Elena Bianca Adamo

Effects of the Phytoestrogen Genistein on Bone Metabolism in Osteopenic Postmenopausal Women: A Randomized Trial

Context Women seeking alternative treatments to preserve bone often use phytoestrogens, but evidence of their effectiveness is lacking. Phytoestrogens are found in soy products. Genistein is a phytoestrogen with a structure similar to that of 17-estradiol. Contribution This randomized trial compared genistein, 54 mg/d, with placebo for 24 months in 389 osteopenic postmenopausal women. Increases in bone mineral density were greater with genistein than with placebo. Genistein also had favorable effects on markers of bone metabolism. Genistein did not increase endometrial thickness, but it did cause gastrointestinal side effects. Implications Genistein appears to have a favorable effect on markers of bone health in osteopenic postmenopausal women. Studies of its effect on fractures are needed. The Editors Postmenopausal osteoporosis is caused by a sharp decrease in estrogen levels that leads to an increased rate of bone remodeling (13). Currently available treatments for postmenopausal osteoporosis include hormone replacement therapy; calcitonin; bisphosphonates; and selective estrogen receptor modulators, such as raloxifene (4, 5). Although hormone replacement therapy is effective in reducing postmenopausal bone loss (68), it is associated with a higher risk for breast, endometrial, and ovarian cancer; cardiovascular disease; venous thromboembolism; and stroke (810). Epidemiologic data indicate that women who ingest high amounts of phytoestrogens, particularly isoflavones in soy products, have less risk for osteoporosis than do those who consume a typical Western diet (1113). Consequently, many women use phytoestrogens to maintain bone density. Genistein, an isoflavone phytoestrogen that is abundant in soybean products, structurally resembles 17-estradiol (14). As a natural selective estrogen receptor modulator, genistein may positively regulate bone cell metabolism without harmful estrogenic activity in the breast and uterus. This safe profile results from the greater affinity of genistein for estrogen receptor-, which is more abundant in bone, than for estrogen receptor-, which is abundant in reproductive tissue. Observational studies suggest that postmenopausal Asian women who consume diets high in isoflavones have a lower rate of fracture than that in other groups (15, 16). However, the mechanism of action of genistein on bone is not yet fully understood. In postmenopausal women, treatment with genistein (54 mg/d) increased bone mineral density (BMD) at the lumbar spine and femoral neck with no clinically significant adverse effects on the breast and uterus (17). In the same cohort, genistein decreased the ratio of soluble receptor activator of nuclear factor-B ligand to osteoprotegerin, which may partly account for its positive effects on BMD (18). These investigations were the first to evaluate the effects of purified, standardized genistein on bone health, but they were short in duration and included only 90 patients. One published trial assessed the effect of isoflavones on BMD in postmenopausal women, but it compared isoflavone-rich soy milk (containing only a small amount of genistein) with natural transdermal progesterone (19). Other studies of pure genistein from our research group have focused on cardiovascular outcomes or menopausal symptoms rather than on bone health (2022). We performed a randomized, placebo-controlled trial of the effects of pure genistein on bone density and bone metabolism over 24 months in a larger cohort of osteopenic postmenopausal women. Methods Design and Setting The study protocol is consistent with the principles of the Declaration of Helsinki, and participants gave written informed consent. Participants were recruited from women reporting to the Center for Osteoporosis in the Department of Internal Medicine and the Center for Menopause in the Department of Obstetrical and Gynecological Sciences, University of Messina (Messina, Italy), and to the Department of Medical Physiopathology, University La Sapienza (Rome, Italy). Three hundred eighty-nine women met the inclusion criteria and agreed to participate (Figure 1). Figure 1. Study flow diagram. Participants Participants were women 49 to 67 years of age who had been postmenopausal for at least 12 months at baseline, were in good general health, had not had a menstrual period in the preceding year, had not undergone surgically induced menopause, and had a follicle-stimulating hormone level greater than 50 IU/L and a serum 17-estradiol level of 100 pmol/L or less (27 pg/mL). At the start of the study, a complete family history was obtained, physical examination and laboratory evaluation (chemical analytes and hematologic measurements) were performed, and BMD was measured at the lumbar spine and femoral neck. Exclusion criteria were clinical or laboratory evidence of confounding systemic diseases, such as cardiovascular, hepatic, or