Molecular response to toxic diatom-derived aldehydes in the sea urchin Paracentrotus lividus.

Diatoms are dominant photosynthetic organisms in the world's oceans and represent a major food source for zooplankton and benthic filter-feeders. However, their beneficial role in sustaining marine food webs has been challenged after the discovery that they produce secondary metabolites, such as polyunsaturated aldehydes (PUAs), which negatively affect the reproductive success of many invertebrates. Here, we report the effects of two common diatom PUAs, heptadienal and octadienal, which have never been tested before at the molecular level, using the sea urchin, Paracentrotus lividus, as a model organism. We show that both PUAs are able to induce teratogenesis (i.e., malformations), as already reported for decadienal, the better-studied PUA of this group. Moreover, post-recovery experiments show that embryos can recover after treatment with all three PUAs, indicating that negative effects depend both on PUA concentrations and the exposure time of the embryos to these metabolites. We also identify the time range during which PUAs exert the greatest effect on sea urchin embryogenesis. Finally, we report the expression levels of thirty one genes (having a key role in a broad range of functional responses, such as stress, development, differentiation, skeletogenesis and detoxification processes) in order to identify the common targets affected by PUAs and their correlation with morphological abnormalities. This study opens new perspectives for understanding how marine organisms afford protection from environmental toxicants through an integrated network of genes.

Neonato afecto del síndrome del ácido valproico fetal / Newborn with fetal valproic acid syndrom

Introducción. El síndrome del ácido valproico fetal consiste en alteraciones congénitas que aparecen en el feto expuesto a tratamiento gestacional con ácido valproico. El ácido valproico, el antiepiléptico más teratogénico, es utilizado ampliamente en gestantes afectas de epilepsia. Las estrategias de prevención, sospecha y detección precoz de este síndrome son de vital importancia, ya que el inicio precoz del estudio y tratamiento permite ofrecer una mejoría en la calidad de vida del paciente. Caso clínico. Se presenta el caso clínico de un recién nacido afecto del síndrome del ácido valproico fetal. En el paciente destacaba una facies peculiar con microtia bilateral. Al alta fue seguido por Otorrinolaringología, para la realización de pruebas audiológicas y colocación de audífonos, y por Cirugía, para realizar la reconstrucción de los pabellones auriculares. Comentarios. El estudio en los casos de microtia consiste en la realización de pruebas de imagen (Tomografía computeritzada craneal), para comprobar el grado de afectación del oído medio e interno, y pruebas audiológicas para conocer el umbral auditivo del paciente, hipoacusia moderada de transmisión en la mayoría de los casos. El tratamiento persigue una estimulación precoz del oído para favorecer el lenguaje mediante la colocación de audífonos de transmisión ósea. El tratamiento quirúrgico se realiza a partir de los 10 años de vida, y consiste en la reconstrucción de un pabellón auricular en tres dimensiones a partir de cartílago costal(AU)

Introduction. The fetal valproic acid syndrome consists of congenital abnormalities occurring in the fetus exposed to gestational treatment with valproic acid; this drug is the most teratogenic antiepileptical treatment that is used in pregnant women suffering from epilepsy. Strategies for prevention and early diagnosis of this syndrome are key since early evaluation and treatment can improve significantly the quality of life of these patients. Case report. We report the case of a newborn affected by fetal valproic syndrome, who presented with an abnormal facies with severe microtia bilaterally. The patient was subsequently followed by otorhinolaryngology for audiology evaluation and placement of hearing aids, followed by reconstructive surgery of the earlobes. Comments. The diagnostic evaluation of microtia involves neuroimaging for assessing the degree of involvement of the middle and inner ear, and audiology evaluation, which shows moderate conductive hearing loss in most cases. Treatment aims to stimulating early hearing to promote language through the placement of bone transmission hearing aids. Surgical treatment is usually not recommended until 10 years of age, and it includes 3-dimensional ear reconstruction using rib cartilage(AU)

Evaluation of cytotoxicity, genotoxicity and teratogenicity of marine sediments from Qingdao coastal areas using in vitro fish cell assay, comet assay and zebrafish embryo test.

