Assessment of the risk of death of Clarias gariepinus and Oreochromis niloticus pulse-exposed to selected agricultural pesticides.

Aquatic organisms are often exposed briefly to high pesticide concentration. Survival time model was used to study risk of death in C. gariepinus and O. niloticus fingerlings exposed to 24 mg/L atrazine, 42 mg/l mancozeb, 1 mg/L chlorpyrifos and 0.75 µg/L lambda cyhalothrin for 15, 30, 45 and 60 minutes and continuously for 96 hours. Mortality, time-to-death, weight, length, and condition factor of the fingerlings were recorded. Results obtained showed tilapia was more susceptible than catfish to continuous exposure but not pulse exposure. The survival probability of both species was similar when exposed for 15, 30 and 45 minutes (p > 0.05) but differed after 60 minutes (p < 0.05). Risk of death of catfish exposed briefly to atrazine, mancozeb and chlorpyrifos for 60 minutes was similar to 96 hours continuous exposure, same for tilapia exposed to 1 mg/L chlorpyrifos (p > 0.05). Survival probability of tilapia exposed to chlorpyrifos for 15, 30, 45 and 60 minutes was similar (p > 0.05) and was not influenced by pulse length. Pesticide hazard and risk of death decreased as fish size (weight, length, and condition factor) increased. Pulse toxicity assessment using survival models could make pesticides exposure assessment more realistic by studying factors that can influence the toxicity of pesticides.

Organochlorine pesticides: Agrochemicals with potent endocrine-disrupting properties in fish.

Organochlorine pesticides (OCPs) are persistent environmental contaminants that act as endocrine disruptors and organ system toxicants. These pesticides (e.g. dichlorodiphenyltrichloroethane (DDT), dieldrin, toxaphene, among others) are ranked as some of the most concerning chemicals for human health. These pesticides (1) act as teratogens, (2) are neuroendocrine disruptors, (3) suppress the immune and reproductive systems, and (4) dysregulate lipids and metabolism. Using a computational approach, we revealed enriched endocrine-related pathways in the Comparative Toxicogenomics Database sensitive to this chemical class, and these included reproduction (gonadotropins, estradiol, androgen, steroid biosynthesis, oxytocin), thyroid hormone, and insulin. Insight from the Tox21 and ToxCast programs confirm that these agrochemicals activate estrogen receptors, androgen receptors, and retinoic acid receptors with relatively high affinity, although differences exist in their potency. We propose an adverse outcome pathway for OCPs toxicity in the fish testis as a novel contribution to further understanding of OCP-induced toxicity. Organochlorine pesticides, due to their persistence and high toxicity to aquatic and terrestrial wildlife as well as humans, remain significant agrochemicals of concern.

Exposição crônica ao cloridrato de metformina e à glibenclamida causa alterações comportamentais, glicêmicas e de mortalidade em Hemigrammus caudovittatus e Danio rerio / Chronic exposure to metformin chloridrate and glibenclamide causes behavioral, blood glucose and mortality changes of Hemigrammus caudovittatus and Danio rerio

Hemigrammus caudovittatus e Danio rerio foram expostos aos hipoglicemiantes orais (HOs) cloridrato de metformina a 40µg/L e 120µg/L e glibenclamida a 0,13µg/L e 0,39µg/L durante 100 dias. Foram avaliados os efeitos tóxicos dos fármacos em relação ao peso, ao comportamento animal, à glicemia e à mortalidade. H. caudovittatus expostos à menor concentração dos fármacos apresentaram aumento significativo (P<0,05) no evento Respiração Aérea. Ainda, foi observado aumento no comportamento Descansar quando os animais foram expostos à glibenclamida a 0,39µg/L. Em D. rerio expostos ao cloridrato de metformina a 120µg/L, foi observado aumento (P<0,05) no comportamento Descansar. A glibenclamida provocou redução (P<0,05) na glicemia de H. caudovittatus. Ambos os fármacos causaram efeito letal na espécie D. rerio, contudo a glibenclamida foi mais tóxica, causando 100% de mortalidade em 30 dias de exposição. Os animais que vieram a óbito apresentaram congestão nos arcos branquiais e hemorragia. Os HOs foram desenvolvidos para apresentarem efeitos fisiológicos em mamíferos, entretanto efeitos tóxicos foram encontrados nas duas espécies de peixe estudadas. Isso levanta a preocupação sobre possíveis efeitos tóxicos de HOs e sobre quais métodos serão utilizados para a sua degradação no ambiente aquático.(AU)

