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Fourier transform infrared (FTIR) spectroscopy is a powerful tool for analyzing the biochemical properties of biological samples such as proteins, cellular materials, and tissues. It provides objective information on samples and has been adopted in many research areas of biomedical and biotechnological interest. FTIR spectroscopy can be performed using different approaches at the macro and micro levels allowing the examination of an incredibly broad class of materials. However, it has become evident that the choice of proper spectra acquisition geometries and the modalities of sample preparation in FTIR spectroscopy analysis require special consideration, especially for certain classes of materials such as cells and tissues. In the present paper, we described the different procedures used for preparing and analyzing different types of biological and biotechnological samples when the more largely available approaches are employed using a commercial FTIR spectrometer. Some basic aspects of data analysis procedures are presented in an Appendix. A certain number of our previous experimental results are reported for demonstrating once more the versatility and the potentiality of FTIR spectroscopy.

Gelatin hydrogels by microbial-transglutaminase crosslinking are being increasingly exploited for tissue engineering, and proved high potential in bone regeneration. This study aimed to evaluate, for the first time, the combination of enzymatically crosslinked gelatin with hyaluronan and the newly developed biotechnological chondroitin in enhancing osteogenic potential. Gelatin enzymatic crosslinking was carried out in the presence of hyaluronan or of a hyaluronan–chondroitin mixture, obtaining semi-interpenetrating gels. The latter proved lower swelling extent and improved stiffness compared to the gelatin matrix alone, whilst maintaining high stability. The heteropolysaccharides were retained for 30 days in the hydrogels, thus influencing cell response over this period. To evaluate the effect of hydrogel composition on bone regeneration, materials were seeded with human dental pulp stem cells and osteogenic differentiation was assessed. The expression of osteocalcin (OC) and osteopontin (OPN), both at gene and protein level, was evaluated at 7, 15 and 30 days of culture. Scanning electron microscopy (SEM) and two-photon microscope observations were performed to assess bone-like extracellular matrix (ECM) deposition and to observe the cell penetration depth. In the presence of the heteropolysaccharides, OC and OPN expression was upregulated and a higher degree of calcified matrix formation was observed. Combination with hyaluronan and chondroitin improved both the biophysical properties and the biological response of enzymatically crosslinked gelatin, fastening bone deposition.

Introduction: Bisphenol A (BPA) exposure has been correlated to non-alcoholic fatty liver disease (NAFLD) development and progression. We investigated, in a clinical model, the effects of the administration of 303 mg of silybin phospholipids complex, 10 g of vitamin D, and 15 mg of vitamin E (RealSIL, 100D, IBI-Lorenzini, Aprilia, Italy) in male NAFLD patients exposed to BPA on metabolic, hormonal, and oxidative stress-related parameters. Methods: We enrolled 32 male patients with histologic diagnosis of NAFLD and treated them with Realsil 100D twice a day for six months. We performed at baseline clinical, biochemical, and food consumption assessments as well as the evaluation of physical exercise, thiobarbituric acid reactive substances (TBARS), plasmatic and urinary BPA and estrogen levels. The results obtained were compared with those of healthy control subjects and, in the NAFLD group, between baseline and the end of treatment. Results: A direct proportionality between TBARS levels and BPA exposure was shown (p &lt, 0.0001). The therapy determined a reduction of TBARS levels (p = 0.011), an improvement of alanine aminotransferase, aspartate aminotransferase, insulinemia, homeostatic model assessment insulin resistance, C reactive protein, tumor necrosis factor alpha (p &lt, 0.05), an increase of conjugated BPA urine amount, and a reduction of its free form (p &lt, 0.0001, p = 0.0002). Moreover, the therapy caused an increase of plasmatic levels of the native form of estrogens (p = 0.03). Conclusions: We highlighted the potential role of BPA in estrogen oxidation and oxidative stress in NAFLD patients. The use of Realsil 100D could contribute to fast BPA detoxification and to improve cellular antioxidant power, defending the integrity of biological estrogen-dependent pathways.

National audience; The objective of this work has been to characterize the estrogenic activity of bisphenol-A (BPA) and the adverse effects on the endocannabinoid system (ECS) in modulating germ cell progression. Male offspring exposed to BPA during the foetal-perinatal period at doses below the no-observed-adverse-effect-level were used to investigate the exposure effects in adulthood. Results showed that BPA accumulates specifically in epididymal fat rather than in abdominal fat and targets testicular expression of 3 beta-hydroxysteroid dehydrogenase and cytochrome P450 aromatase, thus promoting sustained increase of estrogens and a decrease of testosterone. The exposure to BPA affects the expression levels of some ECS components, namely type-1 (CB1) and type-2 cannabinoid (CB2) receptor and monoacylglycerol-lipase (MAGL). Furthermore, it affects the temporal progression of germ cells reported to be responsive to ECS and promotes epithelial germ cell exfoliation. In particular, it increases the germ cell content (i.e., spermatogonia while reducing spermatocytes and spermatids), accelerates progression of spermatocytes and spermatids, promotes epithelial detachment of round and condensed spermatids and interferes with expression of cell-cell junction genes (i.e., zonula occcludens protein-1, vimentin and beta-catenin). Altogether, our study provides evidence that early exposure to BPA produces in adulthood sustained and site-specific BPA accumulation in epididymal fat, becoming a risk factor for the reproductive endocrine pathways associated to ECS.

