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Craniofacial defects are one of the most frequent abnormalities at birth, but their experimental evaluation in animal models requires complex procedures. The aim of the present work is the comparison of different methodologies to identify dose- and stage-related craniofacial malformations in Xenopuslaevis assay (R-FETAX, where the full cartilage evaluation, including flat mount technique, is the gold standard for skeletal defect detection). Different methods (external morphological evaluation of fresh samples, deglutition test, whole mount cartilage evaluation and Meckel–palatoquadrate angle measurements) were applied. Triadimefon (FON) was selected as the causative molecule as it is known to induce craniofacial defects in different animal models, including the amphibian X.laevis.FON exposure (0–31.25 μM) was scheduled to cover the whole 6-day test (from gastrula to free swimming tadpole stage) or each crucial developmental phases: gastrula, neurula, early morphogenesis, late morphogenesis, tadpole. Dose-dependent effects (fusions among craniofacial cartilages) were evident for groups exposed during the morphogenetic periods (neurula, early morphogenesis, late morphogenesis); gastrula was insensitive to the tested concentrations, tadpole group showed malformations only at 31.25 μM. The overall NOAEL was set at 3.9 μM. Results were evaluated applying benchmark dose (BMD) approach. The comparison of relative potencies from different methods showed deglutition as the only assay comparable with the gold standard (cartilage full evaluation).In conclusion, we suggest deglutition test as a reliable method for a rapid screening of craniofacial abnormalities in the alternative model X.laevis. This is a rapid, inexpensive and vital test allowing to preserve samples for the application of further morphological or molecular investigations.

In compliance to animal welfare 3Rs principle there is a great demand for refined tests alternative to classical mammal teratogenicity tests. We propose a refined alternative amphibian method (R-FETAX) to evaluate chemical induced embryotoxicity. The human foetal valproate spectrum disorder (FVSD) characteristics are morphological defects (including cranio-facial, neural tube defects) and behavioural alterations due to valproate (VPA) exposure in pregnancy. Vertebrate assays to evaluate FVSD include classical and alternative mammal (implying adult sacrifice), and non-mammal developmental models (zebrafish, amphibians, chick). Among these latter only zebrafish assays report in the same test both morphological and behavioural examinations. Compared to zebrafish, the amphibian Xenopus laevis excels having a more comparable organ development and morphology to mammalian systems. We used X. laevis embryos exposed during developmental specific windows to VPA therapeutic concentrations. Different VPA effects were observed depending on the exposure window: concentration-related embryo-lethal and teratogenic effects (neural tube, facial, tail defects) were observed in groups exposed at the organogenetic phylotypic stages. Neurobehavioral deficits were described using a functional swimming test at the highest VPA concentration exposure during the phylotypic stages and at any concentration during neurocognitive competent stages. Malformations were compared to those obtained in a mammalian assay (the rat post-implantation whole embryo culture method, WEC), that we used in the past to evaluate VPA teratogenicity. R-FETAX and WEC data were modelled and their relative sensitivity was calculated. We suggest the amphibian R-FETAX as a refined windowed alternative test for the evaluation of chemicals inducing both morphological and behavioural anomalies, including VPA.

Three-dimensional (3D) structured organoids are the most advanced in vitro models for studying human health effects, but their application to evaluate the biological effects associated with microplastic exposure was neglected until now. Fibers from synthetic clothes and fabrics are a major source of airborne microplastics, and their release from dryer machines is poorly understood. We quantified and characterized the microplastic fibers (MPFs) released in the exhaust filter of a household dryer and tested their effects on airway organoids (1, 10, and 50 µg mL

The last decade has witnessed the identification of several families affected by hereditary non-syndromic hearing loss (NSHL) caused by mutations in the SMPX gene and the loss of function has been suggested as the underlying mechanism. In the attempt to confirm this hypothesis we generated an Smpx-deficient zebrafish model, pointing out its crucial role in proper inner ear development. Indeed, a marked decrease in the number of kinocilia together with structural alterations of the stereocilia and the kinocilium itself in the hair cells of the inner ear were observed. We also report the impairment of the mechanotransduction by the hair cells, making SMPX a potential key player in the construction of the machinery necessary for sound detection. This wealth of evidence provides the first possible explanation for hearing loss in SMPX-mutated patients. Additionally, we observed a clear muscular phenotype consisting of the defective organization and functioning of muscle fibers, strongly suggesting a potential role for the protein in the development of muscle fibers. This piece of evidence highlights the need for more in-depth analyses in search for possible correlations between SMPX mutations and muscular disorders in humans, thus potentially turning this non-syndromic hearing loss-associated gene into the genetic cause of dysfunctions characterized by more than one symptom, making SMPX a novel syndromic gene.

