Author Report for: Falugi C

The brine shrimp Artemia was used as a model organism to test toxicity of several neuroactive pesticides (chlorpyrifos (CLP), chlorpyrifos oxon (CLP ox), diazinon (DZN), carbaryl (CBR)) following exposure to far below than lethal doses. Cysts were exposed to the pesticides in order to test a scenario similar to actual coastal environment contamination, by analyzing different responses. Cysts were rehydrated in water containing the pesticides at concentrations ranging from 10(-11) to 10(-5)M, for 72, 96 and 192h, respectively. For these exposure times, morpho-functional and biochemical parameters, such as hatching speed and viability were investigated in the larvae together with cholinesterase (ChE) activity quantification and histochemical localization. Finally, ChE inhibition was also compared with conventional selective ChE inhibitors. Results showed that CLP ox and CBR caused a significant dose-dependent decrease in hatching speed, followed by high percentages of larval death, while CLP and DZN were responsible for irregular hatching patterns. In addition, the pesticides mostly caused larval death some days post-hatching, whereas this effect was negligible for the specific ChE inhibitors, suggesting that part of pesticide toxicity may be due to molecules other than the primary target. ChE activity was observed in the protocerebrum lobes, linked to the development of pair eyes. Such activity was inhibited in larvae exposed to all pesticides. When compared to conventional selective inhibitors of ChE activities, this inhibition demonstrated that the selected pesticides mainly affect acetylcholinesterase and, to a lesser extent, pseudocholinesterases. In conclusion, the brine shrimp is a good model to test the environmental toxicity of long term exposure to cholinergic pesticides, since changes in hatching speed, viability and ChE activity were observed.

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. (c) 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1552-1562, 2016.

The aim of this study was to investigate the potential toxicity of Silica nanoparticles (SiO2 NPs) in seawater by using the sea urchin Paracentrotus lividus as biological model. SiO2 NPs exposure effects were identified on the sperm of the sea urchin through a multidisciplinary approach, combining developmental biology, ecotoxicology, biochemistry, and microscopy analyses. The following responses were measured: (i) percentage of eggs fertilized by exposed sperm; (ii) percentage of anomalies and undeveloped embryos and larvae; (iii) enzyme activity alterations (acetylcholinesterase, AChE) in the early developmental stages, namely gastrula and pluteus. Sperms were exposed to seawater containing SiO2 NPs suspensions ranging from 0.0001mg/L to 50mg/L. Fertilization ability was not affected at any concentration, whereas a significant percentage of anomalies in the offspring were observed and quantified by means of EC50 at gastrula stage, including undeveloped and anomalous embryos (EC50=0.06mg/L), and at pluteus stage, including skeletal anomalies and delayed larvae (EC50=0.27mg/L). Moreover, morphological anomalies were observed in larvae at pluteus stage, by immunolocalizing molecules involved in larval development and neurotoxicity effects - such as acetylated tubulin and choline acetyltransferase (ChAT) - and measuring AChE activity. Exposure of sea urchins to SiO2 NPs caused neurotoxic damage and a decrease of AChE expression in a non-dose-dependent manner. In conclusion, through the multidisciplinary approach used in this study SiO2 NPs toxicity in sea urchin offspring could be assessed. Therefore, the measured responses are suitable for detecting embryo- and larval- toxicity induced by these NPs.

We investigated the effects of three different carbon-based nanomaterials on brine shrimp (Artemia salina) larvae. The larvae were exposed to different concentrations of carbon black, graphene oxide, and multiwall carbon nanotubes for 48h, and observed using phase contrast and scanning electron microscopy. Acute (mortality) and behavioural (swimming speed alteration) responses and cholinesterase, glutathione-S-transferase and catalase enzyme activities were evaluated. These nanomaterials were ingested and concentrated in the gut, and attached onto the body surface of the A. salina larvae. This attachment was responsible for concentration-dependent inhibition of larval swimming, and partly for alterations in the enzyme activities, that differed according to the type of tested nanomaterials. No lethal effects were observed up to 0.5mg/mL carbon black and 0.1mg/mL multiwall carbon nanotubes, while graphene oxide showed a threshold whereby it had no effects at 0.6mg/mL, and more than 90% mortality at 0.7mg/mL. Risk quotients calculated on the basis of predicted environmental concentrations indicate that carbon black and multiwall carbon nanotubes currently do not pose a serious risk to the marine environment, however if uncontrolled release of nanomaterials continues, this scenario can rapidly change.

We examined egg fertilisation in purple sea urchin (Paracentrotus lividus) after sperm exposure to carbon-based nanomaterials, carbon black (CB) and graphene oxide (GO), from 0.0001mg/L to 1.0mg/L. Gastrula stage embryos were investigated for acetylcholinesterase and propionylcholinesterase activities, and their morphological characteristics. Plutei were analysed for morphological abnormalities, with emphasis on skeletal rod formation. Egg fertilisation was significantly affected by CB, at all concentrations tested. Loss of cell adhesion at the gastrula surface was observed in eggs fertilised with sperm treated with CB. However, concentration-dependant morphological anomalies were observed in the gastrulae and plutei formed after sperm exposure to either CB or GO. The activities of both cholinesterases decreased in the gastrulae, although not in a concentration-dependent manner. These effects appear to arise from physical interactions between these carbon-based nanomaterials and the sperm, whereby nanomaterials attached to the sperm surface interfere with fertilisation, which leads to disturbances in the signalling pathways of early embryonic development. Reduced cholinesterase activity in gastrulae from eggs fertilised with nanomaterial-treated sperm confirms involvement of the cholinergic system in early sea urchin development, including skeletogenesis.

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 objective of this study is to examine the toxicity of engineered nanoparticles (NPs) that are dispersed in sea water by using an in vivo model. Because many products of nanotechnology contain NPs and are commonly used and well-established in the market, the accidental release of NPs into the air and water is quite possible. Indeed, at the end of their life cycle, some NPs are inevitably released into waste water and can reach marine ecosystem and affect the organisms there. Although there are few data on the presence of NPs in the marine environment, our awareness of their potential impact on environmental and organismal health is growing. Shallow-water benthonic organisms such as sea urchins provide planktonic larvae as a trophic base for finfish juveniles and are exposed to water from estuaries and precipitation. Such organisms can therefore be directly affected by NPs that are dispersed into those media. We evaluated the effects of exposure to different concentrations of nanosilver, titanium oxide and cobalt NPs on the sperm of the sea urchin Paracentrotus lividus by analyzing the functionality and the morphology and biochemistry of the first developmental stages of the sea urchin. Sperm were exposed to sea water containing suspensions of NPs ranging from 0.0001mg/L to 1mg/L. Fertilization ability was not affected, but developmental anomalies were identified in embryos from the gastrula to pluteus stages, including morphological alterations of the skeletal rods. In addition, the enzymatic activity (cholinesterase, ChE) of the larvae was measured. Acetylcholinesterase (AChE) and propionylcholinesterase activity (PrChE) was affected in all of the exposed samples. The results did not vary consistently with the concentration of NP, but controls were significantly different from exposed samples. Exposure of sea urchin to these NPs may cause neurotoxic damage, and the altered ChE activity may be involved in skeletogenic aberrations. In conclusion, the sea urchin represents a suitable and sensitive model for testing the toxicity and effects of engineered NPs that are dispersed in sea water.

Metal nanosolicoparticles are suspected to cause diseases in a number of organisms, including man. In this paper, we report the effects of nanosilver (Ag, 1-20 nm particles) on the early development of the zebrafish, a well-established vertebrate model. Embryos at the midgastrula stage were exposed to concentrations ranging from 100 to 0.001 mg/L to verify the effects on different endpoints: lethality, morphology, expression of cholinergic molecules, and development of the immune system. (1) Relative risk of mortality was exponential in the range between 0.001 and 10 mg/L. Exposure to 100 mg/L caused 100% death of embryos before reaching the tail-bud stage. (2) Developmental anomalies were present in the 72 h larvae obtained from embryos exposed to nanosilver: whole body length, decreased eye dimension, and slow response to solicitation by gentle touch with a needle tip, with a significant threshold at 0.1 mg/L. (3) Dose-dependent inhibition of acetylcholinesterase activity was significant among the exposures, except between 1 mg/L and 10 mg/L. (4) The distribution of CD41+ cells and of CDF/LIF-like immunoreactivity was altered according to the Ag concentration. The possible effect of nanosilver in impairing immune system differentiation through the inhibition of molecules related to the cholinergic system is discussed.

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 alpha-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 alpha-7 subunit is localized mainly in ganglia, ectoderm ciliary bands and in the motile cells forming the gut wall. Both alpha-bungarotoxin binding sites and alpha-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.

The aim of this study was to assess medium-term toxicity of weathered oil on European seabass. A mesocosm system reproducing an oil spill at sea was applied. Fish were collected after 48 h, 7, 30 and 60 days. Cyp1a gene transcription, EROD and UDPGT activities, bile PAHs metabolites and micronuclei frequency were investigated. A progressive disappearance of low molecular weight n-alkanes and PAHs in the water of the mesocosm occurred during the experimentation. Fishes exposed to oil displayed a significant increase of cyp1a expression and EROD activity during the entire experiment as well as higher concentrations of PAHs metabolites in bile. Micronulei frequency resulted significantly higher during all experiment in oil exposed sea bass compared to controls. The results highlight the environmental risk associated with the release of oil products at sea and confirm the adopted parameters as useful tools for studying the impact of accidental oil spills on fish.