Summary Background Bisphenol A (BPA) exposure has been associated with increased incidence of diabetes and obesity in adults. Objectives To evaluate whether an association between BPA urinary levels and insulin resistance as well as adiponectin and resistin production and serum concentrations may occur in obese children. Methods Clinical and biochemical features of 141 obese children were collected. Serum resistin and adiponectin were evaluated. Insulin resistance and urinary BPA levels were assessed. Moreover, the effect of BPA on adiponectin and resistin gene expression in adipocytes from eight normal weight prepubertal children was investigated by quantitative real-time RT-PCR (qPCR). Results Direct association between BPA and homeostasis model assessment (r = 0.23; p: 0.0069) and a strong inverse association between BPA and adiponectin have been found (r = −0.48; p

Non-alcoholic fatty liver disease (NAFLD) is considered a predominant chronic liver disease worldwide and a component of metabolic syndrome. Due to its relationship with multiple organs, it is extremely complex to precisely define its pathogenesis as well as to set appropriate therapeutic and preventive strategies. Endocrine disruptors (EDCs) in general, and bisphenol A (BPA) in particular, are a heterogeneous group of substances, largely distributed in daily use items, able to interfere with the normal signaling of several hormones that seem to be related to type 2 diabetes mellitus (T2DM), obesity, and other metabolic disorders. It is reasonable to hypothesize a BPA involvement in the pathogenesis and evolution of NAFLD. However, its mechanisms of action as well as its burden in the vicious circle that connects obesity, T2DM, metabolic syndrome, and NAFLD still remain to be completely defined. In this review we analyzed the scientific evidence on this promising research area, in order to provide an overview of the harmful effects linked to the exposure to EDCs as well as to frame the role that BPA would have in all phases of NAFLD evolution. ispartof: INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH vol:16 issue:17 ispartof: location:Switzerland status: published

An innovative complementary approach using a liquid chromatographic-mass spectrometer method and infrared spectroscopy is proposed for measuring internal biological exposure to dangerous chemical contaminants and for monitoring biochemical changes in target organs. The proposed methodologies were validated and applied in the case of rats exposed to low-doses of Bisphenol A (BPA). A liquid chromatographic method coupled to a tandem mass spectrometer was used in order to measure BPA concentration in rat livers. BPA was detected at different levels in all liver samples from BPA-treated rats, although the exposure dose was the same in all treated animals, and also from control rats, highlighting the difficulties in eliminating external uncontrolled exposure and the need for internal biological monitoring. Fourier Transform Infrared analysis was applied to detect structural changes occurring in several molecules (lipids, proteins, carbohydrates and nucleic acids) as well as the presence of specific metabolic processes. The spectroscopic analyses clearly demonstrated a different lipid composition more than an evident lipid accumulation and a glycogen accumulation decrease, revealing a metabolic disturbance in livers with a normal histological aspect. These results demonstrated the potential of an integrated approach based on mass spectrometry and infrared spectroscopy to evaluate at an early stage the hepatotoxic effect of BPA exposure in an animal model. This approach can be usefully exploited in all the investigations aimed to provide better information concerning the interrelationships between contaminant exposure, dose, and health effects.

Bisphenol A (BPA) and silybin are considered xenoestrogens and could interfere with the action of endogenous hormones. It was demonstrated a higher level of BPA in plasma of nonalcoholic steatohepatitis (NASH) patients, compared to those with steatosis (NAFL). We investigated the effect of BPA and silybin, alone or in combination, on proliferation, oxidative stress and steroid metabolism in HepG2 grown in high glucose concentration medium (H-HepG2). Cell viability was assessed by adding 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). TBARS were quantified by spectrophotometry. The effect of BPA, silybin and their combination on the expression of phosphorilized extracellular signal-regulated kinase (ERK), ERK and Caspase 3 was determined by Western blot analysis. The identifications of lipids and steroid hormones was performed by mass spectrometry. BPA elicited in H-HepG2 oxidative stress and steroid hormones oxidation leading to the formation of metabolite with estrogenic and genotoxic potentials. Silybin ameliorates the harmful BPA-induced effect decreasing glucose uptake and lipid peroxidation. Moreover silybin activates the synthesis of vitamin D3 metabolites and prevent the steroid hormones oxidation. BPA could be considered as an important risk factor in worsening and progression of NAFLD. At the same time silybin could be a valid support to counteract these effects in NASH patients.

