Ciguatoxins activate the Calcineurin signalling pathway in Yeasts: Potential for development of an alternative detection tool?

Ciguatoxins (CTXs) are lipid-soluble polyether compounds produced by dinoflagellates from the genus Gambierdiscus spp. typically found in tropical and subtropical zones. This endemic area is however rapidly expanding due to environmental perturbations, and both toxic Gambierdiscus spp. and ciguatoxic fishes have been recently identified in the North Atlantic Ocean (Madeira and Canary islands) and Mediterranean Sea. Ciguatoxins bind to Voltage Gated Sodium Channels on the membranes of sensory neurons, causing Ciguatera Fish Poisoning (CFP) in humans, a disease characterized by a complex array of gastrointestinal, neurological, neuropsychological, and cardiovascular symptoms. Although CFP is the most frequently reported non bacterial food-borne poisoning worldwide, there is still no simple and quick way of detecting CTXs in contaminated samples. In the prospect to engineer rapid and easy-to-use CTXs live cells-based tests, we have studied the effects of CTXs on the yeast Saccharomyces cerevisiae, a unicellular model which displays a remarkable conservation of cellular signalling pathways with higher eukaryotes. Taking advantage of this high level of conservation, yeast strains have been genetically modified to encode specific transcriptional reporters responding to CTXs exposure. These yeast strains were further exposed to different concentrations of either purified CTX or micro-algal extracts containing CTXs. Our data establish that CTXs are not cytotoxic to yeast cells even at concentrations as high as 1µM, and cause an increase in the level of free intracellular calcium in yeast cells. Concomitantly, a dose-dependent activation of the calcineurin signalling pathway is observed, as assessed by measuring the activity of specific transcriptional reporters in the engineered yeast strains. These findings offer promising prospects regarding the potential development of a yeast cells-based test that could supplement or, in some instances, replace current methods for the routine detection of CTXs in seafood products.

LUCS (Light-Up Cell System), a universal high throughput assay for homeostasis evaluation in live cells.

Observations of fluorescent cyanine dye behavior under illumination at 500 nm lead to a novel concept in cell biology allowing the development of a new live cell assay called LUCS, for Light-Up Cell System, measuring homeostasis in live cells. Optimization of the LUCS process resulted in a standardized, straightforward and high throughput assay with applications in toxicity assessment. The mechanisms of the LUCS process were investigated. Electron Paramagnetic Resonance experiments showed that the singlet oxygen and hydroxyl radical are involved downstream of the light effect, presumably leading to deleterious oxidative stress that massively opens access of the dye to its intracellular target. Reversible modulation of LUCS by both verapamil and proton availability indicated that plasma membrane proton/cation antiporters, possibly of the MATE drug efflux transport family, are involved. A mechanistic model is presented. Our data show that intracellular oxidation can be controlled by tuning light energy, opening applications in regulatory purposes, anti-oxidant research, chemotherapy efficacy and dynamic phototherapy strategies.

Comparative cytotoxicity induced by bulk and nanoparticulated ZnO in the fish and human hepatoma cell lines PLHC-1 and Hep G2.

The increasing presence of ZnO nanoparticles (NPs) in consumer products may be having a dramatic impact in aquatic environments. The evaluation of ZnO NP toxicity represents a great challenge. This study aimed at evaluating the cytotoxic effect of micro- and nanosized ZnO in a fish and a mammalian hepatoma cell line. A detailed characterisation of the particles in exposure media showed that ZnO NPs formed large aggregates. ZnO cytotoxicity was evaluated with a battery of in vitro assays including LUCS, a new approach based on DNA alteration measurements. In fish cells, ZnO NP aggregates contributed substantially to the cytotoxic effects whereas toxicity in the human cells appeared to be mainly produced by the dissolved fraction. ROS production did not contribute to the observed cytotoxicity. This work also showed that measuring concentrations of NPs is essential to understand the mechanisms underlying their toxicity.

