of alterations induced by each chemical in the parameters examined as criteria for comparison, the order of myelotoxic potency in F(1) males was: GEN>MXC>NPH>VCZ; the order in females: GEN>NPH>VCZ. Additionally, some of the functional changes induced by these compounds were gender-specific or dimorphic. Overall, the results demonstrated that developmental and adult exposures of F1 rats to these endocrine active chemicals at the concentrations tested had varied degrees of myelotoxicity with GEN being the most potent. Furthermore, the sex-specific effects of these chemicals in F1 male and female rats suggest that there may be interactions between these compounds and sex hormone in modulating these responses. [Guo TL et al; Toxicology 211 (3): 207-19 (2005) ]**PEER REVIEWED** PubMed Abstract
  
• LABORATORY ANIMALS: Developmental or Reproductive Toxicity: ... The aim of this study was to investigate the effects of dietary genistein (8 mg/kg body weight/day) on the reproduction, postnatal development and weight regulation of mice across two generations. Genistein treatment decreased the relative food consumption in females at 1 and 5 weeks and in males at 5 weeks. In female pups, the relative kidney weights were lower due to genistein exposure. Furthermore, the genistein-exposed male pups had greater relative prostate and seminal vesicles weights than the control pups. In adult males, genistein treatment decreased the plasma estradiol concentrations and increased levels of the plasma HDL cholesterol and triglycerides in adult females. Also the plasma ghrelin concentrations decreased in the adult genistein treated female mice. Genistein increased the plasma triglyceride levels of male pups and triiodothyronine levels of female pups. Reproduction of the mice was not endangered due to genistein exposure. [Ryokkynen A et al; Anim Reprod Sci 93 (3-4): 337-48 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• LABORATORY ANIMALS: Developmental or Reproductive Toxicity: ... /The investigators/ report that maternal dietary genistein supplementation of mice during gestation, at levels comparable with humans consuming high-soy diets, shifted the coat color of heterozygous viable yellow agouti A(vy/a) offspring toward pseudoagouti. This marked phenotypic change was significantly associated with increased methylation of six cytosine-guanine sites in a retrotransposon upstream of the transcription start site of the agouti gene. The extent of this DNA methylation was similar in endodermal, mesodermal, and ectodermal tissues, indicating that genistein acts during early embryonic development. Moreover, this genistein-induced hypermethylation persisted into adulthood, decreasing ectopic agouti expression and protecting offspring from obesity. Thus, ... in utero dietary genistein affects gene expression and alters susceptibility to obesity in adulthood by permanently altering the epigenome. [Dolinoy DC et al; Environ Health Perspect 114 (4): 567-72 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• ALTERNATIVE and IN VITRO TESTS: The objectives of the current study were: to establish an in vitro model of granulosa cell culture for studying the intracellular mechanism of phytoestrogen action in porcine ovary; to determine an in vitro effect of genistein on basal and FSH-stimulated P(4) and E(2) production by porcine granulosa cell populations (antral, mural, total) isolated from large, preovulatory follicles. Granulosa cells were isolated from large (> or = 8 mm), preovulatory follicles and separated into antral and mural cell subpopulations. Cells were allowed to attach for 72 hr (37 degrees Celsius, 10% serum, 95% air/5% CO2) and than cultured for next 48 hr with or without serum (0, 5 and 10%), FSH (0, 10 or 100 ng/ml) and genistein (0, 0.5, 5 or 50 microM). Basal P(4) and E(2) production did not differ among antral, mural and unseparated granulosa cells isolated form porcine preovulatory follicles. Only mural cells tended to secrete less P(4) and E(2) than other cell populations. FSH stimulated P(4) production in a dose dependent manner in all cell populations and culture systems. Genistein inhibited in a dose dependent manner basal and FSH-stimulated P(4) production by antral, mural and unseparated granulosa cells. However, genistein did not affect E(2) production by granulosa cells. In addition, viability of porcine granulosa cells was not affected by the pyhytoestrogen except the highest dose of genistein. It appears that genistein may be involved in the regulation of follicular function in pigs. [Nynca A, Ciereszko RE; Reprod Biol 6 (1): 31-50 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• GENOTOXICITY: ... In these studies, genistein was evaluated for mutagenicity and clastogenicity in vitro in the S. typhimurium assay (Ames Test), the mouse lymphoma assay and in vivo in the micronucleus test in mice and rats. There was no evidence for a mutagenic effect in the in vitro S. typhimurium assay with and without metabolic activation (S9). In the in vitro mouse lymphoma assay, genistein increased resistant mutants with and without metabolic activation (S9), which were predominantly small colonies indicating that genistein acts as a clastogen. Three independent in vivo micronucleus tests were performed in Moro mice, RAIf rats and Wistar rats. MORO male and female mice were treated orally for 14 days at doses up to 20 mg/kg/day. RAIf and Wistar male and female rats were treated orally at doses up to 2000 mg/kg without an increase in micronuclei in treated mice or rats. It is concluded that genistein was not mutagenic in the S. typhimurium assay or mutagenic or clastogenic in vivo in the mouse and rat micronucleus test. In the mouse lymphoma assay, genistein induced an increase of predominantly small colonies indicating that genistein acts as a clastogen. This observation is in agreement with published data on the inhibitory action of genistein on topoisomerase II, which is known to lead to chromosomal damage with a threshold dose response. [McClain R et al; Food Chem Toxicol 44 (1): 42-55 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• GENOTOXICITY: With the exception of one weak positive result in one strain of Salmonella following metabolic activation, bacterial tests did not indicate mutagenicity. Positive results were generally observed in mutation, micronuclei, chromosomal aberration, and deoxyribonucleic acid (DNA) strand break tests conducted in mammalian cells; only one of the assays utilized metabolic activation. (The /NTP/ Expert Panel concluded that results of the in vitro tests are irrelevant because they were not conducted with the glucuronidated compound.) Results of in vivo micronuclei tests (4 in mice and 3 in rats) and 1 chromosomal aberration test in mice ... did not suggest that genistein induced micronuclei or chromosomal aberrations. [National Toxicology Program, Center For the Evaluation of Risks To Human Reproduction; NTP-CERHR Expert Panel Report on the Reproductive and Developmental Toxicity of Genistein; (NTP-CERHR-GENISTEIN-06) April 2006. Available at http://cerhr.niehs.nih.gov/chemicals/genistein-soy/genistein/Genistein_Report_final.pdf as of August 14, 2007 ]**PEER REVIEWED**
  