Hemigrammus caudovittatus and Danio rerio were exposed to oral hypoglycemic drugs (HOs) metformin hydrochloride at 40µg/L and 120µg/L and to glibenclamide at 0.13µg/L and 0.39µg/L during 100 days. Toxic effects of the drugs were evaluated based on weight, animal behavior, blood glucose and mortality. H. caudovittatus exposed to lowest concentration of the drugs showed significant increase (P< 0.05) in the Air breathing event. Furthermore, increase in Rest event was observed when animals were exposed to glibenclamide at 0.39µg/L. An increase (P< 0.05) in the frequency of Rest behavior in the D. rerio exposed to metformin hydrochloride at 120µg/L was observed. Glibenclamide caused decrease (P< 0.05) in the blood glucose of H. caudovittatus. Both drugs caused lethal effect against D. rerio. Nevertheless, glibenclamide was more toxic causing 100% of mortality after 30 days of exposure. The animals that died showed congestion on the branchial arches and hemorrhage. The HOs were developed to have physiological effects in mammals. However, toxic effects were found in both species of fish studied. This raises concerns about possible toxic effects of HOs and what methods will be used for their degradation in the aquatic environment.(AU)

The Contribution of In Vivo Mammalian Studies to the Knowledge of Adverse Effects of Radiofrequency Radiation on Human Health.

The proliferation of cellular antennas and other radiofrequency radiation (RFR) generating devices of the last decades has led to more and more concerns about the potential health effects from RFR exposure. Since the 2011 classification as a possible carcinogen by the International Agency for Research on Cancer (IARC), more experimental studies have been published that support a causal association between RFR exposure and health hazards. As regard cancer risk, two long-term experimental studies have been recently published by the US National Toxicology Program (NTP) and the Italian Ramazzini Institute (RI). Despite important experimental differences, both studies found statistically significant increases in the development of the same type of very rare glial malignant tumors. In addition to carcinogenicity, reproductive organs might be particularly exposed, as well as sensitive to RFR. In this work, we reviewed the currently available evidence from in vivo studies on carcinogenicity and reproductive toxicity studies in order to summarize the contribution of experimental research to the prevention of the adverse effects of RFR on human health.

Guideline on safety evaluation of cell-based medicinal products for animal use.

With the increased use of cell therapy in the veterinary sector, there is a growing demand for the development of cell-based medicinal products and the determination of their safety. Currently, the Korean Animal and Plant Quarantine Agency has established a guideline for evaluating the safety of cell-based medicinal products for animal use. The guideline includes items related to definition, classification, management, manufacturing procedure and quality control (standard and test method), stability testing, toxicity testing, pharmacological testing, and performance of clinical trials. In addition, testing protocols related to safety assessment of animal cell-based products such as chromosome karyotyping, tumorigenicity testing, confirmatory testing of biodistribution and kinetics, and target animal safety testing are described in detail. Moreover, because cell-based medicinal products are novel therapies, deviations from traditional designs may be justified in order to obtain relevant safety information on the treatment. Additionally, this guideline can be amended on the basis of new scientific findings.

Profiling of Selected Functional Metabolites in the Central Nervous System of Marine Medaka (Oryzias melastigma) for Environmental Neurotoxicological Assessments.

The simultaneous profiling of 43 functional metabolites in the brain of the small model vertebrate organism, marine medaka (Oryzais melastigma), has been accomplished via dansyl chloride derivatization and LC-MS/MS quantification. This technique was applied to examine effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), one of the most abundant polybrominated diphenyl ether flame retardants in the natural environment, on the central nervous system (CNS) of vertebrates. The model teleosts were fed with bioencapsulated Artemia nauplii for up to 21 days. Multivariate statistical analysis has demonstrated that levels of numerous classical neurotransmitters and their metabolites in the CNS of the fish were perturbed even at the early phase of dietary exposure. Subsequent metabolic pathway analysis further implied potential impairment of the arginine and proline metabolism; glycine, serine and threonine metabolism; D-glutamine and D-glutamate metabolism; alanine, aspartate, and glutamate metabolism; valine, leucine, and isoleucine biosynthesis, and the cysteine and methionine metabolism in the brain of the test organism. Our results demonstrate that targeted profiling of functional metabolites in the CNS may shed light on how the various neurological pathways of vertebrates, including humans, are affected by toxicant/stress exposure.

Cytological Bone Marrow Cell Differential Counts and Morphologic Findings in Healthy Cynomolgus Monkeys ( Macaca fascicularis) from Nonclinical Toxicology Studies.

Cytological bone marrow evaluation is utilized in nonclinical toxicology studies to characterize hematopoietic effects when the combined interpretation of histologic and complete blood count data does not yield sufficient information. Results from cytological bone marrow examination should be interpreted in the context of variability observed in concurrent control animals with consideration of cytologist experience and historical/published data. Cytological bone marrow differential counts and cellular morphologic findings from 130 (66 male, 64 female) healthy control cynomolgus monkeys from nonclinical toxicology studies were retrospectively analyzed. Myeloid to erythroid (M:E) ratios and the percentage of total cells for each cell type were determined from differential cell count data. M:E ratios ranged from 0.6:1 to 2.3:1. Percentages of total granulocytic cells, total erythroid cells, and lymphocytes ranged from 26.6% to 60.6%, 25.7% to 52.2%, and 5.5% to 40.4%, respectively. Monocytes, plasma cells, mast cells, and mitotic figures were typically <1% of total cells. Notable morphologic findings included occasional giant neutrophilic metamyelocytes and band neutrophils, ring-shaped band neutrophil nuclei, metarubricyte nuclear blebbing and binucleation, multiple or nonfused megakaryocyte nuclei, and emperipolesis. These results represent cytological bone marrow findings from healthy control cynomolgus monkeys utilized in nonclinical toxicology studies and provide insight into expected background variability.

