PUF60-related developmental disorder: A case series and phenotypic analysis of 10 additional patients with monoallelic PUF60 variants.

PUF60-related developmental disorder (also referred to as Verheij syndrome), resulting from haploinsufficiency of PUF60, is associated with multiple congenital anomalies affecting a wide range of body systems. These anomalies include ophthalmic coloboma, and congenital anomalies of the heart, kidney, and musculoskeletal system. Behavioral and intellectual difficulties are also observed. While less common than other features associated with PUF60-related developmental disorder, for instance hearing impairment and short stature, identification of specific anomalies such as ophthalmic coloboma can aid with diagnostic identification given the limited spectrum of genes linked with this feature. We describe 10 patients with PUF60 gene variants, bringing the total number reported in the literature, to varying levels of details, to 56 patients. Patients were recruited both via locally based exome sequencing from international sites and from the DDD study in the United Kingdom. Eight of the variants reported were novel PUF60 variants. The addition of a further patient with a reported c449-457del variant to the existing literature highlights this as a recurrent variant. One variant was inherited from an affected parent. This is the first example in the literature of an inherited variant resulting in PUF60-related developmental disorder. Two patients (20%) were reported to have a renal anomaly consistent with 22% of cases in previously reported literature. Two patients received specialist endocrine treatment. More commonly observed were clinical features such as: cardiac anomalies (40%), ocular abnormalities (70%), intellectual disability (60%), and skeletal abnormalities (80%). Facial features did not demonstrate a recognizable gestalt. Of note, but remaining of unclear causality, we describe a single pediatric patient with pineoblastoma. We recommend that stature and pubertal progress should be monitored in PUF60-related developmental disorder with a low threshold for endocrine investigations as hormone therapy may be indicated. Our study reports an inherited case with PUF60-related developmental disorder which has important genetic counseling implications for families.

The COVID-19 pandemic's impact on worry and medical disruptions reported by individuals with chromosome 22q11.2 copy number variants and their caregivers.

BACKGROUND: The world has suffered immeasurably during the COVID-19 pandemic. Increased distress and mental and medical health concerns are collateral consequences to the disease itself. The Genes to Mental Health (G2MH) Network consortium sought to understand how individuals affected by the rare copy number variations of 22q11.2 deletion and duplication syndrome, associated with neurodevelopmental/neuropsychiatric conditions, were coping. The article focuses on worry and disruptions in medical care caused by the pandemic. METHODS: The University of Pennsylvania COVID-19 Stressor List and care disruption questions were circulated by 22 advocacy groups in English and 11 other languages. RESULTS: A total of 512 people from 23 countries completed the survey; most were caregivers of affected individuals. Worry about family members acquiring COVID-19 had the highest average endorsed worry, whilst currently having COVID-19 had the lowest rated worry. Total COVID-19 worries were higher in individuals completing the survey towards the end of the study (later pandemic wave); 36% (n = 186) of the sample reported a significant effect on health due to care interruption during the pandemic; 44% of individuals (n = 111) receiving care for their genetic syndrome in a hospital setting reported delaying appointments due to COVID-19 fears; 12% (n = 59) of the sample reported disruptions to treatments; and of those reporting no current disruptions, 59% (n = 269) worried about future disruptions if the pandemic continued. Higher levels of care disruptions were related to higher COVID-19 worries (Ps < 0.005). Minimal differences by respondent type or copy number variation type emerged. CONCLUSIONS: Widespread medical care disruptions and pandemic-related worries were reported by individuals with 22q11.2 syndrome and their family members. Reported worries were broadly consistent with research results from prior reports in the general population. The long-term effects of COVID-19 worries, interruptions to care and hospital avoidance require further study.

Expression of caveolin-1 in tooth germ, ameloblastoma and ameloblastic carcinoma.

BACKGROUND: The caveolin-1 protein (structural component of membrane caveolae) plays important roles in several biological functions, such as endocytosis, cell adhesion, and cell signaling. However, this protein has been associated with mechanisms of tumorigenesis in several neoplasms. The expression patterns and roles of caveolin-1 in the oral epithelium and in embryonic and odontogenic tumor tissues are still unclear. MATERIAL AND METHODS: The expression of caveolin-1 was evaluated in samples of the normal gingival epithelium (n=7), human tooth germ (TG) (n=12), ameloblastoma (AM) (n=83), and ameloblastic carcinoma (AC) (n=9) by immunohistochemistry. Additionally, AM samples were analyzed by qRT-PCR and Western blot. RESULTS: Most TG (91.7%), AM (73.5%) and AC (100%) samples showed diverse patterns of immunohistochemical positivity for caveolin-1, while only one gingival sample was positive. The transcript levels of cav-1 were significantly upregulated by 14.9-fold in AM tissue (P = 0.0014) compared to those in normal gingival epithelial tissue, as shown by qRT-PCR. Presence of caveolin-1 protein was confirmed by Western blot analysis. The caveolin-1 immunoexpression patterns throughout the stages of TG show its importance during odontogenesis. CONCLUSIONS: The overexpression of caveolin-1 in AM and AC compared to its expression in normal gingival epithelium (adult tissue) suggests a possible role of caveolin-1 in protumoral events, but due to the similar immunoexpression observed in AM and AC, caveolin-1 may not necessarily participate in the malignant transformation process. However, future studies are needed to clarify and confirm these hypotheses.

