Review: Morphofunctional and biochemical markers of stress in sea urchin life stages exposed to engineered nanoparticles.

We describe the use of different life stages of the Mediterranean sea urchin Paracentrotus lividus for the assessment of the possible risk posed by nanoparticles (NPs) in the coastal water. A first screening for the presence of NPs in sea water may be obtained by checking their presence inside tissues of organisms taken from the wild. The ability of NPs to pass from gut to the coelomic fluid is demonstrated by accumulation in sea urchin coelomocytes; the toxicity on sperms can be measured by embryotoxicity markers after sperm exposure, whereas the transfer through the food chain can be observed by developmental anomalies in larvae fed with microalgae exposed to NPs. The most used spermiotoxicity and embryotoxicity tests are described, as well as the biochemical and histochemical analyses of cholinesterase (ChE) activities, which are used to verify toxicity parameters such as inflammation, neurotoxicity, and interference in cell-to-cell communication. Morphological markers of toxicity, in particular skeletal anomalies, are described and classified. In addition, NPs may impair viability of the immune cells of adult specimens. Molecular similarity between echinoderm and human immune cells is shown and discussed. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1552-1562, 2016.

Tricarboxylic acid cycle-sustained oxidative phosphorylation in isolated myelin vesicles.

The Central Nervous System (CNS) function was shown to be fueled exclusively by oxidative phosphorylation (OXPHOS). This is in line with the sensitivity of brain to hypoxia, but less with the scarcity of the mitochondria in CNS. Consistently with the ectopic expression of FoF1-ATP synthase and the electron transfer chain in myelin, we have reported data demonstrating that isolated myelin vesicles (IMV) conduct OXPHOS. It may suggest that myelin sheath could be a site for the whole aerobic degradation of glucose. In this paper, we assayed the functionality of glycolysis and of TCA cycle enzymes in IMV purified from bovine forebrain. We found the presence and activity of all of the glycolytic and TCA cycle enzymes, comparable to those in mitochondria-enriched fractions, in the same experimental conditions. IMV also contain consistent carbonic anhydrase activity. These data suggest that myelin may be a contributor in energy supply for the axon, performing an extra-mitochondrial aerobic OXPHOS. The vision of myelin as the site of aerobic metabolism may shed a new light on many demyelinating pathologies, that cause an a yet unresolved axonal degeneration and whose clinical onset coincides with myelin development completion.

Effects of the neurotoxic thionophosphate pesticide chlorpyrifos on differentiating alternative models.

Studies by researchers worldwide have revealed that, even in industrialised nations, people, infants and the aged in particular, are even more exposed to neurotoxic drugs as a consequence of the increased quantity of pesticide residues in food. This phenomenon, as underlined by The Worldwatch Institute (2006), is linked to the exponential increase in the use of these toxic compounds over the last 40 years, up from 0.49 kg per hectare in 1961 to 2 kg in 2004, with the result that these substances are found in the daily diet. Many studies have demonstrated how the assumption of pesticides in the neonatal period and early infancy can alter the development and function of the nervous, immune, endocrine and reproductive apparatuses. Moreover, the unequivocal relationship between brain tumours, infant leukemia and pesticides are well recognised. On the basis of the above information, the effects of the neurotoxic thionophosphate pesticide chlorpyrifos (CPF) have been tested, considering biomarkers of toxicity and toxicity endpoint, on the biological models Dictyostelium discoideum, Paracentrotus lividus, and NTera2 Cells, as they are compatible with the 3Rs strategy (Reduction, Replacement, and Refinement in animal experiments). Our results have revealed that developing organisms are particularly sensitive to the toxic effects of CPF.

The sea urchin, Paracentrotus lividus, as a model to investigate the onset of molecules immunologically related to the α-7 subunit of nicotinic receptors during embryonic and larval development.

Nicotinic acetylcholine receptors play a major role in the regulation of electrochemical synapses at neuromuscular junctions. During the early stages of Paracentrotus lividus development, the nicotinic receptor-like molecules are found and localized by use of the specific blocker, -bungarotoxin, and by α-7 subunit immunoreactivity. Both the methods identify and localize the nicotinic receptor-like molecules at the sites where active changes in ionic intracellular concentration take place. These are well known to lead either fertilization, sperm propulsion or co-ordinated ciliary movement. After neural differentiation, immunoreactivity for the α-7 subunit is localized mainly in ganglia, ectoderm ciliary bands and in the motile cells forming the gut wall. Both α-bungarotoxin binding sites and α-7 subunits are also localized at the cells linked to the skeletal rods, performing the small movements which drive the swimming direction in the water column. The localization of these molecules paves the way to a speculation on their function and possible role in neurogenesis as well as neurodegeneration.