Background: Bisphenol A is an endocrine disrupting chemical associated with type 2 diabetes mellitus (T2DM), cardiovascular disease and liver enzyme abnormalities. Aim: To evaluate bisphenol A plasma and urine levels in non-alcoholic fatty liver disease (NAFLD) patients compared to healthy subjects. Furthermore, we evaluated, in human HepG2 cells, the effects of exposure to different concentrations of bisphenol A on both oxidative stress induction and cell proliferation. Methods: We enrolled 60 patients with histological diagnosis of NAFLD with or without T2DM and sixty healthy subjects. In vitro, the proliferation of bisphenol A-exposed HepG2 cells at two different concentrations (0.025 and 0.05 μM) was evaluated, both at high (H-HepG2) and at low (L-HepG2) glucose concentrations for 48 h. Lipoperoxidation was assessed by thiobarbituric acid reactive substances (TBARS) assay. Results: Bisphenol A levels were significantly higher in 60 NAFLD subjects, both in urine and in plasma (P < 0.0001) when compared to controls and, in this group, it appeared to be higher in 30 non-alcoholic steatohepatitis patients compared to 30 simple steatosis subjects (P < 0.05), independently from the presence of T2DM. After a bisphenol A-free diet for 1 month, NAFLD patients showed a significant reduction in bisphenol A circulating levels (P < 0.05), without a significant reduction in urine levels. H-HepG2 cells treated with bisphenol A (0.05 μM) increased proliferation compared to controls at 48 h (P < 0.0001). Bisphenol A increased TBARS levels at 48 h versus controls. Conclusions: Our study reveals a possible role of bisphenol A as an environmental factor involved in the promotion of NAFLD, particularly in T2DM patients.

Bisphenol A (BPA), an endocrine disruptor, may affect in situ steroidogenesis and alter steroids levels. The present work proposes a liquid chromatography tandem mass spectrometry method to simultaneously quantify BPA, 17&beta, Estradiol and testosterone in two target tissues: testis and visceral fat mass. Analytes were isolated and lipophilic impurities removed by two serial steps: liquid-liquid and solid phase extraction. All compounds were separated in a single gradient run by Kinetex F5 column and detected via multiple reaction monitoring using a triple quadrupole with a TurboIon electrospray source in both negative and positive modes. The method is selective and very sensitive. In the investigated concentration range, the linearity of the detector response is verified in both tissues. The use of specific SPE cartridges for affinity chromatography purification allows obtaining high percentages of process efficiency (68.0&ndash, 83.3% for testicular tissue, 63.7&ndash, 70.7% for visceral fat mass). Good repeatability and reproducibility was observed. The validated method can be efficiently applied for direct biological monitoring in testis and visceral fat mass from mice exposed to BPA. The quantification of compounds in a single assay could be achieved without a loss of sensitivity.

The performance of S2O82-/UV-C and H2O2/UV-C treatments was investigated for the degradation and detoxification of Bisphenol A (BPA). The acute toxicity of BPA and its degradation products was examined with the Vibrio fischeri bioassay, whereas changes in estrogenic activity were followed with the Yeast Estrogen Screen (YES) assay. LC and LC-MS/MS analyses were conducted to determine degradation products evolving during photochemical treatment. In addition, BPA-spiked real freshwater samples were also subjected to S2O82-/UV-C and H2O2/UV-C treatment to study the effect of a real water matrix on BPA removal and detoxification rates. BPA removal in pure water was very fast (20 min) and TOC removals were considerably retarded (by 40%) in the raw freshwater matrix most probably due to its natural organic matter content (TOC = 5.1 mg L-1). H2O2/UV-C and S2O82-/UV-C treatment in raw freshwater did not result in toxic degradation products. (C) 2014 Elsevier Ltd. All rights reserved.

This study reports the occurrence of some endocrine disrupting chemicals in red mullet samples and sediments collected in two representative sites of the northern Sicilian coast (Italy). For this purpose, an improved method, using solid extraction and high-performance liquid chromatography analyses for the simultaneous determination of bisphenol A (BPA), 4-nonylphenol (4-NP) and 4-t-octylphenol (4-t-OP) in fish tissues and sediments, has been developed and validated. Method performance was demonstrated over the concentration range 0.1–200 ng/mL, with detection limits from 0.06 to 0.1 ng/mL. Recoveries ranged from 83.4% to 102.6%, with relative standard deviations of 7.7–14.0% for the entire procedure. Results showed that BPA, 4-t-OP and 4-NP were detected in all fish samples and sediments from two sampling sites, indicating that these chemicals have contaminated Mediterranean aquatic ecosystem and have accumulated in fish. The study provided more comprehensive fundamental data for risk assessment and contamination control of phenolic EDCs in aquatic environment.