renal disorders; coagulopathy; use of oral or transdermal estrogen, progestin, androgens, selective estrogen receptor modulators, or other steroids; use of biphosphonates, cholesterol-lowering therapy, or cardiovascular medications (including antihypertensive drugs) in the preceding 6 months; smoking habit of more than 2 cigarettes daily; treatment in the preceding year with any drug that could have affected the skeleton; family history of estrogen-dependent cancer; and BMD at the femoral neck greater than 0.795 g/cm2 (which corresponds to a T-score of 1.0 SD). Randomization and Intervention We assigned patients to groups by using a computer-generated randomization sequence with a permuted block size of 4, stratified by center. After a 4-week stabilization period during which participants received a standard low-soy, reduced-fat diet, participants were assigned to receive genistein (n= 198), 54 mg/d in 2 tablets (Laboratori Plants, Messina, Italy), or placebo (n= 191) (Figure 1). The biological effects of phytoestrogen intake are described elsewhere (23). No patient withdrew from the study during the stabilization period. The purity of genistein was 98%. Placebo and genistein tablets were identical in appearance and taste. Both genistein and placebo tablets contained calcium carbonate (500 mg) and vitamin D (400 IU). All participants were counseled on an isocaloric, reduced-fat diet composed of 25% to 30% energy from fat, less than 10% energy from saturated fatty acids, 55% to 60% energy from carbohydrates, and 15% energy from protein, with a cholesterol intake less than 300 mg/d and fiber intake of 35 g/d or greater. Recommended daily caloric intake was based on body size and was calculated by using the HarrisBenedict equation. We used this diet during the stabilization period to ensure that all participants had the same energy intake and to avoid interference with the lipid profile. The intake of soy products, legumes, or other nutrient supplements was prohibited. The isoflavone intake before randomization, as assessed by using a food-frequency questionnaire, was 1 to 2 mg/d. This intake has been shown to be typical in Western populations (24). Participants used this diet throughout the study, and adherence was reinforced by a nutritionist. Diet and body mass index were evaluated in all participants during follow-up. Primary Outcome The BMD at the anteroposterior lumbar spine and femoral neck was measured by using dual-energy x-ray absorptiometry (Hologic QDR 4500 W, Technologic, Turin, Italy) at baseline and after 12 and 24 months of treatment. The instrument was calibrated daily according to the manufacturers instructions. Reproducibility was calculated as a coefficient of variation obtained by weekly measurements of a standard phantom on the instrument and by repeated measurements obtained in 3 patients of different ages. The coefficient of variation of our instrument is 0.5% with the standard phantom; in vivo, we calculated a coefficient of variation of 1.1% for the lumbar spine and 1.5% for the femoral neck. Secondary Outcomes Bone Resorption Markers At baseline, 12 months, and 24 months, a 2-hour fasting morning urine sample was collected at the same time of day to assess urinary excretion of pyridinium crosslinks (pyridinoline and deoxypyridinoline). Pyridinoline (normal range, 26 to 91 pmol/mol creatinine) and deoxypyridinoline (normal range, 3 to 21 pmol/mol creatinine) were measured by using high-performance liquid chromatography (Bio-Rad Laboratories, Hercules, California). Bone Formation and Bone Growth Markers and Other Variables After an overnight fast, venous blood samples were collected between 8 a.m. and 9 a.m. through a polyethylene catheter inserted in a forearm vein. The serum was separated from the blood corpuscles by centrifugation and kept frozen at 70C until analysis for bone formation and bone growth markers, calcium, intact parathyroid hormone, 25-hydroxyvitamin D3, 17-estradiol, follicle-stimulating hormone, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides. Serum bone-specific alkaline phosphatase (normal range, 8.5 to 17.9 g/L) and insulin-like growth factor I (normal range, 9.6 to 21.2 nmol/L) were measured by using an immunoenzymatic assay (Pantec, Turin, Italy). Serum calcium (normal range, 2.25 to 2.75 mmol/L [9 to 11 mg/dL]), serum phosphorus (normal range, 1.13 to 1.45 mmol/L [3.5 to 4.5 mg/dL]), and urinary creatinine (130 to 220 molkg1d1 [14.71 to 24.89 mg/kg of body weight per day]) were measured by using automated routine procedures. Parathyroid hormone (normal range, 12 to 100 pg/dL), 25-hydroxyvitamin D3 (normal range, 1.25 to 7.5 nmol/L), and follicle-stimulating hormone (normal range, 21 to 153 IU/L in the postmenopausal phase) were measured by using high-performance liquid chromatography (Bio-Rad Laboratories). 17-Estradiol (normal range, 37 to 110 pmol/L in the postmenopausal phase) was evaluated by using a solid-phase immunoassay (Roche Diagnostics, Monza, Italy). Total cholesterol,