The biological function of the cholinesterase (ChE) enzymes has been studied since the beginning of the twentieth century. Acetylcholinesterase plays a key role in the modulation of neuromuscular impulse transmission in vertebrates, while in invertebrates pseudo cholinesterases are preeminently represented. During the last 40 years, awareness of the role of ChEs role in regulating non-neuromuscular cell-to-cell interactions has been increasing such as the ones occurring during gamete interaction and embryonic development. Moreover, ChE activities are responsible for other relevant biological events, including regulation of the balance between cell proliferation and cell death, as well as the modulation of cell adhesion and cell migration. Understanding the mechanisms of the regulation of these events can help us foresee the possible impact of neurotoxic substances on the environmental and human health.

The potential toxicity of stannum dioxide (SnO(2)), cerium dioxide (CeO(2)) and iron oxide (Fe(3)O(4)) nanoparticles (NPs) in the marine environment was investigated using the sea urchin, Paracentrotus lividus, as an in vivo model. We found that 5 days after force-feeding of NPs in aqueous solutions, the three NPs presented different toxicity degrees, depending on the considered biomarkers. We examined: 1) the presence of the NPs in the coelomic fluid and the uptake into the immune cells (coelomocytes); 2) the cholinesterase activity and the expression of the stress-related proteins HSC70 and GRP78; 3) the morphological changes affecting cellular compartments, such as the endoplasmic reticulum (ER) and lysosomes. By Environmental Scanning Electron Microscope (ESEM) analysis, coupled with Energy Dispersive X-ray Spectroscopy (EDS) we found that NPs were uptaken inside coelomocytes. The cholinesterases activity, a well known marker of blood intoxication in vertebrates, was greatly reduced in specimens exposed to NPs. We found that levels of stress proteins were down-regulated, matching the observed ER and lysosomes morphological alterations. In conclusion, this is the first study which utilizes the sea urchin as a model organism for biomonitoring the biological impact of NPs and supports the efficacy of the selected biomarkers.

Neural Crest Cells (NCCs) are transient multipotent migratory cells that derive from the embryonic neural crest which is itself derived from the margin of the neural tube. DNA repair genes are expressed in the early stages of mammalian development to reduce possible replication errors and genotoxic damage. Some birth defects and cancers are due to inappropriate or defective DNA repair machinery, indicating that the proper functioning of DNA repair genes in the early stages of fetal development is essential for maintaining DNA integrity. We performed a genome-wide expression analysis combining laser capture microdissection (LCM) and high-density oligo-microarray of murine NCCs at pre-migratory embryonic days 8.5 (E8.5), and at E13.5, as well as on neural crest-derived cells from the adrenal medulla at postnatal day 90. We found 11 genes involved in DNA repair activity (response to DNA damage stimulus, DNA damage checkpoint, base-excision repair, mismatch repair), over-expressed in the early stages of mouse embryo development. Expression of these 11 genes was very low or undetectable in the differentiated adrenal medulla of the adult mouse. Amongst the 11 genes, 6 had not been previously reported as being over-expressed during mouse embryonic development. High expression of DNA repair genes in enriched NCCs during early embryonic development may contribute to maintaining DNA integrity whilst failure of some of these genes may be associated with the onset of genetic disease and cancer. Our model of enriched murine NCCs and neural crest-derived cells can be used to elucidate the key roles of genes during normal embryonic development and in cancer pathogenesis.

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.

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.

The NTera2/D1 (NT2) cell line, which was derived from a human teratocarcinoma, exhibits properties that are characteristics of a committed neuronal precursor at an early stage of differentiation. Its property to express a whole set of molecules related to the cholinergic neurotransmission system, including active acetylcholinesterase (AChE, EC 3.1.1.7) makes it a good alternative model for testing the effects of neurotoxic compounds, such as organophosphorus (OP) insecticides, whose primary target is the inhibition of AChE activity. Recent findings have elucidated the role of AChE in the modulation of apoptosis, but the mechanisms are still rather obscure. NT2 cells exposed to the OP insecticide diazinon at concentrations ranging between 10(-4) and 10(-5)M showed a time-dependent enhancement of cell death. When exposed at 10(-6)M diazinon showed higher cell viability than control samples up to 72 h, followed by a decreasing phase. The cell death caused by the exposures showed a number of features characteristic of apoptosis, including membrane and mitochondrial potential changes. We suggest the hypothesis that such behaviour is due to a dynamic balance between activated and blocked acetylcholine receptors that in turn trigger electrical events and caspase cascade.

Stem cell models offer an opportunity both for therapeutic use and for the assessment of alternative in vitro models. Human lipoaspirate is a source of adult stem cells (pre-adipocytes), which are able to differentiate into various phenotypes, such as neurogenic lineage. Here, we analyse the suitability of these in vitro models in screening exogenous compounds, such as environmental pollutants, that may affect adipose cells and neurogenic development. To evaluate neurogenic differentiation, we analysed expression of cholinergic system and acetylcholinesterase immunoreactivity. Heterocyclic derivatives of polycyclic aromatic hydrocarbons (PAHs) are often significant components of environmental contaminants. As they contain inducers of cytochrome P450 1A1 (CYP1A1), we explored the activity of CYP1A1-related enzymes, i.e. 7-ethoxycoumarin- and 7-ethoxyresorufin-O-deethylase (ECOD and EROD) in both cell systems in basal conditions and after exposure to non-cytotoxic doses of beta-naphthoflavone (BNF), a well-known PAH-type inducer. Both cell models showed basal and inducible levels of ECOD. Analysis of CYP1A1 protein expression and EROD-related enzyme activity confirmed the inducibility of the CYP1A1 isoform by BNF. These results demonstrate that mesenchymal adult stem cells can constitute innovative models. We therefore propose the use of pre-adipocytes and their neurogenic derivates to evaluate the cytotoxic/biological effects of unintended exposure to contaminants.

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.

Presently, a large effort is being made worldwide to increase the sustainability of industrial development, while preserving not only the quality of the environment but also that of animal and human life. In this work, sea urchin early developmental stages were used as a model to test the effects of the organophosphate pesticide (diazinon) on the regulation of gene expression by immunohistochemical localization of the human regulatory protein against the human OTX2. Egg exposure to diazinon did not affect fertilization; however, at concentrations 10(-5)-10(-6) M, it did cause developmental anomalies, among which was the dose-dependent alteration of the intracellular distribution of a regulatory protein that is immunologically related to the human OTX2. The severe anomalies and developmental delay observed after treatment at 10(-5) M concentration are indicators of systemic toxicity, while the results after treatment at 10(-6) M suggest a specific action of the neurotoxic compound. In this second case, exposure to diazinon caused partial delivery of the protein into the nuclei, a defective translocation that particularly affected the blastula and gastrula stages. Therefore, the possibility that neurotoxic agents such as organophosphates may damage embryonic development is taken into account. Specifically, the compounds are known to alter cytoplasmic dynamics, which play a crucial role in regulating the distribution of intracellular structures and molecules, as well as transcription factors. Speculatively, basing our assumptions on Fura2 experiments, we submit the hypothesis that this effect may be due to altered calcium dynamics, which in turn alter cytoskeleton dynamics: the asters, in fact, appear strongly positive to the OTX2 immunoreaction, in both control and exposed samples. Coimmunoprecipitation experiments seem to supply evidence to the hypothesis.

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.

The teratocarcinoma cell line NTERA2 is recently used in a wide range of researches (from developmental biology to toxicology, for their ability to be induced to neural differentiation. In order to study the genetic potential of these cells, it is needed to use methods for gene silencing and/or mRNA interference, allowing cell viability and further differentiation. To check these features, we simultaneously tested the transfection efficiency of NTERA2, A549 and HeLa cells with Metafectene PRO (Biontex, Germany) and another optimal transfection reagent currently used in our Laboratory, using as a reporter gene the DsRed2 vector (Clontech, Mountain View, CA). Under our culture conditions for NTERA2 and HeLa cells, Metafectene PRO transfection method was found to possess high throughput performance, that allows low concentration rate and low exposure time to excitation light source, thus reducing both toxicity and phototoxicity.