Pharmacological characterization of venoms from three theraphosid spiders: Poecilotheria regalis, Ceratogyrus darlingi and Brachypelma epicureanum

Background Tarantulas (Theraphosidae) represent an important source of novel biologically active compounds that target a variety of ion channels and cell receptors in both insects and mammals. In this study, we evaluate and compare the pharmacological activity of venoms from three taxonomically different theraphosid spiders bred in captivity: Poecilotheria regalis, an aggressive arboreal tarantula from southeastern India; Ceratogyrus darlingi, an aggressive tarantula from southern Africa; and Brachypelma epicureanum, a docile tarantula from the Yucatan dry forest of Mexico. Prior to this study, no research had been conducted with regard to the composition and pharmacological activity of these venoms. Methods The pharmacological characterization of the venoms was described for the first time by the assessment of their toxicity in crickets (LD50) along with their nociceptive (by using the formalin test), hyaluronidase, phospholipase A2, edematogenic and caseinolytic activity. Results P. regalis and B. epicureanum venoms induced a similar lethal effect on crickets (LD50 = 5.23 ± 3.1 and 14.4 ± 5.0 μg protein/g 48 h post-injection, respectively), whereas C. darlingi venom (119.4 ± 29.5 μg protein/g 48 h post-injection) was significantly less lethal than the other two venoms. All three venoms induced similar edematogenic activity on rats but did not induce nociceptive behavior. The assessment of enzymatic activity indicated that P. regalis venom induces significantly higher hyaluronidase activity (27.6 ± 0.9 TRU/mg) than both C. darlingi (99.7 ± 1.9 TRU/mg) and B. epicureanum (99.6 ± 1.6 TRU/mg); these latter venoms did not display phospholipase A2or caseinolytic activity. Conclusions This study demonstrates that these theraphosid spiders of different habitats produce venoms with different activities. P. regalis venom displays a high level of hyaluronidase activity, which may be associated with its potentially medically significant bite.

Exposure to 50 Hz electromagnetic field changes the efficiency of the scorpion alpha toxin

Background Extremely low-frequency (50 Hz) electromagnetic field (ELF-EMF) is produced by electric power transmission lines and electronic devices of everyday use. Some phenomena are proposed as first effects of ELF-EMF: the discrete changes in the membrane potential and the increase of the calcium channel activity as well as the intracellular concentration of Ca 2+ . Interaction of the scorpion alpha toxin with the sodium channel depends on the orientation of the charges and may be perturbed by changes in the membrane polarization. The toxin induces overexcitability in the nervous system and an increase in the neurotransmitters released with different consequences, mainly the paralysis of muscles. We assumed that the exposure to ELF-EMF 0.7 mT will change the effects of the insect selective scorpion alpha toxin (recombinant LqhIT from Leiurus quinquestriatus hebraeus) at the level of the cercal nerve function, the synaptic transmission and on the level of entire insect organism. Taking into account the compensatory mechanisms in organisms, we tested in addition ten times higher ELF-EMF on whole insects.Methods Experiments were performed in vivo on cockroaches (Periplaneta americana) and in vitro on isolated cockroach abdominal nerve cord with cerci. In biotests, the effects of LqhIT (10 8 M) were estimated on the basis of the insect ability to turn back from dorsal to ventral side. Three groups were compared: the control one and the two exposed to ELF-EMF 0.7 and 7 mT. Bioelectrical activity of the cercal nerve and of the connective nerve that leaves the terminal abdominal ganglion was recorded using extracellular electrodes. LqhIT (5 × 10 8 M) induced modifications of neuronal activity that were observed in the control cockroach preparations and in the ones exposed to ELF-EMF (0.7 mT). The exposure to ELF-EMF was carried out using coils with a size appropriate to the examined objects.Results The exposure to ELF-EMF (0.7 mT) modified the effects of LqhIT (5 × 108 M) on activity of the cercal nerve and of the connective nerve. We observed a decrease of the toxin effect on the cercal nerve activity, but the toxic effect of LqhIT on the connective nerve was increased. Biotests showed that toxicity of LqhIT (10 8 M) on cockroaches was reduced by the exposure to ELF-EMF (0.7 and 7 mT).Conclusions The exposure to 50 Hz ELF-EMF modified the mode of action of the anti-insect scorpion alpha toxin LqhIT at cellular level of the cockroach nervous system and in biotests. Toxin appeared as a usefull tool in distinguishing between the primary and the secondary effects of ELF-EMF.