Effects of the phytoestrogen genistein on hot flushes, endometrium, and vaginal epithelium in postmenopausal women: a 2-year randomized, double-blind, placebo-controlled study.

Objective: To evaluate in a 24-month, prospective, randomized, double-blind, placebo-controlled study whether pure administration of the phytoestrogen genistein (54 mg/d) might reduce the number and severity of hot flushes in postmenopausal women, with no adverse effect on the endometrium and vagina. Methods: A total of 389 participants met the parent study criteria and were randomly assigned to receive the phytoestrogen genistein (n = 198) or placebo (n = 191). About 40% of participants in both groups did not experience hot flushes, and the evaluation was performed in a subgroup of 236 participants (genistein, n = 119; placebo, n = 117). Reductions from the baseline in the frequency and severity of hot flushes were the principal criteria of efficacy. Endometrial thickness was evaluated by ultrasonography. The maturation value was also used to determine hormonal action on the vaginal cells. Results: There were no significant differences in vasomotor symptoms between groups at the baseline (4.4 ± 0.33 hot flushes per day in the genistein group and 4.2 ± 0.35 hot flushes per day in the control group). After 12 months of genistein therapy, there was a significant reduction (−56.4%) in the mean number of hot flushes, with a significant difference compared with the control group. After 24 months, there was no further decrease in the number of hot flushes in both groups. No significant difference was found in mean endometrial thickness and the maturation value score between the two groups, either at the baseline or after 24 months. Conclusions: The phytoestrogen genistein has been shown to be effective on vasomotor symptoms without an adverse effect on the endometrium and vagina, but after the first year, there was no further improvement in the decrease in hot flushes.

Breast Safety and Efficacy of Genistein Aglycone for Postmenopausal Bone Loss: A Follow-Up Study

CONTEXT Genistein aglycone improves bone metabolism in women. However, questions about the long-term safety of genistein on breast as well as its continued efficacy still remain. OBJECTIVE We assessed the continued safety profile of genistein aglycone on breast and endometrium and its effects on bone after 3 yr of therapy. DESIGN The parent study was a randomized, double-blind, placebo-controlled trial involving 389 osteopenic, postmenopausal women for 24-months. Subsequently, a subcohort (138 patients) continued therapy for an additional year. PATIENTS AND INTERVENTIONS Participants received 54 mg of genistein aglycone daily (n = 71) or placebo (n = 67). Both treatment arms received calcium and vitamin D(3) in therapeutic doses. MAIN OUTCOMES Mammographic density was assessed at baseline, 24 and 36 months by visual classification scale and digitized quantification. BRCA1 and BRCA2, sister chromatid exchange, and endometrial thickness were also evaluated. Lumbar spine and femoral neck bone mineral density were also assessed. Secondary outcomes were biochemical levels of bone markers. RESULTS After 36 months, genistein did not significantly change mammographic breast density or endometrial thickness, BRCA1 and BRCA2 expression was preserved, whereas sister chromatid exchange was reduced compared with placebo. Bone mineral density increases were greater with genistein for both femoral neck and lumbar spine compared to placebo. Genistein also significantly reduced pyridinoline, as well as serum carboxy-terminal cross-linking telopeptide and soluble receptor activator of NF-kappaB ligand while increasing bone-specific alkaline phosphatase, IGF-I, and osteoprotegerin levels. There were no differences in discomfort or adverse events between groups. CONCLUSIONS After 3 yr of treatment, genistein exhibited a promising safety profile with positive effects on bone formation in a cohort of osteopenic, postmenopausal women.