Expression of hMLH1 and hMSH2 proteins in ameloblastomas and tooth germs.

BACKGROUND: Mismatch repair proteins (MMRPs) are a group of nuclear enzymes that participate in the repair of base mismatches that occur during DNA replication in all proliferating cells. The most studied MMRPs are hMSH2 and hMLH1, which are known to be highly expressed in normal tissues. A loss of MMRPs leads to the accumulation of DNA replication errors in proliferating cells. Ki-67 is a biomarker regarded to be the gold-standard tool for determining cell proliferation by immunohistochemical methods. The aim of this study was to investigate the immunohistochemical expression of hMLH1, hMSH2 and Ki-67 proteins in ameloblastomas and tooth germs, to contribute to the understanding of the development of this odontogenic neoplasm. MATERIAL AND METHODS: Immunohistochemical assays to determine the presence of proteins hMSH2, hMLH1 and Ki-67 were performed in 80 ameloblastomas (40 solid and 40 unicystic) and five tooth germs. RESULTS: Unicystic ameloblastomas showed higher MMRP expression (hMLH1: 62.5 ± 43.4; hMSH2: 83.3 ± 47.8) than did solid ameloblastomas (hMLH1: 59.4 ± 13.5; hMSH2: 75.8 ± 40.2). Additionally, the cell proliferation index assessed by Ki-67 was inversely proportional to the expression of MMRP. Comparison between tooth germs and ameloblastoma revealed significantly higher expression of hMLH1, hMSH2 and Ki-67 in tooth germs (p=0.02). CONCLUSIONS: The differences of MMRP and Ki-67 immunoexpression between ameloblastomas and tooth germ suggest that alterations in the MMRP mechanisms could participate in the biological behavior of ameloblastomas.

Primordial odontogenic tumor: Subepithelial expression of Syndecan-1 and Ki-67 suggests origin during early odontogenesis.

Primordial odontogenic tumor (POT) is composed of variably cellular myxoid connective tissue, surrounded by cuboidal to columnar odontogenic epithelium resembling the inner epithelium of the enamel organ, which often invaginates into the underlying connective tissue. The tumor is delimited at least partially by a thin fibrous capsule. It derives from the early stages of tooth development. Syndecan-1 is a heparan sulfate proteoglycan that has a physiological role in several cellular functions, including maintenance of the epithelial architecture, cell-to-cell adhesion and interaction of cells with extracellular matrix, and with diverse growth factors, stimulating cell proliferation. Ki-67 is considered the gold standard as a cell proliferation marker. The aim of this study was to examine the expression of Syndecan-1 and Ki-67 proliferation index in POT and normal tooth germs to better understand the biological behavior of this tumor. Results showed that Syndecan-1 was more intensely expressed in subepithelial mesenchymal areas of POT, in a pattern that resembles the early stages of tooth development. The cell proliferation index (4.1%) suggests that POT is a slow growing tumor. Syndecan-1 expression in tooth germs in late cap and early bell stages was similar to POT, showing immunopositivity in subepithelial mesenchymal condensed areas. The immunohistochemical findings showed a pattern in which the population of subepithelial mesenchymal cells exhibited greater proliferative activity than the central portion of the dental papilla.

Molecular diagnosis in patients with retinoblastoma: Report of a series of cases. / Diagnóstico molecular en pacientes con retinoblastoma: reporte de una serie de casos.

OBJECTIVES: To report the benefits of genetic diagnosis in patients with retinoblastoma. METHOD: Observational study. Patients with retinoblastoma and their families were included. Demographic and clinical data were recorded. Blood and tumour samples were obtained. Next generation sequencing was performed on the samples. When deletion 13 q syndrome was suspected, cytogenetics microarray was performed (Cytoscan® HD, Affymetrix, Santa Clara, CA, USA), with a high density chip of 1.9 million of non-polymorphic probes and 750 thousand SNP probes. RESULTS: Of the 7 cases were analysed 4 were male. The mean age at diagnosis was 21 months (range 5-36). Three cases had bilateral retinoblastoma, and 4 unilateral. None had family history. In all patients, blood was analysed, and a study was performed on the tissue from 2 unilateral enucleated tumours, in which 6 mutations were identified, all de novo. Just one was novel (c.164delC; case 1). One case of unilateral tumour revealed blood mosaicism, showing that his condition was inheritable, and that there is a high risk of developing retinoblastoma in the unaffected eye. The patient also has an increased risk of presenting with other primary tumours. CONCLUSION: Molecular diagnosis of RB1 in patients with retinoblastoma impacts on the decision process, costs, treatment, and prognosis of patients, as well as their families.

