A cadmium toxicity assay using stress responsive Caenorhabditis elegans mutant strains

To test the applicability of Caenorhabditis elegans mutant for toxicity screening, the sensitivity of cadmium (Cd) in C. elegans was investigated on 14 mutant strains using median lethal concentration (LC50) tests, with further analysis on growth and reproduction conducted on five selected strains. The 24 h LC50 of Cd observed on the wildtype and mutant strains of C. elegans was in the order of age-1(hx546) > mtl-2(gk125) > sod-3(gk235) > daf-21(p673) > cyp35a2(gk317) > skn-1(or13) > daf-12(rh62rh157) > hsp-16.2(gk249) > daf-18(e1375) > ctl-2(ok1137) > wildtype(N2) > sod-1(or13) > daf-16(mu86) > cep-1(gk138) > cdr-2(ok1996). Compared to the wildtype response, a decreased reproduction potential was observed in mtl-2(gk125), sod-3(gk235), cdr-2(ok1996) and cep-1(gk138) strains. To gain a mechanistic understanding of different sensitivities of the mutant strains, a time-course gene expression analysis was also performed on the five genes. A dramatic increase in the expression of the mtl-2 gene due to Cd exposure confirmed the importance of this gene in C. elegans Cd toxicity. An increased expression of the sod-3 gene at the longer exposure time period (48 h) suggests that oxidative stress may not be a direct toxic mechanism, but may rather be a consequence of Cd toxicity. Even though, LC50 values for the age-1(hx546) mutant strain were the highest among the tested strains, the response on the reproduction potential in age-1(hx546) mutant was unchanged compared to the wildtype, and the age-1 gene expression remained unaltered on exposure to Cd, which may be interpreted as the maintenance of age-1 expression level is needed for the exertion of Cd toxicity; however, the role of the age-1 gene in Cd toxicity may not be via a reproduction-related pathway. The overall results suggest that the C. elegans mutant assay seems to be a promising tool for the study of toxic mechanisms, as well as for toxicity screening in ecotoxicological research.     

Susceptible genes regulate the adverse effects of TiO2-NPs at predicted environmental relevant concentrations on nematode Caenorhabditis elegans

Contributions from mutations of susceptible genes to TiO₂-NPs toxicity at environmental relevant concentrations (ERCs) and the underlying mechanism are largely unclear. After prolonged exposure, among the examined 19 mutants associated with oxidative stress or stress response, we show that sod-2, sod-3, mtl-2, and hsp-16.48 were susceptible genes for TiO₂-NPs toxicity on reproduction and locomotion behavior, sod-2, sod-3, and mtl-2 were susceptible genes for TiO₂-NPs toxicity on survival and intestinal development, and mtl-2 was susceptible gene for TiO₂-NPs toxicity on development. Mutations of these susceptible genes, together with sensitive endpoints, could be used to evaluate TiO₂-NPs toxicity at the concentration of 0.0001 μg/L. Our results imply the usefulness of identified susceptible genes in assessing the potential nanotoxicity of engineered nanomaterial (ENM) at ERCs. One important mechanism to explain property of identified susceptible genes for TiO₂-NPs toxicity was that mutations of these susceptible genes enhanced the uptake of TiO₂-NPs into body of nematodes.From the Clinical EditorThis team of authors identified susceptibility genes influencing the uptake and consequential toxicity of TiO₂ nanoparticles in a nematode, highlighting the general importance of investigating genetic influence on nanoparticle delivery.     