BackgroundThree-dimensional (3D) structured organoids are the most advanced in vitro models for studying human health effects, but they have been applied only once to evaluate the biological effects associated with microplastic exposure. Fibers from synthetic clothes and fabrics are a major source of airborne microplastics, and their release from dryer machines is still poorly understood.ObjectivesIn this study, we aimed to establish an in vitro organoid model of human lung epithelial cells to evaluate its suitability for studying the effects of airborne microplastic contamination on humans. Furthermore, we aimed to characterize the microplastic fibers (MPFs) released in the exhaust filter of a household dryer and to test their interactions and inflammatory effects on the established lung organoids.MethodsThe polyester fibers emitted from the drying of synthetic fabrics were collected. Morphological characterization of the fibers released into the air filter was performed by optical microscopy and scanning electron microscopy (SEM)/energy dispersive x-ray spectroscopy (EDS). The organoids were exposed to various MPF concentrations (1, 10, and 50 mg L−1) and analyzed by optical microscopy, SEM, and confocal microscopy. Gene expression analysis of lung-specific genes, inflammatory cytokines, and oxidative stress-related genes was achieved by quantitative reverse transcription–polymerase chain reaction (qRT-PCR).ResultsWe successfully cultured organoids with lung-specific genes. The presence of MPFs did not inhibit organoid growth, but polarized cell growth was observed along the fibers. Moreover, the MPFs did not cause inflammation or oxidative stress. Interestingly, the MPFs were coated with a cellular layer, resulting in the inclusion of fibers in the organoid.DiscussionThis work could have potential long-term implications regarding lung epithelial cells undergoing repair. This preliminary exposure study using human lung organoids could form the basis for further research regarding the toxicological assessment of emerging contaminants such as micro- or nanoplastics.

Polyethylene terephthalate (PET) is one of the main plastic polymers contaminating natural ecosystems. Although PET microplastics (PET-MPs) have been found in both aquatic and terrestrial ecosystems, the information concerning their potential toxicity towards terrestrial organisms is limited. The present study aimed at investigating the ingestion and the possible adverse effects induced by a 40-days exposure to irregular shaped PET-MPs toward the giant snail Achatina reticulata. Giant snails were exposed via the diet to two concentrations (1% and 10% w/w; i.e., g of PET-MPs/g of the administered food) of PET-MPs and their capability to ingest and egest PET-MPs was assessed together with an evaluation of their potential effects at biochemical and individual levels. Oxidative stress-related biomarkers (i.e., the amount of reactive oxygen species, the activity of antioxidant enzymes and lipid peroxidation) and DNA fragmentation were measured in the digestive gland isolated from snails as biochemical endpoints. Changes in growth trajectories, in terms of body weight and shell size, were considered as morphometric endpoints. Our results demonstrated that A. reticulata can efficiently ingest and egest PET-MPs. Whilst giant snails did not experience an oxidative stress condition, significant changes in their growth trajectories were observed, with PET-MPs-treated snails grew more and more quickly than the control group. Our results suggest that PET-MPs might represent a risk during early-life stages for terrestrial organisms.

The acute toxicity of three differently shaped carbon nanomaterials (CNMs) was studied on Daphnia magna, comparing the induced effects and looking for the toxic mechanisms. We used carbon nano-powder (CNP), with almost spherical primary particle morphology, multi-walled carbon nanotubes (CNTs), tubes of multi-graphitic sheets, and cubic-shaped carbon nanoparticles (CNCs), for which no ecotoxicological data are available so far. Daphnids were exposed to six suspensions (1, 2, 5, 10, 20 and 50 mg L−1) of each CNM, and then microscopically analyzed. Ultrastructural analyses evidenced cellular uptake of nanoparticle in CNP and CNT exposed groups, but not in samples exposed to CNCs. Despite this difference, very similar effects were observed in tissues exposed to the three used CNMs: empty spaces between cells, cell detachment from the basal lamina, many lamellar bodies and autophagy vacuoles. These pathological figures were qualitatively similar among the three groups, but they differed in frequency a...

Microplastics are practically ubiquitous and pose a serious survival challenge for many species. Most of the exposure experiments for determining the toxicological effects of microplastics were performed with a microplastic varying little in shape and size (often purchased microplastic beads), but few studies deal with non-homogeneous samples. We analyzed the effect on Xenopuslaevis larva on the early development of polyester fibers, PEFs, taken from a dryer machine in which 100% polyester fabrics were dried after washing. Three concentrations were tested. The results showed that the gastrointestinal tract, GIT, was the most affected system by PEFs which modified the normal shape of the intestine with an EC50 96 h value of 6.3 µg mL−1. Fibers were observed to press against the digestive epithelium, deforming the normal architecture of the gut, sometimes pushing deep into the epithelium until piercing it. Physical GIT occlusion was observed in a concentration-dependent manner. However, no other damages were registered. No mortality was observed, but PEF-exposed larvae showed a significant reduction in their mobility. The results of the present paper suggest that environmental samples with their heterogeneity may have adverse effects on X. laevis development.

Microplastic (MP) contamination represents a serious threat for marine organisms. Several lab studies demonstrated adverse effects induced by exposure to different MP polymers toward diverse marine species. However, the information regarding toxicity of polyethylene terephthalate (PET) MPs is largely unknown. The present study was aimed at investigating the adverse effects induced by 7-day exposure to two concentrations (0.125 or 12.5 µg/ml) micronized, irregular shaped and variable size PET microparticles (PET-MPs) toward Manila clam (

Plastic debris and microplastics (MPs) evolve into an urgent problem for environmental contamination. Until now, many sources have been identified and nearly all the environmental compartments are subject to MP contamination. Plastic pollution has become of concern as potential threat to human health, too, as MPs were also in food and drinking water. Sources of MP ingestion by humans have already been discussed in several studies [1, 2, 3]. Moreover, recent studies [4, 5, 6], attracted the attention of researchers and the general public as they confirmed the presence of MPs in bottled drinking water.

