The sea urchin, Paracentrotus lividus, embryo as a "bioethical" model for neurodevelopmental toxicity testing: effects of diazinon on the intracellular distribution of OTX2-like proteins.

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.

Efficiency of two different transfection reagents for use with human NTERA2 cells.

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.

Cell signalling during sea urchin development: a model for assessing toxicity of environmental contaminants.

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.

Interaction between organophosphate compounds and cholinergic functions during development.

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 beta-core fragment of human chorionic gonadotrophin inhibits growth of Kaposi's sarcoma-derived cells and a new immortalized Kaposi's sarcoma cell line.

OBJECTIVE: Kaposi's sarcoma (KS), a condition often associated with HIV infection, is more common in men than in women; pregnancy and sex hormones could be involved. Urinary human chorionic gonadotrophin (hCG) has been reported to inhibit the growth of KS cell lines, with great variability among preparations. Urinary hCG often contains free forms of the hCG subunits and a fragment of the free beta-subunit, the beta-core, which may have biological activity. We compared the effect of the beta-core fragment, the beta-subunit, recombinant and urinary hCG on KS immortal and spindle cells. DESIGN AND METHODS: A new immortal KS cell line was phenotypically and karyotypically characterized. The effects on growth of this cell line and of primary KS spindle cells by hCG and its purified derivatives were tested. Induction of apoptosis was demonstrated using acridine orange/ethidium bromide staining. RESULTS: The beta-core fragment harboured the most potent growth inhibitory activity on a molar basis. After 72 h of treatment with the beta-core, 60-70% of KS cells show apoptotic nuclei. No effects were observed on endothelial cells. CONCLUSIONS: The beta-core fragment of hCG proved to be the most effective part of the hCG molecule, inducing growth inhibition and apoptosis of KS cells. Thus, the beta-core could be the most appropriate hCG derivative for the therapy of KS.

Distinct chemotactic and angiogenic activities of peptides derived from Kaposi's sarcoma virus encoded chemokines.

The vMIPs are chemokine-like proteins expressed by the Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) during the lytic phase of viral infection. vMIP-I activates CCR8, a chemokine receptor expressed by Th2 lymphocytes and cultured monocytes. vMIP-II is an agonist for CCR3, a receptor expressed by eosinophils, and an antagonist for several other chemokine receptors. Both are highly angiogenic in the chick chorio-allantoic membrane. We designed and tested three 26-mer peptides, derived from vMIP-I (pK-I), from vMIP-II (pK-II) and from the control MIP-1alpha (pM), spanning key residues of chemokines. pK-I, pK-II and pM all were able to activate a strong chemotactic response in monocytes, higher than parental vMIP-I and II. This corresponded to induction of calcium fluxes in these cells, typical of chemokines. Interestingly, pK-II and pM were also active on PMN neutrophils. In vivo studies (matrigel sponge and rabbit cornea models) showed that pK-I retains the strong angiogenic potential exerted by vMIP-I, while pK-II and pM induced an inflammatory response, probably mediated by PMN recruitment. Our observations indicate that chemokine-derived peptides can show biological activity at pharmacological concentrations. pK-I, in particular, displays the angiogenic activity of full-length vMIP-I, while all peptides appear to have acquired additional properties, stimulating new cellular targets.

Antiapoptotic and antigenotoxic effects of N-acetylcysteine in human cells of endothelial origin.

N-Acetylcysteine (NAC) is a drug bearing multiple preventive properties that can inhibit genotoxicity and carcinogenicity. NAC also inhibits invasion and metastasis of malignant cells, as well as tumor take. We recently demonstrated the effects of NAC on Kaposi's sarcoma cells supernatant-induced invasion in vitro and angiogenesis in vivo. Many anticancer agents act through cytotoxicity of rapidly proliferating cells and several antineoplastic drugs induce apoptosis of cancer cells. Since endothelial cells are the target for the inhibition of angiogenesis, we wanted to verify that NAC, while inhibiting tumor vascularization and endothelial cell invasion would not induce endothelial cell apoptosis. We tested the ability of NAC to modulate apoptosis and cytogenetic damage in vitro and to promote differentiation on a reconstituted basement membrane (matrigel) in two endothelial cell lines (EAhy926 and HUVE). Treatment with NAC protected endothelial cells from TGF-beta-induced apoptosis and paraquat-induced cytogenetic damage. Therefore, NAC acts as an antiangiogenic agent and, at the same time, appears to prevent apoptosis and oxygen-related genotoxicity in endothelial cells.

The role of the thiol N-acetylcysteine in the prevention of tumor invasion and angiogenesis.

