In vitro estrogenic activity of Achillea millefolium L.

Isolation and biological characterization of pure compounds was used to identify and characterize estrogenic activity and estrogen receptors (ER) preference in chemical components of Achillea millefolium. This medicinal plant is used in folk medicine as an emmenagogue. In vitro assay, based on recombinant MCF-7 cells, showed estrogenic activity in a crude extract of the aerial parts of A. millefolium. After fractionation of the crude extract with increasing polar solvents, estrogenic activity was found in the methanol/water fraction. Nine compounds were isolated and characterized by HR-MS spectra and 1D- and 2D-NMR techniques. In particular, dihydrodehydrodiconiferyl alcohol 9-O-beta-D-glucopyranoside - a glycosyl-neolignan - was isolated for the first time from the genus Achillea in addition to six flavone derivatives, apigenin, apigenin-7-O-beta-D-glucopyranoside, luteolin, luteolin-7-O-beta-D-glucopyranoside, luteolin-4'-O-beta-D-glucopyranoside, rutin, and two caffeic acid derivatives, 3,5-dicaffeoylquinic acid and chlorogenic acid. Apigenin and luteolin, the most important estrogenic compounds among those tested, were studied for their ability to activate alpha or beta estrogen receptors (ERalpha, ERbeta) using transiently transfected cells. Our results suggest that isolation and biological characterization of estrogenic compounds in traditionally used medicinal plants could be a first step in better assessing further (e.g. in vivo) tests of nutraceutical and pharmacological strategies based on phytoestrogens.

Investigating the estrogenic risk along the river Po and its intermediate section.

Different endpoints have been used to investigate the occurrence of estrogenic risk along the Po River, particularly its middle section. An in vitro assay based on recombinant yeast could not detect estrogenic activity in bed sediments of the Italian river or in bile samples of five Cyprinid species, with the only exception being one carp (Cyprinus carpio) captured downstream of the River Lambro, a polluted tributary of the middle River Po. Chemical analyses of fish bile and water samples from the same middle section showed diffuse contamination by moderately low levels of estrogenic chemicals (estrone [E1], 17beta-estradiol, estriol [E3], 17alpha-ethinylestradiol, 4-nonylphenol [NP], 4-tert-octylphenol [tOP], 4-n-octylphenol, and bisphenol A) but they were of limited help in understanding the risk present in the downstream area where intersex barbel were previously found. In contrast, the analyses of River Lambro waters showed that this tributary is a source to the middle River Po of all eight estrogens investigated. Analyses of bed sediments and macroinvertebrates from the same area consistently showed at least two levels of contamination, with the downstream stretch showing higher concentrations of natural steroids (E1 and E3) and xenoestrogens (NP and tOP). Accordingly, new histologic examinations undertaken on young barbel (Barbus sp.) showed intersex gonads only in the individuals captured in the downstream stretch, thereby confirming previous results. Present findings confirm the occurrence of disrupting conditions in the middle River Po and provide the first suggestions of cause-effect relationships.

Antioxidant activity of wild plants collected in Valsesia, an alpine region of Northern Italy.

A selection of wild plants collected in Valsesia (Northwest Italy) was screened for their in vitro antioxidant activity. Aerial parts of selected plants were dried at room temperature and powdered. Then, four sequential extractions were performed with increasing polarity solvents, i.e. n-hexane, chloroform, chloroform-methanol (9:1, v/v) and methanol. By employing different assays, it was shown that all the methanol extracts of the samples collected were endowed with antioxidant activity, though, as expected, their potency varied according to the different tests. In particular, plants of the Thymus and Achillea genus displayed the highest activity. Given that a diet rich in wild plants is associated with a reduced incidence of degenerative diseases, such as atherosclerosis and cancer, this study suggests that some Valsesia plants could be pharmaceutically exploited.

Zea mays L. protein changes in response to potassium dichromate treatments.