• GENOTOXICITY: ... using BB rats, ovariectomized to model menopause and to exclude the effects of intrinsic sex hormones, /the authors/ investigated the effects of feeding diadzein (DZ), genistein (GE), or 17beta-estradiol (E2) on the genotoxicity of the potent rodent carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) in several estrogen-responsive tissues, including mammary, uterus, heart, and liver. Mammary glands and uterus were also analyzed for tumorigenicity... . DZ and GE were found to be biologically active as determined by increased proliferating cell nuclear antigen (PCNA) positive cells and concomitant decrease in atrophy in the OVX rats, as well as levels detected in serum. Feeding rats diets containing DZ or GE was not associated with significant changes in animal or organ weights, although there was a concurrent reduction in animal weights and an increase in uterine weight in the animals fed E2. Both OVX and INT rats fed control diets containing the isoflavones (except DZG in the OVX) did not significantly alter the mutagenicity of DMBA in the hormone-responsive tissues (heart, liver, mammary, and uterus) examined. E2 feeding effectively reduced DMBA-induced mutagenicity in the heart, and molecular analysis of the mutants showed a shift in the mutational spectra of DMBA from the common type (A:T to T:A) to G:C to A:T and A:T to G:C. The DMBA treatment induced tumors only in the mammary gland of INT animals, and neither DZ nor GE given separately resulted in any significant changes in DMBA-induced tumorigenicity. The DZG diet, however, reduced DMBA tumorigenic response in the mammary gland. DMBA treatment alone failed to induce tumors in the uterus; while feeding E2 to rats treated with DMBA resulted in 100% incidence of uterine dysplasia in the OVX rats. In comparison with E2, dietary DZ and GE were associated with low incidence of uterine dysplasia in OVX rats, suggesting weak estrogenic effects of these phytoestrogens. Taken together, DZ and GE given separately resulted in nonsignificant increase in the DMBA-mediated carcinogenicity in the mammary gland, while the mixture reduced DMBA response ... [Aidoo A et al; Regulatory Research Perspectives 7 (1) July 2007 Available at http://www.fda.gov/nctr/science/journals/text/vol7iss1/rrp0707.htm ]**PEER REVIEWED**
  
• IMMUNOTOXICITY: ... the purpose of these reported studies was to determine the potential effects of genistein on the immune system. ... Genistein was procured and administered in feed /to Sprague Dawley rats at 6 doses ranging from 5 to 1250 PPM/ ... . the results from this immunotoxicological evaluation demonstrate that, under the experimental conditions used, exposure to genistein did not impact on the immunocompetence of the F0 generation female rats with exception of an increased natural killer cell activity. However, for the F1 generation male and female rats, exposure to genistein for 77 days resulted in increases of both T cell number and T cell function. [National Toxicology Program; MG96005: NTP Assessment of the Immunotoxicity of Genistein (Endocrine Disruptor) (CAS No. 446-72-0) in Male and Female Sprague-Dawley Rats. Available at http://ntp.niehs.nih.gov/index.cfm?objectid=CAE460E6-F1F6-975E-7B661618456B37EA as of August 14, 2007 ]**PEER REVIEWED**
  
• IMMUNOTOXICITY: ... Female C57BL/6 mice (25- to 27-days-old) were ovariectomized, then placed on phytoestrogen-free feed 5 days later. Seven days after ovariectomy, they were given daily sc injections of either dimethylsulfoxide (DMSO) or genistein (8, 20, 80 mg/kg) for 28 days; some mice were given 80 mg/kg genistein plus the anti-estrogen ICI 182,780 (5 mg/kg/week). Cell-mediated immune response was tested by analyzing the delayed-type hypersensitivity (DTH) response to a hapten, 4-hydroxy-3-nitrophenyl acetyl succinimide (NP-O-SU), at the end of treatment. Reversibility of the effects of genistein was tested by measuring the DTH response in mice that were given genistein (20 or 80 mg/kg) for 28 days, then allowed to recover for 28 days. To determine if dietary genistein could affect cell-mediated immunity, mice ovariectomized as above were fed genistein at 0, 1000 or 1500 parts per million (ppm) for 28 days. There was a 46-67% decrease in the DTH response in the footpads of mice injected with 8-80 mg/kg genistein compared with controls (P<0.05 vs control for all treatment groups); these effects were reversible. On histopathological examination of the feet, there was decreased cell infiltration in genistein-treated animals compared with controls, and the numbers of CD4(+) and CD8(+) T cells in popliteal lymph nodes were reduced. The effects of genistein are mediated through both estrogen receptor (ER) and non-ER pathways, as the anti-estrogen ICI 182,780 only partially blocked the effects of genistein on the DTH response. Dietary genistein (1000 or 1500 ppm) decreased cell-mediated immunity while producing serum genistein concentrations in the physiological range for humans under certain nutritional conditions. [Yellayi S et al; J Endocrinol 176 (2): 267-74 (2003) ]**PEER REVIEWED** PubMed Abstract
  