Hormetic effect of methylmercury on Caenorhabditis elegans

Research has demonstrated the toxic effects of methylmercury (MeHg), yet molecular mechanisms underlying its toxicity are not completely understood. Caenorhabditis elegans (C. elegans) offers a unique biological model to explore mechanisms of MeHg toxicity given many advantages associated with its ease of use and genetic power. Since our previous work indicated neurotoxic resistance of C. elegans to MeHg, the present study was designed to examine molecular mechanisms associated with this resistance. We hypothesized MeHg would induce expression of gst, hsp or mtl in vivo since glutathione (GSH), heat shock proteins (HSPs), and metallothioneins (MTs) have shown involvement in MeHg toxicity. Our studies demonstrated a modest, but significant increase in fluorescence in gst-4::GFP and mtl-1::GFP strains at an acute, low L1 MeHg exposure, whereas chronic L4 MeHg exposure induced expression of gst-4::GFP and hsp-4::GFP. Knockout gst-4 animals showed no alterations in lethality sensitivity compared to wildtype animals whereas mtl knockouts displayed increased sensitivity to MeHg exposure. GSH levels were increased by acute MeHg treatment and depleted with chronic exposure. We also demonstrate that MeHg induces hormesis, a phenotype whereby a sublethal exposure to MeHg rendered C. elegans resistant to subsequent exposure to the organometal. The involvement of gst-4, hsp-4, mtl-1, and mtl-2 in hormesis was examined. An increase in gst-4::GFP expression after a low-dose acute exposure to MeHg indicated that gst-4 may be involved in this response. Our results implicate GSH, HSPs, and MTs in protecting C. elegans from MeHg toxicity and show a potential role of gst-4 in MeHg-induced hormesis.     

The neurotoxic effects of heavy metal exposure on GABAergic nervous system in nematode Caenorhabditis elegans

The number of cell body or synapse made by Caenorhabditis elegans GABAergic neurons is constant during development. The neurotoxic effects of metal (Pb, Hg, Cu, Cd, Cr, and Mn) exposure on GABAergic motor neurons were investigated in C. elegans. Exposure to examined metals could not alter the position of GABA neurons, whereas exposure to high concentrations (75 μM and 200 μM) of metals caused noticeable axonal degeneration and neuronal loss in nerve cords, suggesting neurodegeneration will be induced by metal exposure to different degrees. In addition, exposure to Pb, Hg, Cu, and Cd at the low concentration (2.5 μM) could also induce obviously neuronal loss. Moreover, exposure to high concentrations (75 μM and/or 200 μM) of most of examined metals significantly reduced the relative size and fluorescent intensities of AVL, RMEs, and RIS neurons. Therefore, the neurodegeneration and abnormal structures may be formed in GABAergic motor neurons after metal exposure, and the endpoint of neuronal loss will be useful for the neurotoxicity assessment from trace metal exposure.     

Copper and Chromium toxicity is mediated by oxidative stress in Caenorhabditis elegans: The use of nanoparticles as an immobilization strategy

Environmental contamination by heavy metals (HMs) has impelled searching for stabilization strategies, where the use of zero-valent iron nanoparticles (nZVI) is considered a promising option. We have evaluated the combined effect of Cu(II)-Cr(VI) on two Caenorhabditis elegans strains (N2 and RB1072 sod-2 mutant) in aqueous solutions and in a standard soil, prior and after treatment with nZVI (5% w/w). The results showed that HMs aqueous solutions had an intense toxic effect on both strains. Production of reactive oxygen species and enhanced expression of the heat shock protein Hsp-16.2 was observed, indicating increased HM-mediated oxidative stress. Toxic effects of HM-polluted soil on worms were higher for sod-2 mutant than for N2 strain. However, nZVI treatment significantly diminished all these effects. Our findings highlighted C. elegans as a sensitive indicator for HMs pollution and its usefulness to assess the efficiency of the nanoremediation strategy to decrease the toxicity of Cu(II)-Cr(VI) polluted environments.     

Establishing a novel C. elegans model to investigate the role of autophagy in amyotrophic lateral sclerosis

Aim:

To develop a C. elegans model of amyotrophic lateral sclerosis (ALS) and to evaluate the role of autophagy in the disease.

Methods:

Stable transgenic worms expressing the G93A mutant form of Cu,Zn-superoxide dismutase (SOD1) in GABAergic motor neurons were generated. Axon guidance and protein aggregation in the motor neurons were observed with fluorescence microscopy. A paralysis assay was performed to evaluate the motor function of the transgenic worms. The expression of autophagic genes in daf-2(e1370) mutants was analyzed using real-time PCR. The reporter GFP::LGG-1 was used to verify whether autophagy was induced in motor neurons.

