Francesco Squadrito

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,

The effect of the phytoestrogen genistein on plasma nitric oxide concentrations, endothelin-1 levels and endothelium dependent vasodilation in postmenopausal women

The phytoestrogen genistein improves endothelial dysfunction in ovariectomized rats through a nitric oxide-dependent mechanism. We investigated whether genistein alters the balance between the nitric oxide products and endothelin-1 and influences endothelium-dependent vasodilation in postmenopausal women. Sixty healthy postmenopausal women were enrolled in the study. A double-blind, placebo controlled, randomized design was employed. After a 4-week stabilization on a standard fat-reduced diet, participants to the study were randomly assigned to receive either genistein (n=30; 54 mg/day) or placebo (n=30). Flow-mediated, endothelium-dependent vasodilation of the brachial artery, plasma nitric oxide breakdown products and endothelin-1 levels were measured at baseline and after 6 months of genistein therapy. The mean baseline level of nitrites/nitrates was 22+/-10 micromol/l and increased to 41+/-10 micromol/ml after 6 months of treatment. The mean baseline plasma endothelin-1 level was 14+/-4 pg/ml and decreased to 7+/-1 pg/ml following 6 months of treatment with genistein. The mean baseline ratio of nitric oxide to endothelin also significantly increased at the end of treatment. Flow-mediated, endothelium-dependent vasodilation of the brachial artery was 3.9+/-0.8 mm at baseline and increased to 4.4+/-0.7 mm after 6 months of treatment. Placebo-treated women showed no changes in plasma nitrites/nitrates, endothelin-1 levels and flow-mediated vasodilation. Genistein therapy improves flow-mediated endothelium dependent vasodilation in healthy postmenopausal women. This improvement may be mediated by a direct effect of genistein on the vascular function and could be the result of an increased ratio of nitric oxide to endothelin.

Genistein supplementation and estrogen replacement therapy improve endothelial dysfunction induced by ovariectomy in rats

BACKGROUND We investigated the effect of genistein, a phytoestrogen derived from a soy diet with a flavonoid chemical structure, on endothelial dysfunction induced by estrogen deficiency in rats. METHODS Female mature Sprague-Dawley rats were subjected to a bilateral ovariectomy (OVX rats). Sham-operated animals (Sham OVX rats) were used as controls. Three weeks after surgery animals were randomized to the following treatments: genistein (0.2 mg/kg/day, s.c. for 4 weeks), 17 beta-estradiol (20 micrograms/kg/day, s.c. for 4 weeks) or their respective vehicles. Mean arterial blood pressure (MAP), heart rate (HR), total plasma cholesterol, plasma estradiol, plasma genistein levels and uterine weights were studied. Furthermore, we investigated acetylcholine (ACh 10 nM-10 microM) and sodium nitroprusside: (SN 15-30 nM) induced relaxation of aortic rings as well as NG-L-arginine (L-NMA: 10-100 microM) induced vasoconstriction in phenylephrine precontracted aortic segments and calcium-dependent nitric oxide synthase (cNOS) activity in homogenates of lungs taken from both sham OVX and OVX rats. RESULTS Untreated OVX rats had, compared with sham OVX animals, unchanged body weight, MAP, HR and plasma cholesterol. In contrast ovariectomy impaired endothelial responses, blunted L-NMA induced contraction (L-NMA 100 microM: Sham OVX = 2.1 +/- 0.2 g/mg tissue; OVX = 1.7 +/- 0.4 g/mg tissue) and reduced cNOS activity. Treatment with 17 beta-estradiol increased the hormone plasma levels, reverted the endothelial dysfunction and increased cNOS activity in lung homogenates. Genistein supplementation enhanced the circulating levels of the phytoestrogen and affected NOS activity and endothelial dysfunction to the same extent. CONCLUSIONS Our data suggest that genistein and 17 beta-estradiol show overlapping effects on experimental endothelial dysfunction.