• OTHER TOXICITY INFORMATION: ... To further investigate the molecular effects of genistein on the mammary gland, ... non-tumor bearing, ovariectomized female Wistar rats /were treated/ with this phytoestrogen either sc (10 mg/kg body weight) or orally (100 and 200 mg/kg body weight) for 3 days. Estradiol (E(2), 0.004 mg/kg sc) and ethynylestradiol (EE, 0.1 mg/kg per os) served as reference compounds. In the breast tissue, mRNA and protein expression of the progesterone receptor (marker for estrogenicity) and PCNA (marker gene for proliferation) were examined by quantitative real-time PCR, Western blotting and immunohistochemistry; the uterotrophic response was assessed also. Treatment with genistein per os or sc results in a small but significant stimulation of the uterine wet weight. In the mammary gland, genistein stimulates the expression of progesterone receptor (PR) but, in contrast to E(2), the isoflavone does not stimulate the expression of PCNA. ... /The/ data indicate that in non-malignant breast tissue short-term administration of genistein, in contrast to more potent estrogens like E(2), does not induce proliferation. [Hertrampf T et al; Planta Med 72 (4): 304-10 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• OTHER TOXICITY INFORMATION: Mice with established MCF-7 tumors were fed a basal diet (AIN-93G), divided into 5 groups, and given daily sc injections (10 mg/kg body weight) of either lenterodiol (END), enterolactone (ENL), genistein (GEN), a mixture of these compounds (MIX), or vehicle as a negative control for 22 weeks. Results showed that GEN acts estrogenically in both the uterus and bone by increasing the uterus weight, femur bone mineral density (BMD), and femur biomechanical strength (yield load), while the lignans do not. However, treatment with MIX induced minimal effects on femur biomechanical strength parameters but significantly increased uterus weight. A significant positive correlation was observed between MCF-7 tumor volume and femur BMD and biomechanical strength parameters (femur peak load and yield load) but not with uterus weight, suggesting that the uterus may respond differently to phytoestrogens compared to MCF-7 tumors and bone. It is concluded that GEN induces beneficial effects on bone but has adverse effects on tumors and uterus in this model of postmenopausal breast cancer. ... [Power KA et al; Bone 39 (1): 117-24 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• OTHER TOXICITY INFORMATION: ... /The authors/ evaluated whether dietary genistein can act in an additive manner with low circulating levels of 17beta-estradiol (E2). ... /by inserting/ various concentrations of E2 silastic implants (1:127, 1:63, 1:31, 1:15 and 1:7 = E2:cholesterol) and injected estrogen-dependent human breast cancer (MCF-7) cells into ovariectomized athymic mice. The E2 implants tested (1:127-1:7) generated 30.1-101.6 pM E2 in plasma, which is comparable to the E2 levels observed in postmenopausal women. The E2 implants stimulated MCF-7 tumor growth in a dose-dependent manner. ...The 1:31 ratio of E2 implant /was selected/ to evaluate if dietary genistein acts in an additive manner with low E2 levels to influence the growth of MCF-7 tumors. Ovariectomized mice were divided into four groups: MCF-7 control, 500 ppm genistein, 1:31 E2, and 1:31 E2 + 500 ppm genistein. At week 17, the average tumor sizes were 7.6, 32.1, 67.4 and 106.8 sq mm for these groups, respectively (P < 0.05), demonstrating that 500 ppm genistein additively stimulated MCF-7 tumor growth in the presence of low levels of E2. ... Results from this study suggest that consumption of products containing genistein may not be safe for postmenopausal women with estrogen-dependent breast cancer. [Ju YH et al; Carcinogenesis 27 (6): 1292-9 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• OTHER TOXICITY INFORMATION: ... This paper reports plasma pharmacokinetic analysis and metabolite identification using the parental mouse strain fed ... /isoflavone-supplemented/ diets, which contained genistin, mixed isoflavones, Novasoy, soy molasses, or soy flour plus mixed isoflavones. Whereas the degree of soy processing did affect several parameters reflecting isoflavone bioavailability and gut microflora metabolism of daidzein to equol, stimulation of tumor growth correlated significantly with only the plasma concentration of aglycon genistein produced by the diets. This conclusion is consistent with the known estrogen agonist activity of genistein aglycon on mammary tumor growth. [Allred CD et al; J Agric Food Chem 53 (22): 8542-50 (2005) ]**PEER REVIEWED** PubMed Abstract
  
• OTHER TOXICITY INFORMATION: Phytoestrogens were first associated with adverse effects on mammalian development and fertility from observations of animals consuming phytoestrogen-rich plants. Ewes feeding on Australian clover developed abnormal plasma concentrations of endogenous hormones with subsequent loss of fertility ... . The syndrome was termed “Clover Disease”. Subsequent investigations showed that when pregnant ewes were fed on yarloop clover, plasma progesterone and estrogen concentrations were lowered. This resulted in a substantial reduction (27%) of the mated ewes achieving successful conception, compared with ewes (95%) fed on grass. Similar, but more extreme, effects on fertility were observed in ewes administered large quantities of estradiol (300 mg) for periods of up to 26 months ... . These effects on fertility by a known estrogen led to the hypothesis that estrogenic compounds in the clover were responsible for the adverse effects on fertility. Compounds of similar structure to estradiol were identified in several types of clover: formononetin, biochanin A, and genistein in White clover (Trifolium repens), Subterranean clover (Trifolium subterraneum) and Yarloop (Trifolium yanninicum) and coumestrol in Medicago sativa ... . It is thought that sheep may be particularly susceptible to the estrogenic effects of these phytoestrogens due to the efficient conversion of formononetin to the more estrogenic compound, equol and the limited deactivation of the potent estrogen, coumestrol by metabolism ... . Improved farming practices have since prevented further incidences of “Clover Disease” ... . Additional evidence that dietary exposure to estrogenic compounds could adversely affect reproduction in mammals was shown in research on captive female cheetahs. High concentrations of genistein and daidzein in commercial feed, together with the limited ability of this species to deactivate these compounds metabolically, resulted in veno-occlusive disease and reproductive failure ... . Fertility problems in the females of mammalian species such as the cow, sheep, rabbit, guinea pig and mouse have also been reported... . [Hughs I, Woods HF; Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment Phytoestrogens and Health. UK Food Standards Agency, 444 p. (May 2003). Available at http://www.food.gov.uk/multimedia/pdfs/phytoreport0503 as of August 14, 2007 ]**PEER REVIEWED**
  
