Comparative toxicity of antifouling compounds on the development of sea urchin

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Comparative toxicity of antifouling compounds on the development of sea urchin

In the present study, embryotoxicity experiments using the sea urchin Lytechinus variegatus were carried out to better clarify the ecotoxicological effects of tributyltin (TBT) and triphenyltin (TPT) (the recently banned antifouling agents), and Irgarol and Diuron (two of the new commonly used booster biocides). Organisms were individually examined to evaluate the intensity and type of effects on embryo-larval development, this procedure has not been commonly used, however it showed to be a potentially suitable approach for toxicity assessment. NOEC and LOEC were similar for compounds of same chemical class, and IC10 values were very close and showed overlapping of confidence intervals between TBT and TPT, and between Diuron and Irgarol. In addition, IC10 were similar to NOEC values. Regardless of this, the observed effects were different. Embryo development was interrupted at the gastrula and blastula stages at 1.25 and 2.5 mu g l(-1) of TBT, respectively, whereas pluteus stage was reached with the corresponding concentrations of TPT. Furthermore, embryos reached the prism and morula stages at 5 mu g l(-1) of TPT and TBT, respectively. The effects induced by Irgarol were also more pronounced than those caused by Diuron. Pluteus stage was always reached at any tested Diuron concentration, while embryogenesis was interrupted at blastula/gastrula stages at the highest concentrations of Irgarol. Therefore, this study proposes a complementary approach for interpreting embryo-larval responses that may be employed together with the traditional way of analysis. Consequently, this application leads to a more powerful ecotoxicological assessment tool focused on embryotoxicity.

Effects of ocean acidification on embryo development: Does encapsulation matter?

As the concentration of CO2 in surface seawaters increases (ocean acidification, or OA) the saturation of calcium carbonate decreases, preventing marine organisms from creating shells and other calcified structures. These effects of elevated CO2 on calcification have been previously shown in free-spawning larvae, but are not as well-studied in larvae that spend their early life stages in encapsulation. The focus of our study was to determine what effects CO2 would have on a diversity of encapsulated embryos, and whether different types of encapsulating structures provided different levels of protection against OA. We found only a moderate larval response to low (600 ppm), medium (1050 ppm), and high (1500 ppm) CO2 concentrations across all species taken as a whole, but did observe that several species/ populations exhibited a decline in shell length with no corresponding decline in inorganic content. This suggests that while calcification was not significantly decreased by our OA conditions, perhaps the morphology of certain shells changed, becoming wider and shorter. Our hatch times, which increased with elevated CO2, confirmed that increased CO2 placed embryos under stress during development.

Primary screening of the bioactivity of brackishwater cyanobacteria: toxicity of crude extracts to artemia salina larvae and paracentrotus lividus embryos

Cyanobacteria are a diverse group of Gram-negative bacteria that produce an array of secondary compounds with selective bioactivity against vertebrates, invertebrates, plants, microalgae, fungi, bacteria, viruses and cell lines. The aim of this study was to assess the toxic effects of aqueous, methanolic and hexane crude extracts of benthic and picoplanktonic cyanobacteria isolated from estuarine environments, towards the nauplii of the brine shrimp Artemia salina and embryos of the sea urchin Paracentrotus lividus. The A. salina lethality test was used as a frontline screen and then complemented by the more specific sea urchin embryo-larval assay. Eighteen cyanobacterial isolates, belonging to the genera Cyanobium, Leptolyngbya, Microcoleus, Phormidium, Nodularia, Nostoc and Synechocystis, were tested. Aqueous extracts of cyanobacteria strains showed potent toxicity against A. salina, whereas in P. lividus, methanolic and aqueous extracts showed embryo toxicity, with clear effects on development during early stages. The results suggest that the brackishwater cyanobacteria are producers of bioactive compounds with toxicological effects that may interfere with the dynamics of invertebrate populations.

A key role of TRPC channels in the regulation of electromechanical activity of the developing heart.

