Biocompatibility Analysis of Bio-Based and Synthetic Silica Nanoparticles during Early Zebrafish Development.

During the twenty-first century, engineered nanomaterials (ENMs) have attracted rising interest, globally revolutionizing all industrial sectors. The expanding world population and the implementation of new global policies are increasingly pushing society toward a bioeconomy, focused on fostering the adoption of bio-based nanomaterials that are functional, cost-effective, and potentially secure to be implied in different areas, the medical field included. This research was focused on silica nanoparticles (SiO2-NPs) of bio-based and synthetic origin. SiO2-NPs are composed of silicon dioxide, the most abundant compound on Earth. Due to their characteristics and biocompatibility, they are widely used in many applications, including the food industry, synthetic processes, medical diagnosis, and drug delivery. Using zebrafish embryos as in vivo models, we evaluated the effects of amorphous silica bio-based NPs from rice husk (SiO2-RHSK NPs) compared to commercial hydrophilic fumed silica NPs (SiO2-Aerosil200). We evaluated the outcomes of embryo exposure to both nanoparticles (NPs) at the histochemical and molecular levels to assess their safety profile, including developmental toxicity, neurotoxicity, and pro-inflammatory potential. The results showed differences between the two silica NPs, highlighting that bio-based SiO2-RHSK NPs do not significantly affect neutrophils, macrophages, or other innate immune system cells.

Characterization of microparticles derived from waste plastics and their bio-interaction with human lung A549 cells.

Microplastics (MPs) represent a worldwide emerging relevant concern toward human and environmental health due to their intentional or unintentional release. Human exposure to MPs by inhalation is predicted to be among the most hazardous. MPs include both engineered, or primary MPs, and secondary MPs, materials obtained by fragmentation from any plastic good. The major part of the environmental MPs is constituted by the second ones that are irregular in size, shape and composition. These features make the study of the biological impact of heterogenous MPs of extremely high relevance to better estimate the real toxicological hazards of these materials on human and environmental organisms. The smallest fractions of plastic granules, relying on the micron-sized scale, can be considered as the most abundant component of the environmental MPs, and for this reason, they are typically used to perform toxicity tests using in vitro systems representative of an inhalation exposure scenario. In the present work, MPs obtained from industrial treatment of waste plastics (wMPs < 50 µm) were investigated, and after the physico-chemical characterization, the cytotoxic, inflammatory and genotoxic responses, as well as the modality of wMPs interactions with alveolar lung cells, were determined. Obtained results indicated that, at high concentrations (100 µg/ml) and prolonged exposure time (48 h), wMPs affect biological responses by inducing inflammation and genotoxicity, as a result of the cell-wMP interactions, also including the uptake of the smaller particles.

Microplastics from miscellaneous plastic wastes: Physico-chemical characterization and impact on fish and amphibian development.

Microplastic pollution represents a global problem with negative impacts on aquatic environment and organisms' health. To date, most of the laboratory toxicological studies on microplastics (MPs) have made use of single commercial micro and nano-polymers, which do not reflect the heterogeneity of environmental MPs. To improve the relevance of the hazard assessment, micrometer-sized plastic particles of miscellaneous non-reusable waste plastics, with size <100 µm and <50 µm (waste microplastics, wMPs), were characterized by microscopic and spectroscopic techniques and tested on developing zebrafish and Xenopus laevis by FET and FETAX assays respectively. Moreover, the modalities of wMP interaction with the embryonic structures, as well as the histological lesions, were explored by light and electron microscopy. We have shown that wMPs had very heterogeneous shapes and sizes, were mainly composed of polyethylene and polypropylene and contained metal and organic impurities, as well as submicrometric particle fractions, features that resemble those of environmental occurring MPs. wMPs (0.1-100 mg/L) caused low rate of mortality and altered phenotypes in embryos, but established species-specific biointeractions. In zebrafish, wMPs by adhering to chorion were able to delay hatching in a size and concentration dependent manner. In Xenopus embryos, which open stomodeum earlier than zebrafish, wMPs were accumulated in intestinal tract, where produced mechanical stress and stimulated mucus overproduction, attesting an irritation response. Although wMP biointeractions did not interfere with morphogenesis processes, further studies are needed to understand the underlying mechanisms and long-term impact of these, or even smaller, wMPs.

