Analysis and occurrence of some phenol endocrine disruptors in two marine sites of the northern coast of Sicily (Italy).

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 0.1-200ng/mL, with detection limits from 0.06 to 0.1ng/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.

S2O8(2-)/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.

A developmental hepatotoxicity study of dietary bisphenol A in Sparus aurata juveniles.

Previous studies in rats have indicated that a diet enriched with Bisphenol A adversely effects metabolism and reproductive success. In rats exposed to BPA by maternal gavage, alteration in the developmental programming, higher obesity rates and reproductive anomalies were induced. Starting with this evidence, the aim of this study was to provide important insights on the effects induced by a BPA enriched diet, on the reproductive physiology and metabolism of juvenile fish, simulating the scenario occurring when wild fish fed on prey contaminated with environmental BPA. Seabream was chosen as model, as it is one of the primary commercial species valued by consumers and these results could provide important findings on adverse effects that could be passed on to humans by eating contaminated fish. A novel method for measuring BPA in the food and water by affinity chromatography was developed. Analysis of signals involved in reproduction uncovered altered levels of vtg and Zp, clearly indicating the estrogenic effect of BPA. Similarly, BPA up-regulated catd and era gene expression. A noteworthy outcome from this study was the full length cloning of two vtg encoding proteins, namely vtgA and vtgB, which are differently modulated by BPA. Cyp1a1 and EROD activity were significantly downregulated, confirming the ability of estrogenic compounds to inhibit the detoxification process. GST activity was unaffected by BPA contamination, while CAT activity was down regulated. These results collectively confirm the estrogenic effect of BPA and provide additional characterization of novel vtg genes in Sparus aurata.

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 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.

A high selective and sensitive liquid chromatography-tandem mass spectrometry method for quantization of BPA urinary levels in children.

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.

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.

Biodegradation of bisphenols with immobilized laccase or tyrosinase on polyacrylonitrile beads.

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 τ(50), i.e., the time to obtain the 50% of degradation, and the removal efficiency (RE(90)) 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.

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.

Apple juice clarification by immobilized pectolytic enzymes in packed or fluidized bed reactors.

The catalytic behavior of a mixture of pectic enzymes, covalently immobilized on different supports (glass microspheres, nylon 6/6 pellets, and PAN beads), was analyzed with a pectin aqueous solution that simulates apple juice. The following parameters were investigated: the rate constant at which pectin hydrolysis is conducted, the time (tau(50)) in which the reduction of 50% of the initial viscosity is reached, and the time (tau(comp,dep)) required to obtain complete depectinization. The best catalytic system was proven to be PAN beads, and their pH and temperature behavior were determined. The yields of two bed reactors, packed or fluidized, using the catalytic PAN beads, were compared to the circulation flow rate of real apple juice. The experimental conditions were as follows: pH 4.0, T = 50 degrees C, and beads volume = 20 cm(3). The initial pectin concentration was the one that was present in our apple juice sample. No differences were observed at low circulation rates, while at higher recirculation rates, the time required to obtain complete pectin hydrolysis into the fluidized reactor was found to be 0.25 times smaller than in the packed bed reactor: 131 min for the packed reactors and 41 min for the fluidized reactors.

A computational approach for detecting peptidases and their specific inhibitors at the genome level.

BACKGROUND: Peptidases are proteolytic enzymes responsible for fundamental cellular activities in all organisms. Apparently about 2-5% of the genes encode for peptidases, irrespectively of the organism source. The basic peptidase function is "protein digestion" and this can be potentially dangerous in living organisms when it is not strictly controlled by specific inhibitors. In genome annotation a basic question is to predict gene function. Here we describe a computational approach that can filter peptidases and their inhibitors out of a given proteome. Furthermore and as an added value to MEROPS, a specific database for peptidases already available in the public domain, our method can predict whether a pair of peptidase/inhibitor can interact, eventually listing all possible predicted ligands (peptidases and/or inhibitors). RESULTS: We show that by adopting a decision-tree approach the accuracy of PROSITE and HMMER in detecting separately the four major peptidase types (Serine, Aspartic, Cysteine and Metallo- Peptidase) and their inhibitors among a non redundant set of globular proteins can be improved by some percentage points with respect to that obtained with each method separately. More importantly, our method can then predict pairs of peptidases and interacting inhibitors, scoring a joint global accuracy of 99% with coverage for the positive cases (peptidase/inhibitor) close to 100% and a correlation coefficient of 0.91%. In this task the decision-tree approach outperforms the single methods. CONCLUSION: The decision-tree can reliably classify protein sequences as peptidases or inhibitors, belonging to a certain class, and can provide a comprehensive list of possible interacting pairs of peptidase/inhibitor. This information can help the design of experiments to detect interacting peptidase/inhibitor complexes and can speed up the selection of possible interacting candidates, without searching for them separately and manually combining the obtained results. A web server specifically developed for annotating peptidases and their inhibitors (HIPPIE) is available at http://gpcr.biocomp.unibo.it/cgi/predictors/hippie/pred_hippie.cgi.

Advantages of using non-isothermal bioreactors in agricultural waste water treatment by means of immobilized urease. Study on the influence of spacer length and immobilization method.

The behavior of three different catalytic membranes, obtained by immobilizing urease on nylon sheets chemically grafted with methyl methacrylate, was studied in a bioreactor operating under isothermal and non-isothermal conditions. Membrane activation was carried out by condensation or acyl azide reaction, and spacers of different lengths, such as hexamethylendiamine or hydrazine, were used. Under isothermal conditions, the activities of the catalytic membranes and soluble urease were characterized as a function of pH, temperature, and urea concentration. Both enzyme forms showed the same optimum pH, whereas the optimum temperature was lower for the immobilized enzymes. The spacer length appeared to determine broader pH- and temperature-activity profiles for the urease derivatives. The apparent K(m) values of the insoluble urease were dependent on membrane type and were higher than those of the soluble counterpart, thus indicating an affinity loss for urea. Under non-isothermal conditions, all membranes exhibited an increase of percentage activity proportional to the applied temperature difference and decreasing with the increase of urea concentrations. A decrease of the apparent K(m) was also observed. These results suggest that substrate diffusion limitations due to the immobilization process can be overcome in the presence of temperature gradients. In addition, the remarkable reduction of the production times supports the use of non-isothermal bioreactors for the treatment of urea-polluted waste waters.