Aims It is well established that dysfunction of voltage-dependent ion channels results in arrhythmias and conduction disturbances in the foetal and adult heart. However, the involvement of voltage-insensitive cationic TRPC (transient receptor potential canonical) channels remains unclear. We assessed the hypothesis that TRPC channels play a crucial role in the spontaneous activity of the developing heart.Methods and results TRPC isoforms were investigated in isolated hearts obtained from 4-day-old chick embryos. Using RT-PCR, western blotting and co-immunoprecipitation, we report for the first time that TRPC1, 3, 4, 5, 6, and 7 isoforms are expressed at the mRNA and protein levels and that they can form a macromolecular complex with the alpha 1C subunit of the L-type voltage-gated calcium channel (Cav1.2) in atria and ventricle. Using ex vivo electrocardiograms, electrograms of isolated atria and ventricle and ventricular mechanograms, we found that inhibition of TRPC channels by SKF-96365 leads to negative chrono-, dromo-, and inotropic effects, prolongs the QT interval, and provokes first-and second-degree atrioventricular blocks. Pyr3, a specific antagonist of TRPC3, affected essentially atrioventricular conduction. On the other hand, specific blockade of the L-type calcium channel with nifedipine rapidly stopped ventricular contractile activity without affecting rhythmic electrical activity.Conclusions These results give new insights into the key role that TRPC channels, via interaction with the Cav1.2 channel, play in regulation of cardiac pacemaking, conduction, ventricular activity, and contractility during cardiogenesis.

Seawater carbonate chemistry and protein content, respiration, symbiodinium densities, survivorship of Pocillopora damicornis larvae in a laboratory experiment

Efforts to evaluate the response of coral larvae to global climate change (GCC) and ocean acidification (OA) typically employ short experiments of fixed length, yet it is unknown how the response is affected by exposure duration. In this study, we exposed larvae from the brooding coral Pocillopora damicornis to contrasts of temperature (24.00 °C [ambient] versus 30.49 °C) and pCO2 (49.4 Pa versus 86.2 Pa) for varying periods (1-5 days) to test the hypothesis that exposure duration had no effect on larval response as assessed by protein content, respiration, Symbiodinium density, and survivorship; exposure times were ecologically relevant compared to representative pelagic larval durations (PLD) for corals. Larvae differed among days for all response variables, and the effects of the treatment were relatively consistent regardless of exposure duration for three of the four response variables. Protein content and Symbiodinium density were unaffected by temperature and pCO2, but respiration increased with temperature (but not pCO2) with the effect intensifying as incubations lengthened. Survival, however, differed significantly among treatments at the end of the study, and by the 5th day, 78% of the larvae were alive and swimming under ambient temperature and ambient pCO2, but only 55-59% were alive in the other treatments. These results demonstrate that the physiological effects of temperature and pCO2 on coral larvae can reliably be detected within days, but effects on survival require > or = 5 days to detect. The detection of time-dependent effects on larval survivorship suggests that the influence of GCC and OA will be stronger for corals having long PLDs.

Physiological performance measures and tolerance limits of estuarine indicator species in south Florida

Current water management practices in South Florida have negatively impacted many species inhabiting Florida Bay. Variable and high salinity has been identified as a key stressor in these estuaries. The Comprehensive Everglades Restoration Plan (CERP) includes water redistribution projects that will restore natural freshwater flows to northeastern Florida Bay. My studies focused on the following central theme and hypotheses: Biological performance measures (i.e., growth, reproduction, survival), behavior (i.e., habitat preference and locomotor behavior) and diversity of estuarine fish will be controlled by changes in salinity and water quality that will occur as a result of the restoration of freshwater flow to the bay. A series of acute and subchronic physiological toxicity studies were conducted to determine the effects of salinity changes on the life stages (embryo/larval, juvenile, adult) and fecundity of four native estuarine fish (Cyprinodon variegatus, Floridichthys carpio, Poecilia latipinna, and Gambusia holbrooki). Fish were exposed to a range of salinity concentrations (freshwater to hypersaline) based on salinity profiles in the study areas. Growth (length, weight) and survival were measured. Salinity trials included both rapid and gradual change events. Results show negative effects of acute, abrupt salinity changes on fish survival, development and reproductive success as a result of salinity stress. Other studies targeted reproduction and critical embryo-larval/neonate development as key areas for detecting long-term population effects of salinity change in Florida Bay. Adults of C. variegates and P. latipinna were also examined for behavioral responses to pulsed salinity changes. These responses include changes in swimming performance, locomotor behavior and zone preference. Finally, an ecological risk assessment was conducted for adverse salinity conditions in northeastern Florida Bay. Using the U.S. EPA's framework, the risk to estuarine fish species diversity was assessed against regional salinity profiles from a 17-year database. Based on the risk assessment, target salinity profiles for these areas are recommended for managers.^