Responsiveness of hepatic and cerebral cytochrome P450 in rat offspring prenatally and lactationally exposed to a reconstituted PCB mixture.

Perinatal polychlorinated biphenyl (PCB) exposures still remain a serious health concern because offspring receive PCB burden from mother during vulnerable processes of development. Since cytochrome P450 (CYP) represents a toxicological endpoint, in the present study, representing an extended investigation of a previous multitasked one, we explored the long-term responsiveness of CYP1A and CYP2B isoforms by Western blot analysis in liver and whole brain of lactating (PN12), weaning (PN21), and adult offspring (PN60) rats prenatally and lactationally exposed to a reconstituted PCB mixture (RM) of noncoplanar PCB138, 153, 180, and coplanar PCB126 congeners. We chose highly chlorinated PCBs instead of lower chlorinated one, because their recalcitrance to biotransformation makes easy their accumulation/persistence in tissues and breast milk. Dioxin-like congener PCB126 binding aryl hydrocarbon receptor (AHR) is responsible of many toxic effects. Pregnant Sprague-Dawley dams with high affinity AHR received subcutaneous injection of RM (10 mg/kg body weight) daily during gestation (days 15-19) and twice a week during breast-feeding. The results evidenced a transfer of PCBs to neonates through milk and a significant responsiveness of hepatic CYP in both mothers and offspring. In liver of exposed progeny, CYP isoforms exhibited a significant increment at PN12 (70% over control) and at PN21 (270% over control). Contrary to dams, in adult PCB offspring CYP levels showed a decline up to values similar to those of control. This transient developmental responsiveness of CYP isoforms in offspring liver reflects roughly the time course of hepatic PCB levels previously reported. Even if congeners were detected in brain, we failed in evidencing a responsiveness of CYP isoforms probably because of region-specific CYP expression in this organ. In conclusion, induction of offspring hepatic CYP is index of liver PCB burden, and despite the insensitivity of whole brain CYP we cannot exclude brain vulnerability toward PCB. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 856-866, 2014.

The use of a complex tetra-culture alveolar model to study the biological effects induced by gold nanoparticles with different physicochemical properties.

A substantial increase in engineered nanoparticles in consumer products has been observed, heightening human and environmental exposure. Inhalation represents the primary route of human exposure, necessitating a focus on lung toxicity studies. However, to avoid ethical concerns the use of in vitro models is an efficient alternative to in vivo models. This study utilized an in vitro human alveolar barrier model at air-liquid-interface with four cell lines, for evaluating the biological effects of different gold nanoparticles. Exposure to PEGylated gold nanospheres, nanorods, and nanostars did not significantly impact viability after 24 h, yet all AuNPs induced cytotoxicity in the form of membrane integrity impairment. Gold quantification revealed cellular uptake and transport. Transcriptomic analysis identified gene expression changes, particularly related to the enhancement of immune cells. Despite limited impact, distinct effects were observed, emphasizing the influence of nanoparticles physicochemical parameters while demonstrating the model's efficacy in investigating particle biological effects.

Exposure during embryonic development to Roundup® Power 2.0 affects lateralization, level of activity and growth, but not defensive behaviour of marsh frog tadpoles.