Microplastic (μPs) contamination represents a dramatic environmental problem threatening both aquatic and terrestrial organisms. Although several studies have highlighted the presence of μPs in aquatic environments, the information regarding their toxicity towards organisms is still scant. Moreover, most of the ecotoxicological studies of μPs have focused on marine organisms, largely neglecting the effects on freshwater species. The present study aimed at exploring the effects caused by 21-days exposure to three concentrations (0.125, 1.25 and 12.5 μg/mL) of two differently sized polystyrene microplastics (PμPs; 1 and 10 μm) to the Cladoceran Daphnia magna. The ingestion/egestion capability of daphnids (24 h) and adults, the changes in individual growth and behavior, in terms of changes in swimming activity, phototactic behavior and reproduction, were investigated. Both particles filled the digestive tract of daphnids and adults within 24 h of exposure at all the tested concentrations. Ingested PμPs remained in the digestive tract even after 96 h in a clean medium. For both particles, an overall increase in body size of adults was noted at the end of the exposure to the highest tested concentrations, accompanied by a significant increase in swimming activity, in terms of distance moved and swimming velocity, and by an alteration of the phototactic behavior. A significant increase in the mean number of offspring after the exposure to the highest PμPs concentrations of different size was recorded. Polystyrene μPs can affect behavioral traits of D. magna leading to potentially harmful consequences on population dynamics of this zooplanktonic species.

A growing number of studies have highlighted the contamination and the effects towards organisms of diverse microplastics (μPs) in the marine environment. Surprisingly, although the main sources of μPs for marine environments are inland surface waters, the information on the occurrence and the effects of μPs in freshwater ecosystems is still scant. Thus, the aim of the present work is to investigate the ingestion and possible adverse effects due to the exposure to polystyrene μPs (PSμPs; O = 3 μm) on tadpoles of the Amphibian Xenopus laevis. Larvae at the developmental stage 36, prior to mouth opening, were exposed under semi-static conditions to 0.125, 1.25, and 12.5 μg mL−1 of PSμPs, and allowed to develop until stage 46. At the end of the exposure, the digestive tract and the gills from exposed and control tadpoles were microscopically examined, as well as changes in body growth and swimming activity. PSμPs were observed in tadpoles’ digestive tract, but not in the gills, from each tested concentration. However, neither body growth nor swimming activity were affected by PSμPs exposure. Our results demonstrated that PSμPs can be ingested by tadpoles, but they did not alter X. laevis development and swimming behavior at least during early-life stages, also at high, unrealistic concentrations.

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.

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.

The ultrastructure of mature spermatozoa from three leech species, Haemadipsa zeylanica ( Moquin-Tandon, 1826 ) (Haemadipsidae), Theromyzon tessulatum ( Muller, 1774 ) and Placobdella costata (Fr. Muller, 1846 ) (Glossiphoniidae), was investigated by means of transmission electron microscopy. While the three species showed the general features of hirudinid spermatozoa, considerable variation was found in nearly all characters examined: the anterior acrosome varied in length and in shape; the nuclei also varied in size and show a very complicated shape which is maintained for the whole length in the two glossiphonids, while in the haemadipsid, it showed two different morphological regions. The single mitochondrion, which is surrounded by an electron-dense sheath, is straight in H and T. tessulatum, but twisted in P. costata. The flagellum in the three studied species is of the prominent central sheath type. The basal body is absent in the three species and the central apparatus of the flagellum was observed penetrating to the mitochondrion. By comparing the present data with those from “oligochaeta”, Branchiobdellida and Acanthobdellida, we found that only the presence of an anterior acrosome characterizes the true leeches and at present should be regarded as an autapomorphic character of Euhirudinea.

Plastic particle ingestion has become of concern as a possible threat to human health. Previous works have already explored the presence of microplastic (MP) in bottled drinking water as a source of MP intake. Here, we consider the release of MP particles from single-use PET mineral water bottles upon exposure to mechanical stress utilizing SEM plus EDS, which allows the implementation of morphological and elemental analysis of the plastic material surface and quantification of particle concentrations in sample water. The aim of this study was to better evaluate the sources of MP intake from plastic bottles, especially considering the effect of daily use on these bottles such as the abrasion of the plastic material. For that, we analysed MP release of PET bottlenecks and HDPE caps on their surfaces after a series of bottle openings/closings (1 x, 10 x, 100 x). Furthermore, we investigated, if the inner surface of the PET bottles released MPs, counted particle increase of the water and identified MPs in the PET bottled water after exposing the bottles to mechanical stress (squeezing treatment; none, 1 min, 10 min). The results showed a considerable increase of MP particle occurrence on the surface of PET and HDPE material (bottlenecks and caps) after opening and closing the bottles. After 100 times the effect was impressive, especially on caps. Moreover, great differences exist in cap abrasion between brands which uncovers a discrepancy in plastic behavior of brands. Interestingly, particle concentrations in the bottled mineral water did not significantly increase after exposure to mechanical stress (squeezing treatment). The morphological analysis of the inner wall surface of the bottles supported this observation, as no stress cracks could be detected after the treatment, implying that the bottles itself are not a consistent source of MP particles after this extent of mechanical stress. However, chances of MP ingestion by humans increase with frequent use of the same single-use plastic bottle, though only from the bottleneck-cap system.