Allosteric interactions between receptor site 3 and 4 of voltage-gated sodium channels: a novel perspective for the underlying mechanism of scorpion sting-induced pain

Background BmK I, a site-3-specific modulator of voltage-gated sodium channels (VGSCs), causes pain and hyperalgesia in rats, while BmK IT2, a site-4-specific modulator of VGSCs, suppresses pain-related responses. A stronger pain-related effect has been previously attributed to Buthus martensi Karsch (BmK) venom, which points out the joint pharmacological effect in the crude venom.Methods In order to detect the joint effect of BmK I and BmK IT2 on ND7-23 cells, the membrane current was measured by whole cell recording. BmK I and BmK IT2 were applied successively and jointly, and the synergistic modulations of VGSCs on ND7-23 cells were detected.Results Larger peak I Na and more negative half-activation voltage were elicited by joint application of BmK I and BmK IT2 than by application of BmK I or BmK IT2 alone. Compared to the control, co-applied BmK I and BmK IT2 also significantly prolonged the time constant of inactivation.Conclusions Our results indicated that site-4 toxin (BmK IT2) could enhance the pharmacological effect induced by site-3 toxin (BmK I), suggesting a stronger effect elicited by both toxins that alone usually exhibit opposite pharmacological effects, which is related to the allosteric interaction between receptor site 3 and site 4. Meanwhile, these results may bring a novel perspective for exploring the underlying mechanisms of scorpion sting-induced pain.

Evaluation of the acute and subchronic oral toxicity of ethanol extract from Valeriana pavonii species in Wistar rats / Evaluación de la toxicidad oral aguda y sub-crónica del extracto etanólico de la especie Valeriana pavonii en ratas Wistar

Introduction: One of the most frequent problems found in medicinal plants is the absence of clinical, toxicological, and pharmacological studies. Valeriana pavonii is one of the species used in Colombia as an anxiolytic. Further study of this specie is rendered to add information in the toxicological area. Objective: The acute and subchronic oral toxicity of V. pavonii ethanolic extract was evaluated in Wistar rats of both sexes. Materials and methods: The rats were distributed into four groups: the control group received the vehicle (0.5 mL/100 g of corporal weight) and the other three groups received increasing levels of the dosage for 90 days to evaluate characteristics like physical exam, laboratory test (blood chemistry and haematology), and anatomopathological findings. Results: This study reveals that there were no signs of toxicity, mortality, or significant alterations attributable to the ethanolic extract of V. pavonii. Conclusions: The Not Observed Adverse Effect Levels (NOAEL) of V. pavonii ethanolic extract were 2000 and 1000 mg/kg of body weight for the acute and subchronic toxicity studies, respectively.

Introducción: Uno de los problemas más frecuentes asociados con el uso de plantas medicinales es la ausencia de evidencias farmacológicas, toxicológicas y clínicas. Valeriana pavonii es una de las especies más utilizadas popularmente en Colombia con fines ansiolíticos. Es necesario avanzar en el estudio de esta especie para aportar información en el campo toxicológico. Objetivos: Evaluar la toxicidad oral aguda y sub-crónica del extracto etanólico de V. pavonii en ratas Wistar de ambos sexos. Materiales y métodos: En cada uno de los estudios se distribuyeron ratas en cuatro grupos; un grupo control que recibió únicamente vehículo (0.5 ml/100 g de peso corporal) y tres grupos correspondientes a niveles crecientes de dosis, así: para el estudio de toxicidad aguda se administraron en dosis única 20, 200 y 2000 mg/kg con un período de observación de 14 días y para el de toxicidad sub-crónica, dosis diarias de 250, 500 y 1000 mg/kg durante 90 días. Se evaluaron los parámetros de examen físico, los exámenes de laboratorio (química sanguínea y hematología) y el estudio anatomopatológico. Resultados: No se presentaron signos de toxicidad, letalidad ni alteraciones significativas atribuibles al consumo del extracto etanólico de V. pavonii, según el examen físico, el examen anatomopatológico y el análisis de las pruebas de química sanguínea y hematología. Conclusiones: Los valores de nivel sin efectos adversos observados (NOAEL) del extracto etanólico de V. pavonii, fueron 2000 y 1000 mg/kg de peso corporal para los estudios de toxicidad aguda y sub-crónica, respectivamente. No se encontraron valores de nivel más bajo de efecto adverso observado (LOAEL).