The plant metabolic response to heavy metal stress is largely unknown. The present investigation was undertaken to examine the influence of different concentrations of potassium dichromate on the Zea mays L. plantlets. A clear effect of chromium on maize plantlets growth and seed germination was observed strating from 100-300 ppm up to 1500 ppm. In this concentration range, chromium uptake was dependent on the concentration in the medium. Metallothioneins, involved in heavy metal binding, were measured by capillary electrophoresis (CE), and showed a dose-response induction. Protein profile analyzed by two-dimensional gel electrophoresis showed differential expression of several proteins. Identification of spots of upregulated proteins was performed by MALDI mass spectrometry. Results showed that proteins induced by heavy metal exposure are principally involved in oxidative stress tolerance or in other stress pathways. Induction of proteins implicated in sugar metabolism was also observed. Identification of factors involved in plant response may lead to a better understanding of the mechanisms involved in cell protection and tolerance. This information could be used to improve agricultural production and environmental quality.

Activation of brain estrogen receptors in mice lactating from mothers exposed to DDT.

The insecticide dichlorodiphenyltrichloroethane (DDT) interferes with physiological endocrine processes modulating estrogens receptor activity. Most of the data describing the DDT mechanism of action have been collected in vitro or in reproductive tissues in vivo. Here we use a new transgenic mouse model to investigate the DDT effects on estrogens receptor activation in vivo in non-reproductive tissues. In particular, we demonstrate that DDT is able to activate estrogen receptors in the brain and the liver of adult mice after acute administration, and it is active in lactating mice when accumulated in the mother's milk. Furthermore, we demonstrate that the acute administration of DDT activates estrogen receptors with a different kinetics with respect to 17beta-estradiol. Experiments with a breast cancer cell line engineered to express luciferase under the transcriptional control of activated estrogen receptors reveal that the microsomal metabolization of DDT is required for its full activity on estrogen receptors. Taken together these data lead to hypothesize that the delayed DDT time course on estrogen receptor activation in vivo might be due to a necessary step of metabolism of the compound.

AFLP analysis as biomarker of exposure to organic and inorganic genotoxic substances in plants.

In recent years several plant species have been in use as bioindicators and several tests have been developed to evaluate the toxicity of environmental pollutants in vegetal organisms. In the present paper Arabidopsis thaliana (L.) Heynh. (ecotype Wassilewskija) was used as bioindicators of two genotoxic substances: potassium dichromate and dihydrophenanthrene. Inhibition of seed germination was observed with both pollutants. AFLP analysis (i) indicated that both substances are genotoxic, (ii) showed that dihydrophenanthrene induces DNA changes in different target sequences than potassium dichromate, (iii) quantified the genotoxic effect using cluster analysis by comparing DNA from treated plants with that of control plants. On the basis of these considerations we suggest that AFLP method is a powerful tool for measuring qualitative and quantitative genotoxic activity due to environmental pollutants. AFLP method can be applied to a wide range of bioindicator organisms and may become a universal methodology to identify target genes for specific genotoxic agents. This could open up possibilities for designing specifically targeted assays and new approaches to risk assessment.

Estrogenic activity of Nigella damascena extracts, evaluated using a recombinant yeast screen.

We used the yeast estrogen screen (YES) containing a human estrogen receptor to evaluate the estrogenic activity of extracts obtained from Nigella damascena seeds. Alcohol extracts obtained by direct extraction of seeds showed a low estrogenic activity, while the alcohol extract obtained after extraction with solvents of increasing polarity showed a strong estrogenic activity. This suggests the presence in Nigella of polar components whose activity can be clearly demonstrated after previous elimination of interacting apolar components that may mask the activity of more polar components. The response of both alcohol fractions follow a bell-shaped curve indicating a concentration-dependent relationship.

Estrogenic activity of phenolic compounds from Nigella damascena evaluated using a recombinant yeast screen.