• OTHER TOXICITY INFORMATION: Endometriosis is a disease in which uterine tissue proliferates in extrauterine sites. Using a surgical model to simulate endometriosis, /the authors/ explored the potential for the phytoestrogen genistein, by injection and diet, to sustain endometriosis in rats. Uterine tissue was attached to intestinal mesentery of 8-week-old Sprague Dawley rats. After 3 weeks, the rats were ovariectomized and the implants measured. Following 3 weeks of daily injections or exposure to dietary genistein, animals were necropsied and implants located and measured. Injections of genistein (50 and 16.6 ug/g BW) or estrone (1 ug/rat) sustained the implants; injection of sesame oil (vehicle for estrone), dimethylsulfoxide (DMSO; vehicle for genistein), or genistein at 5.0 ug/g BW did not sustain implants. Dietary genistein (250 or 1000 mg genistein/kg AIN-76A diet) did not support the implants. In ovary-intact rats exposed to 250 mg genistein/kg AIN-76A diet, implant size was not altered, compared to control-fed animals. To assess estrogenic actions of genistein, we measured uterine estrogen receptor alpha (ER-alpha) and progesterone receptor (PR) isoforms A and B by Western blot analyses. Injections of estrone or genistein (50 or 16.6 ug/g BW) significantly reduced uterine ER-alpha compared to vehicle-treated animals. PR (B) was significantly increased by all injected doses of genistein or estrone and by the higher dietary dose (1000 mg genistein/kg AIN-76A). PR (A) was significantly increased by injected doses of genistein (16.6 and 5.0 ug/g BW). /The authors/ conclude that pharmacologic injections, but not dietary physiological concentrations of genistein, support surgically induced endometriosis in rats /and conclude that their/ results suggest a critical role for ER modulation and genistein bioavailability in the maintenance of the implants. [Cotroneo MS, Lamartiniere CA; Toxicol Sci 61 (1): 68-75 (2001) ]**PEER REVIEWED** PubMed Abstract
  

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Human Toxicity Values

• None found

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Non-Human Toxicity Values

• None found

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Absorption, Distribution and Excretion

• ... Genistein is rapidly absorbed in humans following oral intake. Before absorption into the systemic circulation, most genistein is conjugated with glucuronic acid and excreted in the bile to undergo enterohepatic circulation ... . Therefore, genistein bioavailability is very limited. Times to obtain maximum plasma concentrations were reported at 1 to 6 hours for free genistein ... and 3 to 8 hours for total genistein (aglycone + conjugates ...). In one of the studies, the lowest dose used (2 mg/kg bw) was stated to provide more than twice the level of isoflavones ingested in a Japanese daily diet. A study in which menopausal women were given a 50 mg commercial isoflavone extract incorporated into fruit juice, chocolate, or a cookie showed no significant effect of the food matrix on genistein absorption or urinary excretion parameters. In a study in which 8 women were dosed with 0.4 or 0.8 mg/kg bw 13C-labeled genistein, the area under the curve (AUC) at the higher dose was less than double the AUC at the lower dose, suggesting a decrease in fractional absorption with increasing dose. [National Toxicology Program, Center For the Evaluation of Risks To Human Reproduction; NTP-CERHR Expert Panel Report on the Reproductive and Developmental Toxicity of Genistein; (NTP-CERHR-GENISTEIN-06) April 2006. Available at http://cerhr.niehs.nih.gov/chemicals/genistein-soy/genistein/Genistein_Report_final.pdf as of August 14, 2007 ]**PEER REVIEWED**
  
• There is considerable individual variation in the absorption and metabolism of ingested genistin and genistein. There are some data suggesting that genistein may be more bioavailable than genistin. However, other data suggest that the extent of absorption of genistein is similar for the aglycone and the glucoside forms. There are little data available on the tissue distribution of genistein. [Physicians Desk Reference (PDR) for Nutritional Supplements 1st ed, Medical Economics, Thomson Healthcare; Montvale, NJ p.180 (2001) ]**PEER REVIEWED**
  
• A recently completed study has also shown inter-individual variation in the urinary excretion of isoflavones and their metabolites following soy challenge in adults. In this study, 76 volunteers were fed either a high (104+/-24 mg total isoflavones/day) or low (0.5+/-0.5 mg total isoflavones/day) soya diet for 10 weeks. Volunteers on the high soya diet showed extensive urinary excretion of daidzein, genistein and their metabolites. Of the volunteers on the high soya diet 34% were identified as good equol excretors ( 1000 nmol/24 hours). Comparative analysis of the fecal flora between equol and non-equol producers was investigated, however, the microflora (bacteria) responsible for equol production could not be isolated and therefore, were not be identified [Hughs I, Woods HF; Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment Phytoestrogens and Health. UK Food Standards Agency, 444 p. (May 2003). Available at http://www.food.gov.uk/multimedia/pdfs/phytoreport0503 as of August 14, 2007 ]**PEER REVIEWED**
  
• The pharmacokinetics of isoflavones in 10 healthy women were determined from serum appearance/disappearance concentration profiles and urinary excretions after single-bolus ingestion of 10, 20 or 40 g of soy nuts delivering increasing amounts of the conjugated forms of daidzein (6.6, 13.2 and 26.4 mg) and genistein (9.8, 19.6 and 39.2 mg). Peak serum daidzein and genistein concentrations were attained after 4-8 hr, and elimination half-lives were 8.0 and 10.1 hr, respectively. There were no differences in the pharmacokinetics of daidzein and genistein between pre- and postmenopausal women, indicating absorption and disposition of isoflavones to be independent of age or menopausal status. A curvilinear relationship was observed between the bioavailability of daidzein and genistein, apparent from the area under the curve to infinity (AUC(inf)) of the serum concentration-time profiles and the amount of isoflavones ingested. The mean fraction of the isoflavones excreted in urine decreased with increasing intake when expressed as a percentage of the administered dose (63.2 + or - 8.0, 54.4 + or - 8.1 and 44.0 + or - 4.3%, respectively, for daidzein, and correspondingly, 25.2 + or - 5.3, 13.4 + or - 2.1 and 15.8 + or - 2.7% for genistein), underscoring the trend toward nonlinear pharmacokinetics. Equol was identified as a metabolite in 30% of women; it was present consistently in urine and blood from the same subjects. Its delayed appearance was consistent with colonic synthesis. On the basis of the pharmacokinetics, optimum steady-state serum isoflavone concentrations would be expected from modest intakes of soy foods consumed regularly throughout the day rather than from a single highly enriched product. [Setchell KD et al; J Nutr 133 (4): 1027-35 (2003) ]**PEER REVIEWED** PubMed Abstract
  