Results:

Expression of G93A SOD1 in motor neurons caused age-dependent motor defects accompanied by significant SOD1 aggregation and axon guidance failure. After 12 d, over 80% of the G93A worms became paralyzed, whereas less than 10% of the controls showed a paralytic phenotype. In the daf-2(e1370) mutants of C. elegans, the levels of autophagic genes bec-1, atg-7, lgg-1, and atg-18 were upregulated by approximately 1.5-fold, the level of unc-51 increased by approximately fourfold, and autophagosomes in motor neurons was markedly increased. Crossing the daf-2(e1370) mutation into the G93A SOD1 mutant worms significantly ameliorated the motor defects, SOD1 aggregation, and axon guidance failure.

Conclusion:

G93A SOD1 expression in motor neurons of C. elegans results in characteristic alterations of ALS. Increased autophagy protects C. elegans motor neurons against the toxicity of mutant SOD1.     

In vitro-selected resistance to fluoroquinolones in two Brucella strains associated with mutational changes in gyrA

In the present study, Brucella melitensis biovar Abortus 2308 and Brucella abortus 3196 biotype 5 reference strains, which are susceptible to fluoroquinolones, became in vitro-resistant to fluoroquinolones by culture in trypticase soy agar. The quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes of the two reference strains were analysed by polymerase chain reaction sequencing analysis to obtain the wild-type sequence. These sequences were then compared with the corresponding sequences of four in vitro-selected fluoroquinolone-resistant mutants to characterise mutations associated with resistance. Sequencing of the ofloxacin-selected resistant mutant 2308 revealed a transition of GAT to AAT (corresponding to position 87 of Escherichia coli gyrA), leading to substitution of Asp91 → Asn, whilst at the same position the ciprofloxacin-selected resistant mutant 2308 revealed a transition of GAT to TAT (corresponding to the same position of E. coli as above), leading to substitution of Asp91 → Tyr. The ofloxacin-selected resistant mutant 3196 had a transition of GCT to GTT, generating an amino acid change of Ala87 → Val. Amino acid changes were detected in the portion of the Brucella gyrA gene (Ala71 to Gln110) corresponding to the E. coli gyrA QRDR region (Ala67 to Gln110). Amino acid changes were also detected in Ser83, corresponding to the region where fluoroquinolone-associated amino acid changes are most commonly found in other bacterial species.     

Pre-treatment with mild metal exposure suppresses the neurotoxicity on locomotion behavior induced by the subsequent severe metal exposure in Caenorhabditis elegans

Adaptive response to neurotoxicity on locomotion behavior by severe metal exposure was investigated in Caenorhabditis elegans. Exposure to 2.5 μM of metals induced a moderate but significant reduction of locomotion behavior and induction of hsp-16.2::gfp expression. After pre-exposure to 2.5 μM of metals, the reduced locomotion behavior induced by subsequent 50 and 100 μM of metal exposure were significantly prevented, and the induction of hsp-16.2::gfp expression caused by subsequent 50 and 100 μM of metal exposure were significantly suppressed. In contrast, after pre-exposure to 50 μM examined metals, the reduced locomotion behavior induced by subsequent 50 and 100 μM metal exposure were further decreased, and the noticeable induction of hsp-16.2::gfp expression caused by subsequent severe metal exposure were further enhanced. Therefore, pre-treatment with mild metal exposure can activate the adaptive response to neurotoxicity on locomotion behavior induced by subsequent severe metal exposure in nematodes.     