Collaborative study for the detection of toxic compounds in shellfish extracts using cell-based assays. Part II: application to shellfish extracts spiked with lipophilic marine toxins.

Successive unexplained shellfish toxicity events have been observed in Arcachon Bay (Atlantic coast, France) since 2005. The positive mouse bioassay (MBA) revealing atypical toxicity did not match the phytoplankton observations or the liquid chromatography-tandem mass spectrometry (LC-MS/MS) investigations used to detect some known lipophilic toxins in shellfish. The use of the three cell lines (Caco2, HepG2, and Neuro2a) allows detection of azaspiracid-1 (AZA1), okadaic acid (OA), or pectenotoxin-2 (PTX2). In this study, we proposed the cell-based assays (CBA) as complementary tools for collecting toxicity data about atypical positive MBA shellfish extracts and tracking their chromatographic fractionation in order to identify toxic compound(s). The present study was intended to investigate the responses of these cell lines to shellfish extracts, which were either control or spiked with AZA1, OA, or PTX2 used as positive controls. Digestive glands of control shellfish were extracted using the procedure of the standard MBA for lipophilic toxins and then tested for their cytotoxic effects in CBA. The same screening strategy previously used with pure lipophilic toxins was conducted for determining the intra- and inter-laboratory variabilities of the responses. Cytotoxicity was induced by control shellfish extracts whatever the cell line used and regardless of the geographical origin of the extracts. Even though the control shellfish extracts demonstrated some toxic effects on the selected cell lines, the extracts spiked with the selected lipophilic toxins were significantly more toxic than the control ones. This study is a crucial step for supporting that cell-based assays can contribute to the detection of the toxic compound(s) responsible for the atypical toxicity observed in Arcachon Bay, and which could also occur at other coastal areas.

Collaborative study for the detection of toxic compounds in shellfish extracts using cell-based assays. Part I: screening strategy and pre-validation study with lipophilic marine toxins.

Human poisoning due to consumption of seafood contaminated with phycotoxins is a worldwide problem, and routine monitoring programs have been implemented in various countries to protect human consumers. Following successive episodes of unexplained shellfish toxicity since 2005 in the Arcachon Bay on the French Atlantic coast, a national research program was set up to investigate these atypical toxic events. Part of this program was devoted to fit-for-purpose cell-based assays (CBA) as complementary tools to collect toxicity data on atypical positive-mouse bioassay shellfish extracts. A collaborative study involving five laboratories was conducted. The responses of human hepatic (HepG2), human intestinal (Caco2), and mouse neuronal (Neuro2a) cell lines exposed to three known lipophilic phycotoxins-okadaic acid (OA), azaspiracid-1 (AZA1), and pectenotoxin-2 (PTX2)-were investigated. A screening strategy composed of standard operating procedures and a decision tree for dose-response modeling and assay validation were designed after a round of "trial-and-error" process. For each toxin, the shape of the concentration-response curves and the IC(50) values were determined on the three cell lines. Whereas OA induced a similar response irrespective of the cell line (complete sigmoid), PTX2 was shown to be less toxic. AZA1 induced cytotoxicity only on HepG2 and Neuro2a, but not on Caco2. Intra- and inter-laboratory coefficients of variation of cell responses were large, with mean values ranging from 35 to 54 % and from 37 to 48 %, respectively. Investigating the responses of the selected cell lines to well-known toxins is the first step supporting the use of CBA among the panel of methods for characterizing atypical shellfish toxicity. Considering these successful results, the CBA strategy will be further applied to extracts of negative, spiked, and naturally contaminated shellfish tissues.

Image-free assessment of protein translocation in live cells.