As glyphosate-based herbicides, sold under the commercial name Roundup®, represent the most used herbicides in the world, contamination of the freshwater environment by glyphosate has become a widespread issue. In Italy, glyphosate was detected in half of the surface waters monitoring sites and its concentrations were higher than environmental quality standards in 24.5% of them. It can last from days to months in water, leading to exposure for aquatic organisms and specifically to amphibians' larvae that develop in shallow water bodies with proven effects to development and behaviour. In this study, we tested the effects of a 96 h exposure during embryonic development of marsh frog's tadpoles to three ecologically relevant Roundup® Power 2.0 concentrations. As expected, given the low concentrations tested, no mortality was observed. Morphological measurements highlighted a reduction in the total length in tadpoles exposed to 7.6 mg a.e./L, while an increase was observed at lower concentrations of 0.7 and 3.1 mg a.e./L compared to control group. Tadpoles raised in 7.6 mg a.e./L also showed a smaller tail membrane than those raised in the control solution. Regarding behaviour, we tested tadpoles in two different sessions (Gosner stages 25 and 28/29) for lateralization, antipredator response and basal activity. Lower intensity of lateralization was detected in tadpoles raised at the highest Roundup® concentration in the first session of observation, while no significant difference among treatments was observed in the second one. In both sessions, effects of Roundup® Power 2.0 embryonic exposure on antipredator response, measured as the proportional change in activity after the injection of tadpole-fed predator (Anax imperator) cue, were not detected. Tadpoles exposed during embryonic development to Roundup® exhibited lower basal activity than the control group, with the strongest reduction for the 7.6 mg a.e./L treatment. Our results reinforce the concern of Roundup® contamination impact on amphibians.

Recovery ability of human adipose stem cells exposed to cobalt nanoparticles: outcome of dissolution.

Aim: To demonstrate that cobalt nanoparticles doses are safe for use in humans and to understand the consequences of the particulate effects, which may persist inside the cells. Materials & methods: Human adipose stem cells were used. We evaluated cell recovery by viability test, morphology and ultrastructure using electronic and optical microscopy, while gene expression was assessed utilizing real-time PCR. Results: After exposure, most stem cells recovered their normal function. Co3O4-nanoparticles remained inside the cell for the entirety of the considered time. A slight modification of gene expression was observed in the exposed cells. Conclusion: After exposure to 100 M cobalt nanoparticles, most cells returned to normal function. Nanoparticle toxicity was due to ions released by dissolution as well as from the nanoparticles themselves.

Iron nanoparticle bio-interactions evaluated in Xenopus laevis embryos, a model for studying the safety of ingested nanoparticles.

Iron nanoparticles (NPs) have been proposed as a tool in very different fields such as environmental remediation and biomedical applications, including food fortification against iron deficiency, even if there is still concern about their safety. Here, we propose Xenopus laevis embryos as a suitable model to investigate the toxicity and the bio-interactions at the intestinal barrier of Fe3O4 and zerovalent iron (ZVI) NPs compared to Fe(II) and (III) salts in the 5 to 100 mg Fe/L concentration range using the Frog Embryo Teratogenesis Assay in Xenopus (FETAX). Our results demonstrated that, at concentrations at which iron salts induce adverse effects, both iron NPs do not cause acute toxicity or teratogenicity even if they accumulate massively in the embryo gut. Prussian blue staining, confocal and electron microscopy allowed mapping of iron NPs in enterocytes, along the paracellular spaces and at the level of the basement membrane of a well-preserved intestinal epithelium. Furthermore, the high bioaccumulation factor and the increase in embryo length after exposure to iron NPs suggest greater iron intake, an essential element for organisms. Together, these results improve the knowledge on the safety of orally ingested iron NPs and their interaction with the intestinal barrier, useful for defining the potential risks associated with their use in food/feed fortification.

Chronic treatment with polychlorinated biphenyls (PCB) during pregnancy and lactation in the rat: Part 1: Effects on somatic growth, growth hormone-axis activity and bone mass in the offspring.