A selective and highly sensitive analytical methodology for determination of Bisphenol A in human plasma was developed and validated. The method was based on selective liquid/solid extraction, combined with liquid chromatography-electrospray ionization tandem mass spectrometry in the multiple reaction monitoring mode and negative ionization. The linearity of the detector response was verified in human plasma over the concentration range 0.100-200ngmL(-1). The detection limit was 0.03ngmL(-1) and the quantification limit was 0.100ngmL(-1). The analytical features of the proposed in-house validated method were satisfactory: precision was

Bisphenol A (BPA) is an endocrine disruptor (ED) that is abundant in the environment because of its extensive use in human manufactured products. In this study, the BPA concentration was measured in the muscle and liver of five edible fish, characterized by different habitat and habits, caught in two different sites of the Tyrrhenian Sea (Italy). Our results show that: (i) fish livers are about 2.5 times more polluted than muscle; (ii) fish caught in the Gulf of Naples are more polluted than those from the Latium coasts, ranging from 1.2-fold more for White Bream to 6.6-fold for Grey Mullet; and (iii) the percentages of fish found to be BPA-polluted in the Gulf of Naples ranged from 73% (for Bass) to 90% (for Mullet), while the Latium fish range from 60% (for Bass) to 90% (for Mullet). These data indicate that consumers of fish caught in the Gulf of Naples are at a greater risk for BPA-induced endocrine pathologies compared to those who consume fish caught along the Latium coasts.

In view of the heath problems induced by the presence into the environment of endocrine disruptors, laccase from Trametes versicolor was covalently immobilized on a chemically modified polypropylene membrane in order to remove phenol and its derivatives from polluted waters. Using phenol as substrate model the optimal immobilization conditions were determined. The immobilized laccase exhibited maximal enzyme activity at pH 5.5 and optimal temperature at 55 °C. These operative parameters have been compared with those obtained with the soluble laccase in order to ascertain the immobilization effect. When employed in a bioreactor operating under isothermal conditions the immobilized laccase was able to oxidize a wide range of phenolic substrates. In particular it was found that some phenol derivatives (2-CP, 3-CP, 4-CP, NP and chlorophene) were oxidized at a similar rate than phenol, other derivatives (paracetamol, 3-MP and chloroxyphenol) at a smaller rate, while others (2,4-DCP and BPA) at higher rate. When the catalytic membrane was employed in a non-isothermal reactor the reaction rate increased with the increase of the applied temperature difference. Practically the increase of the laccase oxidative power under the non-isothermal conditions followed the same sequence observed under isothermal conditions. Interesting enough, the percentage increase of enzyme reaction rate under non-isothermal conditions resulted higher in the cases in which the isothermal reaction rate was smaller. When the reduction of the production times by the presence of a temperature gradient is considered, the measured values strongly candidate the technology of non-isothermal bioreactors as a useful tool in processes of detoxification of waste waters polluted by endocrine disruptors of phenolic origin. © 2010 Elsevier B.V. All rights reserved.

A hollow-fiber enzyme reactor, operating under isothermal and nonisothermal conditions, was built employing a polypropylene hollow fiber onto which beta-galactosidase was immobilized. Hexamethylenediamine and glutaraldehyde were used as spacer and coupling agent, respectively. Glucose production was studied as a function of temperature, substrate concentration, and size of the transmembrane temperature gradient. The actual average temperature differences across the polypropylene fiber, to which reference was done to evaluate the effect of the nonisothermal conditions, were calculated by means of a mathematical approach, which made it possible to know, using computer simulation, the radial and axial temperature profiles inside the bioreactor and across the membrane. Percent activity increases, proportional to the size of the temperature gradients, were found when the enzyme activities under nonisothermal conditions were compared to those measured under comparable isothermal conditions. Percent reductions of the production times, proportional to the applied temperature gradients, were also calculated. The advantage of employing nonisothermal bioreactors in biotechnological industrial process was discussed.