The chronic toxicity of ZnSO4 and ZnO nanoparticles has been studied in Daphnia magna also considering the life cycle parameters beyond the standard 21-day exposure time. Specimens have been individually followed until the natural end of their life, and some of them sampled for microscopic analyses at 48 h, 9 and 21 days. Despite the low level of exposure (0.3 mg Zn/L), ultrastructural analyses of the midgut epithelial cells revealed efficient internalization of nanoparticles between 48 h and 9 d, and translocation to other tissues as well. At 21 d, the most affected fields have been recorded for both compounds; in particular samples exposed to ZnO nanoparticles showed swelling of mitochondria, while those exposed to ZnSO4 had a great number of autophagy vacuoles. The life cycle parameters resulted altered as well, with a significant inhibition of reproduction in both groups, when compared to controls. After the 21-day exposure, some interesting results were obtained: animals, previously exposed to nanoZnO at low concentrations, showed a complete recovery of the full reproduction potential, while those previously exposed to ZnSO4 presented a dose-dependent and compound-specific reduction in lifespan. Based on the results from the present research and the effects of the same chemicals at higher doses, it can be concluded that the soluble form plays a key role in ZnO nanoparticle cytotoxicity, and that the nanoparticulate form is able to locally increase the amount of Zn inside the cell, even within the ovary. It's worth noting that ZnO nanoparticles have been internalized despite the very low concentration used: this raises concern about the possible environmental implications which may derive from their use, and which in turn must be carefully considered.

The chronic toxicity of ZnSO

Lethal and teratogenic potentials of carbon nanoparticles (CNPs) in their amorphous form were investigated by the standardized Frog Embryo Teratogenesis Assay-Xenopus (FETAX), a 96-h in vitro whole-embryo toxicity test based on the amphibian Xenopus laevis. Embryos were acutely exposed to 1, 10, 100 and 500 mg/L CNP suspensions and evaluated for lethality, malformations and growth inhibition. Larvae were processed for histological and ultrastructural analyses to detect the main affected organs, to look for specific lesions at the subcellular level and to image and track CNPs into tissues. Only the highest CNP suspension resulted in being embryolethal for X. laevis larvae, while malformed larva percentages significantly differed from controls starting from 100 mg/L. The stomach and gut were the preferential CNP accumulation sites, on the contrary, the digestive epithelium remained intact. The analyses showed the presence of isolated nanoparticles and/or aggregates in different secondary target organs. CNPs were found in circulating erythrocytes. The research confirms the good tolerance of X. laevis towards pure elemental carbon in its nanoparticulate amorphous form, but highlights the possibility of CNP transfer toward all body areas.

The aim of the present work is the assessment of teratogenic effects of three triazole-derived fungicides (Triadimefon, FON, Tebuconazole, TEBU, Cyproconazole, CYPRO) on rat and Xenopus laevis embryos cultured in vitro. Rat embryos, exposed to FON 31.25–250 μM, CYPRO 31.25–62.5 μM and to TEBU 62.5–250 μM, showed specific malformations (fusions) at the level of the first and second branchial arches, with a concentration-dependent increase of severity of malformative pictures. After immunostaining, the ectomesenchyme has been identified as the target tissue. X. laevis larvae showed, at the same concentrations, specific malformations at the level of cartilaginous element derived from the first and second branchial arch ectomesenchyme. This work indicates the three tested triazoles as teratogenic both in rodents and in amphibian, inducing ectomesenchymal abnormalities, and suggests, at least for this class of molecules, the X. laevis method as adequate alternative model for teratogenic screening.

BACKGROUND: The use of nonmammal models in teratological studies is a matter of debate and seems to be justified if the embryotoxic mechanism involves conserved processes. Published data on mammals and Xenopus laevis suggest that azoles are teratogenic by altering the endogenous concentration of retinoic acid (RA). The expression of some genes (Shh, Ptch-1, Gsc, and Msx2) controlled by retinoic acid is downregulated in rat embryos exposed at the phylotypic stage to the triazole triadimefon (FON). In order to propose X. laevis as a model for gene-based comparative teratology, this work evaluates the expression of Shh, Ptch-1, Gsc, and Msx2 in FON-exposed X. laevis embryos. METHODS: Embryos, exposed to a high concentration level (500 µM) of FON from stage 13 till 17, were examined at stages 17, 27, and 47. Stage 17 and 27 embryos were processed to perform quantitative RT-PCR. RESULTS: The developmental rate was never affected by FON at any considered stage. FON-exposed stage 47 larvae showed the typical craniofacial malformations. A significant downregulation of Gsc was observed in FON-exposed stage 17 embryos. Shh, Ptch-1, Msx2 showed a high fluctuation of expression both in control and in FON-exposed samples both at stages 17 and 27. CONCLUSION: The downregulation of Gsc mimics the effects of FON on rat embryos, showing for this gene a common effect of FON in the two vertebrate classes. The high fluctuation observed in the gene expression of the other genes, however, suggests that X. laevis at this stage has limited utility for gene-based comparative teratology. Birth Defects Res (Part B) 92:189–194, 2011. © 2011 Wiley-Liss, Inc.