We have extensively studied the effects of N-acetylcysteine (NAC), a cytoprotective drug that can prevent in vivo carcinogenesis. Here we review our findings NAC completely inhibits gelatinolytic activity of metalloproteases and chemotactic and invasive activities of tumor cells. In addition, NAC reduces the number of lung metastases when malignant murine melanoma cells are injected into nude mice. NAC treatment decreases the weight of primary tumors and produces a dose-related increase in tumor latency. Moreover, oral administration of NAC reduces the formation of spontaneous metastases. In experimental metastasis assays, we have found a synergistic reduction in the number of lung metastases after treatment with doxorubicin (DOX) and NAC in nude mice. In tumorigenicity and spontaneous metastasis assays, the combined administration of DOX and oral NAC again has shown synergistic effects on the frequency and weight of primary tumors and local recurrences and completely prevented the formation of lung metastases. The addition of NAC to endothelial cells strongly reduces their invasive activity in response to angiogenic stimuli. NAC inhibited the degradation and release of radiolabeled type IV collagen by activated endothelial cells, indicating that NAC blocks gelatinase activity. Oral administration of NAC reduces the angiogenic response induced by KS tumor cell products, confirming the ability of NAC to inhibit the invasive activity of endothelial cells in vivo and thereby blocking angiogenesis.

Sister-chromatid exchanges, chromosomal aberrations and cytotoxicity produced by topoisomerase II-targeted drugs in sensitive (A2780) and resistant (A2780-DX3) human ovarian cancer cells: correlations with the formation of DNA double-strand breaks.

Doxorubicin, ellipticine and etoposide are antineoplastic drugs with topoisomerase II inhibitory activity. The relationship between drug-induced sister-chromatid exchanges (SCEs) or chromosomal aberrations (CAs) and cytotoxicity, or drug-induced DNA double-strand breaks (DSBs) and cytotoxicity, or drug-induced SCEs and DSBs was investigated in human ovarian cancer cells sensitive (A2780) and resistant (A2780-DX3) to topoisomerase II inhibitors. 30-min drug treatments produced SCEs, CAs and DSBs in sensitive cells, doxorubicin being more potent than etoposide at equimolar concentrations. The same treatments of resistant (A2780-DX3) cells did not produce chromosomal damage (SCEs, CAs, DSBs) and no cytotoxicity was observed. A plot of cytotoxicity versus SCEs indicated a good correlation between these two parameters for topoisomerase II inhibitors and not for mytomicin C. The plot of DSBs versus SCEs also showed a very good correlation.

Acetylcholine synthesis and possible functions during sea urchin development.

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.

Toxicity of metal oxide nanoparticles in immune cells of the sea urchin.

The potential toxicity of stannum dioxide (SnO2), cerium dioxide (CeO2) and iron oxide (Fe3O4) 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.

Apoptosis as a specific biomarker of diazinon toxicity in NTera2-D1 cells.

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.

The cAMP analog 8-Cl-cAMP inhibits growth and induces differentiation and apoptosis in retinoblastoma cells.

Retinoblastomas appear to be derived from a multipotential stem cell of the retina, due to alterations of the Rb1 gene. These tumors arise only within a discrete time frame during childhood, prior to terminal differentiation of the retinal precursor cells. Treatment of retinoblastoma cells with certain agents can induce a partial differentiation of cell types resembling those of the mature retina, such as rod and cone photoreceptors, glia, conventional neurons and pigment epithelia. We have tested the effects of 8-Cl-cAMP, a synthetic analog of cAMP which preferentially binds to and activates the RII subunit of protein kinase A on the Y-79 retinoblastoma cell line in vitro. Y-79 cells treated with 8-Cl-cAMP produced short, branching processes and showed a substantial increase in staining for neuron-specific enolase, a marker for conventional neuronal differentiation. In contrast, dibutyryl-cAMP gives a strong increase in the glial marker glial acidic fibrillary protein. Y-79 cell proliferation was strongly inhibited by 8-Cl-cAMP at concentrations as low as 5-25 microM. 8-Cl-cAMP significantly increased the rate of apoptosis of Y-79 cells in a dose-dependent manner. It also modulated expression of the RI regulatory subunit of intracellular cAMP-dependent protein kinase A, which is produced in abnormal quantities by Y-79 cells. A decrease in protein production was observed, with no clear effect on the RI subunit mRNA expression, suggesting that RI regulation occurs post-transcriptionally.

KSHV sequences in biopsies and cultured spindle cells of epidemic, iatrogenic and Mediterranean forms of Kaposi's sarcoma.

The pathogenesis of Kaposi's sarcoma (KS) is still unclear, and several factors appear to be involved in the onset of the Kaposi's lesion. Epidemiological studies suggest that a common infective agent may contribute to KS. Sequences which appear to represent a new gammaherpesvirus, currently termed KSHV/HHV8, have recently been identified in KS. To further examine the relationship between this virus and KS, we obtained biopsy samples of KS lesions; these samples, the spindle cells cultured from these lesions and the PBMC of the same patients were tested for the presence of KSHV sequences by PCR. In addition, we tested several "late passage" KS spindle cell lines as well as control samples. The biopsy samples were from lesions of the following forms of KS: one sporadic KS, two epidemic KS and three iatrogenic KS, one of which was in the process of regressing after reduction of immunosuppressive therapy, and two that were at different stages (patch and nodular) from a single patient. The sporadic KS specimen was positive, as were the PBMCs from this patient, and cells grown from this biopsy appeared to contain KSHV viral sequences up to the fifth passage. Both epidemic KS biopsies were positive, but in these cases KSHV sequences were not detected in the cultured cells. The biopsy from the regressing iatrogenic KS lesion was negative, as were the cells cultured from this lesion. However, the PBMCs of this patient were weakly positive for KSHV at the time of biopsy, and PBMCs collected from this patient one month later were completely negative. The samples of both the patch and the nodular KS lesions obtained from another immunosuppressed patient showed amplifiable sequences of KSHV, but both the PBMCs of this patient and primary KS cell cultures from these biopsies were negative. Of the late-passage KS lines tested, only one, IST AIDS KS 12, was positive for KSHV. This line is derived from an early angiomatous-macula lesion. Taken together, these data suggest that an active KSHV infection is associated with KS and that elimination of KSHV from the lesion precedes regression of the lesion, strongly correlating KSHV with KS. In addition, early KS lesions may have a higher KSHV burden, or contain cells more susceptible to KSHV infection, further linking KSHV to KS.