• ... After intestinal absorption, circulating genistein and daidzein are eliminated primarily by the kidneys. This study was undertaken to assess the metabolism of genistein and daidzein in patients with end-stage renal disease (ESRD) on hemodialysis therapy, and to test whether this treatment modality can replace the lack of kidney function, with respect to the elimination of the isoflavones. Twenty-three hemodialysis patients and 10 healthy subjects were studied. While consuming a self-selected low isoflavone diet, baseline blood levels were undetectable in eight of 10 healthy subjects and in 14 of 23 dialysis patients. The remaining participants had detectable levels, with the nine dialysis patients displaying much higher blood concentrations than the two healthy control subjects /with detectable levels/. After the evening intake of one dose of an isoflavone-rich soy protein isolate drink, the early morning blood levels of genistein and daidzein were higher in seven dialysis patients than in eight healthy subjects (genistein 1271+/-321 versus 425+/-104, P<0.05; daidzein 1304+/-352 versus 292+/-78, P<0.05). The blood clearance of the isoflavones was studied in two healthy subjects and in three dialysis patients. Genistein and daidzein were eliminated within 2 days in the healthy subjects, but had not returned to baseline in two of three ESRD patients, 7 d after intake. The half-life of both compounds was estimated to be 10-fold longer in the ESRD patients than in the healthy subjects. Finally, genistein and daidzein levels were measured before and after dialysis in five patients, both while on their regular diet and after one dose of a soy protein isolate drink. In both instances, the dialysis treatment did not affect the blood isoflavone levels. [Fanti P et al; J Am Soc Nephrol 10 (4): 864-71 (1999) ]**PEER REVIEWED** PubMed Abstract
  
• Amniotic fluid samples (n=59) from women (n=53) undergoing routine amniocentesis between 15 and 23 weeks of gestation were analyzed by gas chromatography/mass spectrometric (GC/MS). Analytes included the phytoestrogens daidzein, genistein, formononetin, biochanin A, and coumestrol. Dietary phytoestrogens were quantified in 96.2% of second trimester amniotic fluid samples tested. The mean (+/- standard deviation (S.D.)) concentration of daidzein and genistein in amniotic fluid was 1.44 +/- 1.34 and 1.69 +/- 1.48 ng/mL with maximum levels of 5.52 and 6.54 ng/mL, respectively. Second trimester amniotic fluid contains quantifiable levels of dietary phytoestrogens and thus is a marker of mid pregnancy fetal exposure. [Foster WG et al; Toxicol Lett 129 (3): 199-205 (2002) ]**PEER REVIEWED** PubMed Abstract
  
• Three papers reported that genistein is distributed to the human conceptus. Adlercreutz et al. used a GC/MS method to measure maternal plasma, cord plasma, and amniotic fluid phytoestrogen levels in 7 healthy omnivorous Japanese women (20-30 years old) who had just given birth. Only the results for genistein are discussed here. ... Genistein was detected in cord blood and amniotic fluid, and levels were reported to be variable between subjects. Correlations between maternal blood and cord blood or amniotic fluid genistein levels were not statistically significant. Most of the genistein in amniotic fluid was represented by glucuronide or sulfoglucuronide conjugates. [Unconjugated and sulfate conjugates of genistein represented 10-15% of total genistein in cord blood and amniotic fluid.] The study authors concluded that phytoestrogens cross the placenta. Foster et al. measured phytoestrogens in 57 human amniotic fluid samples collected between 15 and 23 weeks of gestation. Samples were collected in Los Angeles [ethnic composition and dietary factors not discussed]. Measurements were made by GC/MS after glucuronidase treatment to hydrolyze the conjugates. Genistein was measurable in 42 of the samples with a mean +/- SD concentration of 1.08 +/-0.91 ng/mL [4.0 +/-3.4 nM] (range 0.4-4.86 ng/mL [1.5-17.9 nM]). In a different paper, these authors reported genistein concentrations in 59 amniotic fluid samples obtained from 53 pregnant women at 15-23 weeks of gestation (4 sets of twins and 1 woman who was sampled 3 times). There were 42 women with measurable amniotic fluid genistein concentrations. The mean +/- SD genistein concentration was 1.69 +/- 1.48 ng/mL [6.25 +/- 5.48 nM] (maximum 6.54 ng/mL [24.2 nM]). [In a table, the mean +/- SD is reported as 1.37 +/- 1.00 ng/mL (5.07 +/- 3.7 nM) with a median of 0.99 ng/mL (3.7 nM). It is not known whether there are any samples represented in both papers.] Engel et al. measured genistein in amniotic fluid samples obtained prior to 20 weeks. The samples were collected for the sole indication of “advanced maternal age” (>35 years). The median (range) genistein concentration was 1.38 (0.20-7.88) g/L. [National Toxicology Program, Center For the Evaluation of Risks To Human Reproduction; NTP-CERHR Expert Panel Report on the Reproductive and Developmental Toxicity of Genistein; (NTP-CERHR-GENISTEIN-06) April 2006. Available at http://cerhr.niehs.nih.gov/chemicals/genistein-soy/genistein/Genistein_Report_final.pdf as of August 14, 2007 ]**PEER REVIEWED**
  