Inactivation of Crotalus atrox venom hemorrhagic activity by direct current exposure using hens' egg assay.

The hemotoxic venoms of Viperidae and Crotalidae are responsible for most of the evenomations in the United States, West Africa, India, South-East Asia, New Guinea, and Latin America. We previously reported that a short exposure of Crotalus atrox venom to direct electric current (dc) from a low-voltage generator, in solution, causes consistent and irreversible inactivation of venom phospholipase A(2) and metalloproteases. Here we report by in vivo assay on chicken embryos at stage 18 of development according to Hamburger and Hamilton that the hemorrhagic activity of C. atrox venom is lost after exposure to dc (from low voltage). Venom was exposed to dc ranging between 0 and 1 mA. dc values above 0.7 mA abolished hemorrhage. Such in vivo data, showing that dc neutralizes C. atrox venom hemorrhagic activity suggest that a deeper knowledge is needed to understand the relationship among dc and biological matter.

Evidence for ectopic aerobic ATP production on C6 glioma cell plasma membrane.

Extracellular ATP plays a pivotal role as a signaling molecule in physiological and pathological conditions in the CNS. In several glioma cell lines, ATP is a positive factor for one or more characteristics important for the abnormal growth and survival of these cells. This work presents immunofluorescence and biochemical analyses suggesting that an aerobic metabolism, besides mitochondria, is located also on the plasma membrane of C6 glioma cells. An ATP synthesis coupled to oxygen consumption was measured in plasma membrane isolated from C6 cells, sensitive to common inhibitors of respiratory chain complexes, suggesting the involvement of a putative surface ATP synthase complex. Immunofluorescence imaging showed that Cytochrome c oxydase colocalized with WGA, a typical plasma membrane protein, on the plasma membrane of glioma cells. Cytochrome c oxydase staining pattern appeared punctuate, suggesting the intriguing possibility that the redox chains may be expressed in discrete sites on C6 glioma cell membrane. Data suggest that the whole respiratory chain is localized on C6 glioma cell surface. Moreover, when resveratrol, an ATP synthase inhibitor, was added to culture medium, a cytostatic effect was observed, suggesting a correlation among the ectopic ATP synthesis and the tumor growth. So, a potential direction for the design of new targets for future therapies may arise.

Characterization of Myelin Sheath F(o)F(1)-ATP synthase and its regulation by IF(1).

F(o)F(1)-ATP synthase is the nanomotor responsible for most of ATP synthesis in the cell. In physiological conditions, it carries out ATP synthesis thanks to a proton gradient generated by the respiratory chain in the inner mitochondrial membrane. We previously reported that isolated myelin vesicles (IMV) contain functional F(o)F(1)-ATP synthase and respiratory chain complexes and are able to conduct an aerobic metabolism, to support the axonal energy demand. In this study, by biochemical assay, Western Blot (WB) analysis and immunofluorescence microscopy, we characterized the IMV F(o)F(1)-ATP synthase. ATP synthase activity decreased in the presence of the specific inhibitors (olygomicin, DCCD, FCCP, valynomicin/nigericin) and respiratory chain inhibitors (antimycin A, KCN), suggesting a coupling of oxygen consumption and ATP synthesis. ATPase activity was inhibited in low pH conditions. WB and microscopy analyses of both IMV and optic nerves showed that the Inhibitor of F(1) (IF(1)), a small protein that binds the F(1) moiety in low pH when of oxygen supply is impaired, is expressed in myelin sheath. Data are discussed in terms of the role of IF(1) in the prevention of the reversal of ATP synthase in myelin sheath during central nervous system ischemic events. Overall, data are consistent with an energetic role of myelin sheath, and may shed light on the relationship among demyelination and axonal degeneration.

Expression pattern of mUBPy in the brain and sensory organs of mouse during embryonic development.