Physiological performance measures and tolerance limits of estuarine indicator species in South Florida

Current water management practices in South Florida have negatively impacted many species inhabiting Florida Bay. Variable and high salinity has been identified as a key stressor in these estuaries. The comprehensive Everglades Restoration Plan (CERP) includes water redistribution projects that will restore natural freshwater flows to northeastern Florida Bay. My studies focused on the following central theme and hypotheses: Biological performance measures (i.e., growth, reproduction, survival), behavior (i.e., habitat preference and locomotor behavior) and diversity of estuarine fish will be controlled by changes in salinity and water quality that will occur as a result of the restoration of freshwater flow to the bay. A series of acute and subchronic physiological toxicity studies were conducted to determine the effects of salinity changes on the life stages (embryo/larval, juvenile, adult) and fecundity of four native estuarine fish (Cyprinodon variegatus, Floridichthys carpio, Poecilia latipinna, and Gambusia holbrooki). Fishe were exposed to a range of salinity concentrations (freshwater to hypersaline) based on salinity profiles in the study areas. Growth (length, weight) and survival were measured. Salinity trials included both rapid and gradual change events. Results show negative effects of acute, abrupt salinity changes on fish survival, development and reproductive success as a result of salinity stress. Other studies targeted reproduction and critical embryo-larval/neonate development as key areas for detecting long-term population effects of salinity change in Florida Bay. Adults of C. variegatus and P. latipinna were also examined for behavioral responses to pulsed salinity changes. These responses include changes in swimming performance, locomotor behavior and zone preference. Finally, an ecological risk assessment was conducted for adverse salinity conditions in northeastern Florida Bay. Using the U.S. EPA's framework, the risk to estuarine fish species diversity was assessed against regional salinity profiles from a 17-year database. Based on the risk assessment, target salinity profiles for these areas are recommended for managers.