It is now widely accepted that assays with protists are relevant to be exploited for the study of environmental modifications due to the presence of xenobiotic compounds. In this work, the possibility of utilizing Euplotes crassus, an interstitial marine ciliate, for the pre-chemical screening of estuarine and coastal sediments was evaluated. For this purpose, the effects of exposure to pollutants were tested on the cell viability, fission rate and lysosomal membrane stability of E. crassus. The following toxicants were used: an organophosphate (OP) pesticide, basudin, an organochlorine hydrocarbon, AFD25, both employed especially for pest control in agricultural sites, a toxic heavy metal, mercury (HgCl2) and different mixtures of the above-mentioned compounds, as they might occur in polluted sites. Exposure to these toxicants affected cell viability at concentrations ranging from 96.6 to 966 x 10(3)mg/l for basudin, from 3.3 to 33 x 10(3)mg/l for AFD25 and from 0.1 to 1mg/l for HgCl2. A significant decrease in the mean fission rate (P<0.001) was found after 24- or 48-h exposures to 9.66 mg/l basudin, 3.3 mg/l AFD25 and 7 x 10(-2)mg/l HgCl2. Furthermore, the Neutral Red Retention Assay showed a significant decrease in lysosomal membrane stability after 60- and 120-min exposures to AFD25 (33 mg/l) and HgCl2 (0.33 mg/l). In addition, as it is well-known that the inhibition of acetylcholinesterase activity represents a specific biomarker of exposure to OP and carbamate pesticides in higher organisms, initially the presence of cholinesterase (ChE) activity was detected in E. crassus, using cytochemical, spectrophotometric and electrophoretic methods. Afterwards, this enzyme activity was characterized spectrophotometrically by its sensitivity to specific ChE inhibitors and to variations in pH and temperature. The ChE activity was inhibited significantly by basudin- (9.66 and 96.6 mg/l) or AFD25-exposure (3.3 mg/l). Conversely, exposure to AFD25 (33 mg/l) or HgCl2 (0.1 and 0.3mg/l) caused a significant increase in this enzyme activity. Moreover, exposure to mixtures containing basudin, AFD25 and HgCl2 was found to affect the cell viability, the mean fission rate and the ChE activity differently, in an unpredictable manner. Our results indicate that E. crassus seems to be a suitable test organism to evaluate the toxicity of marine sediments.

Recently, we showed that Paramecium primaurelia synthesizes molecules functionally related to the cholinergic system and involved in modulating cell-cell interactions leading to the sexual process of conjugation. It is known that nitric oxide (NO) plays a role in regulating the release of transmitter molecules, such as acetylcholine, and that the NO biosynthetic enzyme, nitric oxide synthase (NOS), shows nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity. In this work, we detected the presence of NADPH-d activity in P. primaurelia. We characterized this activity histochemically by examining its specificity for beta-NADPH and alpha-NADH co-substrates, and sensitivity both to variations in chemico-physical parameters and to inhibitors of enzymes showing NADPH-d activity. Molecules immunologically related to NOS were recognized by the anti-rat brain NOS (bNOS) antibody. Moreover, bNOS immunoreactivity and NADPH-d activity sites were found to be co-localized. The non-denaturing electrophoresis, followed by exposure to beta-NADPH or alpha-NADH co-substrates, revealed the presence of a band of apparent molecular mass of about 124 kDa or a band of apparent molecular mass of about 175 kDa, respectively. In immunoblot experiments, the bNOS antibody recognized a single band of apparent molecular mass of about 123 kDa.

A year-round biomonitoring study on blue mussels (Mytilus galloprovincialis) was carried out in 4 selected sites along the Gulf of Oristano (Sardinia, Italy): a commercial port (Port), the outlet of the S'Ena Arrubia and Marceddi lagoons (in the catchment area of intensive agricultural and diary activities, and abandoned mining), and a reference site (North). Heavy metal concentrations in sediments from Marceddi were 2-3 to 10-20 times higher in Pb, Cd and Zn, respectively, than those found at North and S'Ena Arrubia. Higher values (P<0.05) of micronuclei frequency were detected in mussels from Marceddi and Port compared to those detected in mussels from North and S'Ena Arrubia. DNA damage in animals from North was significantly lower than that at the other sites. Results of acetylcholinesterase inhibition consistently showed the strongest effects in mussels from Port and Marceddi. Our results suggest that these biomarkers can be used in coastal marine biomonitoring as early signals of exposure and adverse effects along a pollution gradient.

Previous studies have shown that the cholinergic system plays a pivotal rule in small cell lung cancer (SCLC) cell growth through an autocrine loop that activates the nicotinic cholinergic receptor, which together with the activation of this receptor by nicotine links SCLC evolution with tobacco use. Non-small cell lung cancer (NSCLC) is the most common form of lung cancer and is also linked to tobacco use. Here we describe the presence of molecules of the cholinergic system in NSCLC samples and cell lines and investigate the implications of the cholinergic system in cell growth regulation. Cholino-acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and acetylcholinesterase (AChE) were observed in NSCLC tumor biopsies and in NSCLC cell lines. Polymeric alkylpyridinium salts (poly-APS) are AChE inhibitors isolated from the crude extract of the marine sponge, Reniera sarai. These metabolites were characterized as a mixture of two polymers of 3-octylpyridinium, including 29 and 99 monomeric units. Exposure of normal lung fibroblast and NSCLC cell lines to poly-APS revealed a selective cytotoxicity for cancer cells as compared to the normal fibroblast cell lines. FACS analysis indicated poly-APS induced apoptosis in NSCLC cells but not in normal lymphocytes. Non-toxic doses of poly-APS also potently reduced NSCLC cell-cell adhesion in suspension cultures. The limited toxicity of poly-APS on normal cells was confirmed by injection in the caudal vein of mice. No overt effects on health parameters, such as weight gain and physical behavior, were observed, and histological analysis of major organs did not reveal differences between the treated animals as compared to controls. These data demonstrate that NSCLC cells express cholinergic molecules that may be involved in cell growth regulation and that the cholinesterase inhibitor, poly-APS, shows selective toxicity toward NSCLC cells while having no apparent toxicity towards normal cells and tissue in vitro and in vivo.

Exposure of fertilized eggs of the sea urchin Paracentrotus lividus to an electromagnetic field of 75-Hz frequency and low amplitudes (from 0.75 to 2.20 mT of magnetic component) leads to a dramatic loss of synchronization of the first cell cycle, with formation of anomalous embryos linked to irregular separation of chromatids during the mitotic events. Because acetylcholinesterase (ACHE) is thought to regulate the embryonic first developmental events of the sea urchin, its enzymatic activity was assayed in embryo homogenates and decreased by 48% when the homogenates were exposed to the same pulsed field. This enzymatic inactivation had a threshold of about 0.75 +/- 0.01 mT. The same field threshold was found for the effect on the formation of anomalous embryos of P. lividus. Moreover, ACHE inhibitors seem to induce the same teratological effects as those caused by the field, while blockers of acetylcholine (ACh) receptors are able to antagonize those effects. We conclude that one of the main causes of these dramatic effects on the early development of the sea urchin by field exposure could be the accumulation of ACh due to ACHE inactivation. The crucial role of the membrane in determining the conditions for enzyme inactivation is discussed.

Organophosphate (OP) compounds exert inhibition on cholinesterase (ChE) activity by irreversibly binding to the catalytic site of the enzymes. For this reason, they are employed as insecticides for agricultural, gardening and indoor pest control. The biological function of the ChE enzymes is well known and has been studied since the beginning of the XXth century; in particular, acetylcholinesterase (AChE, E.C. 3.1.1.7) is an enzyme playing a key role in the modulation of neuromuscular impulse transmission. However, in the past decades, there has been increasing interest concerning its role in regulating non-neuromuscular cell-to-cell interactions mediated by electrical events, such as intracellular ion concentration changes, as the ones occurring during gamete interaction and embryonic development. An understanding of the mechanisms of the cholinergic regulation of these events can help us foresee the possible impact on environmental and human health, including gamete efficiency and possible teratogenic effects on different models, and help elucidate the extent to which OP exposure may affect human health. The chosen organophosphates were the ones mainly used in Europe: diazinon, chlorpyriphos, malathion, and phentoate, all of them belonging to the thionophosphate chemical class. This research has focused on the comparison between the effects of exposure on the developing embryos at different stages, identifying biomarkers and determining potential risk factors for sensitive subpopulations. The effects of OP oxonisation were not taken into account at this level, because embryonic responses were directly correlated to the changes of AChE activity, as determined by histochemical localisation and biochemical measurements. The identified biomarkers of effect for in vitro experiments were: cell proliferation/apoptosis as well as cell differentiation. For in vivo experiments, the endpoints were: developmental speed, size and shape of pre-gastrula embryos; developmental anomalies on neural tube, head, eye, heart. In all these events, we had evidence that the effects are mediated by ion channel activation, through the activation/inactivation of acetylcholine receptors (AChRs).

The early development of sea urchins has been thoroughly studied since the beginning of the 20th century thanks to the particular features of the model involving cell signalling, making it easy to follow the complex cell-to-cell interactions that lead to development. In this chapter, the prominent role of cell-to-cell communication in developmental events is discussed, as well as the role of intracellular ion changes that are in turn regulated by signal molecules belonging to the cholinergic system. The results seem to indicate that the zygote stage is the most suitable to study the role of the cholinergic system, as at this stage, a calcium spike can be evoked by exposure to acetylcholine (ACh) or to muscarinic drugs, at any time before the nuclear breakdown. The described outcomes also open a path to a new way of considering biomarkers. In fact, most environmental factors have the capacity to interfere with the cholinergic system: stress, wounds, inflammation and pollution in general. In particular, this offers a way to investigate the presence in the environment and the degree of aggressiveness of neurotoxic contaminants, such as organophosphate and carbamate pesticides, largely used in European countries for many purposes, including agricultural pest control and medical treatment. These drugs exert their function by interfering with the regulation of the cholinergic system and the consequent electrical events. Thus, the sea urchin zygote could represent a reliable model to be used in biosensors with the capacity to translate the effect of neurotoxic pesticides, and generally of stress-inducing contaminants, in living cell responses, such as electrical responses.