Protein translocation is a universal event shared by most cell signalling pathways to transmit signals between cell compartments. In recent years, the use of new fluorescence microscopy technologies combined with fluorescent probes--most often fluorescent proteins--and image analysis software has allowed the visualization and extensive analysis of such dynamic events in the context of the living cell. This review article focuses on emerging fluorescence approaches that tackle live cell protein translocation in the image-free context. Such methods are based on either protein-protein interactions or analysis of spatial diffusion of proteins by fluorescence intensity measurements. The potential benefits of intensity measurement on global cell populations versus image analysis of heterogeneous cell sample are discussed in the context of drug discovery applications.

Biological characterization of rodent and human vasopressin V1b receptors using SSR-149415, a nonpeptide V1b receptor ligand.

[(3)H]SSR-149415 is the first tritiated nonpeptide vasopressin V(1b) receptor (V(1b)R) antagonist ligand. It was used for studying rodent (mouse, rat, hamster) and human V(1b)R from native or recombinant origin. Moreover, a close comparison between the human and the mouse V(1b)R was performed using SSR-149415/[(3)H]SSR-149415 in binding and functional studies in vitro. [(3)H]SSR-149415 binding was time-dependent, reversible, and saturable. Scatchard plot analysis gave a single class of high-affinity binding sites with apparent equilibrium dissociation constant (K(d)) approximately 1 nM and maximum binding density (B(max)) values from 7,000 to 300,000 sites/cell according to the cell line. In competition experiments, [(3)H]SSR-149415 binding was stereospecific and dose-dependently displaced by reference peptide and nonpeptide arginine vasopressin (AVP)/OT ligands following a V(1b) rank order of affinity: SSR-149415 = AVP > dCha > dPen > dPal > dDavp > SSR-126768A > SR-49059 > SSR-149424 > OT > SR-121463B. Species differences between human, rat, mouse, and hamster V(1b)R were observed. Autoradiography studies with [(3)H]SSR-149415 on rat and human pituitary showed intense specific labeling confined to corticotroph cells and absence of labeling in the other tissues examined. SSR-149415 potently and stereospecifically antagonized the AVP-induced inositol phosphate production and intracellular Ca(2+) increase (EC(50) from 1.83 to 3.05 nM) in recombinant cell lines expressing either the mouse or the human V(1b)R. AVP (10(-7) M) exposure of AtT20 cells expressing mouse or human EGFP-tagged V(1b)R induced their rapid internalization. Preincubation with 10(-6) M SSR-149415 counteracted the internalization process. Moreover, recycling of internalized receptors was observed upon 10(-6) M SSR-149415 treatment. Thus SSR-149415/[(3)H]SSR-149415 are unique tools for studying animal and human V(1b)R.

Position 4 analogues of [deamino-Cys(1)] arginine vasopressin exhibit striking species differences for human and rat V(2)/V(1b) receptor selectivity.