The dysmorphogenic activity of the triazole fungicide triadimefon (FON) has been evaluated using Xenopus laevis development. Embryos, exposed to 500 μM FON during early neurulation phases (stages 13-17, Nieuwkoop and Faber), were allowed to develop until stage 47. Larvae revealed serious craniofacial defects, bent forebrain, and abnormal hindbrain segmentation. CRABP and CYP26 (markers related to retinoic acid homeostasis) gene and protein expression and protein distribution have been evaluated at stage 17 and at the end of the branchial arch morphogenesis (stage 27) by real-time PCR, western blot and whole-mount immunostaining. A significant increase of CYP26 transcript has been observed at both embryonic stages. A co-localization of the two markers has been observed at the cephalic region. Embryos exposed to FON showed abnormal distribution of positive tissues. Due to the strict similarity of these results with those previously described in rodents, a FON-related alteration of mechanism conserved during vertebrate evolution is suggested.

Summary In order to investigate the relationships between Italian wild boar and major pig breeds, we studied the genetic variability of four wild boar populations in Italy (Arezzo, Pisa, Parma, Bergamo) using a 533-bp fragment of the mitochondrial control region. Sixty-nine wild boar samples were analysed, allowing the identification of 10 distinct haplotypes, which involve a total of 15 single nucleotide polymorphisms. Phylogenetic and network analyses were performed also considering several sequences of wild and domesticated forms available in the databases. The Bayesian phylogenetic tree and the Median-Joining network analyses show three main groups: the Italian (IT), European (EU) and Asian (AS) clades. The IT clade corresponds to the Maremma endemic wild boar population and also includes Sardinian individuals, while the EU and AS groups include wild boars as well as domestic pig breeds. Only two individuals from Pisa cluster in the IT group, whereas two haplotypes from Bergamo cluster in the AS group and all other samples cluster in the EU clade. These findings suggest that in Italy wild boar populations have a mixed origin, both EU and AS, and that an interbreeding between wild and domesticated strains has probably occurred. Eight of the 10 wild boars coming from the Migliarino-San RossoreMassaciuccoli Regional Park (Pisa) belong to H2 and H3 haplotypes, and cluster into the EU clade, suggesting that this regional park is not anymore exclusive of the endemic Maremma wild boar.

Embryotoxic effects of Carbaryl (CB), a widely used carbamate insecticide, was evaluated by modified Frog Embryo Teratogenesis Assay-Xenopus (FETAX), coupled with a histopathological screening of the survived larvae. X. laevis embryos were exposed to 1, 2, 4, 8, 16 and 24 mg/L CB from stage 8 to stage 47. From an estimated LC50 of 20.28 mg/L and TC50 of 8.43 mg/L a TI of 2.41 was derived, indicating that CB is to be considered teratogenic for X. laevis embryos. The most characteristic terata, classified as abnormal tail flexure, involved a significant percentage of larvae from 1 mg/L CB onward, reaching 100% at 24 mg/L CB. Histopathological screening revealed tail musculature and notochord as the main targets for CB. Skeletal muscle lesions consisted of myotomes reduced in size, showing myocytes with disorganized contractile systems and irregular myosepta, coupled with disarranged myocyte apexes. Notochords from CB exposed larvae appeared wavy or bent, with irregular connective sheaths and histologically characterized by protrusions of fibrous matrix and inclusions of ectopic cell masses. This axial–skeletal damage was hypothesized to be related both to the inhibition of acetylcholinesterase, with consequent muscular tetanic spasms, and to disorders in the organization of the connective tissue matrix surrounding the notochord.

The role of soluble zinc has been determined in Daphnia magna by a morphological approach, integrating a previous paper in which the ultrastructural damages to gut epithelial cells have been studied after ZnO nanoparticles exposure. In the present paper, the toxicity and morphological effects of soluble zinc from ZnSO4 have been determined in a 48-h acute exposure test. Daphnids have been exposed to six nominal zinc concentrations (0.075, 0.15, 0.3, 0.6, 1.2, and 2.4mg Zn/L) and then fixed for microscopic analyses. Data from the acute toxicity tests gave an EC50 value of 0.99mg/L and showed that no immobilization appeared up to 0.3mg Zn/L. Ultrastructural analyses of samples from the two highest concentrations showed large vacuolar structures, swelling of mitochondria, multilamellar bodies, and a great number of autophagy vacuoles. These findings have been compared to those from our previous study, and similarities and/or differences discussed. Based on the overall results it can be concluded that dissolved zinc ions played a key role in ZnO nanoparticle toxicity and that the morphological approach is an extremely useful tool for comparing toxicological effects as well. A possible common toxic mechanism of soluble zinc and ZnO nanoparticles was also proposed.

The zebra mussel Dreissena polymorpha is widely present in Italy, but Italian populations of this bivalve have never been genetically characterized. The mitochondrial Cytochrome Oxidase subunit I gene fragments have been used to determine the genetic structures of 3 populations from Lake Garda (n = 30), the first Italian basin colonized by this species, as well as 2 populations from Lake Constance, Germany (n = 14). A Neighbor-Joining tree and a Maximum Likelihood analysis were performed with 4 new Italian (LG1-LG4) and 3 German (LC1-LC3) haplotypes, also considering all the available Dreissena sequences from GenBank in order to estimate phylogeographic relationships. Almost all Italian haplotypes clustered with German and mid-European samples providing evidences of a mixed origin for Italian mussels. We speculate about Lake Garda colonization, for which the hypothesized "German origin" has been partially confirmed as well as the necessity of more efficient control strategies at least to minimize new colonizations.