The bioremediation of waters polluted by Bisphenol A, taken as a model for endocrine disruptors, has been pursued by means of catalytic membranes in bioreactors operating under isothermal and non-isothermal conditions. Laccase from Trametes versicolor was immobilized on nylon membranes grafted with Glycidyl Methacrylate and using Phenylendiamine as spacer. The behaviour of the catalytic membrane was studied as a function of BPA concentration. Affinities of immobilized laccase towards BPA were found to increase with average temperature and under nonisothermal conditions. Percentage increases of enzyme activity, proportional to the applied temperature differences, were found to decrease with the increase of BPA concentrations. Interestingly, the highest levels of BPA biodegradation occurred at the lowest concentrations, in other words those present in wastewaters given the small water solubility of this compound. The results are discussed in terms of the process of thermodialysis by considering the additional BPA fluxes towards the immobilized enzymes driven by the temperature gradients. (c) 2006 Elsevier B.V. All rights reserved.

Bisphenol A (BPA) is a xenobiotic endocrine-disrupting chemical.In vitroandin vivostudies have indicated that BPA alters endocrine-metabolic pathways in adipose tissue, which increases the risk of metabolic disorders and obesity. BPA can affect adipose tissue and increase fat cell numbers or sizes by regulating the expression of the genes that are directly involved in metabolic homeostasis and obesity. Several studies performed in animal models have accounted for an obesogen role of BPA, but its effects on human adipocytes – especially in children – have been poorly investigated. The aim of this study is to understand the molecular mechanisms by which environmentally relevant doses of BPA can interfere with the canonical endocrine function that regulates metabolism in mature human adipocytes from prepubertal, non-obese children. BPA can act as an estrogen agonist or antagonist depending on the physiological context. To identify the molecular signatures associated with metabolism, transcriptional modifications of mature adipocytes from prepubertal children exposed to estrogen were evaluated by means of microarray analysis. The analysis of deregulated genes associated with metabolic disorders allowed us to identify a small group of genes that are expressed in an opposite manner from that of adipocytes treated with BPA. In particular, we found that BPA increases the expression of pro-inflammatory cytokines and the expression ofFABP4andCD36, two genes involved in lipid metabolism. In addition, BPA decreases the expression ofPCSK1, a gene involved in insulin production. These results indicate that exposure to BPA may be an important risk factor for developing metabolic disorders that are involved in childhood metabolism dysregulation.

The effects of low intensity, low frequency magnetic fields (MFs) on catalytic activity of the calcium dependent protease calpain was determined following the enzyme activation both in “in vitro” and “in vivo” conditions. We have observed that a 0.3 mT MF induces a significant increase in the requirement of the protease for this metal ion. This change is detectable at low [Ca2+] and disappears when the level of Ca2+ is raised to saturating amounts. The observed effects are not due to transient MF− induced conformational changes occurring in calpain, but to direct effects of the MF on Ca2+ ions, which become less available for the binding sites present in calpain. Altogether, these results indicate that exposure to low intensity, low frequency MFs alters the intracellular Ca2+ “availability,” thereby modifying the related cell response. Bioelectromagnetics 27:43–50, 2006. © 2005 Wiley-Liss, Inc.

β-Galactosidase has been immobilised through spacers of different length on nylon membranes grafted with glycidyl methacrylate. Hexamethylendiamine, ethylendiamine or hydrazine have been separately used as spacers. The behaviour of the catalytic membranes has been studied in a bioreactor operating under non-isothermal conditions as a function of the applied temperature difference ΔT. Comparison of the enzyme reaction rates under isothermal and non-isothermal conditions resulted in percentage activity increases (PAI) and reduction of the production time (τr) proportional to the size of the applied ΔT. Both these parameters increased with the increase of the spacer length. Results have been discussed in the frame of reference of the process of thermodialysis which reduces the limitations to the diffusion of substrate and reaction products across the catalytic membrane, limitations introduced by the grafting and immobilisation process. The advantages of employing non-isothermal bioreactors in biotechnological productive processes have been outlined.

The present study aims at assessing Bisphenol A (BPA) level in water used in laboratories. A total of five types of water commonly used in laboratory (tap, softened, distilled, double distilled, commercial LC-MS pure water), were analyzed and BPA was quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Only the samples of ultrapure water showed BPA levels above detection limits (>0.004 ng/mL). The mean BPA level ranged from 0.008 to 0.473 ng/mL. A higher mean BPA level was found in water obtained from ultrafilter process compared to commercial sources. Activated carbon filtration is necessary to achieve a BPA free level in ultrapure water.