Cholinergic neurotransmitter system molecules were found to play a role during fertilisation and early cell cycles of a large number of invertebrate and vertebrate organisms. In this study, we investigated the presence and possible function of choline acetyltransferase (ChAT, the biosynthetic enzyme of acetylcholine) in gametes of the sea urchin, Paracentrotus lividus, through localisation and functional studies. ChAT-like molecules were detected in oocytes, mature eggs and zygotes with indirect immunofluorescence methods. Positive immunoreactivity was found in the ovarian egg cytoplasm and surface as well as at the zygote surface. This suggests the eggs' capacity to autonomously synthesise acetylcholine (ACh), the signal molecule of the cholinergic system. Acetylcholinesterase (AChE, the lytic enzyme of acetylcholine) was also found in ovarian eggs, with a similar distribution; however, it disappeared after fertilisation. Ultrastructural ChAT localisation in sperms, which was carried out with the immuno-gold method, showed immunoreactivity in the acrosome of unreacted sperms and at the head surface of reacted sperms. In order to verify a functional role of ACh during fertilization and sea urchin development, in vivo experiments were performed. Exposure of the eggs before fertilisation to 1 mM ACh + 1 microM eserine caused an incomplete membrane depolarisation and consequently enhanced polyspermy, while lower concentrations of ACh caused developmental anomalies. The exposure of zygotes to 0,045 AChE Units/mL of sea water caused developmental anomalies as well, in 50% of the embryos. Altogether, these findings and other previously obtained results, suggest that the cholinergic system may subserve two different tasks during development, according to which particular type of ACh receptor is active during each temporal window. The first function, taking place in the course of fertilisation is a result of autonomously synthesised ACh in sperms, while the second function, taking place after fertilisation, is due to maternal ChAT molecules, assembled on the oolemma along with egg maturation and fertilisation processes.

Acetylcholine (Ach), one of the most important examples of a neurotransmitter, represents a phylogenetically old molecule, widely distributed from bacteria to humans. The finding that neuronal Ach receptors (nAChRs) are present in non-neuronal cells raised some interesting issues related to their specific activity. In humans, different studies have showed that many lung cancer cells expressed nAchRs and that low concentrations of nicotine blocked the induction of apoptosis in these cells. A recent study presents data that SCLC express a cholinergic autocrine loop that can regulate cell growth. Such work demonstrates that SCLC cells have a cholinergic phenotype and that ACh exerts as an autocrine growth factor in human lung tumors. Recently it has been shown that human malignant pleural mesothelioma express a cholinergic system, involved in cell growth regulation. Hence, mesothelioma cell growth as well as normal mesothelial cells growth is modulated by the cholinergic system in which agonists (i.e. nicotine) has a proliferative effect and antagonists (i.e. curare) has an inhibitory effect. Furthermore apoptosis mechanisms in mesothelioma cells are under the control of the cholinergic system (nicotine antiapoptotic via induction of NF-kappaB complexes and phosphorilation of Bad at Serine(112), curare proapoptotic via G(0)-G(1) arrest p21(waf-1)-dependent, but p53-independent). The involvement of the non-neuronal cholinergic system in lung cancer and mesothelioma appears reasonable and open up new therapeutic strategies.

This study presents data suggesting that both human mesothelioma (cell lines and human mesothelioma biopsies) and human normal mesothelial cells express receptors for acetylcholine and that stimulation of these receptors by nicotine prompted cell growth via activation of nicotinic cholinergic receptors. Thus, these data demonstrate that: (a) human mesothelioma cells and human biopsies of mesothelioma as well as of normal pleural mesothelial cells express functionally alpha-7 nicotinic acethlycholine receptors, evaluated by alpha-bungarotoxin-FITC binding, receptor binding assay, Western blot, and reverse transcription-PCR; (b) choline acetyltransferase immunostaining is present in mesothelioma cells; (c) mesothelioma cell growth is modulated by the cholinergic system in which agonists (i.e., nicotine) has a proliferative effect, and antagonists (i.e., curare) has an inhibitory effect, evaluated by cell cloning, DNA synthesis and cell cycle; (d) nicotine induces Ca(+2) influx, evaluated by [(45)Ca(2+)] uptake, and consequently activation of mitogen-activated protein kinase pathway (extracellular signal-regulated kinase and p90(RSK) phosphorylation), evaluated by Western blot; and (e) apoptosis mechanisms in mesothelioma cells are under the control of the cholinergic system (nicotine antiapoptotic via induction of nuclear factor-kappaB complexes and phosphorylation of Bad at Ser(112); curare proapoptotic via G(0)-G(1) arrest p21(waf-1) dependent but p53 independent). The involvement of the nonneuronal cholinergic system in mesothelioma appears reasonable and open up new therapeutic strategies.

The aim of the present study was to investigate the presence and distribution of cholinergic molecules in Balanus amphitrite cyprids and their possible involvement in settlement and adhesion. Acetylcholinesterase (AChE, the lythic enzyme of acetylcholine) activity was detected, for the first time, by biochemical and histoenzymological methods, in the thoracic muscles, gut wall and cement gland. The immunodetection of choline acetyltransferase-like (ChAT) molecules in the same area and in the neuropil of the central nervous system suggests the presence of a cholinergic innervation, and the involvement of acetylcholine in muscular contraction and cement gland exocytosis. The binding of FITC-conjugate alpha-bungarotoxin in the cement gland cells confirms the latter hypothesis. Acetylcholine involvement in the settlement process was also investigated by laboratory tests employing cholinergic antagonists and agonists. An increase of available acetylcholine due to the partial inhibition of AChE activity produced an increase in cyprid settlement. The data presented support the hypothesis that acetylcholine has a neurotransmitter/neuromodulator role in settlement and adhesion of barnacle cyprids.

Oncogenic Ras proteins have been seen as an important target for novel anticancer drugs. Due to the functional role of Ras farnesylation, fanesyltransferase (FTase) inhibition was thought to be a strategy for interfering with Ras-dependent transformation. When farnesylation is blocked, the function of Ras protein is severely impaired because of the inability of the nonfarnesylated protein to anchor to the membrane. Although it has been clearly demonstrated that FTase inhibitors (FTIs) inhibit Ras farnesylation, it is uncertain whether the antiproliferative effects of these compounds result exclusively from the effects on Ras. Moreover, no consensus has been reached as to the relevant targets(s) of FTIs that can explain their mosaic pharmacology. In searching for downstream targets for FTIs effects, CENP-E and CENP-F/mitosin were identified. Different studies showed that the inhibition of farnesylation interferes with CENP-E-microtubule association. In the presence of FTIs, chromosome alignment to the metaphase plate is delayed, suggesting that farnesylated proteins are involved in a step critical to bipolar spindle formation and chromosome alignment. An important question is whether these biological effects might contribute to the chemotherapeutic effects of the FTIs. However, FTIs, triggering the spindle checkpoint, might elevate the rate of cellular missegregation to levels that are incompatible with cell viability, as well as have a reduced (but still significant?) effect on checkpoint-proficient normal cells. As an example, RPR-115135 induced micronuclei (MN) increase in cancer cells displaying high chromosome instability (CIN) levels, whereas in normal cells it is devoid of activity. Cancer cells showing high CIN level might represent an ideal target for the activity of some FTIs.

Biological effects of neurotoxic insecticides widely used for agricultural purposes were studied using the early development of the Mediterranean sea urchin Paracentrotus lividus as a model. These compounds, dispersed as aerosols or powders in agricultural regions near to the coast, may affect the health of organisms in the marine environment. The biological effects of Basudin (an organophosphate compound containing 20% Diazinon), Diazinon (Dzn, a thionophosphate), Carbaryl and Pirimicarb (carbamates) on the early phases of sea urchin development were thus investigated. Morphological, biochemical, histochemical and immuno histochemical analyses were performed both during embryo and larval development. For the morphological effects on fertilisation and first cleavages, the effective concentration of insecticides was found to be 10(-4) M, while for further stages concentrations between 10(-5) and 10(-7) M were effective: 10(-3) M of any of these insecticides totally arrested development. During embryonic development, the treatment with organophosphates slowed the rate of early mitotic cycles down, affected nuclear and cytoskeletal status as well as DNA synthesis. From the gastrulation stage onwards, the main effects were exerted on the rate of primary mesenchyme cells migration, larval size, perioral arm length, and acetylcholinesterase activity distribution, thus deregulating the cholinergic system, which modulates cell-to-cell communication mediated by the signal molecule acetylcholine.