Tire debris (TD) and its organic components were identified as a main source of PM10 atmospheric and water pollution. Because few data are available on the embryotoxic effects of TD organic components, the lethal and teratogenic potential of tire debris organic extract (TDOE) was evaluated using the frog embryo teratogenesis assay-Xenopus (FETAX), coupled with a histopathological screening of the survived larvae. From stage 8 to stage 47, Xenopus laevis embryos were exposed to TDOE at concentrations of 50, 80, 100, 120 and 140 mg/L. The results showed 50 mg/L TDOE to be the non-observable effect concentration (NOEC). TDOE mortality at 80 mg/L was significantly higher than the control, but did not increase further with higher concentrations. A good concentration-response was observed for percentages of malformed larva and from 80 mg/L on these percentages were significantly higher than the control. Therefore, probit analysis gave a 144.6 mg/L TC50. At 120 and 140 mg/L, many larvae were plurimalformed. The most frequent alterations observed were abnormal gut coiling, microphthalmia, monolateral anophthalmia, and narrowing eyes. The histological screening mainly revealed ocular malformations such as double retina, retina nervous cell layer coiling, and altered lens. Moreover severe vacuolisation and necrosis were scored in liver and axial musculature. These results strongly support the assumption that TDOE is a powerful teratogen for X. laevis. Keywords: Tire debris organic extract, FETAX, Histology, Teratogenicity

The carpal organ is a glandular complex implicated in scent communication. To investigate its ethological significance, morpho‐functional evaluations were conducted on 67 glands collected from animals of different ages and sex during the 2002 hunting season (October–December) in the Bergamo district, Northern Italy. All samples were histologically and morphometrically examined to determine gland surface and thickness, number of secretory tubules per area and outer diameter of the tubules. Comparative analyses revealed significant differences in the above parameters between sub‐adults and adults for both sexes, but no differences were found between adult males and females. However, when all specimens were pooled, males revealed more developed and functional glands than females, with the oldest males having the highest values of gland surface, gland thickness and tubule diameter. Our findings suggest that this organ plays a relevant role in social communication by producing chemical signals involved in terr...

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.

BACKGROUND: Since MYS is a microtubular poison with a reversible activity, Xenopus blastulae were exposed to MYS to verify the eventual drug-related developmental suspension and the reversibility of this effect. METHODS: Lethal and teratogenic effects of myoseverin (MYS) were evaluated using the FETAX. Embryos were exposed to different MYS concentrations from stage 8 to stage 47. RESULTS: Probit analysis gave 12.14 µM LC50 and 7.67 µM TC50 from which 1.58 T.I. is derived. Several malformations were observed such as facial abnormalities, abnormal tail flexure, heart ventricle chamber enlargement and external appendix. MYS led to an arrest of living embryo development. Before MYS removing, exposed blastulae showed the lack of mitotic spindles along with different nuclei alterations. Living embryos, moved in control solution, mainly died around the hatching showing severe malformations likely ascribable to the altered planes of newly occurring mitosis. CONCLUSION: In spite of the low T.I, MYS has to be considered a highly teratogenic compound. Birth Defects Res (Part B) 77, 2006. © 2006 Wiley-Liss, Inc.

BACKGROUND: As previously shown, Paraquat (PQ) treatments of Xenopus developing embryos mainly induce a characteristic developmental alteration we named “abnormal tail flexure.” PQ oxidative activity has been indicated as the cause of this malformation. Since PQ evokes reactive oxygen species (ROS), among which hydroxyl radicals (OH•), and H2O2 can be converted to (OH•) via Fenton reaction, we compared here the lethal and teratogenic potentials of both oxidants by using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX), in order to grasp eventual similarities in their teratogenic activity. METHODS:Xenopus embryos were exposed, from stage 8 to stage 47, at 368, 491, 612, and 735 µM H2O2 and 0.388 µM PQ. The probit analysis of H2O2 mortality and malformed larva percents gave a 598.82 µM Lethal Concentration 50% (LC50) and 536.04 µM Teratogenic Concentration 50% (TC50) from which a 1.11 Teratogenic Index (T.I.) has been calculated. This T.I. value should allow the classification of H2O2 as a non-teratogenic compound. RESULTS: A comparison of H2O2 mortality and malformed larva percents with those obtained from PQ exposure showed the higher embryotoxicity of PQ, but, markedly, both compounds mainly induced the “abnormal tail flexure.” Histological analysis of both H2O2 and PQ malformed embryo tails showed a similar distorted morphology of both somites and myocytes. Some of muscle cells were necrotic and affected by an apical enlargement as well as a detachment from the connective tissue of intersomitic boundaries. CONCLUSIONS: In our opinion, both of the tested chemicals likely weaken the mechanical bridge connecting the myocyte contractile apparatus to the extracellular matrix, therefore causing the detachment of some of tail myocytes from their connectival septum as well as their apical enlargement. This could lead to the unbalance of tail tensional forces and, in turn, to the appearance of the “abnormal tail flexure.” Birth Defects Research (Part B) 77:238–243, 2006. © 2006 Wiley-Liss, Inc.