Understanding the role of CAP proteins in the polarization process of C. elegans

La division cellulaire asymétrique est un processus crucial dans le développement des organismes multicellulaires puisqu’elle permet la génération de la diversité cellulaire. Les cellules qui se divisent de façon asymétrique doivent tout d’abord se polariser et correctement orienter leur fuseau mitotique pour ségréger des déterminants cellulaires en deux entités distinctes. L’embryon du nématode C. elegans est un modèle robuste et largement utilisé pour étudier la division cellulaire asymétrique. Dans cet embryon, le point d'entrée du spermatozoïde détermine l'axe de polarité antéro-postérieur. Suite à la fécondation, le cortex embryonnaire est uniformément contractile et un complexe conservé formé des protéines PAR-3, PAR-6 et PKC-3 (nommé complexe PAR-3 ci-dessous) est localisé sur l'ensemble du cortex. La complétion de la méiose maternelle induit une relaxation corticale au postétieur et un flux cortical vers l’antérieur de l’embryon. Ces contractions corticales asymétriques mènent à la formation d'un domaine antérieur contenant le complexe PAR-3, tandis que le cortex postérieur, dont le complexe PAR-3 s’est délocalisé, est enrichi avec les protéines PAR-2 et PAR-1. Par conséquent, les domaines formés par les protéines PAR définissent un pôle antérieur et un pôle postérieur dans l'embryon suite au remodelage du cytosquelette. Les protéines PAR-4 et PAR-5 restent localisées de façon uniforme dans l'embryon. Curieusement, les protéines PAR exercent une régulation par rétroaction sur la contractilité corticale. Il a été montré qu’une des protéines PAR récemment identifiée, PAR-5, est orthologue à la protéine adaptatrice 14-3-3 et joue un rôle important dans la contractilité corticale. En dépit de son rôle central dans la contractilité corticale et le processus de polarisation cellulaire, le mécanisme par lequel PAR-5 régule la contractilité corticale n’est pas bien compris. Le but de ce projet est de mieux comprendre comment PAR-5 et ses interacteurs contrôlent la régulation des contractions corticales et, de ce fait, la polarité cellulaire. Dans un essai de capture de la protéine GST (GST pull-down), nous avons identifié plusieurs nouveaux interacteurs de PAR-5. Parmi ceux-ci, nous avons trouvé CAP-2 (protéine de coiffage de l'actine), qui a été identifiée dans des éxpériences de capture de 14-3-3 dans trois systèmes modèles différents. CAP-2 est un hétérodimère des protéines CAP, qui sont impliquées dans la régulation de l'actine. Nous avons trouvé que la déplétion des protéines CAP par interférence à l’ARN dans des vers de type sauvage mène à une augmentation létalité embryonnaire, ce qui suggère que ces protéines jouent un rôle important dans le développement embryonnaire. L'imagerie en temps réel d'embryons déplétés pour les protéines CAP montre qu’ils ont une diminution des contractions corticales avec un sillon de pseudoclivage mois stable, suggérant un défaut dans la régulation du cytosquelette d'actine-myosine. Ceci a également été confirmé par la diminution de la vitesse et du nombre de foci de NMY-2::GFP. En outre, ces embryons montrent une légère diminution de la taille du croissant cortical de PAR-2 lors de la phase d’établissement de la polarité. Les embryons déplétés en CAP-2 montrent également un retard dans la progression du cycle cellulaire, mais le lien entre ce phénotype et la régulation des contractions corticales reste à être précisé. La caractérisation des protéines CAP, des régulateurs du remodelage du cytosquelette, permettra d'améliorer notre compréhension des mécanismes qui sous-tendent l'établissement et le maintien de la polarité cellulaire, et donc la division cellulaire asymétrique.

Understanding the role of CAP proteins in the polarization process of C. elegans

La division cellulaire asymétrique est un processus crucial dans le développement des organismes multicellulaires puisqu’elle permet la génération de la diversité cellulaire. Les cellules qui se divisent de façon asymétrique doivent tout d’abord se polariser et correctement orienter leur fuseau mitotique pour ségréger des déterminants cellulaires en deux entités distinctes. L’embryon du nématode C. elegans est un modèle robuste et largement utilisé pour étudier la division cellulaire asymétrique. Dans cet embryon, le point d'entrée du spermatozoïde détermine l'axe de polarité antéro-postérieur. Suite à la fécondation, le cortex embryonnaire est uniformément contractile et un complexe conservé formé des protéines PAR-3, PAR-6 et PKC-3 (nommé complexe PAR-3 ci-dessous) est localisé sur l'ensemble du cortex. La complétion de la méiose maternelle induit une relaxation corticale au postétieur et un flux cortical vers l’antérieur de l’embryon. Ces contractions corticales asymétriques mènent à la formation d'un domaine antérieur contenant le complexe PAR-3, tandis que le cortex postérieur, dont le complexe PAR-3 s’est délocalisé, est enrichi avec les protéines PAR-2 et PAR-1. Par conséquent, les domaines formés par les protéines PAR définissent un pôle antérieur et un pôle postérieur dans l'embryon suite au remodelage du cytosquelette. Les protéines PAR-4 et PAR-5 restent localisées de façon uniforme dans l'embryon. Curieusement, les protéines PAR exercent une régulation par rétroaction sur la contractilité corticale. Il a été montré qu’une des protéines PAR récemment identifiée, PAR-5, est orthologue à la protéine adaptatrice 14-3-3 et joue un rôle important dans la contractilité corticale. En dépit de son rôle central dans la contractilité corticale et le processus de polarisation cellulaire, le mécanisme par lequel PAR-5 régule la contractilité corticale n’est pas bien compris. Le but de ce projet est de mieux comprendre comment PAR-5 et ses interacteurs contrôlent la régulation des contractions corticales et, de ce fait, la polarité cellulaire. Dans un essai de capture de la protéine GST (GST pull-down), nous avons identifié plusieurs nouveaux interacteurs de PAR-5. Parmi ceux-ci, nous avons trouvé CAP-2 (protéine de coiffage de l'actine), qui a été identifiée dans des éxpériences de capture de 14-3-3 dans trois systèmes modèles différents. CAP-2 est un hétérodimère des protéines CAP, qui sont impliquées dans la régulation de l'actine. Nous avons trouvé que la déplétion des protéines CAP par interférence à l’ARN dans des vers de type sauvage mène à une augmentation létalité embryonnaire, ce qui suggère que ces protéines jouent un rôle important dans le développement embryonnaire. L'imagerie en temps réel d'embryons déplétés pour les protéines CAP montre qu’ils ont une diminution des contractions corticales avec un sillon de pseudoclivage mois stable, suggérant un défaut dans la régulation du cytosquelette d'actine-myosine. Ceci a également été confirmé par la diminution de la vitesse et du nombre de foci de NMY-2::GFP. En outre, ces embryons montrent une légère diminution de la taille du croissant cortical de PAR-2 lors de la phase d’établissement de la polarité. Les embryons déplétés en CAP-2 montrent également un retard dans la progression du cycle cellulaire, mais le lien entre ce phénotype et la régulation des contractions corticales reste à être précisé. La caractérisation des protéines CAP, des régulateurs du remodelage du cytosquelette, permettra d'améliorer notre compréhension des mécanismes qui sous-tendent l'établissement et le maintien de la polarité cellulaire, et donc la division cellulaire asymétrique.