Aspectos clínicos da intoxicação experimental pelas favas de Stryphnodendron fissuratum (Leg. Mimosoideae) em caprinos / Clinical aspects of the experimental poisoning by the pods of Stryphnodendron fissuratum (Leg. Mimosoideae) in goats

Com o objetivo de caracterizar o quadro clínico da intoxicação por Stryphnodendron fissuratum Mart. (Leg. Mimosoideae) em caprinos, administraram-se as favas dessa planta a oito caprinos, por via oral forçada em doses únicas e a outros dois caprinos, em doses fracionadas. A menor dose que causou sinais clínicos e morte foi a de 10g/kg. Doses de 20g/kg e 40g/kg causaram sinais acentuados e doses únicas de 5g/kg não provocaram sinais. Doses fracionadas de 5g/kg durante quatro dias, totalizando 20g/kg provocaram sinais acentuados e morte. Em ambos os grupos, os primeiros sinais de intoxicação foram observados a partir do primeiro dia de experimento e a evolução variou de 4-25 dias. A doença caracterizou-se principalmente por alterações digestórias e nervosas que consistiram em anorexia, desidratação, hipomotilidade e atonia ruminal, timpanismo, gemidos constantes, dor à percussão abdominal, fezes com muco, ranger de dentes, apatia, ataxia, dismetria, tremores de cabeça, tremores musculares, fraqueza com o andar cambaleante e trôpego, acentuada depressão e decúbito esternal ou lateral prolongado e morte. Alguns animais apresentaram acentuada queda de pêlos na região dorsal; apenas um caprino apresentou fezes líquidas, marrom-escuras e fétidas. Outros sinais incluíram perda de fluido ruminal durante a ruminação, sialorréia, exsudato nasal seroso e lacrimejamento. As provas de função hepática e renal revelaram alterações discretas. As concentrações séricas de aspartato aminotransferase encontraram-se levemente aumentadas e as de creatinofosfocinase muito aumentadas.

In order to confirm the susceptibility of goats to the poisoning by Stryphnodendron fissuratum Mart. (Leg. Mimosoideae) and to characterize the clinical disease, the pods of the plant were given orally to each of eight young goats and in fractioned doses to other two. The lowest lethal dose was 10g/kg. The same dose was the lowest that induced disease. Doses of 20g/kg and 40g/kg caused pronounced clinical signs and doses of 5g/kg did not caused signs. Fractioned doses of 5g/kg during four days also caused pronounced signs. In each groups the first signs of poisoning were observed from the first day of experiments and the changes ranged from 4-25 days. The disease was characterized mainly by digestive and nervous disorders. Clinical signs were partial to complete anorexia, dehydration, decrease in ruminal activity up to atonia, tympanism, constant vocalizations, grinding of the teeth pain up on abdominal palpation, apathy, ataxia, depression, dysmetria, head and muscle tremors, weakness, difficulty in rising, sternal or lateral recumbency and death. Some goats presented extense hair loss in the skin of the dorsum; one goat presented liquid and black fetid feces. Other signs included loss of ruminal fluid during rumination, drooling, serous nasal and ocular discharges. Liver and kidney function tests had resulted in slight changes. AST serum levels were slightly increased and creatine phosphokinase levels were highly increased. These changes can associated to the effects of triterpenic saponins contained in the S. fissuratum pods.

Aspectos clínicos da intoxicação experimental pelas favas de Stryphnodendron fissuratum (Leg. Mimosoideae) em caprinos / Clinical aspects of the experimental poisoning by the pods of Stryphnodendron fissuratum (Leg. Mimosoideae) in goats

Com o objetivo de caracterizar o quadro clínico da intoxicação por Stryphnodendron fissuratum Mart. (Leg. Mimosoideae) em caprinos, administraram-se as favas dessa planta a oito caprinos, por via oral forçada em doses únicas e a outros dois caprinos, em doses fracionadas. A menor dose que causou sinais clínicos e morte foi a de 10g/kg. Doses de 20g/kg e 40g/kg causaram sinais acentuados e doses únicas de 5g/kg não provocaram sinais. Doses fracionadas de 5g/kg durante quatro dias, totalizando 20g/kg provocaram sinais acentuados e morte. Em ambos os grupos, os primeiros sinais de intoxicação foram observados a partir do primeiro dia de experimento e a evolução variou de 4-25 dias. A doença caracterizou-se principalmente por alterações digestórias e nervosas que consistiram em anorexia, desidratação, hipomotilidade e atonia ruminal, timpanismo, gemidos constantes, dor à percussão abdominal, fezes com muco, ranger de dentes, apatia, ataxia, dismetria, tremores de cabeça, tremores musculares, fraqueza com o andar cambaleante e trôpego, acentuada depressão e decúbito esternal ou lateral prolongado e morte. Alguns animais apresentaram acentuada queda de pêlos na região dorsal; apenas um caprino apresentou fezes líquidas, marrom-escuras e fétidas. Outros sinais incluíram perda de fluido ruminal durante a ruminação, sialorréia, exsudato nasal seroso e lacrimejamento. As provas de função hepática e renal revelaram alterações discretas. As concentrações séricas de aspartato aminotransferase encontraram-se levemente aumentadas e as de creatinofosfocinase muito aumentadas.