The β-adrenergic receptor-adenyl-cyclase system of rat reticulocytes

Summary Non-nucleated red blood cells from rats contain adenyl cyclase, the activity of which is predominantly localized in the reticulocytes. Basal enzyme activities in membrane preparations from reticulocyte-rich blood (pretreatment of rats with acetyl-phenylhydrazide: about 60% reticulocytes) are about 5 times higher than in preparations from reticulocyte-poor blood (untreated animals: 2–3% reticulocytes). The enzyme activities are stimulated 10-fold by sodium fluoride (10^–2 M) and 6 to 8-fold by isoprenaline (10^–4 M).Adenyl cyclase activities in membrane preparations from reticulocyte-rich and reticulocyte-poor blood can be ascribed to identical enzymes since identical apparent K _m (ATP; 3×10^–4 M), K _a (isoprenaline; 3×10^–6 M) and K _i (propranolol vs. isoprenaline; 3×10^–7 M) values were obtained in both preparations.Besides NaF, only phenylethanolamine derivatives with -adrenergic receptor stimulant properties were effective as stimulators of adenyl cyclase activity. The affinities (apparent K _a values) of the investigated compounds decreased in the order isoprenaline—hexoprenaline—fenoterol—salbutamol—adrenaline—terbutaline—noradrenaline—phenylephrine. For maximal intrinsic activity, the catechol structure was essential; the relative intrinsic activities of resorcinol derivatives did not exceed 0.6.The isoprenaline-stimulated adenyl cyclase activities in erythrocyte membrane preparations were competitively inhibited by -adrenergic blocking drugs, the affinities (apparent K _i values) decreasing in the order prindolol—penbutolol—propranolol—practolol. The dextrorotatory enantiomers of penbutolol and propranolol were 1/100 to 1/200 as active as the resp. levorotatory enantiomers.From experiments with -adrenergic agonists (e.g. phenylephrine) and antagonists (e.g. phentolamine), it is concluded that -adrenergic receptors do not interfere with the -adrenergically-mediated cAMP formation in these particular membranes.A variety of hormones and drugs known to stimulate adenyl cyclase activities in various tissues, e.g. ACTH, glucagon, STH, erythropoietin, prostaglandin E ₁ etc. did not affect adenyl cyclase activity in reticulocyte-rich erythrocyte membrane preparations.In contrast to adenyl cyclase activity, phosphodiesterase activities in erythrocyte membrane and cytoplasmic fractions were only twice as high in reticulocyterich as in reticulocyte-poor preparations.From the experiments described, it is obvious that the adenyl cyclase of the rat reticulocyte is subject to monovalent-hormonal, i.e. -sympathomimetic stimulation. Moreover, the premature red blood cell provides a useful model for quantitative studies of the interaction of drugs with the -adrenergic receptor.This study was supported by the Deutsche ForschungsgemeinschaftPreliminary accounts were presented at meetings of the Deutsche Pharmakologische Gesellschaft (Gauger et al., 1972; Gauger and Palm, 1973; Quiring et al., 1974a).     

Potential analgesic and anti-inflammatory activities of Panax ginseng head butanolic fraction in animals

The present study reports the potential anti-rheumatoid activity of Panax ginseng head part. P. ginseng-head part BuOH fraction (PGHB) was safe in acute toxicity (LD₅₀ > 5000 mg/kg) and inhibited the partially acetic acid-induced writhes (approximately 32%, P < 0.05) in mice. PGHB (500 mg/kg) inhibited the acetic acid-induced extravasation of Evan’s blue dye in mice by approximately 20.6% (P < 0.05), and was similar to the suppressive effect of ibuprofen (27.7%) as a positive control drug. Also, PGHB reduced the carrageenan-induced paw edema at 3 h after oral administration, and suppressed the production of serum IL-6 in CIA mice. This suggests that PGHB has potential analgesic and anti-inflammatory activities, and will be the supporting evidence for the potential anti-rheumatoid activity of Korean P. ginseng-head.     