Polychlorinated biphenyls (PCBs) are pollutants detected in animal tissues and breast milk. The experiments described in the present paper were aimed at evaluating whether the four PCB congeners most abundant in animal tissues (PCB-138, -153, -180 and -126), administered since fetal life till weaning, can induce long-term alterations of GH-axis activity and bone mass in the adult rat. We measured PCB accumulation in rat brain and liver, somatic growth, pituitary GH expression and plasma hormone concentrations at different ages. Finally, we studied hypothalamic somatostatin expression and bone structure in adulthood, following long-term PCB exposure. Dams were treated during pregnancy from GD15 to GD19 and during breast-feeding. A constant reduction of the growth rate in both male and female offspring from weaning to adulthood was observed in exposed animals. Long-lasting alterations on hypothalamic-pituitary GH axis were indeed observed in PCB-exposed rats in adulthood: increased somatostatin expression in hypothalamic periventricular nucleus (both males and females) and lateral arcuate nucleus (males, only) and decreased GH mRNA levels in the pituitary of male rats. Plasma IGF-1 levels were higher in PCB-exposed male and female animals as compared with controls at weaning and tended to be higher at PN60. Plasma testosterone and thyroid hormone concentrations were not significantly affected by exposure to PCBs. In adulthood, PCBs caused a significant reduction of bone mineral content and cortical bone thickness of tibiae in male rat joint to increased width of the epiphyseal cartilage disk. In conclusion, the developmental exposure to the four selected PCB compounds used in the present study induced far-reaching effects in the adult offspring, the male rats appearing more sensitive than females.

A glyphosate micro-emulsion formulation displays teratogenicity in Xenopus laevis.

Glyphosate is the active ingredient in broad-spectrum herbicide formulations used in agriculture, domestic area and aquatic weed control worldwide. Its market is growing steadily concurrently with the cultivation of glyphosate-tolerant transgenic crops and emergence of weeds less sensitive to glyphosate. Ephemeral and lentic waters near to agricultural lands, representing favorite habitats for amphibian reproduction and early life-stage development, may thus be contaminated by glyphosate based herbicides (GBHs) residues. Previous studies on larval anuran species highlighted increased mortality and growth effects after exposure to different GBHs in comparison to glyphosate itself, mainly because of the surfactants such as polyethoxylated tallow amine present in the formulations. Nevertheless, these conclusions are not completely fulfilled when the early development, characterized by primary organogenesis events, is considered. In this study, we compare the embryotoxicity of Roundup® Power 2.0, a new GBH formulation currently authorized in Italy, with that of technical grade glyphosate using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). Our results evidenced that glyphosate was not embryolethal and only at the highest concentration (50 mg a.e./L) caused edemas. Conversely, Roundup® Power 2.0 exhibited a 96 h LC50 of 24.78 mg a.e./L and a 96 h EC50 of 7.8 mg a.e./L. A Teratogenic Index of 3.4 was derived, pointing out the high teratogenic potential of the Roundup® Power 2.0. Specific concentration-dependent abnormal phenotypes, such as craniofacial alterations, microphthalmia, narrow eyes and forebrain regionalization defects were evidenced by gross malformation screening and histopathological analysis. These phenotypes are coherent with those evidenced in Xenopus laevis embryos injected with glyphosate, allowing us to hypothesize that the teratogenicity observed for Roundup® Power 2.0 may be related to the improved efficacy in delivering glyphosate to cells, guaranteed by the specific surfactant formulation. In conclusion, the differences in GBH formulations should be carefully considered by the authorities, since sub-lethal and/or long-term effects (e.g. teratogenicity) can be significantly modulated by the active ingredient salt type and concentration of the adjuvants. Finally, the mechanistic toxicity of glyphosate and GBHs are worthy of further research.

Teratogenic hazard of BPEI-coated silver nanoparticles to Xenopus laevis.