A therapeutic strategy that relies on the use of c-myc antisense in combination with a farnesyltransferase inhibitor, RPR-115135 (C31H29NO4), was studied in human cancer cell lines carrying different mutations (Ras, p53, myc amplification). Cell proliferation was strongly inhibited by the combination and was observed when c-myc oligo (at a concentration that down-regulates c-myc expression) was followed by RPR-115135. Cell cycle analysis demonstrated an accumulation in G0-G1 phase and a tendency to apoptosis (not detectable in cells treated with a single agent). Morphological examination and DNA fragmentation assays (filter binding and enzyme-linked immunosorbent assay DNA fragmentation) confirmed the induction of apoptosis. Apoptosis was not p53- and/or p21(waf-1)-dependent, and the key effector was caspase activation. The combination induced Bax expression and Bcl-2 inhibition. Down-regulation of c-myc amplification carried out a specific role exclusively when Ras was mutated. Exposure of human proliferating lymphocytes to combination did not result in cytotoxicity, suggesting that mechanisms regulating c-myc gene expression during normal T cell proliferation might not be involved. Because of the high percentage of human tumors overexpressing c-myc mRNA and/or protein and, simultaneously, harboring oncogenic Ras mutants (i.e., colon cancers), interrupting the myc- and Ras-signaling pathway would be one of the major focuses on therapy of these types of tumors.

AIMS: A case-control study was performed to investigate the relationship between cervical cancer and TP53 polymorphism at codon 72 in young black African women from The Gambia. MATERIALS AND
METHODS: The TP53 polymorphism at codon 72 was examined by PCR amplification and SSCP analysis in 40 patients with primary cervical cancer and in 20 healthy women of the same age and from the same geographical area. The occurrence of TP53 polymorphism in combination with the HPV-16 E6 genotype (assayed by PCR) was evaluated.
RESULTS: The distribution of TP53 genotypes in cervical cancer patients and in the control group was not statistically different (p = 0.45) and homozygosity for argine at residue 72 was not associated with cervical cancer (odds ratio: 1.24; 95% confidence interval 0.21-9.16). Similarly, a different genotype distribution, cervical cancer and presence of HPV-16 E6 were not observed.
CONCLUSIONS: These results cannot rule out an association between TP53 polymorphism at codon 72, HPV infection and the etiology of cervical cancer in this population sample.

Comprehensive and contemporary evaluations of physical, chemical and toxicological endpoints have been performed on bed sediments of the Po River, the major Italian watercourse. Two extensive sampling campaigns were conducted in summer and winter low-flow conditions. Composite sediment samples were collected from ten reaches of the main river: the first was located in the upper region (ambient control), and the others downstream of the confluences of nine principal tributaries. The two sampling programs were paralleled by contemporary investigations on the macroinvertebrate community. The particle-size composition along the Po River showed a relatively uniform distribution of fine sand, a progressive downstream decrease of coarse sands and a corresponding increase of fine materials. The levels of polychlorinated biphenyls (PCB), polycyclic aromatic hydrocarbons (PAH), extractable organo halides (EOX), Cd, Cr, Cu, Hg, Ni, Pb and Zn were determined in sediment fine particles (< 63 microm), and showed marked changes across the ten river reaches. Their longitudinal trends, as those of organic carbon and total nitrogen, were very similar and largely independent of the survey season. Sediment quality benchmarks were used to evaluate sediment chemistry, and, although the overall level of contamination was from moderate to low, the reaches located downstream of the tributaries Dora Riparia, Dora Baltea, Lambro and Oglio were considered to be at risk. Sediments were tested for toxicity on Oncorhynchus mykiss, Ceriodaphnia dubia, Raphidocelis subcapitata and Vibrio fischeri. The toxicity tests were conducted both with sediment extracts and whole samples. Sediment extracts showed toxic potentials that were consistent with the spatial distribution of contaminants. Whole-sediment toxicity showed moderate/low effects which also included false positives and negatives. Alterations of the macroinvertebrate community were found for many kilometers downstream of Dora Riparia, and with a seasonal dependence, also in other reaches of the Italian river. Principal component analysis (PCA) was used to describe the longitudinal and temporal changes of the Po River, and allowed the selection of the most useful and discriminating indicators.

Gamma-aminobutyric acid (GABA)-related molecules were identified in Paramecium primaurelia by immunocytochemical methods, and GABA(A) receptors by their histochemical BODIPY-binding sites. Confocal microscope analysis showed different localizations according to the stages of the developmental cycle. A comparison was made with the cholinergic molecules, such as the acetylcholine biosynthetic enzyme (choline acetyltransferase), in double-labelled cells by confocal microscopy. In vivo experiments suggested the involvement of GABA-related molecules in cell-cell interaction.

Confocal laser scanning microscopy (CLSM) was used to examine molecules related to the cholinergic neurotransmission system and detected at all the larval stages of Paracentrotus lividus, by histochemical and immunohistochemical methods. CLSM, providing spatial resolution of the cells located both at the larval surface and at depth, allows a complete mapping in a three-dimensional volumetric frame. At early larval stages acetylcholinesterase- as well as choline acetyltransferase-like molecules were found mainly in the gut wall cells, and along the ciliary bands of the arms, together with muscarinic acetylcholine receptors. At perimetamorphic stages, cholinergic molecules were present in the ciliate strands along the arms, in the larval body and in the rudiment. At metamorphosis, positivity to cholinergic molecules translocated to the juvenile, where a high frequency of mAChR- and ChAT-like positive cells was found.

Recently, we found acetylcholinesterase (AChE) activity in the ciliate protozoan Paramecium primaurelia. As in the slime mould Dictyostelium discoideum the presence of a serine esterase was found with strong sequence identity to Torpedo AChE, we extended to D. discoideum the investigation on the characterization and possible functions of cholinesterases (ChEs). In amoeboid cells, histochemical, biochemical, and electrophoresis analyses evidenced both a ChE activity able to hydrolyze the substrate PrTChI, and AChE (E.C. 3.1.1.7.) activity similar to Electrophorus electricus AChE. Conversely, butyrylcholinesterase activity was nearly absent, according to our previous results on P. primaurelia. Moreover, the possibility to utilize D. discoideum in a bioassay for the pre-chemical screening both of moist environments and fresh waters, in relation to the occurrence of the neurotoxic organophosphate drugs, such as "basudin", inhibiting ChE activity, was investigated. Exposure to basudin inhibited propionylcholinesterase (PrChE) activity in a dose-dependent manner in the range 10(-1)-10(-7) M (60% at 10(-4) M), without any significant effect on AChE activity. PrChE activity was inhibited slightly by 10(-5) M eserine, and reduced significantly both by 10(-5) M iso-OMPA and BW284C51, classically used to discriminate the different ChE molecular forms. The effects on cell morphology, cell density, and differentiation were evaluated in cultures exposed to PrTChI 10(-5) M or basudin 10(-4) M for a three-day period. The PrTChI-exposed sample exhibited cell morphology, cell density, ability to aggregate, and to form fruiting bodies similar to the control; whereas, the basudin-exposed sample showed anomalies in cell morphology and lower cell density than the control, together with inability to aggregate.

The existence of a response to acetylcholine (ACh) and cholinomimetic drugs in sea urchin eggs and zygotes was investigated in two sea urchin species: Paracentrotus lividus and Lytechinus pictus. The calcium sensitive fluorescent probe, Fura-2 dextran, was employed to investigate the regulation of cytosolic free calcium concentration ([Ca(2+)](i)) by cholinomimetic drugs in unfertilised and fertilised eggs of both the sea urchin species. Exposure to cholinomimetic agonists/antagonists, either extracellularly or intracellularly, had no effect either on resting [Ca(2+)](i) levels in the unfertilised sea urchin egg, or on the transient [Ca(2+)](i) increase at fertilisation. However, following fertilisation, extracellular application of ACh receptors agonists, such as ACh and carbachol, predominantly muscarinic agonist, but not nicotine, induced a significant increase in [Ca(2+)](i), which was partially inhibited by atropine. As a consequence of exposure after fertilisation to the agonists of ACh receptors, chromatin structure was transiently affected. The hypothesis is proposed that muscarinic receptors may be involved in the (presumably Ca(2+)-dependent) modulation of the nuclear status during the first cell cycles.

A protocol for detecting hepatic micronuclei in fish was performed to check genotoxic damage, as an indicator of environmental hydrocarbons exposure, in relation to the "Haven" oil spill. As target fish, we have chosen three demersal species with different habitats and feeding behaviour (i.e., Lepidorhombus boscii, Merluccius merluccius and Mullus barbatus) collected from two differently impacted areas and a control site. Additional analysis was performed by histological detection of hepatic tissue damages such as the presence of necrotic and tumour-like aspects. The three studied species showed different sensitivity to environmental pollutants exposure, L. boscii resulting the more sensitive in terms of both micronuclei incidence and tissue damage. The results of this study show that: (1) the micronucleus test could be an effective and fast method to detect oil pollution; (2) a clear response of L. boscii only to oil contamination for both micronucleus test and liver tissue alterations.