Marine sediments are often a final sink for numerous anthropogenic contaminants and may impose serious effects on benthic organisms and ecosystem. An in vitro cell assay using a cell line derived from flounder gill (FG) cells, an in vitro comet assay in FG cells, and an in vitro zebrafish embryo assay were used to evaluate the in vitro cytotoxicity (measured by MTT reduction), genotoxicity and teratogenicity of crude sediment extracts of Li Cang (LC), Zhan Qiao (ZQ) and Olympic Sailing Center (OSC) from Qingdao coastal area. Sediments from the three sites displayed different cytotoxicity, genotoxicity and teratogenicity potencies; however, all three assays yielded similar LOECs (lowest observed effect concentration) for each site, suggesting that the assays were equally sensitive to and suitable for initial screening of the LOECs of marine sediments. The cytotoxicity, genotoxicity and teratogenicity for these three sampling sites were in the same order of LC>ZQ>OSC, indicating different degrees of contamination. Interestingly, trials with the three sediment extracts at the doses inducing a similar cytotoxicity as evaluated with MTT reduction did not produce similar genotoxicity and teratogenicity, with the genotoxic and teratogenic activities of LC and ZQ extracts being markedly higher than those of OSC sediments. These findings indicate that cytotoxicity does not form a fully equivalent toxicity index with that of genotoxicity and teratogenicity. Therefore, in order to assess the true toxic potential of marine sediments, all three assays should be performed. Analysis of 16 EPA (US Environmental Protection Agency) priority PAHs in these three sediment samples showed a clear correlation between PAH concentrations and sediment toxicities, with a higher PAH content corresponding to higher toxicity although PAHs are surely not the only cause.

Evaluation of pyridoacridine alkaloids in a zebrafish phenotypic assay.

Three new minor components, the pyridoacridine alkaloids 1-hydroxy-deoxyamphimedine (1), 3-hydroxy-deoxyamphimedine (2), debromopetrosamine (3), and three known compounds, amphimedine (4), neoamphimedine (5) and deoxyamphimedine (6), have been isolated from the sponge Xestospongia cf. carbonaria, collected in Palau. Structures were assigned on the basis of extensive 1D and 2D NMR studies as well as analysis by HRESIMS. Compounds 1-6 were evaluated in a zebrafish phenotype-based assay. Amphimedine (4) was the only compound that caused a phenotype in zebrafish embryos at 30 muM. No phenotype other than death was observed for compounds 1-3, 5, 6.

Teratogenic effects of diatom metabolites on sea urchin Paracentrotus lividus embryos.

The diatom-derived polyunsaturated aldehydes (PUAs), 2-trans,4-trans-decadienal, 2-trans,4-trans-octadienal, 2-trans,4-trans,7-octatrienal, 2-trans,4-trans-heptadienal, as well as tridecanal were tested on early and later larval development in the sea urchin Paracentrotus lividus. We also tested the effect of some of the more abundant diatom polyunsaturated fatty acids (PUFAs) on development, in particular 5,8,11,14,17-eicosapentaenoic acid (EPA), one of the main precursors of diatom PUAs, as well as 4,7,10,13,16,19-docosahexaenoic acid (DHA), 6,9,12,15-octadecatetraenoic acid (stearidonic acid), 6,9,12-octadecatrienoic acid (gamma-linolenic acid) and 9,12-octadecadienoic acid (linoleic acid). PUAs blocked sea urchin cell cleavage in a dose dependent manner and with increasing chain length from C7 to C10 PUAs, with arrest occurring at 27.27 microM with heptadienal, 16.13 microM with octadienal, 11.47 microM with octatrienal and 5.26 microM with decadienal. Of the PUFAs tested, only EPA and stearidonic acid blocked cleavage, but at much higher concentrations compared to PUAs (331 microM for EPA and 181 microM for stearidonic acid). Sub-lethal concentrations of decadienal (1.32-5.26 microM) delayed development of embryos and larvae which showed various degrees of malformations depending on the concentrations tested. Sub-lethal concentrations also increased the proportion of TUNEL-positive cells indicating imminent death in embryos and larvae. Using decadienal as a model PUA, we show that this aldehyde can be detected spectrophotometrically for up to 14 days in f/2 medium.

A cross-sectional observation of effect of exposure to N-methyl-2-pyrrolidone (NMP) on workers' health.

This study was aimed at clarifying the effect of exposure to N-methyl-2-pyrrolidone (NMP) on workers' health. Fifteen male NMP-exposed workers and 15 referent male workers were recruited for this study. Exposure concentrations were assessed by determining NMP in the breathing zones and urinary NMP. Clinical examinations, motor and sensory nerve conduction velocities in the dominant arm, and neurobehavioral tests were carried out. The subjects were asked to complete self-administered questionnaires for subjective symptoms and psychological assessment. The mean NMP exposure concentrations ranged from 0.14 to 0.26 ppm, and urinary NMP levels at the end of each workday ranged from 0.17 to 0.22 mg/l, throughout the work week. In terms of clinical data, motor and sensory nerve conduction velocities, neurobehavioral tests, and subjective symptom assessments, there were no differences and no dose-dependent changes in either the means or the prevalence of abnormal findings between NMP-exposed and referent workers.

Cyclopamine-induced synophthalmia in sheep: defining a critical window and toxicokinetic evaluation.