The developmental toxicity of nanostructured materials, as well as their impact on the biological barriers, represents a crucial aspect to be assessed in a nanosafety policy framework. Nanosized metal oxides have been demonstrated to affect Xenopus laevis embryonic development, with nZnO specifically targeting the digestive system. To study the mechanisms of the nZnO-induced intestinal lesions, we tested two different nominally sized ZnO nanoparticles (NPs) at effective concentrations. Advanced microscopy techniques and molecular marker analyses were applied in order to describe the NP-epithelial cell interactions and the mechanisms driving NP toxicity and translocation through the intestinal barrier. We attributed the toxicity to NP-induced cell oxidative damage, the small-sized NPs being the more effective. This outcome is sustained by a marked increase in anti-oxidant genes' expression and high lipid peroxidation level in the enterocytes, where disarrangement of the cytoskeleton and cell junctions' integrity were evidenced. These events led to diffuse necrotic changes in the intestinal barrier, and trans- and paracellular NP permeation through the mucosa. The uptake routes, leading NPs to cross the intestinal barrier and reach secondary target tissues, have been documented. nZnOs embryotoxicity was confirmed to be crucially mediated by the NPs' reactivity rather than their dissolved ions. The ZnO NPs' ability to overwhelm the intestinal barrier must be taken into high consideration for a future design of safer ZnO NPs. © 2014 Informa UK, Ltd.

The teratogenic potential of commercially available copper oxide (CuO), titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles (NPs) was evaluated using the standardized FETAX test. After characterization of NP suspensions by TEM, DLS and AAS, histopathological screening and advanced confocal and energy-filtered electron microscopy techniques were used to characterize the induced lesions and to track NPs in tissues. Except for nCuO, which was found to be weakly embryolethal only at the highest concentration tested, the NPs did not cause mortality at concentrations up to 500 mg/L. However, they induced significant malformation rates, and the gut was observed to be the main target organ. CuO NPs exhibited the highest teratogenic potential, although no specific terata were observed. ZnO NPs caused the most severe lesions to the intestinal barrier, allowing NPs to reach the underlying tissues. TiO2 NPs showed mild embryotoxicity, and it is possible that this substance could be associated with hidden biological effects. Ions from dissolved nCuO contributed greatly to the observed embryotoxic effects, but those from nZnO did not, suggesting that their mechanisms of action may be different. © 2012 Informa UK, Ltd.

The teratogenic potential of commercially available copper oxide (CuO), titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles (NPs) was evaluated using the standardized FETAX test. After characterization of NP suspensions by TEM, DLS and AAS, histopathological screening and advanced confocal and energy-filtered electron microscopy techniques were used to characterize the induced lesions and to track NPs in tissues. Except for nCuO, which was found to be weakly embryolethal only at the highest concentration tested, the NPs did not cause mortality at concentrations up to 500 mg/L. However, they induced significant malformation rates, and the gut was observed to be the main target organ. CuO NPs exhibited the highest teratogenic potential, although no specific terata were observed. ZnO NPs caused the most severe lesions to the intestinal barrier, allowing NPs to reach the underlying tissues. TiO₂ NPs showed mild embryotoxicity, and it is possible that this substance could be associated with hidden biological effects. Ions from dissolved nCuO contributed greatly to the observed embryotoxic effects, but those from nZnO did not, suggesting that their mechanisms of action may be different.

The zebra mussel Dreissena polymorpha was used to follow the recently reported DDT pollution of Lake Iseo (N. Italy). Histopathological analyses were performed on mussels sampled from March 2005 to April 2006, when high DDT levels were found, and results were compared to those from mussels sampled in 2001/2002, before the pollution event. During the 2005/2006 reproductive season, the first male gamete release happened one month later than the onset of spawning in females who showed a high number of specimens with degenerating oocytes, despite a regular pattern of gametogenesis. These results indicated a disrupting action of DDT on the mechanisms involved in sperm release, and a disturbance in the gametogenic phases of the ovary. Pathological pictures in the digestive gland of many mussels from both 2001/2002 and 2005/2006 have also been observed, but DDT pollution is unable to explain the presence of pathological fields in mussels during 2001/2002, for which a previously reported contamination seems to be the main cause. A possible role of DDT in skewing the sex ratio towards a predominance of females was also discussed, considering the high number of females sampled in 2005/2006.

The oxidative agent paraquat induced tail abnormalities during Xenopus laevis development. Specimens exposed from blastula to the tadpole stage revealed pear-shaped myocytes and irregular intersomitic boundaries. The histological feature of the axial musculature was evaluated in embryos sampled at significant stages of the primary myogenesis. During the somitogenesis PQ-treated embryos showed normal appearing myotomes, but reduced PAS activity in the post-rotating myotomal cells, and myoblasts with slight vacuolations. Once etched from the vitelline envelope, embryos showed severely altered myoblasts with irregular cellular apexes, heavy sarcoplasmic vacuolations, pyknotic nuclei and disorganizing intersomitic boundaries. Myotomes with many necrotic myocytes containing disorganized contractile material and heavily malformed intersomitic boundaries characterized the late myogenic stages. Our results evidence the heaviest PQ histopathological effects to affect myogenesis of post-etched embryos, suggesting a possible linkage between the swimming activity and the oxidative damage to muscle tissue.

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 g/l), but both exhibited a powerful teratogenicity. The probit analysis of malformed larva percentages showed a TC50 of 161.54g/l for CPF, and a TC50 of 2394.01g/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 laevismyogenesis suggest a possible role played by OPs on induction of congenital muscular dystrophy. © 2004 Elsevier B.V. All rights reserved.