A new non peptidic farnesyltransferase inhibitor, RPR-115135, in combination with 5-FU was studied in 10 human colon cancer cell lines (HCT-116, RKO, DLD-1, Colo-320, LoVo, SW-620, HT-29, HCT-15, Colo-205 and KM-12) carrying several mutations but well characterized for p53 and Ras status. We found that there was a slight tendency (not statistically significant) for the p53 inactivated cells to be less sensitive to 5-FU after 6 days continuous treatment. Simultaneous administration of RPR-115135 and 5-FU, at subtoxic concentrations, resulted in a synergistic enhancement of 5-FU cytotoxicity in the p53 wildtype cells (HCT-116, RKO, DLD-1, Colo-320, LoVo). In the p53 mutated cells (SW-620, HT-29, HCT-15, Colo-205, KM-12) the effect was very complicated. In HCT-15 the combination resulted in antagonism, in KM-12 in antagonism or in synergy (at different concentrations) and in SW-620, HT-29 and Colo-205 cells in synergy but only when 5-FU was administered at high concentrations. Growth inhibition could be accounted for on the basis of a specific cell cycle arrest phenotype (G2-M arrest), as assayed by flow cytometry, only in the p53 functioning cell lines. The combination RPR-115135 + 5-FU increases apoptotic events only in these cell lines. In the mutated cell lines no major alterations on cell cycle arrest phenotype and no induction of apoptosis was observed. Although RPR-115135 can potentiate the effect of 5-FU in cells in which p53 function is disrupted, these data suggest strongly that RPR-115135 significantly enhances the efficacy of 5-FU only when p53 is functioning.

In our study, we show that expression of HPV-16 E6 sensitizes TNF-induced cytotoxicity of human ovarian cancer cell line A2780. This effect is not related to a different number of TNF receptors present on cell membrane. The major induction of massive apoptosis induced by TNF is not p53- and p21(waf-1)-dependent but it is principally related to NF-kappaB inhibition in A2780/E6 cells. Consistently to NF-kappaB inhibition a rapidly release of cytochrome c and severe induction of DNA fragmentation are seen in A2780/E6 cells. Also in human colon cancer cell line HCT-116/E6 the expression of HPV-16 E6 enhances TNF-cytotoxicity. This effect is not present in the HCT-116/mu-p53 clone (transfected with a dominant-negative mutated p53 transgene). Thus, taken together all these observations suggest that HPV-16 E6 sensitizes A2780 and HCT-116 cells to TNF; this effect is not p53-dependent, but it is essentially mediated through an inhibition in activating NF-kappaB activities.

We recently discovered, in mating-competent Paramecium primaurelia, the presence of functionally related molecules of the cholinergic system: the neurotransmitter acetylcholine (ACh), both its nicotinic and muscarinic receptors and its lytic enzyme acetylcholinesterase (AChE). Our results on the inhibition of mating-cell pairing in vivo in mating-competent cells treated with cholinomimetic drugs support the hypothesis that the cholinergic system plays a role in cell-to-cell adhesion. To investigate the possible function of the signal molecule ACh in conjugation in P. primaurelia, we attempted to detect the intracellular sites of ACh synthesis by localizing the ACh biosynthetic enzyme choline acetyltransferase (ChAT). Using immunocytochemical and histochemical methods, we have demonstrated the presence and activity of ChAT principally on the surface membrane of mating-competent cells and of mature but non-mating-competent cells. No evidence for ChAT activity was found in immature cells. Immunoblot analysis revealed the presence of immunoreactive bands, ranging in molecular mass from 42 to 133 kDa, as reported for ChAT isolated from higher organisms. In vivo experiments showed that inhibition of ChAT activity by Congo Red, known to be a potent competitive inhibitor of acetyl coenzyme A, did not affect mating-cell pairing. Conversely, inhibition of AChE with BW 284c51 or eserine, which block enzyme activity by reacting with a specific serine within the catalytic centre, significantly inhibited mating-cell pairing. Our results suggest that ACh has a negative modulating effect on conjugation in P. primaurelia.

The variation of the two main aromatic compounds in Ocimum basilicum cv Genovese Gigante grown in greenhouse and "in vitro" was analyzed. The content of methyleugenol and eugenol was correlated to the plants' height rather than to the plants' age and the growth site. Particularly, methyleugenol was prevalent in plants up to 6.5 cm, as plants grew it was replaced by eugenol that was dominant in taller plants. Analysis of basil 20 cm in height showed that methyleugenol is prevalently localized in the low part while eugenol is prevalent in the upper part of the plant. Moreover, a chronic and acute toxicity of methyleugenol was evidentiated in an assay using chicken embryos.

To investigate the relationship between oncogene activation and induction of micronuclei by a new non-peptidic mimetic farnesyltransferase inhibitor, RPR-115135, two isogenic cell lines, human colon cancer line HCT-116, which harbors a K-ras mutation, and spontaneously immortalized human breast epithelial cell line MCF-10A, were utilized. HCT-116 cells were transfected with an empty control pCMV vector (clone CMV-2) or with a dominant negative mutated p53 transgene (clone Mu-p53-2) to disrupt p53 function. In both clones RPR-115135 induced a significant increase in the frequency of micronucleation at concentrations that did not affect cell membrane integrity. RPR-115135 produced a significant increase in the ratio of CREST+ to CREST- micronuclei. MCF-10A cells were stably transfected with either c-Ha-ras or c-erbB-2 or both H-ras + c-erbB-2. No induction of micronuclei was observed. No induction of micronuclei was reported in human lymphocytes and in primary spinal cells obtained from 7-day chick embryos. In conclusion, RPR-115135 acts as an aneugenic agent in a complex manner, dependent upon the complement of mutations in cell regulatory genes in tumour cells and this activity may be independent of ras genotype.

In recent years, the finding of a complete set of molecules related to the cholinergic neurotransmitter system in male gametes of different invertebrate and vertebrate species has opened the question of the possible involvement of this signaling system in the regulation of intracellular ion change, functional to sperm motility and probably also to interaction with the egg. In this work, we localized molecules immunologically related to muscarinic acetylcholine receptors (mAChRs) in sea urchin (Paracentrotus lividus) eggs and zygotes, by use of the monoclonal antibody M35. The ultrastructural analysis of immunoreactivity revealed that the localization of M35-positive molecules is different between unfertilized and fertilized egg. In the unfertilized eggs, immunoreactive molecules were localized inside and associated to the membrane of vesicles, scattered in a thick zone of the cortical cytoplasm. Following fertilization, the oolemma was decored along the surface, and positive vesicles were seldom seen in the cytoplasm. At the surface of fertilized eggs, evident pinocytotic vesicles, recognized by the surrounding coat, indicate that the positive sites correspond to receptorial activity, able to rescue ligand portions. The presence of a cholinergic signaling pattern was also suggested by the presence of molecules immunologically related to acetylcholinesterase (AChE), performing AChE enzyme activity, detected by both immunofluorescent and histochemical methods in membranous organelles belonging to the cortical region of unfertilized eggs.

The modifications of several biomarkers were investigated in flounder (Platichthys flesus) when exposed in the laboratory to sediment samples collected from the Northern Adriatic Sea. Besides clean sand used as a control substrate, fish were exposed to sediments sampled offshore the delta of the Po River, the harbour of Trieste, and from the industrial harbour of Venice (Porto Marghera). After six days of exposure, the enzyme activities ethoxyresorufin-O-deethylase (EROD), UDP glucuronyltransferase (UDPGT), glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx), were assayed in fish liver. In addition, the contents of reduced glutathione (GSH), nonprotein thiols (SH), total sugars and extractable lipids were also determined in hepatic tissue, as well as the number of micronucleated hepatocytes and biliary concentrations of fluorescent aromatic compounds (FAC). Despite some variability within treatment groups, differences among exposed organisms were evident and consistent with known contaminant levels of sampled areas. Microsomal activities (EROD, UD-PGT) and FAC levels were the most sensitive exposure indicators. Variations in the other biomarkers showed only tendencies which although not statistically significant were generally consistent with the contamination pattern.

By histochemical and immunohistochemical methods, the presence of cholinergic-like molecules has previously been demonstrated in Paramecium primaurelia, and their functional role in mating-cell pairing was suggested. In this work, both true acetylcholinesterase (AChE) and butyrylcholinesterase (BCHE) activities were electrophoretically investigated, and the presence of molecules immunologically related to BCHE was checked by immunoblotting. The AChE activity, shown in the membrane protein fraction of mating-competent cells and in the cytoplasmic fraction of immature cells, is due to a 260-kDa molecular form, similar to the membrane-bound tetrameric form present in human erythrocytes. This AChE activity does not appear in either the cytoplasmic fraction of mating-competent cells or in the membrane protein fraction of immature cells. No evidence was found for the presence or the activity of BCHE-like molecules. The role of AChE in P. primaurelia developmental cycle is discussed.

The presence of cholinergic molecules was recently found in male gametes of different animal species; very little is known from this point of view about the somatic component of the gonad. In the present study, a contribution is given to the localization of some cholinergic-like molecules, such as acetylcholinesterase (AChE), nicotinic acetylcholine and muscarinic acetylcoline (nAchR and mAChR, respectively) receptors during postnatal development of the testis. Maturation stages were checked by use of histochemical stainings, such as DAPI for nuclear shape changes, and PSA binding to reveal acrosomal maturation. The distribution of cholinergic-like molecules, revealed by immunocytochemical methods in both gametes and somatic cells, varied with development. Generally, during early stages, molecules immunologically related to AChE, and to mAChR's were mainly found in the interstitial and peritubular compartment, while, during maturation, they were found in Sertoli cells and in differentiating germ cells. nAChR's were not found in the somatic compartment, except in the blood vessel walls, and were distributed in mature germ cells, mainly in the flagella. The presence of cholinergic molecules in somatic as well as germ cells could play a role in cell-to-cell communications affecting testicular cell differentiation and function.