Effect of genistein on endothelial function in postmenopausal women: a randomized, double-blind, controlled study

PURPOSE Genistein, a phytoestrogen found in soybeans, corrects endothelial dysfunction induced by oophorectomy in animals. Using a double-blind, controlled, randomized design, we evaluated its effects on endothelial function in women. METHODS We enrolled 79 healthy postmenopausal women (mean [+/- SD] age, 56 +/- 4 years) and randomly assigned them to receive continuous estrogen/progestin therapy (n = 26; 17beta-estradiol [1 mg/d] combined with norethisterone acetate [0.5 mg/d]), genistein (n = 27; 54 mg/d), or placebo (n = 26). Brachial artery flow-mediated, endothelium-dependent vasodilation and plasma levels of nitrites/nitrates (a marker of nitric oxide metabolism) and endothelin-1 were measured at baseline and after 1 year of therapy. RESULTS Treatment with genistein increased levels of nitrites/nitrates (mean increase, 21 micromol/L; 95% confidence interval [CI]: 15 to 26 micromol/L; P <0.001 vs. placebo); estrogen/progestin therapy caused similar changes (P <0.001 vs. placebo). Plasma endothelin-1 levels decreased following 12 months of genistein (mean decrease, 7 pg/mL; 95% CI: 3 to 10 pg/mL; P <0.001 vs. placebo) and after 12 months of estrogen/progestin (P <0.001 vs. placebo). When compared with placebo, brachial artery flow-mediated dilation was improved by genistein (mean increase, 5.5%; 95% CI: 3.9% to 7.0%; P <0.001) and by estrogen/progestin (P <0.001). There were no significant differences between estrogen and genistein for any of these parameters (all P >0.4). CONCLUSION One year of genistein therapy improves endothelium function in postmenopausal women to a similar extent as does an estrogen/progestin regimen.

Nuclear factor kappa-B blockade reduces skeletal muscle degeneration and enhances muscle function in Mdx mice

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease due to a mutation in the dystrophin gene and the consequential protein deficiency in muscle. How the lack of the sarcolemmal protein dystrophin gives rise to the final disease status is still not clear. Several evidences suggest a role of nuclear factor kappa-B (NF-kappaB), a pleiotropic transcription factor, in muscle degeneration and regeneration in DMD patients and mdx mice. We investigated the effects of NF-kappaB blocking by pyrrolidine dithiocarbamate (PDTC), a well-known NF-kappaB inhibitor, on dystrophic process in mdx mice. Five-week-old mdx and wild-type mice received three times a week for 5 weeks either PDTC (50 mg/kg) or its vehicle. PDTC treatment: (i) increased forelimb strength (+20%; P < 0.05) and strength normalized to weight (+24%; P < 0.05) and a decreased fatigue percentage (-61%; P < 0.05) in mdx mice, (ii) blunted the augmented NF-kappaB nuclear binding activity and the enhanced TNF-alpha expression in dystrophic muscles (P < 0.01), (iii) at a quantitative morphological evaluation of extensor digitorum longus (EDL) and biceps muscles, increased area with normal fibers (P < 0.05, in EDL), reduced muscle necrosis (P < 0.05 in biceps; P < 0.01 in EDL), and enhanced muscle regeneration (P < 0.01, in biceps). Our data support the hypothesis that NF-kappaB contributes to the perpetuation of the dystrophic damage and show that its blockade produces beneficial effects on functional, biochemical, and morphological parameters in mdx mice. Most importantly, these new findings may have clinical implications for the pharmacological treatment of patients with DMD.

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.

Adeno-associated viral vector-mediated human vascular endothelial growth factor gene transfer stimulates angiogenesis and wound healing in the genetically diabetic mouse.