The DDT contamination of Lake Maggiore (Northern Italy) has been monitored since a serious pollution event occurred in 1996. To assess the environmental risk associated with this contamination, bioaccumulation data coupled with histopathological markers were evaluated on zebra mussel populations from two different contaminated sites from April 2001 to April 2002. Biomonitoring results showed high DDT pollution in 2001 because of a flood which transported DDTs still contained in the sediments of a polluted river to the lake. DDT concentrations reached values of 4–5 μg/g lipids, higher than those recorded in other industrialized countries but comparable to levels measured in developing ones. In the ovaries of the most highly polluted mussels, histological analyses showed a delay in oocyte maturation and a high incidence of pathological pictures mainly referable to oocyte degeneration and haemocytic infiltration. Moreover, despite the presence of mature sperms, in 2001 first male gamete release occurred about 2 months later than in females. These results indicated a neuroendocrine interference of DDT on Dreissena polymorpha reproduction and also showed that these invertebrates can be successfully used to evaluate ecological implications due to exposure to endocrine disruptors in freshwater environments.

Histological techniques were used to monitor Dreissena polymorpha reproductive biology during 2001 in Lake Maggiore (N. Italy). Considering the DDT pollution which occurred during the mid 1990s in this lake and the high contamination level registered in 2001, we focused on the course of spawning events in zebra mussels and possible modified reproductive behavior induced by this pollution. Specimens were collected from the lake at Baveno as the polluted station, and at Villa Taranto as the control site. At Villa Taranto, morphological analyses of gonadal changes showed a gamete developing and maturing period until April, spawning phases from May to August, the restart of gametogenesis in females and an inactive period in males, immediately after the end of spawnings. This pattern was perturbed at Baveno where the mussels showed a delay in oocyte maturation with a significant percentage of specimens showing oocyte degeneration. Moreover, the onset of gamete release seemed to shift from May to July in the males at Baveno. Our findings agreed with the well recognized endocrine disrupting properties of DDT, even though the physiological mechanisms involved must be studied further.

The freshwater snail Physa fontinalis was used as a bioindicator to study the effects of the herbicide Paraquat (PQ) in laboratory assays. The test solutions used, 0.125, 0.25, and 0.5 mg/l PQ, were in the range of the concentrations recommended for aquatic weed control. The study was carried out in two stages to determine the influence of PQ on the ovipository activity of Physa fontinalis, and the histological effects on these snails. Specimens exposed to PQ continued to be reproductively active, but the number of egg masses and eggs laid decreased significantly. Mortality was almost the same in all the experimental lots, but was significantly related to the production of egg masses only in the controls. The histological analysis showed a clear trend among PQ concentrations and degenerating oocytes, but no visible effects on the male sex-line were observed. By interfering with fertility, PQ has an action that may go well beyond its lethal effect on individuals, suggesting that this herbicide should be strictly regulated in weed control programmes. Moreover, since PQ was observed to interfere with the reproductive process, its endocrine disrupting action must not be excluded.

The high Paraquat (PQ, 1-1′-dimethyl-4,4′bipyridylium dichloride) embryotoxicity in Xenopus laevis has been shown to be due to its rapid reduction and instantaneous re-oxidation which produces a reactive oxygen species, ROS. Nevertheless, PQ did not show any effects before hatching, stage 32, which showed a resistance, in early X. laevis development, to oxidative damage. Moreover, in view of its genotoxic properties in several experimental models, we studied PQ in the X. laevis cleavage phase that, characterized by a series of rapid mitotic divisions, might be damaged by genotoxic compounds. Embryos were exposed to 20, 40, 60, and 80 mg/l PQ concentrations from stage 2 to stage 9, and then left to develop in control FETAX solution until stage 47. The 80 mg/l PQ concentration gave 19% embryo mortality at the end of the exposure time, and 16.7% larvae mortality at the end of the test; both values were statistically different from the control, 5 and 6.8% respectively. These results confirmed the high resistance in early X. laevis development to PQ oxidative damage. The malformed larva percentages in the PQ exposed groups were higher as regards the control value but did not show any concentration–response; the most frequent malformed larvae found were affected by abnormal tail flexure coupled with abnormal gut coiling. A further experiment was carried out using the same methodology, but exposing embryos only to the 80 mg/l PQ concentration. The surviving blastulae were embedded in Paraplast, then the slides were stained with 4′,6-diamidino-2-phenylindole (DAPI) and the nuclei were examined with a confocal microscope. This new preliminary procedure did not reveal any significant presence of micronucleated micromeres in PQ exposed blastulae with respect to the control. Nevertheless, the mechanism by which PQ induced abnormal tail flexure after cleavage exposure remained unknown. PQ seemed to pass through the jelly coats and vitelline membrane, but it expressed teratogenicity between the 2nd and 3rd day. PQ might be accumulated in the embryos during the exposure, and might express teratogenicity later, but it did not seem to induce genotoxicity during the cleavage phase of X. laevis even at very high concentrations.

Zebra mussel (Dreissena polymorpha) is commonly used as contaminant bioindicator in Europe and North America. We used the zebra mussel to follow DDT pollution trends from 1996 to 1997 in Pallanza bay, Lake Maggiore, near the inlet of the River Toce, after a DDT-manufacturing plant discharging residues into a tributary of the River Toce had closed down. DDT contamination fell off sharply outside the bay, but tissue concentrations of the parent compound and residues remained high in molluscs sampled within the bay a year later. Molluscs collected in Pallanza bay in June 1997 released gametes earlier than those sampled at a nearby reference station. Histological studies showed that a significant percentage of these specimens showed marked oocyte degeneration suggesting that DDTs have endocrine-disrupting effects in this species.