During neurogenesis, markers of the cholinergic system are present in the eye and visual cortex of vertebrates. In adult vertebrates, a role for these molecules, including muscarinic acetylcholine receptors (mAChRs), in eye growth non-accommodative regulation is also known. In order to understand the biological mechanisms triggered by the cholinergic system in these events, we analysed the effects of a cholinergic agonist (10(-4) M carbachol) and an antagonist (10(-4) M atropine) of the muscarinic receptors, on early chick development. To establish if the cholinergic system also plays a role in the regulation of early neurogenetic signals, the drug treatments were made at stage 5-6 HH, during the formation of the cephalic process. Specific effects on forehead, and in particular on eye development were found; carbachol treated embryos presented huge and well pigmented eyes, significantly different from controls. The eyes of atropine-exposed embryos presented anomalies with different phenotypes ranging from strongly affected features to normal-like appearance. Generally, the eyes were smaller as compared to the controls, with a number of anomalies, also in the normal-like phenotype, including retina and lens defects. In these structures, distribution of cholinesterase activities was checked by histochemical methods, and the amount of cells undergoing nuclear disgregation was revealed by DAPI staining. We propose that the drugs affected the known nervous and pre-nervous functions of the cholinergic markers, such as cell signalling during primary induction, and regulation of cell death by ACh receptors.

During metamorphosis, ascidian tadpoles settle on appropriate substrates, by secreting adhesive substances from the papillae. The papillae were shown by different authors to possess neural structures, probably for substrate recognition. In free-swimming tadpoles, AChE activity was localized by histochemical methods at the cell surface of the adhesive papillae, suggesting the possible presence of a cholinergic system in these structures. In vivo experiments showed that the treatment with AChCl caused dose-dependent enhancement of metamorphosis, shortening of the exploratory period of the larvae, and secretion within a few minutes of sConA-binding glycoconjugates in the tunica, beginning from the adhesive papillae.

Dose-dependent terathogenic effects of an organophosphate insecticide were found during early sea urchin development. This biological assay is low cost, easy to measure, and allows to detect the effects of the exposure of organisms to the active principle at concentrations lower than the acceptable daily intake (ADI) for man. Effects were found independently from the stage of exposure, and were major as earlier exposure occurred. The stronger effects were exerted on the elongation of the skeletal rods, that was easily measured by following the migration of primary mesenchyme cells, labelled by WGA (wheat germ agglutinin).

Glycoconjugates are generally involved in cell adhesion and morphogenetic movements. To investigate their distribution and expression changes during amphibian development, several FITC-or HRP-labeled lectins were used in Rana dalmatina embryos and larvae. During gastrulation, WGA, ConA, HPA, GSA-I, PNA and RCA-I labeled either the envelopes on the perivitelline space-contained substances, while ectodermal cells and/or extracellular matrix were mainly labeled by WGA, GSA-I, DBA and LTA. In these structures fibronectin-like molecules were also found. ConA, HPA, RCA-I, SBA also labeled ectoderm, but with lower affinity. At larval stages, ectodermal derivatives, such as larval skin, central nervous system, eye and ear, were also strongly labeled by WGA, DBA and LTA, while the affinity to ConA, HPA, GSA-I, SBA, appeared less visible; the affinity sites to RCA-I and PNA were totally lacking. Fibronectin-like immunoreactivity was hardly present at the larval stages. These results point out that glycoconjugate expression changes in cell surface and in extracellular matrix might be developmentally regulated. For this reason the different glycoconjugates seem to play stage-specific roles in early development.

The distribution of different classes of acetylcholine (ACh) receptor-like molecules in sperms of different invertebrate and vertebrate species is described. ACh receptor molecules belong to one of two classes: muscarinic receptors (mAChRs), associated with signal transduction mechanisms in the inner domain of the cell, and nicotinic receptors (nAChRs), capable of opening Na+ channels when activated by the ligand. Molecules immunologically related to mAChRs and to ACh can be identified by specific antibodies, and revealed by immunofluorescent or immunogold staining; the nicotinic receptor-like molecules are localised as curare-sensitive affinity sites for alpha-bungarotoxin. In all species studied, both classes of receptors were found, with a similar distribution. Muscarinic-like molecules were found mainly in the sperm head regions of most species; such a localisation may be correlated to a function in sperm-egg interaction, for instance in the regulation of the block to polyspermy. Nicotinic-like molecules are present mainly in the tail and in the post-acrosomal region of most animals, thus confirming their function in the regulation of sperm propulsion, but are also present at the acrosomal region of most species. The distribution patterns of the different classes of molecules indicate that both may be involved in sperm-egg interactions, in addition to their known function in the regulation of sperm propulsion.

The possible correlation between cholinesterase (ChE) activity and ion channels antagonist drug effects was studied in sea urchin early development, showing that ChE activity is different when cation channels are affected: generally, drugs maintaining channels in the open state, such as nicotine, cause an increased ChE activity, while antagonist drugs to receptors (such as curare, butx, ttx), cause a decreased activity, showing a direct effect of membrane depolarization on the enzymic activity, also in these non-neuromuscular stages.

Neurotransmission in Cnidarians is known to occur mainly by peptidergic neuronal pathways; the role of cholinergic neurotransmission, that in other animals this involves both neurotransmission and developmental regulation, has never been shown with certainty. This histochemical-immunohistochemical study was performed, in different developmental stages of some hydroidomedusan species (characterized by different reproductive and developmental patterns) to investigate the presence and distribution of cholinesterase (ChE) activity, acetylcholine receptors, biogenic amines, and molecules immunologically related to secretory peptides (FMRF amide and VIP). The results showed that ChE (revealed by all the kinds of activities: AChE, PrChE, BCHE) is mainly present in developing or 'juvenile' structures. Biogenic amines are revealed by aldehyde-induced fluorescence at every stage studied, while peptide-like immunoreactivities are mainly present in differentiated structures. These findings support the hypothesis that all the chemical messengers here investigated arose very early in evolution, although, in some cases, with different functions. For most of the investigated species, similarities in the distribution pattern of markers generally reflect similarities in life cycle, rather than phylogenetic affinities.

Single oral doses of norfloxacin (4, 2, 1, 0.5 mmole/kg) administered to pregnant rats significantly increase the frequency of micronucleated polychromatic erythrocytes both in fetal liver and in maternal bone marrow. Therefore norfloxacin in this work has been found to be a genotoxic agent.

Data on the non-nervous location of cholinergic molecules are reviewed to give a general indication of their possible functions. Cholinergic or immunologically related molecules were detected and localized mainly in three classes of differentiative events supported by intracellular ion concentration changes. I: during gamete maturation, activation and interaction; II: during the early development of invertebrate and vertebrate embryos. In this case cholinergic molecules are located mainly in moving cells and tissues engaged in relevant morphogenetic events, such as gastrulation and limb bud differentiation, and are often codistributed with special extracellular matrix molecules (fibronectin); III: during inductive communications between mesenchyme and other tissues. The cholinergic system thus seems to be a multifunctional cell communication system. It appeared early during evolution as a regulator of intercellular communications mediated by ion dynamics, before becoming involved in highly specialized communication structures, such as synapses and nerve endings.

This histochemical-immunohistochemical study was performed on the earthworm Eisenia foetida at different developmental stages, to investigate the presence and distribution of cholinergic molecules (AChE, BCHE, alpha-bungarotoxin-binding sites), several biogenic amines (5HT and catecholamines), and some immunologically-related peptides (somatostatin. FMRF-amide, VIP, substance-P, bombesin). The results showed that the pattern of labelling for the markers is different at different stages. In summary, cholinesterases appeared widely distributed in the early embryonic stages. They then were localized in particular areas of the developing nerve and muscle tissues, whereas in newborn and adult earthworms they were restricted to ventral muscular fibers and to some CNS cells. Biogenic amines were constantly present in the embryonic and adult nervous tissues. Immunologically-related peptides were detectable after organogenesis. Our results provide indirect evidence for a role of cholinesterases in regulating early intercellular communications, neurogenesis and myogenesis, and support the hypothesis that some conservative sequences of messenger peptides arose very early in evolution.

We have investigated the ability of the neutral ionophore ETH 129 to translocate Ca2+ across artificial and biological membranes. ETH 129 induces Ca2+ transport across planar lipid bilayer. The zero-current membrane potential in a gradient of Ca2+ concentration exhibits Nernst behavior. The dependence of the membrane conductance on ionophore and Ca2+ concentration indicates that three ionophore molecules are needed to transfer one Ca2+ across the hydrophobic region of the membrane. In mitochondria the neutral Ca2+ ionophore can move Ca2+ inside in response to a negative membrane potential under conditions in which the endogenous uniporter is blocked by ruthenium red. This electrophoretic transport of Ca2+ by ETH 129 occurs at a concentration much lower than the one previously reported with the neutral Ca2+ ionophore ETH 1001. Using sea urchin eggs, we have also shown that the efficiency of ETH 129 in inducing egg activation, as revealed by cortical granules exocytosis, is four orders of magnitude higher than that of the commonly used Ca2+ ionophore A21387. ETH 129 is a very efficient and useful tool for use in the investigation of Ca(2+)-dependent biological processes.

