Paride Mantecca

Axial-skeletal defects caused by Carbaryl in Xenopus laevis embryos.

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.

Toxic effects and ultrastructural damages to Daphnia magna of two differently sized ZnO nanoparticles: Does size matter?

The toxic effects of two differently sized ZnO nanopowders have been studied in Daphnia magna using advanced microscopy techniques. Five nanoZnO suspensions (0.1, 0.33, 1, 3.3 and 10 mg/L) were tested. The results of the 48-h acute toxicity tests performed with ZnO < 100 nm (bZnO) and ZnO < 50 nm (sZnO) showed slight effects, with EC₅₀ values of 3.1 and 1.9 mg/L for bZnO and sZnO, respectively. Specimens exposed to 1 and 3.3 mg/L have been microscopically analysed and nanoparticles (NPs) from both concentrations have been found into midgut cells: i) in the microvilli; ii) in endocytic vesicles near the upper cell surface; iii) in some endosomes, as well as in mitochondria, in multivesicular and multilamellar bodies; iv) into the enterocytes nuclei; v) free in the cytoplasm; vi) in the paracellular space between adjacent cells; vii) into the folded basal plasma membrane, and viii) in the gut muscolaris, suggesting that not only both nanoZnOs are able to interact with the plasmatic membrane of D. magna enterocytes, but also that they are capable to cross epithelial barriers. The ultrastructural changes increased with increasing concentrations and the worst morphological fields came from samples exposed to 3.3 mg/L of both nanoZnOs. Morphological effects were qualitatively similar between the two nanomaterials, but they appear to be much more frequent for sZnO NPs. Data from ICP-OES analyses demonstrated that the maximum Zn(++) concentration in our tested suspensions was 0.137 mg/L, which is well below the reported NOEC for the soluble Zinc. The corresponding Zn-salt exposures (0.1 mg/L Zn(++)) gave 0% of immobilized daphnids for both NPs suggesting that in our test medium nanoZnO toxicity is not driven by their solubilized ions. The large presence of NPs inside midgut cells after only 48-h exposure to nanoZnOs and their effects on the intestinal cells highlighted the toxic potential of these nanomaterials, also suggesting that studies on chronic effects are needed.

Evidence and uptake routes for Zinc oxide nanoparticles through the gastrointestinal barrier in Xenopus laevis

Abstract 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.

DDT polluted meltwater affects reproduction in the mussel Dreissena polymorpha

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.

Zebra mussels in Italy: where do they come from?

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 (nxa0=xa030), the first Italian basin colonized by this species, as well as 2 populations from Lake Constance, Germany (nxa0=xa014). 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.

Sexual maturity in young Dreissena polymorpha from Lake Como (N. Italy)

Summary The reproductive biology of the zebra mussel Dreissena polymorpha was studied during 1999 in a natural population living in Lake Como (N. Italy), with particular reference to the differences in behavior between young and adult mussels. Histological analysis of gonads was used to study the gametogenic cycles and to evaluate when the young reached sexual maturity. Signs of active gametogenesis were detected in ≥4 mm shell length mussels. Mature gametes and the first spawning events were observed in ≥5 mm males and in ≥6 mm females. The gonad feature of ≥7 mm mussels was similar to that of the adults. Size frequency distributions revealed that the new generation (0+) first spawned in the year following birth, but only after winter. The length of larval time seemed to vary greatly depending on the spawning period, in addition to the environmental conditions. The first settled mussels ≥500 μm in shell length were observed 3–4 months after the first depositions. Water temperature seemed to be the most important factor which regulated sexual phenomena of both young and adult mussels. Its role in the onset of the spawning events was confirmed, but it is important to point out that temperature is not enough to trigger reproduction since gamete release is impossible without sufficient energy, especially for young mussels.

Mitochondrial DNA sequence variations in some Italian wild boar populations.

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 Rossore-Massaciuccoli 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.

SPIO@SiO2–Re@PEG nanoparticles as magneto-optical dual probes and sensitizers for photodynamic therapy

A dual magneto-optical nanoprobe, endowed with properties useful for photodynamic therapy, has been prepared. It is constituted by a superparamagnetic iron oxide (SPIO) core (diameter size distribution centered at ca. 10 nm), obtained by thermal decomposition of iron oleate, coated by a compact silica shell, grown in a reverse microemulsion environment. Luminescent [Re(phen)(CO)3(py)]CF3SO3 complexes were covalently anchored to the silica shell, by functionalizing the pyridine ligand with a triethoxysilane moiety. Finally, the surface of the nanoparticles (NPs) was coated with a layer of polyethylene glycol (PEG), functionalized with triethoxysilane, to improve stability and stealthiness in physiological conditions. Transmission electron microscopy of these SPIO@SiO2–Re@PEG nanocomposites showed a single population, with size distribution centered at ca. 40 nm. NPs showed nuclear relaxivity values that guarantee an appreciable contrast in magnetic resonance imaging (r2 > 30 s−1 mM−1 at frequencies higher than 5 MHz). The presence of the Re complexes imparted photoluminescence to the NPs, with blue shifted emission and higher photoluminescence quantum yields with respect to the free [Re(phen)(CO)3(py-upts)]+ complex (λem 553 vs. 580 nm, Φ 0.060 vs. 0.038, in aerated water solution). The complexes embedded into the NPs maintained a satisfactory efficiency toward 1O2 generation (quantum yields 0.21 vs. 0.26 for the free complex, as assessed using 1,5-dihydroxynaphthalene as indirect marker of the 1O2 presence). Preliminary cell penetration tests were performed on human lung adenocarcinoma A549 cells. Two photon excitation confocal microscopy showed that the NPs were easily internalized and accumulated in the perinuclear region of the cells already after 4 h of incubation. Increased cytotoxicity upon irradiation with respect to the dark was also observed, showing the potential of the nanocomposite for photodynamic therapy applications.

The carpal gland in wild swine: Functional evaluations

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 territorial definition as well as reproduction.