Arginine vasopressin (AVP) mediates a wide variety of biological actions by acting on three distinct G-protein coupled receptors, termed V(1a) (vascular), V(1b) (pituitary) and V(2) (renal). It also binds to the oxytocin (OT) receptor. As part of a program aimed at the design of selective agonists for the human V(1b) receptor, we recently reported the human V(1b), V(1a), V(2) and OT receptor affinities of the following position 4 substituted analogues of [deamino-Cys(1)] arginine vasopressin (dAVP)-(1) d[Leu(4)]AVP, (2) d[Orn(4)]AVP, (3) d[Lys(4)]AVP, (4) d[Har(4)]AVP, (5) d[Arg(4)]AVP, (6) d[Val(4)]AVP, (7) d[Ala(4)]AVP, (8) d[Abu(4)]AVP, (9) d[Nva(4)]AVP, (10) d[Nle(4)]AVP, (11) d[Ile(4)]AVP, (12) d[Phe(4)]AVP, (13) d[Asn(4)]AVP, (14) d[Thr(4)]AVP: (15) d[Dap(4)]AVP. With the exception of Nos. 7 and 12, all peptides exhibit very high affinities for the human V(1b) receptor. Furthermore, peptides 1-4 exhibit high selectivities for the human V(1b) receptor with respect to the V(1a), V(2) and OT receptors and, with d[Cha(4)]AVP, in functional tests, are the first high affinity selective agonists for the human V(1b) receptor (Cheng LL et al., J. Med. Chem. 47: 2375-2388, 2004). We report here the pharmacological properties of peptides 1-4, 5 (from a resynthesis), 7, 9-13, 15 in rat bioassays (antidiuretic, vasopressor and oxytocic) (in vitro: no Mg(++)) with those previously reported for peptides 5, 6, 8, 14. We also report the rat V(1b), V(1a), V(2) and OT receptor affinities of peptides 1-5 and the rat V(2) receptor affinities for peptides: 7-15.The antidiuretic activities in units/mg of peptides 1-15, are: 1=378; 2=260; 3=35; 4=505; 5=748; 6=1150; 7=841; 8=1020; 9=877; 10=1141; 11=819, 12=110; 13=996; 14=758; 15=1053. Peptides 1-4 exhibit respectively the following rat and human (in brackets) V(2) receptor affinities: 1=3.1 nm (245 nm); 2=3.4 nm (1125 nm); 3=24.6 nm (11,170 nm); 4=0.6 nm (1386 nm). Their rat V(1b) receptor affinities are 1=0.02 nm; 2=0.45 nm; 3=9.8 nm; 4=0.32 nm. Their rat V(1a) receptor affinities are 1=1252 nm; 2=900 nm; 3=1478 nm; 4=32 nm. Their rat oxytocin (OT) receptor affinities are 1=481 nm; 2=997 nm; 3=5042 nm; 4=2996 nm. All four peptides have high affinities and selectivities for the rat V(1b) receptor with respect to the rat V(1a) and OT receptors. However, in contrast to their high selectivity for the human V(1b) receptor with respect to the human V(2) receptor, they are not selective for the V(1b) receptor with respect to the V(2) receptor in the rat. These findings confirm previous observations of profound species differences between the rat and human V(2) receptors. Peptides 1-4 are promising leads to the design of the first high affinity selective agonists for the rat V(1b) receptor.

Comparative pharmacology of bovine, human and rat vasopressin receptor isoforms.

In this study, we characterized the bovine vasopressin V(1a), V(1b), V(2) receptor isoforms and compared their pharmacological properties to those of corresponding rat and human vasopressin receptor subtypes. Specific binding sites of high affinity for vasopressin were found in all bovine tissues tested (kidney, liver and pituitary). Using a large series of recent peptidic and non-peptidic selective vasopressin agonists or antagonists, we demonstrated the presence of vasopressin V(2), V(1a) or V(1b) receptors in the kidney, liver and pituitary bovine tissues, respectively. This extensive characterization of bovine vasopressin receptor isoforms validates the pharmacological vasopressin receptor classification earlier established for the rat and human species. As expected, the bovine vasopressin receptors look much more like human receptors than rat ones. Interestingly, among the three vasopressin receptor isoforms studied, the vasopressin V(1b) receptor subtype is the best conserved for the three species studied.

Design of potent and selective agonists for the human vasopressin V1b receptor based on modifications of [deamino-cys1]arginine vasopressin at position 4.