Cyclopamine, a steroidal alkaloid, from the plant Veratrum californicum is teratogenic, causing a range of different birth defects. The critical window for cyclopamine-induced synophthalmia formation has been reported to be gestational day (GD) 14. The objectives of this study were to better describe cyclopamine-induced craniofacial deformities, to better define the window of susceptibility to synophthalmia formation, and to characterize cyclopamine toxicokinetics in sheep. Ewes were dosed i.v. with purified cyclopamine for toxicokinetic analysis. Another four groups of ewes were dosed orally twice daily with 0.88 g/kg of V. californicum on GD 13, 14 or 15 or consecutively on GD days 13-15. Pregnancy and pre-partum fetal malformations were determined by ultrasound imaging on GD 60. At parturition lambs were assessed for gross malformations. The elimination half-life of cyclopamine in ewes was determined to be 1.1 +/- 0.1 h. The rapid clearance of cyclopamine indicates that ingestion of V. californicum must occur during a very narrow window for synophthalmia formation to occur. Ewes dosed with V. californicum on GD 13 or 14 had lambs with various craniofacial malformations including cyclopia, maxillary dysplasia and mandibular micrognathia. Ewes dosed on GD 15 delivered normal lambs. Ewes dosed consecutively on GD 13-15 were not pregnant at GD 60 and Veratrum-induced embryonic death was assumed to be the cause. Interestingly, lambs with cyclopia were smaller, under-developed and appeared premature even though their twin appeared fully developed. Initial evaluations suggest this was due to placental dysplasia.

Teratogenic effects of azaspiracid-1 identified by microinjection of Japanese medaka (Oryzias latipes) embryos.

Azaspiracid-1 (AZA-1) is a newly identified phycotoxin that accumulates in commercially important bivalve molluscs harvested in several European countries and causes severe human intoxications. Molluscan shellfish are known vectors for accumulation and subsequent transfer of phycotoxins such as brevetoxin and domoic acid through various trophic levels within food webs. Finfish can also accumulate phycotoxins, both directly from toxic algae or from consumption of contaminated shellfish and smaller intoxicated fish. To evaluate the teratogenic potential of AZA-1 and its relevancy to toxin accumulation in finfish, we have utilized a microinjection technique to mimic the maternal-egg toxin transfer of an AZA-1 reference standard and a shellfish extract containing azaspiracids in an embryonic Japanese medaka (Oryzias latipes) fish model. Microinjection of purified AZA-1 caused dose-dependent effects on heart rate, developmental rate, hatching success, and viability in medaka embryos. Within 4 days of exposure to doses > or = 40 pg AZA-1/egg, substantial retardation in development was observed as reduced somatic growth and yolk absorption, and delayed onset of blood circulation and pigmentation. Embryos treated to > or =40 pg AZA-1/egg had slower heart rates (bradycardia) for the 9 days in ovo period, followed by reduced hatching success. Microinjection of a contaminated mussel (Mytilus edulis) extract containing AZAs (AZA-1, -2, and -3), okadaic acid, and dinophysistoxin-2 resulted in similar responses from the fish embryos at equivalent doses. These studies demonstrate that AZA-1 is a potent teratogen to finfish. This work will complement future investigations on AZA-1 accumulation in marine food webs and provide a basis for understanding its toxicity at different trophic levels.

The fungal teratogen secalonic acid D is an inhibitor of protein kinase C and of cyclic AMP-dependent protein kinase.

The teratogenic metabolite secalonic acid D deriving from the ergot-producing, rye-infecting ascomycete fungus Claviceps purpurea and from Penicillum oxalicum is an inhibitor of Ca2+- and phospholipid-dependent protein kinase C (PKC) and of the catalytic subunit of cyclic AMP-dependent protein kinase (cAK) (C50 values 15 microM and 12 microM, respectively). Secalonic acid D also inhibits Ca2+-calmodulin-dependent myosin light chain kinase (MLCK) and plant Ca2+-dependent protein kinase (CDPK). The inhibition of cAK by secalonic acid D is competitive with respect to both peptide substrate and ATP. However, secalonic acid D does not inhibit a high-affinity nucleotide-binding phosphatase from potato. A variety of other naturally-occurring teratogenic agents are not inhibitors of the protein kinases examined.

The direct embryotoxicity of immunoglobulin G fractions from patients with systemic lupus erythematosus.

PROBLEM: To determine if IgG fractions from sera of individuals with systemic lupus erythematosus (SLE) were toxic to cultures of whole rat embryos. METHODS: Head-fold stage rat embryos (9.5 days of gestation) were cultured on media consisting of 50% rat serum containing IgG fractions isolated from plasmapheresis plasma of six subjects with SLE and six with other autoimmune diseases. Each fraction was tested at 11 mg/ml and those toxic were also tested at 7.5 and 4 mg/ml. RESULTS: Of the six SLE IgG fractions, four were embryotoxic (embryolethal or teratogenic) while only one of the six non-SLE fractions were embryotoxic. CONCLUSION: IgG fractions from subjects with SLE can be toxic to cultures of whole rat embryos in the absence of maternal tissues or influence. Such cultures of whole embryos may be useful to identify those antibodies that represent a risk for fetal loss as well as to understand their mechanisms of embryotoxicity.