In order to confirm the susceptibility of goats to the poisoning by Stryphnodendron fissuratum Mart. (Leg. Mimosoideae) and to characterize the clinical disease, the pods of the plant were given orally to each of eight young goats and in fractioned doses to other two. The lowest lethal dose was 10g/kg. The same dose was the lowest that induced disease. Doses of 20g/kg and 40g/kg caused pronounced clinical signs and doses of 5g/kg did not caused signs. Fractioned doses of 5g/kg during four days also caused pronounced signs. In each groups the first signs of poisoning were observed from the first day of experiments and the changes ranged from 4-25 days. The disease was characterized mainly by digestive and nervous disorders. Clinical signs were partial to complete anorexia, dehydration, decrease in ruminal activity up to atonia, tympanism, constant vocalizations, grinding of the teeth pain up on abdominal palpation, apathy, ataxia, depression, dysmetria, head and muscle tremors, weakness, difficulty in rising, sternal or lateral recumbency and death. Some goats presented extense hair loss in the skin of the dorsum; one goat presented liquid and black fetid feces. Other signs included loss of ruminal fluid during rumination, drooling, serous nasal and ocular discharges. Liver and kidney function tests had resulted in slight changes. AST serum levels were slightly increased and creatine phosphokinase levels were highly increased. These changes can associated to the effects of triterpenic saponins contained in the S. fissuratum pods.

Hydrogenated castor oil nanoparticles as carriers for the subcutaneous administration of tilmicosin: in vitro and in vivo studies.

Tilmicosin-loaded solid lipid nanoparticles (SLN) were prepared with hydrogenated castor oil (HCO) by o/w emulsion-solvent evaporation technique. The nanoparticle diameters, surface charges, drug loadings and encapsulation efficiencies of different formulations were 90 approximately 230 nm, -6.5 approximately -12.5 mV, 40.3 approximately 59.2% and 5.7 approximately 11.7% (w/w), respectively. In vitro release studies of the tilmicosin-loaded nanoparticles showed a sustained release and the released tilmicosin had the same antibacterial activity as that of the free drug. Pharmacokinetics study after subcutaneous administration to Balb/c mice demonstrated that a single dose of tilmicosin-loaded nanoparticles resulted in sustained serum drug levels (>0.1 microg/mL) for 8 days, as compared with only 5 h for the same amount of tilmicosin phosphate solution. The time to maximum concentration (Tmax), half-life of absorption (T(1/2) ab) and half-life of elimination (T(1/2) el) of tilmicosin-loaded nanoparticles were much longer than those of tilmicosin phosphate solution. Tissue section showed that drug-loaded nanoparticles caused no inflammation at the injection site. Cytotoxicity study in cell culture and acute toxicity test in mice demonstrated that the nanoparticles had little or no toxicity. The results of this exploratory study suggest that the HCO-SLN could be a useful system for the delivery of tilmicosin by subcutaneous administration.

Investigating the relationship between embryotoxic and genotoxic effects of benzo[a]pyrene, 17alpha-ethinylestradiol and endosulfan on Crassostrea gigas embryos.

Genotoxicity biomarkers are widely measured in ecotoxicology as molecular toxic endpoints of major environmental pollutants. However, the long-term consequences of such damage still have to be elucidated. Some authors have suggested that the accumulation of unrepaired DNA lesions could explain the embryotoxicity of certain chemical pollutants. As embryotoxicity exerts a direct impact on the recruitment rate, genotoxicity could be closely related to disturbances of ecological concern and produce a possible impact upon population dynamics. The aim of the present work was to study the genotoxicity and the embryotoxicity of three relevant pollutants for oyster embryos: the polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP), the synthetic estrogenic hormone, 17alpha-ethinylestradiol (EE2), and the organochlorine pesticide, endosulfan (ES). For each substance, gamete fertilization was performed and embryo development followed in contaminated reference seawater. Following exposure, embryotoxicity was evaluated by calculating the percentage of abnormal D-larvae obtained at 20 h development. Genotoxicity was measured in parallel by conducting a comet assay on enzymatically dissociated cells of pre-shelled larvae (16 h development). The oxidized DNA base, 8-oxodGuo, was also measured by HPLC coupled to electrochemical detection. For each contaminant, the relationship between genotoxicity and embryotoxicity was then studied to check for the possible significance of genotoxicity in the population dynamics of marine bivalves from polluted areas. For BaP, embryotoxicity and DNA strand breakage were both observed from the lowest tested concentration of 0.2 nM. Induction of 8-oxodGuo was significant from 20 nM. Endosulfan exposure resulted in similar effects for oyster embryos but from higher concentrations and followed a concentration-dependent manner. Embryotoxicity and genotoxicity in terms of DNA strand breaks were observed for endosulfan from 300 and 150 nM, respectively. No change in 8-oxodGuo level was observed following endosulfan exposure. EE2 displayed no toxic effect for oyster embryos within the range of tested concentrations (from 0.02 to 1.7 nM). Taking into account all the data collected during this study, a positive and significant correlation was demonstrated in oyster embryos between genotoxicity as measured by the comet assay and embryotoxicity.