Aims/hypothesisWe studied the gene therapy efficacy of diabetes-associated wound healing disorder with an adeno-associated virus (AAV) vector expressing the 165-amino acid isoform of human vascular endothelial growth factor-A (VEGF-A) by using an incisional skin-wound model produced on the back of female diabetic C57BL/KsJ db+/db+ mice and their normal littermates (db+/+m).MethodsAnimals were randomized to receive intradermally into the wound edges either rAAV-LacZ (a control gene), or rAAV-VEGF165. Animals were killed on different days (7 and 14 days after skin injury) and wounded skin tissues were used for gene marker studies, histological evaluation and immunohistochemistry, and wound breaking strength analysis. Furthermore we studied the VEGF mature protein in the wounds.ResultsWe found that AAV vectors are highly efficient for gene transfer to the mouse skin, displaying an exquisite tropism for the panniculus carnosus by using the beta-galactosidase activity assay. We confirmed the increased expression of the angiogenic factor at day 7 by measuring the wound content of the mature protein. Delivery of VEGF165 to incisional skin wounds of diabetic mice resulted in a remarkable induction of new vessel formation with consequent improvement in the wound healing process. The rAAV-VEGF165 gene improved wound healing in diabetic mice through the stimulation of angiogenesis, reepithelization, synthesis and maturation of extracellular matrix. Moreover the recombinant AAV encoding the human VEGF165 increased the breaking strenght of the wound and enhanced the wound content of VEGF.Conclusion/interpretationOur study suggests that VEGF gene transfer might represent a new approach to treat wound healing disorders associated with diabetes.

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.

Anorectic activity of NG-nitro-L-arginine, an inhibitor of brain nitric oxide synthase, in obese Zucker rats

We investigated the effects of NG-nitro-L-arginine (L-NO Arg) administration (12.5, 25 and 50 mg/kg i.p.) on food consumption and body weight of male obese Zucker rats (fa/fa) and in their lean age-matched controls (FA/?; FA/FA), both groups aged 14 weeks. Acute or repeated administration of L-NO Arg reduced food intake and body weight in both obese and lean rats. However the lean rats showed tolerance to the L-NO Arg effects after 5 days of treatment. L-NO Arg anorexia was suppressed by pretreatment with metergoline. These results suggest that L-NO Arg may represent a new anorectic drug.

17β-oestradiol reduces cardiac leukocyte accumulation in myocardial ischaemia reperfusion injury in rat

Abstract We investigated whether oestrogens modulate the phenomenon of leukocyte accumulation during ischaemia and reperfusion of the myocardium. Anaesthetized rats were subjected to total occlusion (1 h) of the left main coronary artery followed by 1 h reperfusion. Sham myocardial ischaemia–reperfusion rats (Sham) were used as controls. Myocardial necrosis, myocardial myeloperoxidase activity, serum creatinine phosphokinase activity, serum and macrophages tumour necrosis factor (TNF-α) and the myocardial staining of intercellular adhesion molecule-1 (ICAM-1) were evaluated. Myocardial ischaemia plus reperfusion in untreated rats produced marked myocardial necrosis, increased serum creatinine phosphokinase activity (348±38 U/ml) and cardiac myeloperoxidase activity, a marker of polymorphonuclear leukocyte accumulation, both in the area at risk and in the necrotic area (MPO; 9±1.1 U×10−3/g tissue and 8.2±1 U×10−3/g tissue, respectively), and induced a marked increase in the macrophage (156±14 U/ml at the end of reperfusion) and serum (344±12 U/ml, at the end of reperfusion) levels of TNF-α. Finally, myocardial ischaemia–reperfusion injury increased ICAM-1 staining in the myocardium. Administration of 17β-oestradiol (5, 10 and 20 μg/kg, i.m. 5 min after induction of myocardial ischaemia–reperfusion injury), lowered myocardial necrosis and myeloperoxidase activity in the area at risk and in the necrotic area, reduced serum and macrophages TNF-α (20±3 U/ml and 9±3 U/ml, respectively) and decreased serum creatinine phosphokinase activity (67±3 U/ml). Oestrogen treatment also blunted the increased staining of ICAM-1 in the injured myocardium. Finally, 17β-oestradiol added in vitro to peritoneal macrophages collected from untreated rats subjected to myocardial ischaemia–reperfusion injury, significantly reduced TNF-α production. Our results suggest that 17β-oestradiol, by inhibiting TNF-α production, limits the deleterious ICAM-1-mediated binding of leukocytes to injured mycardium and protects against myocardial ischaemia–reperfusion injury.