Silver nanoparticles (AgNPs) are among the most exploited antimicrobial agents and are used in many consumer products. Size and surface reactivity are critical physico-chemical properties responsible for NPs toxicity, and surface coatings, often used to functionalize or stabilize AgNPs, can influence their toxic profile and biocompatibility. In the current study the developmental toxicity of (1) negatively charged citrate-coated AgNPs (Cit-AgNPs), (2) positively charged branched polyethylenimine-coated AgNPs (BPEI-AgNPs), and (3) Ag+ (from 0.0625 to 0.75 mg Ag/L) was investigated by the standard Frog Embryo Teratogenesis Assay - Xenopus (FETAX). In order to identify the most sensitive developmental phase, embryos were also exposed during different embryonic stages. Morphological and bio-physical studies were performed to characterize tissue lesions and NP uptake. The results suggest that Ag+ was strongly embryo-lethal. Contrary to Cit-AgNPs, the positively charged BPEI-AgNPs exert a concentration-dependent effect on lethality and malformations of embryos. The BPEI-AgNPs showed the highest teratogenic index (TI = 1.6), pointing out the role of functional coating in determining the developmental hazard. The highest susceptibility to BPEI-AgNPs was during early embryogenesis, when embryos are still enclosed in the fertilization envelope, and the post-stomodeum opening stages, when NPs ingestion occurs. In BPEI-AgNPs treated larvae, the histological examination revealed irregular intestinal diverticula coupled with edematous connective tissue. Small NPs aggregates are mapped throughout the intestinal mucosa and secondary target organs by two-photon excitation microscopy. We conclude that a teratogenic risk may be associated with BPEI-AgNPs exposure, but the modality of NP-tissue interactions and the teratogenic mechanism need further investigations to be better defined.

Exposure to the organophosphorus pesticide chlorpyrifos inhibits acetylcholinesterase activity and affects muscular integrity in Xenopus laevis larvae.

The effect of organophosphate pesticide chlorpyrifos (CPF) on acetylcholinesterase (AChE) activity and on skeletal muscle development in Xenopus laevis larvae was studied. To achieve our purpose embryos were exposed to 100, 250 and 3000 microg/l CPF concentrations from late blastula stage (8 h postfertilization, p.f.) to stage 47 (120 h p.f.) and the appearance of AChE activity was monitored every 24 h. Compared with control, CPF treated larvae showed a dose dependent AChE inhibition during the early stages (beginning from 24 h until 120 h p.f.) that are crucial for neuromuscular development. The amount of AChE activity that can still be measured in treated larvae at stage 47 relative to that of the control, ranged from 28% in CPFs 100 microg/l to 4% in CPFs 3000 microg/l. These low AChE activities were associated with muscular damages such as reduced myotome size and hypertrophies coupled with extensive vacuolated regions in myocytes. The occurrence of this tissue-specific injury was related to CPF concentrations and was most pronounced in CPFs 3000 microg/l which revealed a very severe AChE inhibition during the exposure. Since AChE is the major neurotransmitter of the neuromuscular system, this initial descriptive study will be an useful starting-point to ongoing and future subcellular/molecular studies that correlate the morphological damage with changes in AChE activity.

Do Nanoparticle Physico-Chemical Properties and Developmental Exposure Window Influence Nano ZnO Embryotoxicity in Xenopus laevis?

The growing global production of zinc oxide nanoparticles (ZnONPs) suggests a realistic increase in the environmental exposure to such a nanomaterial, making the knowledge of its biological reactivity and its safe-by-design synthesis mandatory. In this study, the embryotoxicity of ZnONPs (1-100 mg/L) specifically synthesized for industrial purposes with different sizes, shapes (round, rod) and surface coatings (PEG, PVP) was tested using the frog embryo teratogenesis assay-Xenopus (FETAX) to identify potential target tissues and the most sensitive developmental stages. The ZnONPs did not cause embryolethality, but induced a high incidence of malformations, in particular misfolded gut and abdominal edema. Smaller, round NPs were more effective than the bigger, rod ones, and PEGylation determined a reduction in embryotoxicity. Ingestion appeared to be the most relevant exposure route. Only the embryos exposed from the stomodeum opening showed anatomical and histological lesions to the intestine, mainly referable to a swelling of paracellular spaces among enterocytes. In conclusion, ZnONPs differing in shape and surface coating displayed similar toxicity in X. laevis embryos and shared the same target organ. Nevertheless, we cannot exclude that the physico-chemical characteristics may influence the severity of such effects. Further research efforts are mandatory to ensure the synthesis of safer nano-ZnO-containing products.