Mechanism of acute silver toxicity in the euryhaline copepod Acartia tonsa.

Acute silver effects on whole-body ion regulation and Na(+),K(+)-ATPase activity were evaluated in the euryhaline copepod Acartia tonsa. Experiments were run at 20 degrees C, three different salinities (5, 15 and 30 ppt), in either the absence or the presence of food (diatom Thalassiosira weissflogii; 2 x 10(4)cells/mL). Standard static-renewal procedures were used. Copepods were acutely (48 h) exposed to silver (AgNO(3)) concentrations equivalent to the 48-h EC10 (dissolved Ag=3, 49, and 94 microg/L), 48-h EC30 (dissolved Ag=5, 71, and 125 microg/L) or 48-h EC50 (dissolved Ag=7, 83, and 173 microg/L) values in the absence of food or to the 48-h EC50 (dissolved Ag=35, 90, and 178 microg/L) values in the presence of food. These values were previously determined under the same experimental conditions at salinities 5, 15 and 30 ppt, respectively. Endpoints analyzed were whole-body ion concentrations (Na(+), Cl(-), and Mg(2+)) and Na(+),K(+)-ATPase activity. In starved copepods, lower whole-body Na(+) and Mg(2+) concentrations were observed in salinities 5 and 30 ppt, respectively. Also a higher whole-body Na(+),K(+)-ATPase activity was observed in all salinities tested. Data from fed copepods indicate that all these salinity effects were completely associated with starvation. Silver exposure induced a decrease in the whole-body Mg(2+) concentration in starved copepods in salinities 5 and 30 ppt and a Na(+),K(+)-ATPase inhibition in both starved and fed copepods in all salinities tested. Thus, food addition in the experimental media completely protected against silver effects on Mg(2+) concentration, but not against those on Na(+),K(+)-ATPase activity. In starved copepods, enzyme inhibition was dependent on silver concentration and a relationship between this parameter and mortality was observed in all salinities tested. Therefore, Na(+),K(+)-ATPase molecules seem to be a key site for acute silver toxicity in marine invertebrates, as reported for freshwater fish and crustaceans.

Evaluation of outer membrane proteins of Pseudomonas aeruginosa as a protective agent in mice model.

The crude Outer Membrane Protein (OMP) from a strain of P. aeruginosa isolated from burn patient was purified by two different methods. One procedure involved separation of Sodium Dodecyle Sulphate (SDS) and Triton X-100, where as the other involved using lysozyme enzyme. Both methods showed very similar polypeptide pattern and the major peptide band with molecular weight of 37 KD was common in both procedures. The protein estimation of OMP extracted by lysosyme was 3 mg mL(-1) compared to 5.5 mg mL(-1) extracted by Triton-X100 method. The latter was chosen to examine for the immunogenicity study in a mice model. The efficacy of immunization with OMP and challenge with homologous strain in mice showed a very good protection compared to control mice injected with saline. The passive haemoagglutination test (PHA) in mice, injected with OMP showed increased level of antibody after the second injection and stayed constant after repeated injection. The results of this study showed that the crude OMP extracted from P. aeruginosa induced a significant protection in mice against Pseudomonas infections and could be used as a vaccine candidate.

Cadmium toxicity to two marine phytoplankton under different nutrient conditions.

Cd accumulation and toxicity in two marine phytoplankton (diatom Thalassiosira weissflogii and dinoflagellate Prorocentrum minimum) under different nutrient conditions (nutrient-enriched, N- and P-starved conditions) were examined in this study. Strong interactions between the nutrients and Cd uptake by the two algal species were found. Cd accumulation as well as N and P starvation themselves inhibited the assimilation of N, P, and Si by the phytoplankton. Conversely, N starvation strongly inhibited Cd accumulation but no influence was observed under P starvation. However, the Cd accumulation difference between nutrient-enriched and N-starved cells was smaller when [Cd(2+)] was increased in the medium, indicating that net Cd accumulation was less dependent on the N-containing ligands at high-Cd levels. As for the subcellular distribution of the accumulated Cd, most was distributed in the insoluble fraction of T. weissflogii while it was evenly distributed in the soluble and insoluble fractions of P. minimum at low-Cd levels. A small percentage of cellular Cd (<15%) was adsorbed on the cell surface for both algae at the lowest [Cd(2+)], which increased when the [Cd(2+)] increased. Cd toxicity in phytoplankton was quantified as depression of growth and maximal photosynthetic system II quantum yield, and was correlated with the [Cd(2+)], intracellular Cd concentration, and Cd concentrations in the cell-surface-adsorbed, soluble, and insoluble fractions. According to the estimated median inhibition concentration (IC50) based on the different types of Cd concentration, the toxicity difference among the different nutrient-conditioned cells was the smallest when the Cd concentration in the soluble fraction was used, suggesting that it may be the best predictor of Cd toxicity under different nutrient conditions.