Inhibition of CXCR4-dependent HIV-1 infection by extracellular HIV-1 Tat.

Certain chemokines inhibit HIV replication through binding to cell surface receptors which also act as viral coreceptors. Based on our previous observations that HIV-1 Tat can interact with alpha- and beta-chemokine receptors, we investigated the potential effect of extracellular Tat (ecTat) on infection and replication of CCR5-dependent (R5) and CXCR4-using (X4) HIV-1 strains in primary activated peripheral blood mononuclear cells (PBMC) of uninfected donors. Receptor desensitization and binding competition studies were used to determine chemokine receptor binding by ecTat. Standard HIV replication assays based on reverse transcriptase (RT) activity determination in culture supernatants of PBMC and real time PCR for HIV-1 gag DNA were used to determine potential effects on early (entry or RT) steps of infection. ecTat bound to CXCR4 expressing monocytes and mitogen-activated PBMC, and competed with the natural ligand of CXCR4, SDF-1alpha (stromal cell-derived factor-1alpha) in calcium mobilization assays. EcTat inhibited replication of the X4 HIV-1 (LAI/IIIB strain) in activated PBMC at concentrations close to those of SDF-1alpha, whereas it only modestly interfered with R5 HIV-1 (BaL) replication in PBMC. Both SDF-1alpha and ecTat inhibited accumulation of X4 HIV-1 gag DNA, indicating interference with viral entry and/or RT. Our data show the surprising and counter-intuitive observation that ecTat selectively represses X4 HIV replication. This could favour spreading of R5 viruses, a condition observed in vivo immediately after transmission and in the early asymptomatic phase of infection.

N-acetylcysteine inhibits endothelial cell invasion and angiogenesis.

The thiol N-acetylcysteine (NAC) is a chemopreventive agent that acts through a variety of mechanisms and can prevent in vivo carcinogenesis. We have previously shown that NAC inhibits invasion and metastasis of malignant cells as well as tumor take. Neovascularization is critical for tumor mass expansion and metastasis formation. We investigated whether a target of the anti-cancer activity of NAC could be the inhibition of the tumor angiogenesis-associated phenotype in vitro and in vivo using the potent angiogenic mixture of Kaposi's sarcoma cell products as a stimulus. Two endothelial (EAhy926 and human umbilical vein endothelial [HUVE]) cell lines were utilized in a panel of assays to test NAC ability in inhibiting chemotaxis, invasion, and gelatinolytic activity in vitro. NAC treatment of EAhy926 and HUVE cells in vitro dose-dependently reduced their ability to invade a reconstituted basement membrane, an indicator of endothelial cell activation. Invasion of HUVE cells was inhibited with an ID50 of 0.24 mM NAC, whereas inhibition of chemotaxis required a 10 fold higher doses, indicating that invasion is a preferential target. NAC inhibited the enzymatic activity and conversion to active forms of the gelatinase produced by endothelial cells. The matrigel in vivo assay was used for the evaluation of angiogenesis; NAC strongly inhibited neovascularization of the matrigel sponges in response to Kaposi's sarcoma cell products. NAC prevented angiogenesis while preserving endothelial cells, implying that it could be safely used as an anti-angiogenic treatment.

HIV-1 Tat protein mimicry of chemokines.

The HIV-1 Tat protein is a potent chemoattractant for monocytes. We observed that Tat shows conserved amino acids corresponding to critical sequences of the chemokines, a family of molecules known for their potent ability to attract monocytes. Synthetic Tat and a peptide (CysL24-51) encompassing the "chemokine-like" region of Tat induced a rapid and transient Ca2+ influx in monocytes and macrophages, analogous to beta-chemokines. Both monocyte migration and Ca2+ mobilization were pertussis toxin sensitive and cholera toxin insensitive. Cross-desensitization studies indicated that Tat shares receptors with MCP-1, MCP-3, and eotaxin. Tat was able to displace binding of beta-chemokines from the beta-chemokine receptors CCR2 and CCR3, but not CCR1, CCR4, and CCR5. Direct receptor binding experiments with the CysL24-51 peptide confirmed binding to cells transfected with CCR2 and CCR3. HIV-1 Tat appears to mimic beta-chemokine features, which may serve to locally recruit chemokine receptor-expressing monocytes/macrophages toward HIV producing cells and facilitate activation and infection.