Toxicity Evaluation of a New Zn-Doped CuO Nanocomposite With Highly Effective Antibacterial Properties.

The increased resistances to conventional antibiotics determine a strong need for new antibacterials, and specific syntheses at the nanoscale promise to be helpful in this field. A novel Zinc-doped Copper oxide nanocomposite (nZn-CuO) has been recently sonochemically synthesized and successfully tested also against multi-drug resistant bacteria. After synthesis and characterization of the physicochemical properties, the new nZn-CuO is here evaluated by the Frog Embyo Teratogenesis Assay-Xenopus test for its toxicological potential and this compared with that of nCuO and nZnO synthesized under the same conditions. No lethal effects are observed, while malformations and growth retardation slightly increase after nZn-CuO exposure. Nevertheless, these effects are smaller than those of nZnO. NP uptake by embryo tissues increase significantly with increasing NP concentrations, while no significant accumulation and adverse effects are seen after exposure to soluble Cu(2+) and Zn(2+) at the concentrations dissolved from the NPs. Key oxidative response genes are upregulated by nZn-CuO, as well as by nCuO and nZnO, suggesting the common mechanism of action. Considering the enhanced biocidal activity shown by the nanocomposite, together with the results presented in this study, we can affirm that the doping of the metal oxide nanoparticles should be considered a useful tool to engineer a safer nano-antibacterial.

Differential modulation of cytochrome P-450 1A and P-glycoprotein expression by aryl hydrocarbon receptor agonists and thyroid hormone in Xenopus laevis liver and intestine.

Several defence mechanisms, such as cytochrome P450 1A (CYP1A) enzymes and P-glycoprotein (Pgp), may influence the intracellular concentration and consequently the toxicity of xenobiotics. The parallel expression of CYP1A and Pgp has been investigated in mammals and, to a lesser extent in fish, in search for evidence of co-ordinated responses to xenobiotic exposure. The aryl hydrocarbon receptor (AHR) agonists are well known CYP1A inducers but some of them resulted not to have a uniquely defined action on Pgp levels in mammalian and fish species. To the best of our knowledge, no detailed studies have been carried out so far on amphibians Xenopus laevis. For this reason, in this work, the time dependent responses of the hepatic CYP1A and Pgp, to the prototypical CYP1A inducers, benzo(a)pyrene (B(a)P), 3-methylcholanthrene (3MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in X. laevis have been assessed at the protein level and compared. The responsiveness of Xenopus intestinal Pgp to these compounds has also been analysed, as the epithelial cells lining the lumen of intestine represent another preferential site of Pgp expression. In addition, since the thyroid hormone has been demonstrated to down regulate the mdr gene during Xenopus development and in primary culture of Xenopus intestinal epithelial cells, the effects of 3,3',5-triiodo-L-thyronine (T(3)) on CYP1A and Pgp protein levels have been investigated in adult organisms. Western blot evidenced that a single injection of B(a)P (100 mg/kg), 3MC (20 mg/kg), and TCDD (3 microg/kg) elicited a statistically significant induction of hepatic CYP1A at all time points considered (72, 120 and 168 h) which decreased in time. The same trend of liver CYP1A induction was observed in T(3) treated Xenopus (15 microg/kg). Unlike CYP1A induction, the modulation of hepatic and intestinal Pgp expression exhibits an heterogeneous pattern. The basal levels of hepatic and intestinal Pgp were not statistically significant affected by treatments. In particular, the hepatic Pgp levels seem not to be induced by TCDD and T(3) at all times considered in comparison to control. For the first time the modulation of CYP1A and Pgp levels by B(a)P, 3MC and in particular by TCDD and T(3) in Xenopus has been demonstrated and the results herewith indicate that the two target defence mechanisms respond to AHR agonists in a dissimilar way in terms of proteins induction in Xenopus. Moreover, these data suggest additional experiments in order to clarify the complex mechanism, which adjusts the parallel expression of CYP1A and Pgp in Xenopus.