Mouse UBPy (mUBPy) belongs to the family of ubiquitin-specific processing proteases (UBPs). In this study we have investigated the expression of mUBPy in the brain and sensory organs of mouse at different embryonic stages (E9, E11, E13, E15, E17, E19) and during the postnatal stages P0, P1, P2, P4 and P5 using Western blot and immunohistochemistry. mUBPy-immunoreactive cell bodies first appeared at stage E11 in several brain regions, particularly in the walls surrounding the vesicles and the ventricles. Subsequently, at stage E13, new mUBPy-positive cells appeared in the corpus striatum, the caudate nucleus, the thalamus, the epithalamus, the hypothalamus and the pons. At E15 the mUBPy pattern was very similar to that observed at E13, whereas at stage E17 mUBPy-immunoreactivity significantly decreased and a high number of mUBPy-immunoreactive cells was found only to line the third ventricle and within the mantle layer of the fourth ventricle. At E19 and P0, no mUBPy-immunoreactive element was found in the brain. At the postnatal stages P2 and P5, mUBPy-positive cells were detected in all subdivisions of the brain, with high concentrations in several cortex regions. Double labeling with the mUBPy antiserum and antisera against specific cell markers showed that the enzyme is expressed both in neurons and astrocytes. Outside the brain, mUBPy was detected, from stage E11, in the eye, within the lens and the cornea, in the inner ear, at the level of the cochlear and vestibular systems and in the olfactory epithelium. The spatio-temporal expression of mUBPy suggests that the enzyme may be involved in neuroregulatory processes during embryogenesis.

Dose-dependent effects of chlorpyriphos, an organophosphate pesticide, on metamorphosis of the sea urchin, Paracentrotus lividus.

The effect of exposures to the insecticide chlorpyrifos on the larval stages of Paracentrotus lividus (Echinodermata, Euechinoidea) up to metamorphosis was investigated with the aim to identify novel risk biomarkers and a new promising model for toxicity tests. The planktonic sea urchin larvae have the ability to undergo a variable exploratory period, up to the choice of a suitable substrate for adult benthonic life. The juvenile bud (called rudiment) is built inside the larval body that, on environmental cues represented by a variety of signal molecules, is reabsorbed by apoptosis and releases the juvenile on the substrate. In this dialogue between larvae and environment, contaminants interfere with the signals reception, and may alter in dose-dependent way the correct regulation of environment-larva-rudiment interaction. Such interaction is shown by larval plasticity, i.e. the ability of the larva to change body proportions according to the environmental conditions. When exposed to low doses of chlorpyriphos (10(-7) to 10(-10) M) since 2-days after fertilization, the larvae showed altered size and shape, but all reached the metamorphosis at the same time as controls, and in the same percentage. Exposures to high concentrations such as 10(-4) to 10(-6) M since 2-days after fertilization did not allow larval growth and differentiation. Exposures at later stages caused reabsorption of larval structures within a few hours and precocious release of the immature rudiments, followed by death of the juveniles. Although the mechanism of chlorpyriphos toxicity in sea urchin larvae is still rather unclear, the measurable stress biomarkers can constitute the basis for new toxicity tests.

Evidence for aerobic metabolism in retinal rod outer segment disks.

The disks of the vertebrate retinal rod Outer Segment (OS), devoid of mitochondria, are the site of visual transduction, a very energy demanding process. In a previous proteomic study we reported the expression of the respiratory chain complexes I-IV and the oxidative phosphorylation Complex V (F(1)F(0)-ATP synthase) in disks. In the present study, the functional localization of these proteins in disks was investigated by biochemical analyses, oxymetry, membrane potential measurements, and confocal laser scanning microscopy. Disk preparations, isolated by Ficoll flotation, were characterized for purity. An oxygen consumption, stimulated by NADH and Succinate and reverted by rotenone, antimycin A and KCN was measured in disks, either in coupled or uncoupled conditions. Rhodamine-123 fluorescence quenching kinetics showed the existence of a proton potential difference across the disk membranes. Citrate synthase activity was assayed and found enriched in disks with respect to ROS. ATP synthesis by disks (0.7 micromol ATP/min/mg), sensitive to the common mitochondrial ATP synthase inhibitors, would largely account for the rod ATP need in the light. Overall, data indicate that an oxidative phosphorylation occurs in rod OS, which do not contain mitochondria, thank to the presence of ectopically located mitochondrial proteins. These findings may provide important new insight into energy production in outer segments via aerobic metabolism and additional information about protein components in OS disk membranes.

Evidence for aerobic ATP synthesis in isolated myelin vesicles.