• In reviews that primarily addressed genistein exposure through soy product intake, it was reported that most ingested genistein is excreted in urine, with very little excreted in feces. Isoflavone excretion has been reported at about 30% in urine and 1 to 4% in feces. These fecal excretion data are in contrast to experimental animal data, which show fecal excretion of 14C-genistein and/or derivatives at 30 to 36% of dose. (A strong possibility must be entertained that some of the material escaped detection due to bacterial degradation ... ). [National Toxicology Program, Center For the Evaluation of Risks To Human Reproduction; NTP-CERHR Expert Panel Report on the Reproductive and Developmental Toxicity of Genistein; (NTP-CERHR-GENISTEIN-06) April 2006. Available at http://cerhr.niehs.nih.gov/chemicals/genistein-soy/genistein/Genistein_Report_final.pdf as of August 14, 2007 ]**PEER REVIEWED**
  
• .. This study describes the internal exposures of post-natal day 10 (PND10) /Sprague-Dawley/ rat pups due to lactational transfer of genistein. Conjugated and aglycone forms of genistein were measured by using LC/MS/MS in serum (PND10) and milk (PND7) from lactating dams consuming a genistein-fortified soy-free diet, and in serum from their pups at a time when milk was the only food source (PND10). This study shows that limited lactational transfer of genistein to rat pups occurs and that internal exposures to the active aglycone form of genistein are generally lower than those measured previously in the fetal period. [Doerge DR et al; Reprod Toxicol 21 (3): 307-12 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• ... The goal of the present study was to measure placental transfer of genistein in rats as a possible route of developmental exposure. Pregnant Sprague-Dawley rats were administered genistein orally, either by diet or by gavage. Concentrations of genistein aglycone and conjugates were measured in maternal and offspring serum and brain using HPLC with isotope dilution electrospray tandem mass spectrometry. Although fetal or neonatal serum concentrations of total genistein were approximately 20-fold lower than maternal serum concentrations, the biologically active genistein aglycone concentration was only 5-fold lower. Fetal brain contained predominately genistein aglycone at levels similar to those in the maternal brain. These studies show that genistein aglycone crosses the rat placenta and can reach fetal brain from maternal serum genistein levels that are relevant to those observed in humans. [Doerge DR et al; Reprod Toxicol 15 (2): 105-10 (2001) ]**PEER REVIEWED** PubMed Abstract
  
• Mass balance, plasma pharmacokinetics, tissue distribution, and metabolism of carbon-14-genistein were investigated in male and female rats (n = 5) following an oral dose of (14-C)genistein (4 mg/kg) to determine potential sites and mechanisms of biological action. Mean total excretion of radioactivity in urine and feces for both sexes was 66 and 33% of the dose respectively at 166 hr after administration. Mean and maximal concentrations of radioactivity in plasma were significantly (P < 0.02) higher in male than female rats, with half-lives of 12.4 and 8.5 hr, respectively. The concentration of radioactivity was significantly (P < 0.002) higher in liver from females than males and in reproductive (vagina, uterus, ovary, and prostate) compared with other peripheral organs. Analysis of plasma extracts by radio-HPLC-MS indicated that the predominant metabolites were genistein glucuronides, 4-hydroxyphenyl-2-propionic acid, and trace amounts of parent compound (<5%). Radioactive residues in uterus and prostate were predominantly parent compound and 4-hydroxyphenyl-2-propionic acid, respectively. Significantly (p < 0. 05) increased retention of (14-C)genistein or metabolites was associated with reproductive organs, such as vagina, uterus, ovary, and prostate, likely to contain relatively high concentrations of estrogen receptors or binding proteins compared with other peripheral tissues. Factors liable to influence bioavailability of biologically active genistein or metabolites, such as dietary intake, warrant further investigation to determine the risks or benefits for different consumer groups of phytoestrogen-containing foods. [Coldham NG et al; Toxicol Appl Pharmacol 164 (2): 206-15 (2000) ]**PEER REVIEWED** PubMed Abstract
  
• Genistein (4 or 40 mg/kg) was administered to pregnant Sprague-Dawley rats by oral gavage daily from gestation day (GD) 5 through 19 or on GD 19 alone. Maternal and GD 19 fetal tissues were collected 0.5, 1, 2, 4, 6, 8, 12, and 24 hr following administration of the final dose on GD 19. Concentrations of genistein, genistein glucuronide, and genistein sulfate were quantitated by LC-MS/MS. In maternal plasma, genistein glucuronide was the predominant metabolite. In the fetal plasma, genistein glucuronide and genistein sulfate were the primary metabolites. Genistein levels in maternal and fetal plasma were much lower than its conjugates. The concentration of genistein in placental tissue was higher than either conjugate. Fetal concentrations of unconjugated genistein following administration of 40 mg/kg were above the EC50 for ERbeta activation. Repeated administration of 40 mg/kg genistein resulted in minor changes in genistein kinetics in the pregnant rat compared to single administration of the same dose. These data suggest that conjugated forms of genistein are not transported across the placenta. High placental concentrations of genistein indicate the placenta is a potential target organ for genistein action during gestation. [Soucy NV et al; Toxicol Sci 90 (1): 230-40 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• Genistein, the principal soy isoflavone, was administered in the diet to male and female Sprague-Dawley rats as part of a multigeneration study of potential endocrine modulation. The rats were exposed to genistein in utero, through maternal milk, and as adults through postnatal day 140 via essentially isoflavone-free feed (approximately 0.5 ug/g) fortified at 5, 100 and 500 ug/g with genistein aglycone. Analytical methods based on liquid chromatography, mass spectrometry and the use of deuterated genistein were developed and validated for use in measuring genistein in serum and tissues. Pharmacokinetic analysis of serum genistein showed a significant difference (P < 0.001) in the elimination half-life and area under the concentration-time curve between male [2.97 +/- 0.14 hr and 22.3 +/- 1.2 umol/(L. hr), respectively] and female rats [4.26 +/- 0.29 hr and 45.6 +/- 3.1 umol/(L. hr), respectively, +/- SEM]. Endocrine-responsive tissues including brain, liver, mammary, ovary, prostate, testis, thyroid and uterus showed significant dose-dependent increases in total genistein concentration. Female liver contained the highest amount of genistein (7.3 pmol/mg tissue) and male whole brain contained the least (0.04 pmol/mg). The physiologically active aglycone form was present in tissues at fractions up to 100%, and the concentration was always greater than that observed in serum in which conjugated forms predominated (95-99%). These results for measured amounts of genistein, present as aglycone and conjugates, in putative target tissues provide a link with other studies in which blood concentrations and physiologic effects of genistein are measured. [Chang HC et al; J Nutr 130 (8): 1963-70 (2000) ]**PEER REVIEWED** PubMed Abstract
  