Purpose Combined treatment based on cisplatin-loaded Poly(D,L-lactic-co-glicolic)acid (PLGA) nanoparticles (NP-C) plus the NSAID piroxicam was used as novel treatment for mesothelioma to reduce side effects related to cisplatin toxicity. Methods PLGA nanoparticles were prepared by double emulsion solvent evaporation method. Particle size, drug release profile and in vitro cellular uptake were characterized by TEM, DLS, LC/MS and fluorescence microscopy. MSTO-211H cell line was used to analyse NP-C biological efficacy by FACS and protein analysis. Results Cisplatin was encapsulated in 197 nm PLGA nanoparticles with 8.2% drug loading efficiency and 47% encapsulation efficiency. Cisplatin delivery from nanoparticles reaches 80% of total encapsulated drug in 14 days following a triphasic trend. PLGA nanoparticles in MSTO-211H cells were localized in the perinuclear space NP-C in combination with piroxicam induced apoptosis using a final cisplatin concentration 1.75 fold less than free drug. Delivered cisplatin cooperated with piroxicam in modulating cell cycle regulators as caspase-3, p53 and p21. Conclusions Cisplatin loaded PLGA nanoparticles plus piroxicam showed a good efficacy in exerting cytotoxic activity and inducing the same molecular apoptotic effects of the free drugs. Sustained cisplatin release allowed to use less amount of drug, decreasing toxic side effects. This novel approach could represent a new strategy for mesothelioma treatment. © 2014 Springer Science+Business Media New York.

Serratia rubidiae, Pseudomonas aeruginosa and Escherichia coli K12 have been studied for their ability of Bisphenol A removal from aqueous systems and biofilm formation on activated granule carbon. Mathematical equations for biodegradation process have been elaborated and discussed. Pseudomonas aeruginosa was found the best strain to be employed in the process of Bisphenol A removal. The yield in BPA removal of a Pseudomonas aeruginosa biofilm grown on GAC and operating in a fluidized bed reactor has been evaluated. The results confirm the usefulness in using biological activated carbon (BAC process) to remove phenol compounds from aqueous systems.

Bisphenol A (BPA) is used as basic chemical compound in the production of polycarbonate food containers or epoxy resins coating metallic cans for food and beverages conservation. Its xeno-estrogenic activity alters endocrine-metabolic pathways modulating glucose metabolism and increasing the risk of developing diabetes, insulin resistance, and obesity. Based onin vitroandin vivoexperimental research, here we report some of the major BPA adverse effects on tissues that play a key role in the regulation on the whole body’s metabolism. Evidences have shown that BPA is able to exert its endocrine disrupting action altering glucose metabolism and contributing to the onset of metabolic disorders, acting on liver functions and affecting insulin production by the pancreas. Exposure to BPA has been reported also to modulate glucose utilization in muscles, as well as to interfere with adipose tissue endocrine function. In addition, to peripheral tissues, recent studies have shown that BPA by acting in the Central Nervous System affects neuroendocrine regulation of glucose metabolism, promoting glucose metabolism dysfunction such as glucose intolerance and insulin resistance. Thus, exposure to BPA seems to be an important risk factor in the onset of obesity and metabolic syndrome. However, its mechanisms of action need to be further investigated to provide a major evaluation of risk assessment.

Biodegradable polymer nanoparticles have recently received considerable attention for drug delivery. One of the most investigated system is represented by cisplatin-loaded poly-(D, L-lactic-co-glycolic)acid (PLGA) nanoparticles. FT-IR microspectroscopy has been used for characterizing these particles. In addition an univariate analysis procedure has been adopted for interpreting experimental results when the spectrum modifications are relatively weak.

The catalytic behaviour under isothermal conditions of two different membranes loaded with β-galactosidase was investigated. One membrane (M1) was constituted by a nylon sheet grafted with methylmethacrylate by means of chemical grafting. The other, (M2), was prepared by a double chemical grafting: the first one with styrene (Sty) and the second one with methylmethacrylate. Membrane activity was characterized as a function of temperature, pH and substrate concentration. The role of Sty in increasing membrane hydrophobicity has been discussed. Membrane M2 was found to be better suited for employment in non-isothermal bioreactors.

The effect of the microenvironment and immobilization method on the activity of immobilized β-galactosidase was investigated. Immobilization was done on Teflon membranes grafted with different acrylic monomers by γ-radiation and activated by two different coupling agents through the functional groups of the grafted monomers. 2-Hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAA) were grafted on the membrane, and 1,6-hexamethylenediamine (HMDA) was used as a spacer. Glutaraldehyde or cyanuric chloride were used as coupling agents to bind the enzyme to the membrane. Four different catalytic membranes were obtained using the same solid support. Direct comparison between the isothermal behaviour of the biocatalyst in its free and immobilized form was carried out. In particular the dependence of the isothermal activity on the temperature and pH was studied and the kinetic parameters determined. The influence of the microenvironment on the observed activity of the four membranes was evidenced and discussed. The way of improving the yield of these catalytic membranes is discussed also.