We used a yeast estrogen screen (YES) containing human estrogen receptor to evaluate the estrogenic activity of both crude extracts and simple pure phenolic compounds from Nigella damascena seeds. Estrogenic activity was established in the methanolic and aqueous extracts of the seeds as well as in two simple phenolic compounds isolated from the methanolic extract, 2,4-dihydroxyphenylacetic acid, 3,4-dihydroxy-beta-phenethyl alcohol.

Environmental contaminants and biochemical response in eel exposed to Po river water.

Groups of eels (Anguilla anguilla) were exposed to Po river water under control conditions at "la Casella" Fluvial Hydrobiology Station for 30-day periods between November and May. At the end of the exposure period, fish were sacrificed and cytochrome P4501A was evaluated in liver microsomes using both catalytic (EROD) and semiquantitative immunodetection (ELISA) assays. At the same time, water samples were taken for chemical analyses of PCBs, PAHs and pesticides. Eel was chosen as bio-indicator on the bases of experiments on cytochrome P450 expression and activity under basal or induced conditions and in consideration of the ecological and economic relevance and ease of handling. Low levels of cytochrome P450 during Winter were followed by a step increase during Spring, associated with a substantial concentration increase of agrochemicals. A good correlation was found between measurements of cytochrome P4501A by EROD and ELISA in dose-effect experiments, however, ELISA showed a higher sensitivity. Immunochemical techniques may be used in addition to enzyme activity measurements both to detect lower levels of cytochrome P450 induction (where they proved more sensitive) and as quality control. These results suggest that measurement of cytochrome P4501A under controlled conditions, using eel as bio-indicator, can be a useful tool in monitoring Po river ecosystems.

Regulatory relevance of lipoproteins in interleukin-2 stimulated lymphocytes in vitro.

The regulatory role of very low density lipoproteins (VLDL) in de novo protein synthesis and in natural killer (NK) mediated cytotoxicity against cancer cells was evaluated in IL-2 activated peripheral blood mononuclear cells (PBL). Supplementing of VLDL to the incubation medium induced a potent dose-related inhibitory effect on the synthesis and secretion of (35S) labelled proteins. The maximal inhibitory response was observed at 50 micrograms/ml cholesterol VLDL. The effect of VLDL on the IL-2 induced ability of lymphocytes to kill cancer cells and to interfere with target cell proliferation was then evaluated. Maximal cytotoxicity was observed at 50 micrograms VLDL/ml as cholesterol. This VLDL-dependent stimulatory effect was associated with a significant decrease in the proliferative effect of the medium conditioned by PBL. The possibility that VLDL mediate the regulation of immune functions by interacting with metabolic patterns expressed by IL-2 stimulated lymphocytes is discussed in light of these results.

A comparison of highly unsaturated fatty acid levels in wild and farmed eels (Anguilla anguilla).

1. Absolute and relative amounts of eicosapentaneoic acid (EPA) and docohexaenoic acid (DHA) in muscle of eels from four different fish farms were compared with samples from wild eels from two different areas of northern Italy. 2. Farmed eels were richer in DHA and EPA than wild animals. 3. The addition of cod liver oil to the diet of farmed eels led to a significant accumulation of EPA and DHA, but no change in total lipid content. 4. The calculation of two indices related to highly unsaturated fatty acid (HUFA) content (FLQ = fish lipid quality; AI = aterogenic index), indicated the higher nutritional value of farmed vs wild fish. 5. We conclude that farmed eels are an adequate source of fish products for human nutrition and propose use of the above-mentioned indices as an effective means for assessing fish nutritional quality for populations at high risk of chronic degenerative disease.

Comparison of nutritional status after total or subtotal gastrectomy.