In vivo toxicity studies of europium hydroxide nanorods in mice

Lanthanide nanoparticles and nanorods have been widely used for diagnostic and therapeutic applications in biomedical nanotechnology due to their fluorescence and pro-angiogenic properties to endothelial cells, respectively. Recently, we have demonstrated that europium (III) hydroxide [Eu^III(OH)₃] nanorods, synthesized by the microwave technique and characterized by several physico-chemical techniques, can be used as pro-angiogenic agents which introduce future therapeutic treatment strategies for severe ischemic heart/limb disease, and peripheral ischemic disease. The toxicity of these inorganic nanorods to endothelial cells was supported by several in vitro assays. To determine the in vivo toxicity, these nanorods were administered to mice through intraperitoneal injection (IP) everyday over a period of seven days in a dose dependent (1.25 to 125 mg kg^− 1 day^− 1) and time dependent manner (8–60 days). Bio-distribution of europium elements in different organs was analyzed by inductively coupled plasma mass spectrometry (ICPMS). Short-term (S-T) and long-term (L-T) toxicity studies (mice euthanized on days 8 and 60 for S-T and L-T, respectively) show normal blood hematology and serum clinical chemistry with the exception of a slight elevation of liver enzymes. Histological examination of nanorod-treated vital organs (liver, kidney, spleen and lungs) showed no or only mild histological changes that indicate mild toxicity at the higher dose of nanorods.     

A comparison of the stability of doxorubicin and daunorubicin in solid state

The degradation of doxorubicin and daunorubcin in the solid state was studied using an HPLC method with UV detection (LiChrospher RP-18, 5 μm, 250 mm × 4 mm; mobile phase: acetonitrile-solution A 1:1, v/v (solution A: 2.88 g of laurisulfate sodium and 1.6 ml of phosphoric acid(V) in 1000 ml); flow rate – 1.4 ml min⁻¹; UV detection – 254 nm). The degradation of doxorubicin was a first-order reaction depending on the substrate concentration and daunorubicin degraded according to the kinetic model of autocatalysis. The dependence ln k_i = f(1/T) was described by the equations ln k_DOX = 40.0 ± 15.6 – (19804 ± 5682) (1/T) and ln k_DAU = 35.9 ± 11.3 – (16581 ± 3972) (1/T) at 76.4% RH. The dependence ln k_i = f(RH%) was described by the equations ln k_DOX = (8.80 ± 3.60) × 10⁻² (RH%) – (21.50 ± 2.57) and ln k_DAU = (6.63 ± 1.22) × 10⁻² (RH%) – (13.35 ± 1.68). The thermodynamic parameters (E_a, ΔH^≠a, ΔS^≠a) of the degradation of doxorubicin and daunorubicin were calculated. Although the degradation of doxorubicin was slower at increased temperature (353–373 K) and relative air humidity (50.9–90.0%), the differences between the influence of temperature and relative air humidity on the stability doxorubicin and of daunorubicin were not significant.     

A comparison of the short-term toxicity of cadmium to indigenous and alien gammarid species

Amphipods play an important role in many aquatic ecosystems and are commonly used in ecotoxicology and ecosystem health assessment. Several alien gammarids have been introduced in many regions of the world during the last decades. In this study, we investigated if differences in cadmium sensitivity occurred between (1) different species belonging to the family Gammaridae and (2) different populations of the same species originating from a polluted or a non-polluted site. The acute cadmium toxicity to two indigenous (Gammarus pulex and Gammarus fossarum) and four alien (Dikerogammarus villosus, Echinogammarus berilloni, Gammarus roeseli and Gammarus tigrinus) gammarids occurring in Belgium was tested. Significant differences (P < 0.05) in median lethal concentrations (LC₅₀) were found between the different species, with 72 h-LC50s ranging from 6.3 to 268 μg/l and 96 h-LC50s from 4.7 to 88.9 μg/l. No clear trend in Cd sensitivity was found when comparing indigenous and alien gammarids. D. villosus, an alien invasive species, was the most sensitive to Cd toxicity and E. berilloni, another alien species, the least sensitive. In addition, larger Gammarid species were more sensitive to Cd toxicity than smaller ones. No significant differences were found between populations of the same species originating from metal polluted sites or non-polluted sites. Overall, our results showed that considerable differences in Cd sensitivity exist between gammarid species, which should be taken into consideration in environmental risk assessment and water quality standard setting. Finally, our data suggest that alien gammarids would not have an advantage over indigenous gammarids in Cd contaminated environments.     