Biological Effects and Dose-Response Assessment of Diesel Exhaust Particles on In Vitro Early Embryo Development in Mice

An increased risk of early pregnancy loss in women briefly exposed to high levels of ambient particulate matter during the preconceptional period was recently observed. The effects of this exposure on early embryo development are unknown. This study was designed to assess the dose-response and biological effects of diesel exhaust particles (DEP) on in vitro embryo development using the in vitro fertilization (IVF) mouse model. Zygotes obtained from superovulated mice after IVF were randomly cultured in different DEP concentrations (0, 0.2, 2, and 20 mu g/cm(2)) for 5 days and observed for their capacity to attach and develop on a fibronectin matrix until day 8. Main outcome measures included blastocyst rates 96 and 120 h after insemination, hatching discriminatory score, total cell count, proportion of cell allocation to inner cell mass (ICM) and trophectoderm (TE), ICM morphology, attachment rate and outgrowth area, apoptosis and necrosis rates, and Oct-4 and Cdx-2 expression. Multivariate analysis showed a negative dose-dependent effect on early embryo development and hatching process, blastocyst cell allocation, and ICM morphology. Although blastocyst attachment and outgrowth were not affected by DEP, a significant impairment of ICM integrity was observed in day 8 blastocysts. Cell death through apoptosis was significantly higher after DEP exposure. Oct-4 expression and the Oct-4/Cdx-2 ratio were significantly decreased in day 5 blastocysts irrespective of DEP concentration. Results suggest that DEP appear to play an important role in disrupting cell lineage segregation and ICM morphological integrity even at lower concentrations, compromising future growth and viability of the blastocyst.

EFFICACY OF NITAZOXANIDE AGAINST Toxocara canis: LARVAL RECOVERY AND HUMORAL IMMUNE RESPONSE IN EXPERIMENTALLY INFECTED MICE