• The intestinal absorption, biliary excretion and metabolism of genistein ... was examined in anesthetized, adult female rats fitted with indwelling biliary cannulas. 4-14C-Genistein, when infused into the duodenum, was rapidly absorbed from the intestine, taken up by the liver and excreted into the bile as its 7-O-beta-glucuronide conjugate. Cumulative recovery of 14C-radioactivity in the bile over a 4-hr period was 70-75% of the dose. When genistein was infused into the portal vein, it was also taken up efficiently by the liver, conjugated with glucuronic acid and transported into bile. However, portal blood collected after duodenal infusions of genistein contained mostly genistein 7-O-beta-glucuronide, suggesting that in vivo glucuronidation occurred in the intestinal wall rather than the liver. This was confirmed using everted intestinal sac preparations. Reinfusion of genistein 7-O-beta-glucuronide into the duodenum or into the mid small intestine resulted in its reappearance in the bile, albeit more slowly than when genistein was infused. Over a 4-hr collection period, the cumulative recovery of 14C-radioactivity in bile was 27 and 70-75% of the administered dose for duodenal and ileal infusions, respectively. These data indicate that genistein is highly bioavailable in rats and because of its enterohepatic circulation may accumulate within the gastrointestinal tract. [Sfakianos J et al; J Nutr 127 (7): 1260-8 (1997) ]**PEER REVIEWED** PubMed Abstract
  
• The present study describes an in vivo bioavailability experiment for genistein and its glycoside genistin, either as pure compounds or from a soy protein isolate extract, using freely moving unanesthetized rats with a cannulation in the portal vein. The results show that genistein is readily bioavailable, being observed in portal vein plasma at the first point of detection at 15 min after dosing. The AUC(0-24hr) values for total genistein and its conjugates were 54, 24, and 13 uM hr for genistein, genistin, and an enriched protein soy extract, respectively. These results indicate that the bioavailability of genistein is higher for the aglycon than for its glycoside. Genistin is partly absorbed in its glycosidic form. It is concluded that bioavailability studies based on portal vein plasma levels contribute to insight into the role of the intestine and liver in deglycosylation and uptake characteristics of glycosylated flavonoids. [Steensma A et al; J Agric Food Chem 54 (21): 8006-12 (2006) ]**PEER REVIEWED** PubMed Abstract
  

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Metabolism/Metabolites

• Toxicokinetic and metabolism data in humans and experimental animals indicate that genistein is absorbed into the systemic circulation of infants and adults. Genistein ... circulates as its glucuronide conjugate, and a much smaller percentage circulates as the aglycone. Genistein can be glucuronidated in the intestine or liver, but the intestine appears to play the major role in glucuronidation. Genistein glucuronides undergo enterhepatic cycling, and in the process can be deconjugated by intestinal bacteria. The role of gut bacteria in the metabolism of genistein has been clearly established. Genistein can be metabolized through a pathway that ultimately leads to the formation of 6'-hydroxy-O-demethylangolensin. Once absorbed, genistein glucuronide, and to a smaller extent genistien aglycone, are widely distributed to organ systems and the conceptus. The majority of a genistein dose is excreted in urine within 24 hours. [National Toxicology Program, Center For the Evaluation of Risks To Human Reproduction; NTP-CERHR Expert Panel Report on the Reproductive and Developmental Toxicity of Genistein; (NTP-CERHR-GENISTEIN-06) April 2006. Available at http://cerhr.niehs.nih.gov/chemicals/genistein-soy/genistein/Genistein_Report_final.pdf as of August 14, 2007 ]**PEER REVIEWED**
  
• Prior to entering the systemic circulation, most genistein is conjugated with glucuronic acid by uridine diphosphate (UDP)-glucuronosyltransferase (UDPGT); a much smaller amount is conjugated to sulfate by sulfotransferase enzymes. Conjugation of genistein occurs in the intestine, although it also has been reported to occur in liver. One study demonstrated that the ability to catalyze glucuronidation of genistein was greatest with microsomes from kidney > colon > liver. UDPGT isoenzymes including 1A1, 1A4, 1A6, 1A7, 1A9, and 1A10 were observed to catalyze the glucuronidation of genistein. The UGT 1A10 isoform, which is present in colon, gastric, and biliary epithelium but not in liver, was observed to have the highest activity and specificity for genistein. Based on those observations, the study authors concluded that the intestine plays a major role in the glucuronidation of genistein. The glucuronide and sulfate conjugates can enter the systemic circulation, and the majority of isoflavone compounds in the circulation are present in conjugated form. In studies where humans were exposed to genistein alone or in combination with other isoflavone aglycones (calculated as genistein doses of 1-16 mg/kg bw), most of the genistein was present in plasma in conjugated form; free genistein represented 1-3% of total plasma genistein levels. The conjugated isoflavones undergo enterohepatic circulation, and upon return to the intestine, they are deconjugated by bacteria possessing beta-glucuronidase or arylsulfatase activity. The metabolites may be reabsorbed or further metabolized by gut microflora. One review reported that about 10% of isoflavonoids are circulated in plasma unconjugated. [National Toxicology Program, Center For the Evaluation of Risks To Human Reproduction; NTP-CERHR Expert Panel Report on the Reproductive and Developmental Toxicity of Genistein; (NTP-CERHR-GENISTEIN-06) April 2006. Available at http://cerhr.niehs.nih.gov/chemicals/genistein-soy/genistein/Genistein_Report_final.pdf as of August 14, 2007 ]**PEER REVIEWED**
  