By use of several lectins (ConA, WGA, SBA, GS I, PNA), a study was carried on gametes and developing embryos of the sea urchin Paracentrotus lividus, to investigate developmental changes in cell surface, leading to changements in cell-environment interactions. ConA, WGA, and SBA, with high affinity, bind to the vitelline membrane of unfertilized eggs, while PNA labelling at the same site is weak; GS I-binding is only present in the cytoplasm and cortical region of the unfertilized eggs. Immediately after fertilization, no ConA-binding is present in the membrane, while WGA- and SBA-binding molecules are located in the fertilization layer. In zygotes, 40 min after fertilization, ConA affinity sites were again present in both cytoplasm and cortical region. During cleavages and gastrulation, ConA binds to the blastomere cytoplasm and cortical region, to the intercellular matrix, and to the cytoplasm of mesenchyme cells. WGA binds to the cortical region of cleaving blastomeres, including the hyaline layer, up to the unhatched blastula. Then it labels the gastrula inner and outer surfaces. SBA binds to the blastomere membranes; no GS I- and PNA-binding was detected during embryonic development. Sperms are bound by all the lectins, except GS I. Mannose and glucose conjugates are the most represented throughout the whole development of P. lividus, and their origin and locations are developmentally regulated. Galacto-residues are scarcely represented or are masked by other terminal sugars (e.g. sialic acid), and become functional during particular developmental events (cell movements).

In the present work we tested the effects of cholinergic drugs on the early development of P. lividus. Fertilization was performed in the presence of cholinergic drugs, and the embryos were fixed after 2 hours, when the controls (untreated) completed the second segmentation cleavage. We identified four classes of stages in the development of the embryos affected by the drugs, i.e.: unhatched zygotes, first cleaved stage, second cleaved stage (= unaffected), and anomalous embryos. Statistical analysis indicated that that drugs with analogous action mechanism caused similar alterations in the distribution of the treated embryos in these four classes. This finding supports the hypothesis of the presence of a complete "pre-nervous" cholinergic system, with a true cholinergic role. It is tempting to speculate that this system is involved in the first cell to cell interactions during early segmentation and possibly in the block to polyspermy.

The effects of the spider toxin alpha-latrotoxin (alpha-LTX) on gametes, zygotes, and early embryos of the sea urchin Paracentrotus lividus have been investigated by in vivo experiments and by histochemical studies of acetylcholinesterase (AChE) activity. Treatment of unfertilized eggs with nanomolar amounts (1 to 0.3 nmol/l) of alpha-LTX neither triggered cortical granule exocytosis, nor prevented the elevation of the fertilization layer by sperms. Instead, fertilized eggs exposed to alpha-LTX showed noticeable alterations in cell surface topography, including the appearance of prominent membrane-limited blebs. Moreover, the zygotes treated with 1 nmol/l alpha-LTX failed to cleave. The histochemical staining of treated zygotes revealed a very strong AChE activity in the cortical region, including blebs. An enzyme reaction was also found in the perivitellin space. Our results suggest the hypothesis that some alpha-LTX receptors may appear after fertilization, supporting the awareness that fertilized eggs display excitable cell features.

Experiments are described, showing the presence of putative nicotinic cholinoreceptors in the egg after fertilization. The experiments were carried out on gametes and early embryos of the sea urchin Paracentrotus lividus, by using nicotinic agonists and antagonists. 1 mM Acetylcholine (ACh), 100 microM nicotine, 100 nM alpha-bungarotoxin (alpha-BuTx) and 100 microM curare inhibit sperm motility and fertilization, while they have no effect on unfertilized eggs. The drugs added within 1 min. after the raising of the fertilization layer had stronger effects on cleavage and development; when added more than 15 min. after the raising of the fertilization layer, they had lesser effects on further development up to pluteus stage. In all the experiments, nicotine was the most effective drug. The binding of fluorescein-labelled alpha-BuTx did not point out any affinity sites on unfertilized eggs, while they were localized on the sperms and on the eggs fertilized by sperms, but not on the eggs activated artificially. The binding was prevented by pretreatment of sperms and activated eggs with 10 nM native alpha-BuTx and 10 microM curare. We conclude that, in the fertilized egg, putative nicotinic cholinoreceptors are present, which are able to bind alpha-BuTx and curare. Fertilization by sperms is needed to trigger the formation of alpha-BuTx receptors.

The effects of specific inhibitors of cholinesterases on chick development were studied. Inhibitors were injected into the eggs, at final concentration ranging between 1 mM and 10 nM. Their effects were depending on inhibitor concentration, and detectable at stages as more advanced as more diluted were the inhibitors. The strongest teratogenic effects on gastrulation, neurulation and morphogenesis were caused by BW 284c51, specific inhibitor of acetylcholinesterase. Its effects were compared to those of ion channels blockers. The inhibitors action seems to be correlated to an altered cholinergic system and to consequently altered intercellular communications.

The effects of weak electromagnetic signals on the early development of the sea urchin Paracentrotus lividus have been studied. The duration and repetition of the pulses were similar to those used for bone healing in clinical practice. A sequence of pulses, applied for a time ranging from 2 to 4 h, accelerates the cleavages of sea urchin embryo cells. This effect can be quantitatively assessed by determining the time shifts induced by the applied electromagnetic field on the completion of the first and second cleavages in a population of fertilized eggs. The exposed embryos were allowed to develop up to the pluteus stage, showing no abnormalities.

Acetylcholinesterase (AChE) activity was determined in normal and malignant human cell lines by histochemical methods. In normal human fibroblasts, no AChE activity could be demonstrated by any histochemical technique or substrate. Enzymic activity was observed in HT-1080 human fibrosarcoma cells, RD 2 human rhabdomyosarcoma cells, and SW 311 human colon carcinoma cells. Activity was localized around the nuclear envelope, in the cytoplasm and associated with the cortical region of most cells. The specificity of the reaction was shown through the use of specific cholinesterase inhibitors.

Cholinesterase activity was detected by histochemical methods in normal human blood smears. In erythrocytes, acetylcholinesterase was found to be localized in the cortical region of the cells and, to a lesser degree, in the inner cytoplasm. In leucocytes, cholinesterase activity was found around the nuclear membrane and in the cytoplasm. The specificity of the enzymic activity was ascertained by using selective inhibitors.

The distribution of acetylcholinesterase (AChE) and pseudocholinesterase (BCHE) activities was studied by histochemical, quantitative and electrophoretical methods during the early development of chick limbs, from stage 16 to stage 32 H.H. (Hamburger & Hamilton, 1951). By quantitative methods, true AChE activity was found, and increased about threefold during the developmental period, together with a smaller amount of BCHE which increased more rapidly in comparison with the AChE activity from stage 25 to 32 H.H. Cholinesterase activity was histochemically localized mainly in interacting tissues, such as the ectoderm (including the apical ectodermal ridge) and the underlying mesenchyme. True AChE was histochemically localized around the nuclei and on the plasma membrane of ectodermal (including AER) and mesenchymal cells, and at the plasma membrane of mesenchymal cell processes reaching the basal lamina between the ectoderm and the mesenchyme. AChE together with BCHE activity was found in the basal lamina between the ectoderm and the mesenchyme, in underlying mesenchymal cells and in deeper mesenchymal cells, especially during their transformation into unexpressed chondrocytes. During limb morphogenesis, the cellular and regional localization of the enzyme activities showed variations depending on the stage of development and on the occurrence of interactions. The possibility of morphogenetic functions of the enzyme id discussed.

The presence of "embryonic" acetylcholinesterase activity, as described by Drews (1975) was investigated during early chick embryonic development, mainly in the following systems: a) primitive streak and Hensen's node during gastrulation movements; b) area opaca during blood islets and vessels differentiation; c) mesoderma of lateral laminae, during delamination movements. The demonstration of enzymic activity was performed with slightly modified histochemical methods. The enzyme was thus localized around the nuclei, in the cytoplasm and associated to plasma membrane of cells engaged in morphogenetic movements. The enzyme activity localized at the plasma membrane was supposed to be involved in the regulation of membrane functions concerning intercellular communications, such as inductive message, perhaps mediated by ion fluxes.

The presence of different kinds of cells in the gastric tubular glands of the Elasmobranchian Cartilaginous fish "Raja asterias" was investigated by histochemical and ultrastructural methods. Our study demonstrates that the gastric glands, unlike those of the other non-Mammalian Vertebrates, contain in the distal portion, near the neck zone, the oxyntic-like cells, and in the proximal portion the zymogenic-like cells. The former presents histochemical and ultrastructural patterns similar to the Mammalian oxyntic cells, the latter to the Mammalian peptic cells. Between these two types there are always cells with intermediate aspects. These features support the presence of two different stages in the secretory activity: at first the gastric tubular cell is implicated in the secretion of hydrochloric acid, later on, in the secretion of pepsinogen.

A modified histochemical procedure was used to detect specific acetylcholinesterase localization in cultured human cells. Enzymic activity was found in cytoplasm in the perinuclear zone and in the plasma membrane of 20% to 40% of HT-1080 human fibrosarcoma cells. The presence of pseudocholinesterase was excluded using specific substrates and inhibitors.