Comparative teratogenicity of chlorpyrifos and malathion on Xenopus laevis development.

The embryotoxic potential of chlorpyrifos (CPF) and malathion (MTN), two organophosphorus insecticides (OPs), was evaluated by modified Frog Embryo Teratogenesis Assay-Xenopus (FETAX). CPF and MTN were not embryolethal even at the highest concentration tested (6000 microg/l), but both exhibited a powerful teratogenicity. The probit analysis of malformed larva percentages showed a TC(50) of 161.54mug/l for CPF, and a TC(50) of 2394.01 microg/l for MTN. Therefore, CPF teratogenicity was about 15 times higher than MTN. Larvae of both exposed groups were mainly affected by ventral and/or lateral tail flexure coupled with abnormal gut coiling. Histopathological diagnosis displayed abnormal myotomes and myocytes with marked hypertrophies localized at the cell extremity, probably due to a break away of myofibril extremities at the intersomitic junction level. We speculate that this muscular damage was related to inhibition of acetylcholinesterase that showed a clear concentration-response in CPF and MTN exposed larvae. The teratogenic effects of these anti-cholinesterase compounds on Xenopus laevis myogenesis suggest a possible role played by OPs on induction of congenital muscular dystrophy.

Non-neuronal cell modulation relieves neuropathic pain: efficacy of the endogenous lipid palmitoylethanolamide.

We have previously shown that the endogenous lipid palmitoylethanolamide (PEA) induced relief of neuropathic pain through an action upon receptors located on the nociceptive pathway. Recently, it has been proposed that immune cells, in particular mast cells, and microglia, by releasing algogen mediators interact with neurons to alter pain sensitivity thereby contributing to the development and maintenance of chronic pain states. The aim of this work was to explore whether the anti-nociceptive properties of PEA might be accompanied by modulation of these non-neuronal cells. Mice were subjected to a chronic constriction injury model of neuropathic pain and treated with PEA. The data show that at the earlier (3 days) time-point after nerve injury there was a substantial recruitment of mast cells whose activation was not yet pronounced. In contrast, at the later time point (8 days) there was no further increase in mast cell number, but rather a marked activation of these cells. An up-regulation of activated microglia was found in the spinal cord of neuropathic pain mice. PEA delayed mast cell recruitment, protected mast cells against degranulation and abolished the nerve growth factor increase in sciatic nerve concomitantly preserving the nerve from degeneration, while reducing microglia activation in the spinal cord. These findings support the idea that non-neuronal cells may be a valuable pharmacological target to treat neuropathic pain since the current neuronal-direct drugs are still unsatisfactory. In this context PEA could represent an innovative molecule, combining a dual analgesic activity, both on neurons and on nonneuronal cells.

First evidences of the occurrence of polycyclic synthetic musk fragrances in surface water systems in Italy: spatial and temporal trends in the Molgora River (Lombardia Region, Northern Italy).