Development of chronic tests for endocrine active chemicals. Part 1. An extended fish early-life stage test for oestrogenic active chemicals in the fathead minnow (Pimephales promelas).

An extended early-life stage test (based on OECD test guideline 210) was developed to allow the evaluation of a weak environmental oestrogen, 4-tert-pentyphenol (4TPP), on sexual differentiation and gonadal development. Fathead minnow (Pimephales promelas) embryos were exposed to three concentrations of 4TPP (56, 180 and 560 microg l(-1)) in a flow-through system, at 25+/-1 degrees C, for <107 days post-hatch (dph). In addition, some embryos were exposed to 180 microg 4TPPl(-1) until 30 or 60 dph, after which they were exposed to dilution water only until 107 dph. At 30, 60 and 107 dph fish were evaluated for growth and gonadal development (via histology), and at 107 dph fish were also evaluated for secondary sexual characteristics (SSC), gonadosomatic index (GSI) and plasma vitellogenin (VTG). There were no effects of 4TPP on hatching success or survival, however, there was a delay in the time taken for embryos to hatch (560 microg 4TPPl(-1)). No treatment-related effects were observed on fish growth, with the exception of at 107 dph when the condition factor in female fish was reduced in all 4TPP continuous exposure treatments. Plasma VTG was only elevated in female fish exposed to 180 microg 4TPPl(-1) and inhibition of gonadal growth (GSI) occurred only in females exposed to 560 microg 4TPPl(-1). Histological examination of the gonads revealed delays and disruption in male sexual differentiation and development (180 microg 4TPPl(-1)) and no testicular tissue was observed in any fish exposed to 560 microg 4TPPl(-1). Mixed gonads (predominately testes with a scattering of primary oocytes) were present in fish exposed to all doses of 180 microg 4TPPl(-1) at 107 dph. Feminisation of the reproductive ducts (formation of an ovarian like cavity) occurred in the testis of all males exposed to 180 microg l(-1), regardless of length of 4TPP exposure. Results indicate that the period of 30-60 dph appears to be the sensitive window for disruption of formation of the reproductive duct and this effect is not reversible when the fish are transferred to dilution water. The data also show that this integrative test is suitable for the detection of a weak environmental oestrogen and comparisons of these results with that of a fish full life-cycle, in medaka, indicate that this test could be a suitable surrogate for a fish full life-cycle.

Toxic actions of dinoseb in medaka (Oryzias latipes) embryos as determined by in vivo 31P NMR, HPLC-UV and 1H NMR metabolomics.

Changes in metabolism of Japanese medaka (Oryzias latipes) embryos exposed to dinoseb (2-sec-butyl-4,6-dinitrophenol), a substituted dinitrophenol herbicide, were determined by in vivo (31)P NMR, high-pressure liquid chromatography (HPLC)-UV, and (1)H NMR metabolomics. ATP and phosphocreatine (PCr) metabolism were characterized within intact embryos by in vivo (31)P NMR; concentrations of ATP, GTP, ADP, GDP, AMP and PCr were determined by HPLC-UV; and changes in numerous polar metabolites were characterized by (1)H NMR-based metabolomics. Rangefinding exposures determined two sublethal doses of dinoseb, 50 and 75 ppb, in which embryos survived from 1-day post fertilization (DPF) through the duration of embryogenesis. In vivo (31)P NMR data were acquired from 900 embryos in 0, 50, and 75 ppb dinoseb at 14, 62, and 110 h (n = 6 groups) after initiation of exposure. After 110 h, embryos were observed for normal development and hatching success, then either preserved in 10% formalin for growth analysis or flash frozen and extracted for HPLC-UV and (1)H NMR analysis. Dinoseb exposure at both concentrations resulted in significant declines in [ATP] and [PCr] at 110 h as measured by in vivo (31)P NMR (p < 0.01), HPLC-UV (p < 0.001) and NMR-based metabolomics. Reduced eye growth and diminished heart rate occurred in a concentration-dependent fashion. Metabolic effects measured by in vivo (31)P NMR showed a significant increase in orthophosphate levels (P(i); p < 0.05), and significant decreases in [ATP], [PCr] and the PCr/P(i) ratio (p < 0.05). Metabolomics revealed a dose-response relationship between dinoseb and endogenous metabolite changes, with both dinoseb concentrations producing significantly different metabolic profiles from controls (p < 0.05). Metabolic changes included decreased concentrations of ATP, PCr, alanine and tyrosine, and increased concentrations of lactate with medaka embryotoxicity. This study demonstrated that medaka embryos respond to dinoseb with significant changes in metabolism, reduced growth and heart rates, and increased abnormal development and post-exposure mortality. All three analytical methods confirmed similar trends, and utilization of PCr to compensate for ATP loss was found to be a consistent indicator of sublethal stress-one that could be used to quantify stress associated with medaka embryotoxicity.