A method based on solid-phase extraction followed by liquid chromatography, coupled to UV-visible and fluorescence spectrophotometry, has been developed for determination of bisphenol A (BPA) in canned tomatoes. The limit of quantification (LOQ) of the procedure used is 0.03 μM (0.26 μg BPA/kg tomato). For each of three different tomato based products (peeled, cherry and concentrated paste), 16 samples belonging to six commercial brands, retailed in Italian markets, were tested for migration of BPA epoxy-coating cans. All the tomato samples exhibited migration levels below 0.4 μg/kg, while samples subjected to heating process and/or can's damage by denting, exhibited a significant increase in the migration levels. In any case, no sample contained BPA exceeding the European Union limit for migration, set at 600 μg/kg of food. By comparing the results for each brand, no relevant difference in BPA concentration was found depending on the kind of tomato products. © 2014 Elsevier Ltd. All rights reserved.

A selective and highly sensitive liquid chromatography-tandem mass spectrometry method has been developed and validated for determination of Bisphenol A (BPA) in human urine using labeled d6-BPA as internal standard. BPA was purified from human urine by affinity chromatography on solid extraction AFFINIMIP® Bisphenol A cartridges, based on molecularly imprinted polymers. After purification, the samples were analyzed on a Phenomenex Kinetex 100 × 4.6 mm, 2.6 μm particle PFP reversed-phase HPLC column, coupled to a triple quadrupole mass spectrometer by an electrospray ion source. Analyses were performed in the multiple reaction monitoring mode and negative ionization; the product ions at 133.2 and 212.1 m/z for BPA and at 138.2 and 215.0 m/z for d6-BPA were monitored to assess unambiguous identification. The linearity of the detector response was verified in human urine over the concentration range 0.100-200 ng/mL. The detection limit was calculated as 0.03 ng/mL and the limit of quantification of the method is 0.10 ng/mL. This LC/ESI-MS/MS method was in-house validated evaluating specificity, trueness, within-day and between-days precision. The mean recoveries of BPA from spiked urine samples were higher than 94 % and good reproducibility (relative standard deviations ≤ 8.1 %) was observed. The developed method was applied to a pilot study involving 105 children, aged from 6 to 14 years (16 normal weight and 89 obese children), from the Regione Campania (Southern Italy). The aim of this study was to determine the concentrations of BPA in urine of children and possible correlations with childhood obesity. © 2013 Springer-Verlag Berlin Heidelberg.

A fluidized bed reactor, filled with laccase-based beads, has been employed to bioremediate aqueous solutions polluted by endocrine disruptors belonging to the alkylphenols (APs) class. In particular Octylphenol and Nonylphenol have been studied. The catalytic activity of free and immobilized laccase from Trametes versicolor has been characterized as a function of pH, temperature and substrate concentration in the reaction medium. In view of practical applications for each substrate concentration the removal efficiency (RE), the time to halve the initial concentration (tau(50)), and the t(c-0), i.e. the time to reach complete pollutant removal, have been calculated. The immobilized laccase exhibited a lower affinity for octylphenol (K-m = 1.11 mM) than for Nonylphenol (K-m = 0.72 mM), but all the other parameters of applicative interest resulted more significant for octylphenol. For example, the times to reach the complete removal of octylphenol compared to those for nonylphenol at the same concentration is shorter of about 15% (at low concentrations) up to 40% (at high concentrations). The study of cell proliferation with MPP89 cells, a human mesothelioma cell line, and the assay with the YES test indicated the loss of estrogenic activity of the APs solutions after laccase treatment. (C) 2013 Elsevier B.V. All rights reserved.

Pregnant adult Balb-C mice were exposed daily to two different doses of Bisphenol A (BPA) by subcutaneous injection beginning on gestational day 1 through the seventh day after delivery. The mothers were sacrificed on postpartum day 21, and the offspring were sacrificed at 3 months of age. Control mice were subjected to the same experimental protocol but received saline injections. The liver, muscles, hindbrain and forebrain of the offspring were dissected and processed using HPLC to assess the level of BPA in the tissues and to determine its dependence on the exposure dose and gender. For comparison, the same tissues were dissected from the mothers and analysed. We report the following results: (1) the level of BPA that accumulated in a given tissue was dependent on the exposure dose; (2) the rank order of BPA accumulation in the various tissues was dependent on the gender of the offspring; (3) the average BPA concentrations in the liver and muscle of the female offspring were higher than in the males; and (4) the average BPA concentration in the central nervous system (i.e., the hindbrain and forebrain) of the male offspring was higher than in the females. (C) 2011 Elsevier B.V. All rights reserved.