Forty-four disease-free patients were evaluated a mean of three years after total gastrectomy (TG) or subtotal gastrectomy (SG) for cancer. The investigation encompassed nutritional assessment by standard anthropometric and biochemical indices; evaluation of the nutritional intake based on 24 h recall and of appetite status on a visual analogue scale; and anamnestic analysis of postcibal symptoms and syndromes. Body weight had declined progressively until the 15th postoperative month after both TG and SG. Weight loss, as well as the general nutritional status index (actual body weight/usual body weight +/- actual body weight/desirable body weight + measured arm muscle circumference/reference arm muscle circumference x 33), had dropped more significantly in patients undergoing TG than those having SG (p less than 0.05). The principal body compartment change was observed in the fat content which was severely depleted, whereas the somatic proteins were relatively spared and the visceral proteins and remaining biochemical variables were in the normal range. Protein intake was not significantly different in the two groups, but caloric intake was significantly lower and the number of meals significantly higher after TG (p less than 0.05). These data suggest that malnutrition after TG is relatively mild and that this operation causes only a limited impairment of the nutritional state, and spares most of the nutritional variables of clinical interest in comparison with SG. These findings argue in favor of TG when clinically indicated without excessive concern about postoperative nutrition.

Effect of plasma from cancer patients on rat skeletal muscle protein degradation and PGE2 release in vitro.

Cachexia in tumour-bearing patients involves loss of skeletal muscle proteins. In order to elucidate the mechanisms underlying this phenomenon, we tested the hypothesis of the presence of a circulating proteolytic factor (possibly interleukin-1, acting through an increased PGE2 release) in the plasma of cancer patients, because such a mechanism has been demonstrated in patients with sepsis or trauma and in animals with bacteraemia or viraemia. The effect of plasma from 13 malnourished cancer patients and 14 controls on PGE2 release and protein degradation (assessed as Tyr release) in rat diaphragm in vitro was evaluated; human recombinant interleukin-1 alpha (IL-1) was used for comparison. IL-1 increased PGE2 release (+44% at 5 U/ml), but did not greatly affect proteolysis. On the contrary, human plasma (125 microliters/ml) from both control and tumour-bearing individuals did not affect PGE2 release significantly, but greatly reduced Tyr release. The decrease in Tyr release by plasma was dose-dependent. In conclusion, our data indicate that, at variance with what was demonstrated in patients with trauma or sepsis, loss of skeletal muscle proteins in cancer patients is not mediated by a circulating factor. In addition, evidence is provided of dissociation between PGE2 and Tyr release and of lack of proteolytic activity for IL-1.

Decrease of polyunsaturated fatty acids and elevation of the oleic/stearic acid ratio in plasma and red blood cell lipids of malnourished cancer patients.

The fatty acids profiles of plasma and red blood cell lipids have been evaluated in 12 malnourished cancer patients in comparison with samples from eight healthy controls. In such patients, significantly lower levels of linoleic acid (LA) as percentage of total fatty acids were observed in plasma phospholipids (PL) and cholesterol esters (CE), and in red blood cells PL. The levels of arachidonic acid (AA) and the unsaturation index of the two lipid classes were also reduced in plasma CE but not in PL. In spite of the marked reduction of LA and, more generally, of total polyunsaturated fatty acids (PUFA), no elevation of eicosatrienoic acid (20:3 n-9) was observed, such acid being considered a typical index of essential fatty deficiency. Moreover, no modification of the parameters indicating impairment of the fatty acid desaturation activity was shown. In addition, the levels of palmitic and oleic acids were significantly higher in both plasma PL and CE and in red blood cells PL. The reported elevation of the oleic to stearic acid ratio in lipids of red blood cells from malnourished cancer patients, already observed by other authors, was confirmed in our study. This ratio was even more markedly elevated in plasma lipids of the patients. A very good correlation was found between the reduction of linoleic acid levels, especially in plasma CE, and weight loss, suggesting enhanced utilization of this fatty acid in association with extensive depletion of lipid stores, in this pathological state.

Anorexia in cancer patients: prevalence and impact on the nutritional status.