SUMMARY The efficacy of nitazoxanide (NTZ) against toxocariasis was investigated in an experimental murine model and results were compared to those obtained using mebendazole. Sixty male BALB/c mice, aged six to eight weeks-old, were divided into groups of 10 each; fifty were orally infected with 300 larvaed eggs of T. canisand grouped as follows, G I: infected untreated mice; G II: infected mice treated with MBZ (15 mg/kg/day) 10 days postinfection (dpi); G III: infected mice treated with NTZ (20 mg/kg/day) 10 dpi; G IV: infected mice treated with MBZ 60 dpi; G V: infected mice treated with NTZ 60 dpi; GVI: control group comprising uninfected mice. Mice were bled via retro-orbital plexus on four occasions between 30 and 120 dpi. Sera were processed using the ELISA technique to detect IgG anti- Toxocaraantibodies. At 120 dpi, mice were sacrificed for larval recovery in the CNS, liver, lungs, kidneys, eyes and carcass. Results showed similar levels of anti- ToxocaraIgG antibodies among mice infected but not submitted to treatment and groups treated with MBZ or NTZ, 10 and 60 dpi. Larval recovery showed similar values in groups treated with NTZ and MBZ 10 dpi. MBZ showed better efficacy 60 dpi, with a 72.6% reduction in the parasite load compared with NTZ, which showed only 46.5% reduction. We conclude that administration of these anthelmintics did not modify the humoral response in experimental infection by T. canis. No parasitological cure was observed with either drug; however, a greater reduction in parasite load was achieved following treatment with MBZ.

Human immune response to triatomine embryo extract

Dipetalogaster maximus embryo extracts were used to stimulate peripheral blood mononuclear cells (PBMC) and in ELISA with sera either from Trypanosoma cruzi infected or non-infected individuals. The results showed that there was significant proliferative response and high antibody titers in sera of chagasic patients.

Experimental lagochilascariosis: histopathological study of inflammatory response to larval migration in the Murine model

The goal of this study was to investigate the pattern of inflammatory response induced by Lagochilascaris minor in murine experimental model. For this purpose 115 mice were given 1000-3000 L. minor infective eggs "per os" and 51 uninfected mice were considered as controls. Four hours post-inoculation (PI), 3rd stage larvae were seen passing through the mucosa of terminal ends of small intestine. Six hours PI larvae were observed as an embolus inside the portal vein and also migrating through the liver parenchyma. During the first 24 h larvae-containing eggs of L. minor were observed in the lumen of intestinal tract. Two days PI larvae were seen migrating through lung parenchyma associated with an initial neutrophilic perivasculitis. From the 13th day of this experimental study, L. minor larvae were found mainly in skeletal muscles, in the center of granulomas. Concentric fibrosis with mixed inflammatory infiltrate involved the larvae after the 47th day PI, persistently. This experimental murine study with L. minor indicated that the 3rd stage larvae penetrated via ileum-cecal mucosa reaching the liver and probably other tissues through the hematogenic via. Throughout its pathway the larvae induced a granulomatous reaction, with abundant polimorphonuclear cells.

Ovarian superstimulatory response and embryo production in mares treated with equine pituitary extract twice daily

Equine pituitary extract (EPE), has been reported to induce multiple ovulation in mares, however ovulation rates are poor in comparison to those obtained in other species. Attempts to improve the effectiveness of EPE for induction of superovulation in cyclic mares has focused on daily frequency of EPE treatment. Two experiments were performed to compare the ovarian response of cyclic mares given EPE once or twice-daily. Mares were assigned to one of two treatment groups 6 to 8 days after ovulation: prostaglandin was given once and EPE (25 mg) was given once daily (Group 1) or twice daily (Group 2). In Experiment 1, more (P < 0.05) follicles 35 mm were detected in mares treated with EPE twice daily (6.1 +/- 3.1) than in mares treated once a daily (2.0 +/- 0.6). In a second experiment, the embryo recovery rates of mares given the two EPE protocols used in Experiment 1 were compared. The number of ovulations per mare was higher (P < 0.05) for mares treated twice-daily (7.1 +/- 5.1, range 3 to 18) than for mares treated once daily (2.4 +/- 1.8, range 1 to 6). The number of embryos produced per mare was higher (P < 0.05) in mares in Group 2 (3.5) than in Group 1 (1.6).Although it is not clear whether the increased ovulation rate is due specifically to dose or frequency, twice-daily administration of a high dose of EPE significantly improved follicular development, ovulation and embryo recovery over the standard treatment of once-daily injection. (C) 2001 by Elsevier B.V.