Involvement of Multidrug Resistance-Associated Protein 1 in Intestinal Toxicity of Methotrexate

Purpose  Methotrexate (MTX) causes dose-limiting gastrointestinal toxicity due to exposure of intestinal tissues, and is a substrate of the multidrug resistance-associated protein (MRP) 1. Here we examine the involvement of MRP1, which is reported to be highly expressed in the proliferative crypt compartment of the small intestine, in the gastrointestinal toxicity of MTX. Methods  MTX was intraperitonealy administered to mrp1 gene knockout (mrp1 ^(−/−)) and wild-type (mrp1 ^(+/+)) mice. Body weight, food and water intake were monitored, intestinal histological studies and pharmacokinetics of MTX were examined. Results   mrp1 ^(−/−) mice more severely decreased body weight, food and water intake than mrp1 ^(+/+) mice. Almost complete loss of villi throughout the small intestine in mrp1 ^(−/−) mice was observed, whereas the damage was only partial in mrp1 ^(+/+) mice. Plasma concentration and biliary excretion profiles of MTX were similar in mrp1 ^(−/−) and mrp1 ^(+/+) mice, though accumulation of MTX in immature proliferative cells isolated from mrp1 ^(−/−) mice was much higher compared to mrp1 ^(+/+) mice. Immunostaining revealed localization of Mrp1 in plasma membrane of the intestinal crypt compartment in mrp1 ^(+/+) mice, but not in mrp1 ^(−/−) mice. Conclusion  Mrp1 determines the exposure of proliferative cells in the small intestine to MTX, followed by gastrointestinal toxicity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Viscosimetric investigation of the interaction between sodium dodecylsulfate micelles and a polymer drug carrier

The viscosities of aqueous sodium dodecyl sulfate solutions with and without α,β-poly(N-hydroxyethyl)-dl-aspartamide (PHEA), at 15, 25 and 35°C are reported. The viscosities of SDS and of PHEA aqueous solutions are discussed in terms of the parameter − 1)/φ] describing the non-ideal behavior of SDS micelles and of PHEA macromolecules. The viscosities of SDS plus PHEA aqueous solutions, discussed in terms of the parameter F [F = η_rel(PHEA) + η_rel(SDS) − η_rel(SDS + PHEA)]M, demonstrate the occurrence of interactions between SDS micelles and the PHEA macromolecule. Both D and F are scarcely influenced by temperature variation.     

The effects of dietary nickel exposure on growth and reproduction of Daphnia magna

Although there is growing evidence that dietborne metals can be toxic to various aquatic species, there is still insufficient knowledge to integrate this information in environmental risk assessment procedures. In this study, we investigated the effects of a 21-day exposure of Daphnia magna to a control diet (i.e. the green alga Pseudokirchneriella subcapitata containing <4.0 μg Ni/g dry wt) and five diets with elevated Ni concentrations (i.e. the same alga contaminated with Ni burdens between 33.7 and 837 μg Ni/g dry wt). A significant accumulation of dietborne Ni in D. magna, i.e. between 49.6 and 72.5 μg Ni/g dry wt, was observed when they were fed with diets containing between 85.6 and 837 μg Ni/g dry wt. This was paralleled by a significant reduction of reproduction (by 33.1%), measured as the total number of juvenile offspring per female and growth (by 9.1%), measured as the carapax length of 21-day-old females. Life-history analysis showed that the time to first brood of Ni exposed organisms was between 7.8 and 8.2 days, and occurred 0.7–1.1 days earlier than for the control organisms (time to first brood = 8.9 days). The number of offspring in the first brood was significantly reduced (by 21–33% compared to the control) in all dietary treatments. Longer exposure (≥8.9 days, i.e. from the second brood onwards) led to a reduction of brood size only when given diets containing 85.6 and 837 μg Ni/g dry wt. The results suggest that a variety of mechanisms may be involved in the effects of dietary Ni exposure, including altered resource allocation or targeted reproductive inhibition. While Ni exposure clearly altered the quality of the diet (measured as essential ω3 polyunsaturated fatty acid content and C:P ratio), we found no conclusive evidence that these diet quality shifts could have affected growth or total reproductive output. More research is required to fully understand the mechanisms of Ni toxicity associated with the dietary exposure route.     

