Rolando Marini

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.

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.

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.

Gene transfer of IκBα limits infarct size in a mouse model of myocardial ischemia-reperfusion injury

Nuclear factor-κB (NF-κB) plays a central role in myocardial ischemia-reperfusion (MI/R) injury. The inhibitory protein IκBα prevents its activation. We investigated the effects of adeno-associated viral vector-mediated IκBα gene transfer in MI/R injury. Male C57BL/6 mice were randomized to receive a recombinant adeno-associated virus (rAAV) encoding the gene for the NF-κB inhibitory protein IκBα (rAAV- IκBα) or the β-galactosidase gene (a control and inert gene; rAAV-LacZ), both at a dose of 1011 copies. Four weeks later anesthetized animals were subjected to total occlusion (45 minutes) of the left main coronary artery followed by 5 hours of reperfusion. MI/R produced a wide infarct size (IF/area-at-risk = 56 ± 8%; IF/left ventricle = 44 ± 5%) and tissue neutrophil infiltration, studied by means of elastase activity (area-at-risk = 2.5 ± 0.4 μg/gm tissue; infarct area = 2.9 ± 0.6 μg/gm tissue). Furthermore MI/R caused peak message for intercellular adhesion molecule-1 (ICAM-1) in the area-at-risk at 3 hours of reperfusion (1.2 ± 0.4 relative amount of cardiac ICAM-1 mRNA). NF-κB activation was evident at 0.5 hours of reperfusion and reached its maximum increase at 2 hours of reperfusion. rAAV-IκBα injection reduced infarct size (IF/area-at-risk = 19 ± 3%; IF/left ventricle = 10 ± 2%; p < 0.001), blocked NF-κB activation, diminished cardiac ICAM-1 expression (0.4 ± 0.02 relative amount of cardiac ICAM-1 mRNA; p < 0.001), and blunted leukocyte accumulation (area-at-risk = 0.6 ± 0.05 μg/gm tissue; infarct area = 0.4 ± 0.02 μg/gm tissue; p < 0.001). Our data indicate that rAAV-IκBα may be useful for MI/R gene therapy.

Spontaneous electromechanical activity in the rat duodenum in vitro

Isolated rat duodenum shows spontaneous mechanical and electrical activities. Mechanical activity consists in changes both in endoluminal pressure and in isometric tension. Electrical activity is characterized by slow waves with superimposed bursts. This spontaneous activity is tetrodotoxin (TTX) resistant and therefore it is myogenic in origin. Indeed, TTX pretreatment, even in the presence of atropine and guanethidine, caused an increase in amplitude and in frequency of the electrical and mechanical activities. This finding indicates the presence of tonically active inhibitory intramural non adrenergic, non cholinergic (NANC) nerves. Duodenal longitudinal strips showed a spontaneous mechanical activity resembling that one recorded from isolated segment. Instead, circular strips are quiescent under resting condition and a contractile activity can be detected only after TTX pretreatment suggesting that: i) the circular smooth muscle layer is tonically inhibited by intramural NANC nerves and, ii) the contractions observed in the rat duodenum are due to the activity of the longitudinal one.

Influence of nerve plexus on distensibility and spontaneous activity in the isolated guinea pig gallbladder

Correspondence: R. Mancinelli, Institute of Human Physiology, Catholic University of Rome, Rome, Italy. The studies of the physiological role of the intrinsic innervation are somewhat contradictory [2,3,6,7]. Recent studies on pressure-volume relationship in our laboratory [4] have demonstrated that guinea pig gallbladder distensibility decreases after pharmacological blocking of the nerve plexus. Also, during pressure-volume recording, phasic contraction waves appear spontaneously having max imum ampli tude and duration with volumes in the range of 0.6-1.4 ml. In order to investigate whether the nerve plexus can directly influence the mechanical properties of the whole gallbladder, lengthtension (distensibil-