One hundred and eightysix cancer patients with different types, site and stage of disease, consecutively admitted to the Istituto Nazionale Tumori, Milan, were examined. The purpose of the study was to assess the prevalence of subjective anorexia and to determine whether some nutrition-related indices are different within homogeneous categories of cancer patients, anorectic and non-anorectic. Anorexia was measured on the basis of an appetite score, evaluated with a visual analogue rating scale. Nutritional intake was evaluated by a computerised programme of the habitual food consumption. Nutritional assessment included anthropometric evaluations (triceps skinfold, arm circumference, arm muscle circumference) and biochemical indices (serum albumin, and total iron binding capacity). A nutritional index was then calculated: GNS = P + I + M x 100, where P: actual weight/premorbid weight; I: actual weight/ideal weight; M: actual arm muscle mass circumference/ideal arm muscle mass circumference. The evaluation of the data showed that: (1) The prevalence of subjective anorexia ranged from 1 3 to 2 5 of the patients depending on the type and site of tumour, and stage of disease. (2) Most of the nutritional-related variables were significantly worse in anorectic compared with non-anorectic patients. (3) Within groups of patients with different tumour location, but homogeneous for the presence or absence of subjective anorexia, there is no difference with respect to nutritional indices; except for patients with cancer of the oesophagus and stomach who were always more depleted. (4) Anorexia did not seem to influence the nutritional status in patients with cancer at an early stage (except in cancer of oesophagus and stomach) but it was associated to malnutrition as the disease progressed to more advanced stages.

Hospital malnutrition: incidence and prospective evaluation of general medical patients during hospitalization.

The incidence of malnutrition and the effect of hospitalization was evaluated in 100 consecutive admissions to the Clinical Pathology R Department of the University of Genoa. Nutritional deficiencies were evaluated at the time of admission and discharge from the Hospital, among patients hospitalized 2 weeks or longer, using the following nutrition-related parameters: body fat, muscle proteins, weigh, rate of weight loss plasma proteins level, vitamin B12 and folic acid plasma level, plasma iron and ferritin. We also considered the nutritional alterations in malnourished patients with relation to appetite decrease and to pathological status. At the admission to the hospital, the findings showed a high incidence (79%) of alterations in some nutritional parameters. In patients with nutritional impairments we observed a worsening of most of the nutritional parameters during hospitalization, especially in patients with severe appetite decrease and those affected by sepsis, neoplastic, gastric and renal diseases.

Effect of retinol on macromolecular synthesis in cultured cell lines.

The effects of retinol on SK Mel 28 and HeLa cell lines were studied with regard to DNA and protein synthesis. It was found that vitamin A added to the culture media at a 50 microM concentration causes a 60% inhibition of DNA synthesis, that the inhibition is reversible and that this treatment does not select retinol-resistant clones. The synthesis of cytoplasmic, acid-soluble nuclear proteins and that of non-histone chromosomal proteins is inhibited as soon as the vitamin reaches the cell.

[Clinical study of a prepared reducing diet: adequacy of the vitamin-mineral contribution in comparison to the nutritional and immunity status]. / Studio clinico di un preparato dietetico dimagrante: adeguatezza dell'apporto vitaminico-minerale nei confronti dello stato di nutrizione ed immunitario.

The Authors studied the effectiveness and safety of a commercial hypocaloric diet on 11 obese postmenopausal women. During the experimental period 1 meal/day has been replaced with a chemically defined low calories product. Different parameters have been evaluated to assess the nutritional status 1), anthropometric: weight, skinfold thickness, arm muscle circumference; 2) biochemical total plasma proteins, transferrin, vitamins A, E, C, B12, folic acid; plasma iron, hemoglobin, MCV, RBC; 3) immune status (T lymphocytes and immunoglobulins). Moreover blood sugar, cholesterol and triglyceride levels, as well as blood pressure have been taken into account. All the possible side effects and the diet acceptance for all the patients have been scored. After 1 month all the abnormal conditions (weight, cholesterol, blood pressure) improved, while the nutritional status and immune response remained at an optimal level.