The oxidative biodegradation of 2- and 4-chlorophenol (CPs) catalyzed by immobilized laccase from Trametes versicolor was conducted in a bed reactor. The effects of reaction conditions (reaction time, dosage of immobilized enzyme, flow rate, pH value and temperature, initial concentration) on CPs removal efficiency (RE) were investigated. By using in the immobilization solution an enzyme dosage of 2.5 mg free enz g carrier − 1 , the maximum ratio of laccase to PAN-beads is resulted 0.25 mg imm enz g carrier − 1 . RE values greater than 70% were obtained when 40 mL of 1 mM substrate solution were circulated for 90 min at 75 mL min−1 in the bed reactor filled with 12 g of laccase-PAN beads. The optimal pH for CPs biodegradation was 5 and it was not related to substituent position on aromatic ring. Experiments with immobilized laccase demonstrated good removal at high initial substrate concentrations and high thermal stability. The affinity of 2-CP was higher than that of 4-CP. Finally, the 2-CP was also biodegraded as tertiary mixtures with 2,3,4,5-tetrachlorophenol (TCP) and pentachlorophenol (PCP). The presence of TCP and PCP decreased the removal efficiency of 2-CP. The order of the relative degrading capacity becomes TCP > PCP > 2-CP.

The biodegradation of waters polluted by some bisphenols, endowed with endocrine activity, has been studied by means of laccase or tyrosinase immobilized on polyacrylonitrile (PAN) beads. Bisphenol A (BPA), Bisphenol B (BPB), Bisphenol F (BPF) and Tetrachlorobisphenol A (TCBPA) have been used. The laccase-PAN beads system has been characterized as a function of pH, temperature and substrate concentration. The biochemical parameters so obtained have been compared with those of the free enzyme to evidence the modification induced by the immobilization process. Once characterized, the laccase-PAN beads have been employed in a fluidized bed reactor to determine for each of the four bisphenols the degradation rate constant (k); the τ50, i.e., the time to obtain the 50% of degradation, and the removal efficiency (RE90) after 90 min of enzyme treatment. The same parameters have been measured for each of the four pollutants with the same fluidized bed bioreactor loaded with tyrosinase-PAN beads. The internal comparison, i.e., in each of the two catalytic systems, has shown that both enzymes exhibit a removal efficiency in the following order BPF>BPA>BPB>TCBPA. The external comparison, i.e., the comparison between the two catalytic system, has shown that the catalytic power of laccase were higher than that of tyrosinase. The operational stability of both catalytic systems resulted excellent, since they maintained more than 80% of the initial activity after 30 days of work. © 2010 Springer Science+Business Media B.V.

Octylphenol (OP) is an endocrine-disrupting chemical that accumulates in various organs. It has also been shown to exert noxious effects on the central nervous system. In the present study, we measured in Sprague–Dawley rats the degree of OP accumulation in different areas of the brain and investigated the effect of OP in pain modulation. Two groups of male Sprague–Dawley rats were treated for 20 days with 50 mg/kg BW/day of OP (group 1) or vehicle (group 2). At the end of the treatment, the formalin test was performed to evaluate the effect of OP exposure on pain. Soon after, rats were sacrificed, and the accumulation of OP in the cerebral cortex, hippocampus, hypothalamus, cerebellum, thalamus, striatum, mesencephalus and ventral hindbrain was measured by HPLC analysis. The results showed a greater accumulation of OP in the cerebral cortex compared to all the other areas; there was also more accumulation in the cerebellum compared to the mesencephalus and thalamus. No accumulation was found in the striatum. These results suggest that there is a preferential accumulation of OP in different areas of the brain with consequences to neural behaviour. On the contrary, experiments on facial grooming did not show significant effects of OP on pain.

Endometriosis is a chronic gynecological disease characterized by the growth of endometrial tissue outside the uterine cavity. Exposure to endocrine disruptors during critical period of development causes long-lasting effects, being the genital system one of the targets. This study describes the effects on female genital system caused by developmental exposure to the endocrine-disrupting chemical bisphenol A (BPA) during pre- and pen-natal development in mice. To this end, timed pregnant Balb-C mice were treated from day 1 of gestation to 7 days after delivery with BPA (100, or 1000 mu g/kg/day). After delivery, pups were held for 3 months; then, pelvic organs were analyzed in their entirety and livers of both pups and moms were studied for the presence of BPA. We found in the adipose tissue surrounding the genital tracts of a consistent number of treated animals, endometriosis-like structure with the presence of both glands and stroma and expressing both estrogen receptor and HOXA-10. Moreover, cystic ovaries, adenomatous hyperplasia with cystic endometrial hyperplasia and atypical hyperplasia were significantly more frequent in treated animals respect to the controls. Finally, BPA was found in the livers of exposed moms and female offspring. In conclusion, we describe for the first time an endometriosis-like phenotype in mice, elicited by pre-natal exposition to BPA. This observation may induce to thoroughly reconsider the pathogenesis and treatment of endometriosis, considering the high incidence of endometriosis and the problems caused by associated infertility. (C) 2010 Elsevier Inc. All rights reserved.