El registro de malformaciones congenitas monitorea la eficacia del diagnostico prenatal / Congenital malformations register monitoring prenatal diagnostic efficiency

Se analizo la eficacia de los programas de Genetica para la prevencion d defectos al nacimiento entre 1988 y 1993, en los que la provincia Villa Clara participo en el Registro Cubano de malformaciones Congenitas (RECUMAC), lo que ha permitido monitorear al nacimiento la prevalencia de defectos del tubo neural y de anomalias cromosomicas, para las cuales se llevaron a cabo en la provincia programas de diagnostico prenatal. La eficacia en la prevencion de defectos del tubo neural fue del 93 porciento, la prevencion del sindrome de Down en mujeres de mas de 35 anos fue de 29 porciento y de 25 porciento en gestantes de mas de 38 anos

El Registro de malformaciones congénitas monitorea la eficacia del diagnóstico prenatal / Congenital malformations register monitoring prenatal diagnostic efficiency

Se analizó la eficacia de los programas de Genética para la prevención d defectos al nacimiento entre 1988 y 1993, en los que la provincia Villa Clara participó en el Registro Cubano de malformaciones Congénitas (RECUMAC), lo que ha permitido monitorear al nacimiento la prevalencia de defectos del tubo neural y de anomalías cromosómicas, para las cuales se llevaron a cabo en la provincia programas de diagnóstico prenatal. La eficacia en la prevención de defectos del tubo neural fue del 93 porciento, la prevención del síndrome de Down en mujeres de más de 35 años fue de 29 porciento y de 25 porciento en gestantes de más de 38 años (AU)

Estudios citogenéticos en linfocitos y su relación con formas anómalas de los espermatozoides en varones teratospérmicos / Cytogenetical studies in lymphocytes and its relation with anomalies of the spermatozoa in teratospermic male

Se estudió 24 varones de edad promedio 29,4 años, derivados de diferentes Centros de Infertilidad con el diagnóstico de infertilidad conyugal de etiología atribuible al factor masculino. Se descartaron pacientes portadores de causas conocidas de teratospermia. A todos los pacientes se les requirió 3 espermiogramas según normas dadas por la Organización Mundial de la Salud, detallando el porcentaje de cada forma espermática. El estudio genético consistió en una anamnesis dirigida a su historia reproductiva y el examen físico buscando estigmas genéticos. A partir de cultivo de linfocitos se determinó el cariotipo a cada paciente utilizando técnicas citogenéticas de rutina. Al analizar el resultado del cariotipo se obtiene que el 91,7% de los pacientes se rotuló normal para sexo masculino. Dentro de este grupo (20,8%) se observó aumento aparente de asociaciones entre cromosomas acrocéntricos. En el 8,3% restante se describe dos pacientes portadores de un polimorfismo en el tamaño del cromosoma Y. No fue posible establecer una relación entre porcentaje total de teratospermia y alteraciones cromosómicas. Al relacionar cada forma anómala del espermatozoide con presencia de anormalidad cromosómica, se obtiene por el test de correlación Spearman positividad para la forma piriforme (rs=0,46p<0,03) y al aplicar regresión múltiple se encontró significación para igual forma (r=0,53, p<0,05)

Population surveillance of congenital malformations. Possibilities and limitations.

The paper discusses the possibility of detecting newly introduced teratogenic agents by a population surveillance of congenital malformations. In practice, only outbreaks of previously rare malformations or combinations of malformations can be detected by such systems. The most important aspect of malformation surveillance is therefore a detailed reporting of malformations whereas statistical techniques play a minor role. For ongoing surveillance, a high ascertainment level is less important than speed and amount of detail. For studies of long-time changes in malformation rates, ascertainment must be as complete as possible. Such changes have been identified repeatedly, but are usually explained by changes in diagnosis, ascertainment or population characteristics.

Isolation of teratogenic alkaloids by reversed-phase high-performance liquid chromatography.

Reversed-phase high-performance liquid chromatography was used for both analytical and preparative separations of several steroidal alkaloids which occur in extracts of Veratrum californicum. The inclusion of 0.1% trifluoroacetic acid in the mobile phase improved the efficiency of the chromatography and the solubility of the compounds in aqueous acetonitrile. Nuclear magnetic resonance was used to assist the identification of the isolated steroidal alkaloids. The effect of the interaction of trifluoroacetic acid with the alkaloids could be clearly seen by changes in the chemical shifts in the nuclear magnetic resonance spectra.