The glutamine(4) residue in [deamino-Cys(1)]arginine vasopressin (dAVP) was replaced by a broad series of aliphatic, aromatic, polar, and charged amino acids to give the following peptides: d[Gly(4)]AVP (1), d[Ala(4)]AVP (2), d[Abu(4)]AVP (3), d[Nva(4)]AVP (4), d[Nle(4)]AVP (5), d[Leu(4)]AVP (6), d[Ile(4)]AVP (7), d[Thi(4)]AVP (8), d[Phe(4)]AVP (9), d[Tyr(4)]AVP (10), d[Trp(4)]AVP (11), d[Asn(4)]AVP (12), d[Ser(4)]AVP (13), d[Thr(4)]AVP (14), d[Dap(4)]AVP (15), d[Dab(4)]AVP (16), d[Orn(4)]AVP (17), d[Lys(4)]AVP (18), d[Arg(4)]AVP (19), d[Har(4)]AVP (20), and d[Glu(4)]AVP (21). All peptides were synthesized by solid-phase methods using BOC chemistry for all but one peptide (8), which required the use of Fmoc chemistry. The binding and functional properties of these position 4 substituted analogues of dAVP (d[X(4)]AVP) and the previously reported d[Cha(4)]AVP (Derick et al. Endocrinology 2002, 143, 4655-4664) were evaluated on human arginine vasopressin (AVP) V(1a), V(1b), and V(2) receptors and on the human oxytocin (OT) receptor expressed in living Chinese hamster ovary (CHO) cells. Binding studies revealed that broad modifications of the fourth residue of dAVP do not significantly alter affinity for the human V(1b) receptor. Only aromatic (Phe, Tyr, Trp) or negatively charged (Glu) residues reduce V(1b) affinity. By contrast, the human V(1a) and more particularly the human V(2) and the OT receptors are more sensitive to many of these modifications. Thus, the replacement of the Gln(4) residue of dAVP by aliphatic (Leu, Cha) or positively charged (Orn, Lys, Arg, Har) amino acids led to analogues exhibiting drastic reductions of their affinity for the human V(1a), V(2), and OT receptors. Consequently, in addition to the previously reported d[Cha(4)]AVP, peptides 6 and 17-20 display excellent selectivities for the human V(1b) receptor. The key structural requirement responsible for optimal V(1b) selectivity appears to be the length and branching of the aliphatic side chain of the fourth residue of dAVP. Functional studies performed on CHO cells expressing the different human AVP/OT receptors confirm the V(1b) selectivity of peptides 6, 17, 18, 20, and d[Cha(4)]AVP. However, d[Arg(4)]AVP (19), which triggers an excellent coupling between the human V(2) receptor and adenylyl cyclase, was found to exhibit both V(1b) and V(2) agonism in functional tests. More interestingly, these functional experiments revealed that, depending on the AVP/OT receptor, a given d[X(4)]AVP analogue may behave as a full agonist or as a partial agonist. This strongly suggests that the fourth residue of dAVP plays an important role in the coupling between the hormone-receptor complex, the heterotrimeric G protein, and the effectors. In conclusion, the synthesis of these d[X(4)]AVP analogues led to the discovery of new V(1b) agonists with high affinity and greatly enhanced selectivities. Thus, in addition to d[Cha(4)]AVP, d[Leu(4)]AVP (6), d[Orn(4)]AVP (17), d[Lys(4)]AVP (18), and d[Har(4)]AVP (20) are useful new tools for studying the structure and the function of the human V(1b) receptor.

Functional and pharmacological characterization of the first specific agonist and antagonist for the V1b receptor in mammals.

By activating three distinct vasopressin receptor isoforms called V1a-R, V1b-R (V3-R) and V2-R, vasopressin (VP) mediates a wide number of biological effects in mammals and may be involved in several pathological states. Up to now only specific V1a and V2 receptor agonists and antagonists have been successfully designed. The role of the V1b-R still remains partially unknown, due to the lack of selective V1b-R ligands and orally-active molecules, which are crucial tools for investigating the central and peripheral functions or pathological disorders associated with this receptor. In this review, we report the biological and pharmacological properties of the first two specific V1b-R ligands: d[Cha4] AVP, a high affinity V1b-R agonist and SSR149415, a potent orally-active V1b-R antagonist with good selectivity with respect to other VP/OT receptor isoforms and able to control ACTH secretion in vitro and in vivo. Indeed, these molecules constitute invaluable tools for exploring the central and peripheral roles of VP mediated via V1b receptors. Interestingly, SSR149415 displays potent anxiolytic and antidepressant-like activities, indicating that this new class of drugs has a promising therapeutical potential in the treatment of stress-related disorders, anxiety and depression.