Effects of the phytoestrogen genistein on cardiovascular risk factors in postmenopausal women.

Objective: The phytoestrogen genistein has been shown to be the most efficacious in clinical and experimental studies. We studied whether genistein treatment affects some cardiovascular risk markers in postmenopausal women. Design: Sixty healthy postmenopausal women, who were 52 to 60 years of age, were enrolled in a 6-month double-blind, placebo-controlled, randomized study. After a 4-week stabilization on a standard fat-reduced diet, participants were randomly assigned to receive either genistein (n = 30; 54 mg/d) or placebo (n = 30). At baseline and after a 6-month treatment, we measured fasting glucose, insulin, insulin resistance (HOMA-IR), osteoprotegerin (OPG), fibrinogen, and sex hormone-binding globulin (SHBG). Results: By comparison with placebo, genistein treatment decreased significantly fasting glucose (genistein = −8.7 ± 2.3%; placebo = 3.2 ± 2.3%; P < 0.001), fasting insulin (genistein = −12 ± 3.33%; placebo = 36 ± 3.29%; P < 0.001), and HOMA-IR (genistein = −14 ± 5.8%; placebo = 42 ± 0.6%; P < 0.001). After genistein-treatment, fibrinogen decreased (genistein = 3.18 ± 0.12 g/L; placebo = 3.83 ± 0.04 g/L; P < 0.001) with respect to placebo. In the genistein group, serum OPG was lower (−2 ± 0.3%) than in placebo (9 ± 1.5%; P < 0.001), and serum SHBG was higher (63 ± 3.8 nmol/L) compared with placebo (53 ± 2.9 nmol/L; P < 0.05). Conclusion: Our study suggests that genistein may have a favorable effect on some cardiovascular markers.

OPG and sRANKL Serum Concentrations in Osteopenic, Postmenopausal Women After 2‐Year Genistein Administration

Introduction: RANKL and its decoy receptor osteoprotegerin (OPG) constitute a complex physiological mediator system involved in the regulation of bone resorption and may be responsible for the homeostatic mechanism of normal bone remodeling. Genistein, an isoflavone representing 1–5% of total phytoestrogen content in soybean products, may positively regulate cellular bone metabolism, but its mechanism of action on bone is not yet fully understood.