• Biotransformations by gut microflora play a pivotal role in determining the biological activity of isoflavones that occur in soya-based foods predominantly as betaglycosyl conjugates. Microflora prepared from rat caeca and human feces were used to investigate the metabolic fate of genistein beta-glycosides extracted from soya flour. The end-products of such metabolism were determined by parallel incubations of microflora with [2',3,5',6'-3H] and [4-14C]-labelled genistein. ... Quantitative analysis by LC-MS/IS indicated very rapid and complete degradation of genistin, which was associated with a transient increase in genistein. Qualitative studies indicated that the malonyl and acetyl glycosides of genistein were also degraded by the microflora. ... Incubation of caecal and fecal microflora with (3)H and (14)C genistein yielded similar radiolabelled metabolites, which were identified by radio-LC-MS(n) as the intermediates dihydrogenistein and 6'-hydroxy-O-desmethylangolensin and end-product 4-hydroxyphenyl-2-propionic acid. This profile of genistein metabolites indicated selective hydrolysis of 6'-hydroxy-O-desmethylangolensin between carbon atoms 1' and 1 to yield the end-products 4-hydroxyphenyl-2-propionic acid and 1,3,5-trihydroxybenzene. ... The biological significance of the products of genistein metabolism warrant further investigation since they may play an important role in mediating the beneficial antioxidant health effects associated with the consumption of isoflavones in food. [Coldham NG et al; Xenobiotica 32 (1): 45-62 (2002) ]**PEER REVIEWED** PubMed Abstract
  
• Biotransformation of the phytoestrogen (14-C)genistein was investigated in male and female rats by application of narrow-bore radio-HPLC-MSn (LCQ, Finnigan) to determine intermediates in metabolism. Urine contained five metabolites, Gm1-Gm5, 24 hr after dosing by gavage with [14C]genistein (4 mg kg(-1)). Structural analysis following ESI revealed molecular ions (M+H)+ of m/z 447, 449, 273, and 271 for metabolites Gm2, Gm3, Gm5 and genistein, respectively and an [M-H]- of m/z 349 for Gm4. Metabolite structure was deduced by evaluation of product ion spectra derived from unlabelled and (14)C-labelled ions and sensitivity to treatment with beta-glucuronidase. These studies indicated identity of metabolites with genistein glucuronide (Gm2), dihydrogenistein glucuronide (Gm3), genistein sulphate (Gm4) and dihydrogenistein (Gm5). Detection of the beta-glucuronidase resistant major metabolite Gm1 by ESI was poor and so was analysed by negative ion APCI; this revealed a deprotonated molecular ion of m/z 165 which had chromatographic and mass spectral properties consistent with authentic 4-hydroxyphenyl-2-propionic acid, a novel metabolite of genistein. In vitro metabolism studies with anaerobic caecal cultures derived from male and female rats revealed metabolism of genistein to Gm1 via Gm5 and an additional metabolite (Gm6) which was identified from product ion spectra as 6'-hydroxy-O-desmethylangolensin. Biotransformation of genistein by both isolated hepatocytes and precision-cut liver slices was limited to glucuronidation of parent compound. Commonality of genistein metabolites found in rats with those reported in man suggest similar pathways of biotransformation, primarily involving gut micro-flora. [Coldham NG et al; J Steroid Biochem Mol Biol 70 (4-6): 169-84 (1999) ]**PEER REVIEWED** PubMed Abstract
  
• ... In the large intestine, bacterial beta-glucosidases hydrolyze genistin to genistein. Genistein is either absorbed or further metabolized in the large intestine to dihydrogenistein and 6'-hydroxy-O-desmethylangolensin. Genistein, which is absorbed from the large intestine and small intestine, is eventually transported to the liver. There, it undergoes conjugation with glucuronate and sulfate via hepatic phase II enzymes (UDP-glucuronosyltransferases and sulfotransferases). The glucuronate and sulfate conjugates of genistein are excreted in the urine and in the bile. The genistein conjugates may be deconjugated to release genistein, which may be reabsorbed via the enterohepatic circulation. [Physicians Desk Reference (PDR) for Nutritional Supplements 1st ed, Medical Economics, Thomson Healthcare; Montvale, NJ p.180 (2001) ]**PEER REVIEWED**
  
• Genistein (4 or 40 mg/kg) was administered to pregnant Sprague-Dawley rats by oral gavage daily from gestation day (GD) 5 through 19 or on GD 19 alone. Maternal and GD 19 fetal tissues were collected 0.5, 1, 2, 4, 6, 8, 12, and 24 hr following administration of the final dose on GD 19. Concentrations of genistein, genistein glucuronide, and genistein sulfate were quantitated by LC-MS/MS. In maternal plasma, genistein glucuronide was the predominant metabolite. In the fetal plasma, genistein glucuronide and genistein sulfate were the primary metabolites. [Soucy NV et al; Toxicol Sci 90 (1): 230-40 (2006) ]**PEER REVIEWED** PubMed Abstract
  
• The intestinal absorption, biliary excretion and metabolism of genistein ... was examined in anesthetized, adult female rats fitted with indwelling biliary cannulas. 4-14C-Genistein, when infused into the duodenum, was rapidly absorbed from the intestine, taken up by the liver and excreted into the bile as its 7-O-beta-glucuronide conjugate. ... When genistein was infused into the portal vein, it was also taken up efficiently by the liver, conjugated with glucuronic acid and transported into bile. However, portal blood collected after duodenal infusions of genistein contained mostly genistein 7-O-beta-glucuronide, suggesting that in vivo glucuronidation occurred in the intestinal wall rather than the liver. [Sfakianos J et al; J Nutr 127 (7): 1260-8 (1997) ]**PEER REVIEWED** PubMed Abstract
  

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TSCA Test Submissions

• None found

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Footnotes

¹ Source: the National Library of Medicine's Hazardous Substance Database, 10/28/2007.