Even though brain represents only 2-3% of the body weight, it consumes 20% of total body oxygen, and 25% of total body glucose. This sounds surprising, in that mitochondrial density in brain is low, while mitochondria are thought to be the sole site of aerobic energy supply. These data would suggest that structures other than mitochondria are involved in aerobic ATP production. Considering that a sustained aerobic metabolism needs a great surface extension and that the oxygen solubility is higher in neutral lipids, we have focused our attention on myelin sheath, the multilayered membrane produced by oligodendrocytes, hypothesizing it to be an ATP production site. Myelin has long been supposed to augment the speed of conduction, however, there is growing evidence that it exerts an as yet unexplained neuro-trophic role. In this work, by biochemical assays, Western Blot analysis, confocal laser microscopy, we present evidence that isolated myelin vesicles (IMV) are able to consume O(2) and produce ATP through the operation of a proton gradient across their membranes. Living optic nerve sections were exposed to MitoTracker, a classical mitochondrial dye, by a technique that we have developed and it was found that structures closely resembling nerve axons were stained. By immunohistochemistry we show that ATP synthase and myelin basic protein colocalize on both IMV and optic nerves. The complex of data suggests that myelin sheath may be the site of oxygen absorption and aerobic metabolism for the axons.

Pre-adipocytes commitment to neurogenesis 1: preliminary localisation of cholinergic molecules.

A great effort has recently been made to obtain human stem cells able to differentiate into cholinergic neurons, as a number of diseases are associated to the cholinergic neuron loss, degeneration or incorrect function (Alzheimer's disease and motor neuron disease). A stem cell population (i.e. pre-adipocytes) is present in the adipose stromal compartment. Pre-adipocytes, like the mesodermic derivative cells, retain high plasticity and potentiality to convert in vitro from one phenotype into many others, and they can be isolated from adult adipose tissue. Pre-adipocytes committed in vitro to neural differentiation were followed up to the acquisition of neural morphology. Acetylcholinesterase and choline acetyltransferase are expressed from the native cell stage, with different localisations and roles during neural commitment. Western blots show the beginning of a new synthesis of these enzymes at 4 weeks of culture of neurogenic pre-adipocytes, in parallel with neural morphology. The passage of the choline-acetyltransferase immunoreactivity from cytoplasmic to membrane localisation shows the possible onset of catalytic activity and the histochemical reaction confirms the activity of acetylcholinesterase. This explains the possibility of obtaining cholinergic-like phenotype from pre-adipocytes.

Sea urchin development: an alternative model for mechanistic understanding of neurodevelopment and neurotoxicity.

Echinoderm early developmental stages might supply a good tool for toxicity testing in different fields, ranging from environment to food contamination, and in full respect of the 3Rs objectives (reduction, refinement, and replacement of animal experiments) that will eventually lead to the replacement of high vertebrate animal testing in toxicology. Sea urchin is one of the few organismic models considered by the European Agency for Alternative models. Actually, sea urchin embryonic development has been studied for over a century, and the complex nets of intercellular communications leading to the different events are well known, as well the possibility for environmental molecules and their residuals to interfere with such communications, causing developmental anomalies. In particular, the main goal of toxicologists since several years has been to establish a correlation between the cell-to-cell communications occurring during different developmental events and the signals occurring during neurogenesis, with the aim to pursue a mechanistic understanding of these processes and their deviations caused by stressors from different sources.

Distribution of choline acetyltransferase immunoreactivity in the alimentary tract of the barnacle Balanus amphitrite (Cirripedia, Crustacea).

To date only peptidergic innervation has been described in the alimentary tract of barnacles. In the present work the presence and distribution of choline acetyltransferase (ChAT), the acetylcholine (ACh) synthesizing enzyme, was investigated by immunohistochemistry in the alimentary tract of the adult barnacle Balanus amphitrite. Numerous ChAT-immunoreactive (IR) cells and a net of ChAT-IR cytoplasmic processes were localized inside the epithelium of the posterior midgut, close to the basement membrane; no IR nerve endings were detected in the midgut longitudinal and circular muscle bundles. Epithelial neurons or endocrine cells in the gut epithelium have been described in some invertebrate species belonging to different taxa and their peptidergic features are reported in the literature. Our results point out the presence of neuroepithelial cells also in the gut epithelium of barnacles; moreover, for the first time, a cholinergic feature is suggested for this cell type. These data seem to indicate the involvement of ACh in the gut functions of barnacle and suggest that the barnacle alimentary tract is more complex than previously thought and requires further study.