The polycyclic synthetic musks (PCMs) such as galaxolide (HHCB), tonalide (AHTN) and celestolide (ABDI) are important ingredients in fragrances for consumer products because of their typical musky scent. In EU, PCMs are classified as HPVC (High Production Volume Chemicals). Furthermore, it has been recognized that these substances are only partially degraded in domestic sewers. For both reasons these chemicals are considered ubiquitous contaminants of aquatic systems. Monitoring data are available for the Northern region of the EU, but it is not known whether they are also representative for the Southern EU countries. The lack of data upon the environmental exposure in Southern EU can be significant, since use patterns and volumes differ from country to country. This is particularly true for Italy that has the largest detergent consumption per capita in EU. Due to this, the objective of the present study was to investigate the occurrence of selected PCMs in the Molgora River (Lombardia region, Italy). To our knowledge it represents the first overview of PCM occurrence in the Italian water bodies. Water samples were collected seasonally in seven sampling stations located before and after the 3 sewage treatment plants present along the river, which serve about 300,000 inhabitants. The spatial and temporal profiles of contamination are described. A comparison of the results with existing monitoring data of other European regions indicated a significant higher level of PCM pollution of the Molgora River and the need to extend the monitoring campaigns to other Italian water bodies, in order to achieve a better knowledge of the levels of PCM contamination in this country.

The effects of accumulation of an environmentally relevant polychlorinated biphenyl mixture on cytochrome P450 and P-glycoprotein expressions in fetuses and pregnant rats.

The aim of this study was to improve knowledge about transplacental transfer of an environmentally relevant PCB mixture by evaluating congener levels in livers and brains of rat dams and fetuses after maternal exposure, and correlating them to the levels of CYP450 and P-glycoprotein, involved in biotransformation and xenobiotics export, respectively. Pregnant dams were injected daily from gestation day (GD) 15 to 19 with 10mgkg(-1) of a reconstituted mixture (RM) composed of PCB138, 153, 180 and 126. Our data indicate that at GD20 RM is partitioned among maternal tissues, and that fetuses are not excluded from this distribution, evidencing a placental transfer of PCBs. Considering the ratio of maternal and fetal PCB concentrations based on lipid-weight, the amounts of congeners were 7-fold lower in fetal livers than in maternal livers and 25-30-fold higher in fetal brains than in maternal ones. Moreover, in dams the congeners were able to induce hepatic CYP450 response (total CYP450, CYP1A and CYP2B), but failed to increase P-170 expression, while in fetuses the constitutive expression of CYP450 and P-170 was not induced by treatment. Pearson Product-Moment Correlation applied to treated group data suggests that PCB accumulation in fetal livers, but not in brains, depended principally on their mothers' intoxication pattern. On the whole, these results emphasize the maternal liver and the fetal brain as depot organs for PCB sequestration and their susceptibility towards PCB toxicological risk. Moreover they highlight the lack of a coordinated response between the investigated defence mechanism.

Effects of TCDD on spermatogenesis related factor-2 (SRF-2): gene expression in Xenopus.

2,3,7,8-Tetra-chlorodibenzo-p-dioxin (TCDD) is one of the most toxic dioxins belonging to the wide family of Endocrine Disruptors (EDs), environmental chemicals that adversely interfere with endocrine processes and upset normal function of some target systems. It has been hypothesized that EDs enter cellular cytosol, bind to the Aryl Hydrocarbon Receptor (AhR) and form a heterodimer with the AhR nuclear translocator; this complex binds xenobiotic responsive elements that drive activation of the so-called "Ah gene battery". Spermatogenesis Related Factor-2 (SRF-2) is one of the most recently cloned genes involved in germ cell division and differentiation, whose expression seems to be affected by treatment with TCDD. With the aim to try to clarify the underlying mechanism of TCDD and to investigate if SRF-2 gene represents a good biomarker for ED exposure, we used Xenopus laevis as an animal model, considered to be almost insensitive toward TCDD effects. In this study we reported the partial cloning of SRF-2 cDNA in X. laevis; we then evaluated the SRF-2 expression in embryos exposed to TCDD 0.62 microM by real-time PCR. We also analyzed SRF-2 expression in several adult control tissues and in testis after perilymphatic injection of a single dose of 10 microg/kg body weight. Although SRF-2 expression does not seem to be affected by the treatment, exposed embryos died within 15 days. In the light of these results, we can conclude that SRF-2 is not a good candidate in signalling EDs exposure and that the molecular mechanism for TCDD toxicity in Xenopus likely involves the AhR signalling cascade, as in other vertebrate species.