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Bisphenol A is a widely used compound found in large amount of consumer products. As concerns have been raised about its toxicological and public health effect, the use of alternatives to bisphenol A are now increasing. Bisphenol S is one of the analogues being used as a replacement for bisphenol A despite the fact that little is known about the effects of bisphenol S on living organisms. In this study, we investigated the potential endocrine and genotoxic effects of bisphenol A and bisphenol S in juvenile brown trout (Salmo trutta). The fish were exposed to the compounds for either 2 weeks or 8 weeks via sustained-release cholesterol implants containing doses of 2 mg/kg fish or 20 mg/kg fish of the substances. The effects on the thyroid hormone levels and the estrogenic disrupting marker vitellogenin were evaluated, along with the genotoxic markers micronucleated cells and erythrocyte nuclear abnormalities. An increase in plasma vitellogenin was observed in fish exposed to the high dose of bisphenol A for 2 weeks. At this experimental time the level of the thyroid hormone triiodothyronine (T3) in plasma was elevated after bisphenol S exposure at the high concentration, and paralleled by an increase of micronucleated cells. Moreover, bisphenol A induced an increase of micronuclei frequency in fish erythrocytes after the exposure at the lowest dose tested. Taken together the results indicate that both bisphenol A and its alternative bisphenol S cause endocrine disrupting and genotoxic effects in brown trout, although suggesting two different mechanisms of damage underlying bisphenol A and bisphenol S activity.

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.

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.

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.

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.

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.

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.

Novel bicomponent microfibrous mats containing targeted amount of reactive maleic anhydride groups were prepared by electrospinning of mixed solutions of poly(styrene- alt -maleic anhydride) and poly(styrene- co -maleic anhydride). Then, amino-functionalized P(St- alt -MA)/P(St- co -MA) mats were obtained by reaction with p -phenylenediamine. ATR-FTIR and XPS spectroscopy were used to characterize pristine and modified P(St- alt -MA)/P(St- co -MA) mats. On the next step, laccase from Trametes versicolor was covalently attached onto the modified mats; the average amount of immobilized enzyme was 40 ± 0.7 mg/g mat. The catalytic activity of the immobilized enzyme was studied in respect to bisphenol A (BPA) endocrine disruptor. The optimum activity of the immobilized enzyme was reached at maximum flow rate of 1.3 mL/s. After 90 min the BPA concentration was reduced by 60% and the catalytic activity of microfibrous mats remained stable for about 30 successive reuses. In addition, the relative activity of laccase immobilized on the microfibrous mats was displayed in a broader pH range as compared to that of the free one.

Endocrine disruptors are chemicals able to induce adverse effects into wildlife and humans owing to their ability of interfering with the endocrine system. Bisphenol A (BPA) has been chosen as model of endocrine disruptors. To reduce the BPA pollution in waters we proposed the employment of the process of thermodialysis. Two different catalytic membranes have been prepared by covalently immobilizing laccase (from Trametes versicolor ) by means of a diazotation process or tyrosinase (from mushroom) by condensation. The support was a nylon membrane. The bioremediation power of both catalytic membranes has been analysed under isothermal and non-isothermal conditions. The advantages in using non-isothermal bioreactors were discussed in terms of reduction of the bioremediation times.

Cardiopulmonary bypass induces a systemic inflammatory response (SIR), characterized by the activation of cellular and humoral elements, with concomitant release of neutrophil elastase and matrix-metallo proteinases. In the present study, the protease release during extracorporeal circulation in 28 patients undergoing cardiac surgical operations was monitored using casein zymography. A peak in protease activity was found in all patients at the end of cardiopulmonary bypass. Plasma samples of patients were allowed to interact with different traps obtained by immobilizing different protease inhibitors on specific carriers. α1-Antitpypsin, Bovine Pancreatic Trypsin Inhibitor, Elastatinal or Leupeptin were used as inhibitors and were covalently immobilized by diazotization or by condensation. A reduction in the proteolytic activity of the plasma samples was observed after interaction with the different traps. The most efficient traps, i.e. the ones displaying greatest power to inhibit protease activity, were those obtained by immobilizing Bovine Pancreatic Trypsin Inhibitor and Leupeptin. The biocompatibility of traps was also tested. Results show that protease activity in blood can be decreased by our protease traps.

Laccase from Trametes versicolor was immobilized by diazotization on a nylon membrane grafted with glycidil methacrylate, using phenylenediamine as spacer and coupling agent. The behavior of these enzyme derivatives was studied under isothermal and nonisothermal conditions by using syringic acid as substrate, in view of the employment of these membranes in processes of detoxification of vegetation waters from olive oil mills. The pH and temperature dependence of catalytic activity under isothermal conditions has shown that these membranes can be usefully employed under extreme pH and temperatures. When employed under nonisothermal conditions, the membranes exhibited an increase of catalytic activity linearly proportional to the applied transmembrane temperature difference. Percentage activity increases ranging from 62% to 18% were found in the range of syringic acid concentration from 0.02 to 0.8 mM, when a difference of 1 degrees C was applied across the catalytic membrane. Because the percentage activity increase is strictly related to the reduction of the production times, the technology of nonisothermal bioreactors has been demonstrated to be an useful tool also in the treatment of vegetation waters from olive oil mills.

Laccase from Rhus vernicifera was immobilised on a nylon membrane chemically grafted with glycidyl methacrylate (GMA). Hexamethylenediamine (HMDA) and glutaraldehyde (GLU) were used as spacer and bifunctional coupling agent, respectively. Quinol was used as substrate. To know how the immobilisation procedures affected the enzyme reaction rate the catalytic behaviour of soluble and insoluble laccase was studied under isothermal conditions as a function of pH, temperature and substrate concentration. From these studies, two main singularities emerged from the experimental data: (i) the narrower pH-activity profile of the insoluble enzyme in comparison to that of the soluble counterpart; (ii) the increase of the affinity of the immobilised enzyme for its substrate. The behaviour of the catalytic membrane was also studied in a non-isothermal bioreactor as a function of substrate concentration and size of the applied transmembrane temperature difference. It was found that, under non-isothermal conditions and keeping constant the average temperature of the bioreactor, the enzyme reaction rate linearly increases with the increase of the temperature difference. These results have been discussed in the frame of reference of the process of thermodialysis driving thermodiffusive transmembrane substrate fluxes, which add to the diffusive ones. The advantages of the catalytic process carried out under non-isothermal conditions have been thrown in relief through the evaluation of the reduction of the production times and of the percentage increases of the enzyme activity.

In view of the treatment of agricultural waste waters, urea polluted, a catalytic and hydrophobic membrane was constructed by immobilizing urease on a nylon sheet grafted with cyclohexyl methacrylate (CHMA). Hexamethylenediamine (HMDA) and glutaraldehyde (GA) were used as spacer and crosslinking agent, respectively. With reference to the soluble counterpart immobilized urease was found to exhibit (i) a shift of the optimum pH towards more acidic values; (ii) a shift of the optimum temperature towards higher temperatures; (iii) higher values of Km. The latter result indicated an apparent loss of affinity of immobilized urease towards urea. To recovery this affinity loss, the catalytic membranes were employed in a bioreactor operating under non-isothermal conditions. Under these conditions the catalytic membranes exhibited reaction rates higher and apparent Km smaller than those measured under comparable isothermal conditions. As a consequence, percentage increases of enzyme activity and reduction of the production times, proportional to the magnitude of the applied temperature difference were observed. Results have been discussed in the frame of reference of the process of thermodialysis. The technology of the non-isothermal bioreactors confirmed its usefulness also in the reduction of urea concentration in aqueous solutions.