Nadia Diano

A thionine-modified carbon paste amperometric biosensor for catechol and bisphenol A determination.

A thionine-modified carbon paste electrode for catechol and Bisphenol A (BPA) detection is presented. Graphite powder was modified by adsorbing thionine as electrochemical mediator. The electrochemical response of the modified carbon paste electrode (CPE) was determined before electrode modification with tyrosinase. Then, tyrosinase was added in order to assemble a biosensor. Once established the best operative conditions, an interelectrode reproducibility around 7% was obtained and the resulting biosensor showed improved sensitivities and (S=139.6+/-1.1 nA/microM for catechol and S=85.4+/-1.5 nA/microM for BPA) in comparison with the biosensor constructed without thionine (S=104.4+/-0.5 nA/microM for catechol and S=51.1+/-0.6 nA/microM for BPA) and low detection limits (0.15 microM for both the electrodes and analytes). Also the comparison with the results reported in the literature showed higher sensitivity and lower detection limit for our biosensor. Moreover the functioning of the thionine-tyrosinase CPE was validated following a biodegradation process of water polluted by BPA and comparing the time changes of BPA concentration inferred by the biosensor calibration curve and those determined by means of HPLC measurements.

Pre-natal exposure of mice to bisphenol A elicits an endometriosis-like phenotype in female offspring

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 peri-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 microg/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.

Influence of the spacer length on the activity of enzymes immobilised on nylon/polyGMA membranes: Part 1. Isothermal conditions

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.

Enzymatic removal of estrogenic activity of nonylphenol and octylphenol aqueous solutions by immobilized laccase from Trametes versicolor

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 (τ50), and the tc=0, i.e. the time to reach complete pollutant removal, have been calculated. The immobilized laccase exhibited a lower affinity for octylphenol (Km=1.11mM) than for Nonylphenol (Km=0.72mM), 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.

Effects of some endocrine disruptors on cell cycle progression and murine dendritic cell differentiation

Endocrine disruptor chemicals (EDCs), which are predominantly present in the environment, are able to mimic or antagonise the biological activity of hormones primarily through the interaction with specific receptors. The main consequences are adverse effects on the growth and development of reproductive organs, the induction of cancer and effects on neuronal differentiation. In this study, we investigated the ability of certain EDCs, Bisphenol A (BPA), Bisphenol B (BPB), Bisphenol F (BPF), 4-n Nonylphenol (NP) and Octylphenol (OP), belonging to a homogeneous group of phenol origin, to interfere with specific cellular processes, namely, proliferation, by using MCF-7 breast carcinoma cells, and differentiation, by using murine bone marrow dendritic cells. We correlated the data on cell growth with the stimulation of cell cycle progression, which could become a step in the development of cancer, and we established a proliferation ranking between the tested EDCs: NP>BPA>OP>BPB>BPF. In addition, we investigated the ability of NP, BPA and OP to induce the differentiation of dendritic cells, the powerful antigen-presenting cells of the immune system. The differentiation and activation of these cells could affect a well-regulated immune response and determine an allergic sensitisation. We found that BPA and NP were active in determining differentiation.

Effect of Bisphenol A with or without enzyme treatment on the proliferation and viability of MCF-7 cells

Recently, aqueous solutions polluted by BPA have been bioremediated by us using laccase immobilized on hydrophobic membranes in non-isothermal bioreactors. BPA degradation was checked using analytical methods. To assess in vitro the occurred bioremediation, the proliferation and viability indexes of MCF-7 cells incubated in the presence of aqueous solutions of BPA, or of enzyme-treated BPA solutions, have been measured as a function of the initial BPA concentration. The results demonstrated that: i) at each initial BPA concentration used, both the proliferation and viability indexes are a function of the duration of enzyme treatment; ii) proliferation and viability are uncoupled biological processes with respect to BPA enzyme treatment. Non-isothermal bioreactors are a useful tool for the bioremediation of aqueous solutions polluted by BPA, which is an example of an endocrine disruptor that belongs to the alkyl phenol family.

Bisphenol A content in fish caught in two different sites of the Tyrrhenian Sea (Italy)

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.

S2O82−/UV-C and H2O2/UV-C treatment of Bisphenol A: Assessment of toxicity, estrogenic activity, degradation products and results in real water

The performance of S2O8(2-)/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 S2O8(2-)/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 (⩽7 min) and complete via both H2O2/UV-C and S2O8(2-)/UV-C treatment, accompanied with rapid and significant mineralization rates ranging between 70% and 85%. V.fischeri bioassay results indicated that degradation products being more toxic than BPA were formed at the initial stages of H2O2/UV-C whereas a rapid and steady reduction in toxicity was observed during S2O8(2-)/UV-C treatment in pure water. UV-C treatment products exhibited a higher estrogenic activity than the original BPA solution while the estrogenicity of BPA was completely removed during H2O2/UV-C and S2O8(2-)/UV-C treatments parallel to its degradation. 3-methylbenzoic and 4-sulfobenzoic acids, as well as the ring opening products fumaric, succinic and oxalic acids could be identified as degradation products. BPA degradation required extended treatment periods (>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 S2O8(2-)/UV-C treatment in raw freshwater did not result in toxic degradation products.

Migration of bisphenol A into canned tomatoes produced in Italy: Dependence on temperature and storage conditions

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 cans 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.

The influence of the support nature on the kinetics parameters, inhibition constants and reactivation of immobilized acetylcholinesterase

Acetylcholinesterase (AChE) was immobilized on two different composite membranes constituted by a chemically modified poly-acrylonitrile (PAN) membrane plus a layer of tethered chitosan of different molecular weight, 10 kDa or 400 kDa. AChE was also directly immobilized on a chemically modified PAN membrane with NaOH and ethylenediamine (EDA) without chitosan. To know how the different supports affected the enzyme activity and the kinetic parameters, the AChE activity was studied in the soluble form and in the insoluble form with all the three types of modified PAN membranes. The best performance was obtained by the modified PAN membrane having the chitosan with the lower molecular weight. The results concerning the AChE inhibition by methyl-paraoxon and the subsequent reactivation by pyridine-2-aldoxime methochloride (2-PAM) are presented and discussed. The composite membrane having chitosan with the lower molecular weight appeared to be potentially useful for applications in the field of biosensors.