Genistein in the Metabolic Syndrome: Results of a Randomized Clinical Trial

CONTEXT This study was performed to evaluate the effects of genistein on metabolic and cardiovascular risk factors in Caucasian postmenopausal subjects with metabolic syndrome (MetS). OBJECTIVE Our objective was to assess the effects of genistein on surrogate endpoints associated with diabetes and cardiovascular disease. DESIGN AND SETTING This was a randomized, double-blind, placebo-controlled trial at 3 university medical centers in Italy. PATIENTS Patients included 120 postmenopausal women with MetS according to modified Third Report of the National Cholesterol Education Program (NCEP), Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) criteria. INTERVENTION After a 4-week stabilization period, postmenopausal women with MetS were randomly assigned to receive placebo (n = 60) or 54 mg genistein daily (n = 60) for 1 year. MAIN OUTCOME MEASURES The primary outcome was homeostasis model assessment for insulin resistance (HOMA-IR) at 1 year. Secondary outcomes were fasting glucose, fasting insulin, total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides, visfatin, adiponectin, and homocysteine levels. Data on adverse events were also recorded. RESULTS At 1 year in genistein recipients, fasting glucose, fasting insulin, and HOMA-IR (mean from 4.5 to 2.7; P < .001) decreased and were unchanged in placebo recipients. Genistein statistically increased HDL-C (mean from 46.4 to 56.8 mg/dL) and adiponectin and decreased total cholesterol, LDL-C (mean from 108.8 to 78.7 mg/dL), triglycerides, visfatin, and homocysteine (mean from 14.3 to 11.7 μmol/L) blood levels. Systolic and diastolic blood pressure was also reduced in genistein recipients. Genistein recipients neither experienced more side adverse effects than placebo nor discontinued the study. CONCLUSION One year of treatment with genistein improves surrogate endpoints associated with risk for diabetes and cardiovascular disease in postmenopausal women with MetS.

Enhancement of expression of vascular endothelial growth factor after adeno-associated virus gene transfer is associated with improvement of brain ischemia injury in the gerbil.

Angiogenesis induced by growth factors may represent a rational therapy for patients with stroke. Vascular endothelial growth factor (VEGF) plays a pivotal role in angiogenesis and VEGF expression is enhanced in the post-ischemic brain. VEGF induced by brain hypoxia can lead to the growth of new vessels and may represent a natural protective mechanism improving survival after stroke. In the light of these findings we investigated changes of VEGF expression in different brain regions after intracerebroventricular injection of adeno-associated virus transferring gene for VEGF (rAAV-VEGF) in the gerbil, and after transient brain ischemic injury, we studied the effects of rAAV-VEGF injection on survival, brain edema, delayed neuronal death in the CA1 area and learning ability. Treatment with rAAV-VEGF 6 days or 12 days before ischemia significantly improves survival, brain edema and CA1 delayed neuronal death and post-ischemic learning evaluated by passive avoidance test. Animals treated with rAAV-VEGF showed in the thalamus and the cortex, a significant positive immunostaining for VEGF similar to those subjected to brain ischemia and not treated with rAAV-VEGF. These data represent a further contribution to a possible employment of gene therapy by using rAAV-VEGF in brain ischemia and indicate that thalamus and cortex may be targets for neuroprotective effects of VEGF.

Genistein Aglycone Does Not Affect Thyroid Function: Results from a Three-Year, Randomized, Double-Blind, Placebo-Controlled Trial

CONTEXT AND OBJECTIVE Genistein aglycone positively affects postmenopausal symptoms. However, questions about its long-term safety on the thyroid gland still remain. DESIGN The parent study was a randomized, double-blind, placebo-controlled trial involving 389 osteopenic, postmenopausal women for 24 months. A subcohort (138 patients) continued therapy for an additional year. SETTING Patients received ambulatory care. PATIENTS AND INTERVENTIONS Participants received 54 mg of genistein aglycone daily (n = 71) or placebo (n = 67), plus calcium and vitamin D(3) at therapeutic doses. Circulating thyroid hormones (TSH, free T(3), free T(4)) and autoantibodies (thyroid peroxidase, thyroglobulin, and thyroid microsomal antigen) were assessed in 40 genistein and 37 placebo subjects who completed 3 yr. Thyroid hormone receptor (THRalpha and THRbeta) and retinoid receptor (RARalpha, RARgamma, and RXRalpha) expression from peripheral blood monocytes was also evaluated at baseline, 12, 24, and 36 months in all 3-yr completers. RESULTS Genistein administration over 3 yr did not affect serum thyroid hormones or autoantibodies. In addition, there were no differences in THRalpha, THRbeta, RARalpha, RARgamma, or RXRalpha mRNA expression between groups. CONCLUSION These data suggest that genistein aglycone intake does not significantly increase the risk of clinical or subclinical hypothyroidism at the dose of 54 mg/d.