Stimulation of dopamine release by nicotinic acetylcholine receptor ligands in rat brain slices correlates with the profile of high, but not low, sensitivity α4β2 subunit combination

α4β2 neuronal nicotinic receptors (nAChRs) can exist in high and low sensitivity states possibly due to distinct stoichiometries during subunit assembly: (α4)₂(β2)₃ pentamer (high sensitivity, HS) and (α4)₃(β2)₂ pentamer (low sensitivity, LS). To determine if there is a linkage between HS or LS states and receptor-mediated responses in brain, we profiled several clinically studied α4β2* nAChR agonists for the displacement of radioligand binding to α4β2 [³H]-cytisine sites in rat brain membranes, effects on stimulation of [³H]-dopamine release from slices of rat prefrontal cortex and striatum, and activation of HS and LS human α4β2 nAChRs expressed in Xenopus laevis oocytes. Binding affinities (pK_i) and potency (pEC₅₀) values for [³H]-dopamine release closely correlated with a rank order: varenicline > (−)-nicotine > AZD3480 > dianicline  ABT-089. Further, a good correlation was observed between [³H]-dopamine release and HS α4β2 pEC₅₀ values, but not between [³H]-dopamine release and LS α4β2. The relative efficacies of the agonists ranged from full to partial agonists. Varenicline behaved as a partial agonist in stimulating [³H]-dopamine release and activating both HS and LS α4β2 nAChRs expressed in oocytes. Conversely, ABT-089, AZD3480 and dianicline exhibited little efficacy at LS α4β2 (<5%), were more effective at HS α4β2 nAChRs, and in stimulating cortical and striatal [³H]-dopamine release ≥30%. In the presence of α-conotoxin MII to block α6β2* nAChRs, the α4β2* α-conotoxin-insensitive [³H]-dopamine release stimulated by these ligands correlates well with their interactions at HS, but not LS. In summary, this study provides support for HS α4β2* nAChR involvement in neurotransmitter release.     

The occurrence of domoic acid linked to a toxic diatom bloom in a new potential vector: The tunicate Pyura chilensis (piure)

The tunicate Pyura chilensis (Molina, 1782); Phylum Chordata; Subphylum Urochordata; Class Ascidiacea, common local name “piure” or sea squirt; a filter-feeder (plankton and suspended particles) sessile species; may play an important role in monitoring domoic acid (DA) the principal toxic component of Amnesic Shellfish Poisoning (ASP). Significant DA concentrations have been determined in tunicate samples, collected during a recent ASP outbreak in Bahía Inglesa, an important scallop (Argopecten purpuratus) farming area. Several infaunal species were tested for the presence of DA, in addition to the usual scallop monitoring programme. DA was found at sub-toxic levels in filtering bivalves such as mussels (Mytilus chilensis), large mussels (Aulacomya ater) and clams (Protothaca thaca) (6.4, 5.4 and 4.7 μg DA/g tissue respectively). Of particular interest was the observation of significant accumulations of toxic Pseudo-nitzschia sp. diatoms in the internal siphon and atrium spaces of the tunicate.Toxin distribution within major tunicate organs was heterogeneous with 8.7–15.5 μg DA/g in edible tissues, 14.9–17.9 μg DA/g in the fecal material and 13.6–32.7 μg DA/g in the gut content. DA was determined by HPLC–UV and confirmed by diode-array detection and LC–MS/MS analysis. This is the first report of the presence of DA in a tunicate that is regularly consumed by coastal populations. These results confirm the need to include these organisms in sanitation programs for marine toxins.