Efficacy of genistein aglycone on some cardiovascular risk factors and homocysteine levels: A follow-up study *

BACKGROUND AND AIM Recent evidence suggests that genistein aglycone may act beneficially on surrogate cardiovascular risk markers in postmenopausal women. We assessed the effects of genistein aglycone on some cardiovascular risk factors and homocysteine levels after 3-years of continued therapy in a cohort of osteopenic, postmenopausal women. METHODS AND RESULTS The parent study was a randomized, double-blind, placebo-controlled trial involving 389 postmenopausal women with low bone mass for 24 months. Subsequently, a subcohort (138 patients) continued therapy for an additional year. Participants received 54mg of genistein aglycone (n=71) or placebo (n=67), daily. Both arms received calcium and vitamin D(3) in therapeutic doses. Moreover, 4 weeks before randomization procedures and during our follow-up study, all patients received dietary instructions in an isocaloric fat-restricted diet. Blood lipid profiles, fasting glucose and insulin, insulin resistance (HOMA-IR), fibrinogen, osteoprotegerin (OPG) and homocysteine at baseline and after 24 and 36 months of treatment were measured. Compared to placebo, genistein significantly decreased fasting glucose and insulin, HOMA-IR, fibrinogen and homocysteine after 24 and 36 months of treatment. By contrast, isoflavone administration did not affect high-density lipoprotein cholesterol and triglycerides though serum OPG was higher in the genistein recipients. There were no differences in adverse events or discomfort between groups. Results on routine biochemical, liver function, and hematologic testing did not change over time in placebo or genistein group. CONCLUSIONS After 3-years of treatment, genistein aglycone plus calcium, vitamin D(3) and a healthy diet showed positive effects on some cardiovascular risk factors and homocysteine levels in a cohort of postmenopausal women with low bone mass.

Modulation of IL-1 β gene expression by lipid peroxidation inhibition after kainic acid-induced rat brain injury

Brain injury was induced by intraperitoneal administration of kainic acid (KA, 10 mg/kg). Animals were randomized to receive either IRFI 042 (20 mg/kg i.p.), a lipid peroxidation inhibitor, or its vehicle (NaCl 0.9% DMSO 10% 1 ml/kg i.p.) 30 min before KA administration. A first set of animals was sacrificed 6 h after KA injection to measure malondialdehyde (MDA) content, glutathione-reduced (GSH) levels and the mRNA for interleukin-1beta (IL-1beta) in the cortex and in the hippocampus. A second set of animals was sacrificed 48 h after KA administration for histological analysis. All animals were observed for monitoring the behavioral sequelae and for evaluating latency of convulsions. Sham brain injury rats were used as controls. Intraperitoneal administration of IRFI 042 significantly decreased brain MDA (cortex: KA + vehicle = 0.285 +/- 0.04 nmol/mg protein; KA + IRFI 042 = 0.156 +/- 0.02 nmol/mg protein, P < 0.005; hippocampus: KA + vehicle = 0.350 +/- 0.03 nmol/mg protein; KA + IRFI 042 = 0.17 +/- 0.04 nmol/mg protein, P < 0.005), prevented the brain loss of GSH in both cortex (KA + vehicle = 7.81 +/- 1 micromol/g protein; KA + IRFI 042 = 12.1 +/- 1 micromol/g protein; P < 0.005) and hippocampus (KA + vehicle = 5 +/- 0.8 micromol/g protein; KA + IRFI 042 = 9.4 +/- 1.8 micromol/g protein; P < 0.005), reduced both brain IL-1beta mRNA expression and oedema, and increased latency of convulsions. Histological analysis showed a reduction of cell damage in IRFI 042-treated samples. The present data indicate that lipid peroxidation inhibition reduces IL-1beta gene expression and protects against kainic acid-induced brain damage.