Estrategias de identificación de planteamientos alternativos a la experimentación animal / Strategies for the identification of alternative approaches to animal experimentation

Los investigadores deben asegurarse de que la información que podrían obtener con su experimentación no está ya disponible, que no existe otro procedimiento para llevarlo a cabo sin emplear animales y que el protocolo se ha diseñado teniendo en cuenta consideraciones de protección animal. Sin embargo, la identificación de procedimientos alternativos empleados por otros científicos sigue siendo un proceso muy complejo, debido, sobre todo, a la deficiente indexación de las publicaciones en las bases de datos bibliográficas. Una búsqueda eficiente debe basarse en emplear siempre varias bases de datos, en revisar al menos los documentos de los últimos 5-10 años. En primer lugar debe evitarse la duplicación inútil de investigaciones, es decir, asegurarse de que la información que pudiera obtenerse en el estudio no está ya disponible. A continuación se realiza la búsqueda de posibles alternativas de reemplazo. Si ésta no fuera productiva, se identificarían alternativas de reducción y refinamiento, tratando de mejorar, en lo posible, cada una de las fases de la experimentación animal. Los estudios toxicológicos de finalidad reguladora presentan la exigencia de utilizar protocolos oficiales, por lo que deben localizarse en sus directorios específicos. Las alternativas en la enseñanza y entrenamiento, como los modelos mecánicos, audiovisuales y de simulación, se encuentran recogidas en bases de datos específicas. Finalmente, cuando no se encuentran opciones válidas en otras fuentes, es posible recurrir a expertos, tanto directamente como en foros especializados. Todo ello se facilita con el buscador Buscaalternativas.com (http://buscaalternativas.com) (AU)

The researchers should be sure that the information obtainable with the experiments is not yet available, that there is no other possible procedure without the use of animals or that the protocol was designed taking into account animal protection considerations. However, the identification of alternative procedures employed by other scientists is a very complex process, mainly due to the deficient indexation of the articles. An efficient search must be based on the use of several data bases and the review of documents of the last 5-10 years. The search strategy presents several phases. Firstable, the unnecessary duplication of studies should be avoided, assuring the information obtainable in the study is not yet available. A search for replacement alternatives is then carried out. If it is not productive, reduction and refinement alternatives are identified to improve every phase of animal research. Toxicological regulatory studies must use official protocols, which should be localized in specific directories. Alternatives in education and training, including mechanical models, audiovisuals and simulations are included in specific databases. Finally, when no valid options are found in other sources, it is possible to ask experts, directly or through specialized debate lists. The procedure is facilitated thanks to the Web Buscaalternativas.com (http://buscaalternativas.com) (AU)

Necesidades docentes en Evaluación y Prevención del Riesgo Tóxico: de la Toxicología Experimental a la Reguladora / Educational needs in Toxic Prevention and Risk Assessment: From experimental to Regulatory Toxicology

Se revisan las necesidades formativas para una serie de raas aplicadas de la toxicología derivadas de las áreas fundamentales mecanística y de evaluación toxicológica, que comprenden fundamentalmente la toxicología de sistemas, la evaluación del riesgo y la toxicología reguladora. Son materias necesarias para el desempeño profesional en los organismos nacionales, autonómicos y locales responsables de la regulación e inspección de la comercialización y uso de compuestos industriales, medicamentos, productos cosméticos, alimentarios o fitosanitarios, o de contaminantes ambientales o laborales, así como en industrias y consultorías. Se han identificado más de 25 actividades científicas de gran relevancia realizadas en España desde 1995, con una media de 1,6 actividades por año. Los principales grados de las ciencias de la salud incluyen una asignatura troncal de Toxicología, que puede aparecer como optativa en otros, generalmente de 6 créditos ECTS, con un programa muy uniforme, que incluye entre sus descriptores la estimación del riesgo, la evaluación toxicológica, el empleo de métodos alternativos y las bases de la toxicología reguladora. En relación con los estudios de postgrado, se identifican másteres con una gran variedad de contenidos y enfoques, que en algunos casos cubren un abanico muy amplio de disciplinas, incluyendo las toxicológicas, así como otros que cubren las áreas específicas de la toxicología. De la revisión transversal de los programas docentes de pregrado, postgrado y doctorado en España, se deduce la paulatina adaptación al EEES y la adecuada inclusión en los mismos de materias sobre evaluación experimental de la toxicidad, prevención del riesgo tóxico y toxicología reguladora (AU)

The training needs for a range of applied toxicology branches arising from the fundamental areas of mechanistic and toxicological evaluation, which mainly comprise of systems toxicology , risk assessment and regulatory toxicology , are reviewed. Thet are necessary for professional performance in national, regional and local authorities responsible for regulation and inspection of the marketing and use of industrial chemicals, pharmaceuticals, cosmetics, food or plant protection products or environmental or occupational pollutants, as well as industries and consultancies. There are more than 25 highly relevant scientific activities carried out in Spain since 1995, with an average of 1.6 activities per year . The main bachelor's degrees of the health sciences include a very uniform obligatory subject of T oxicology , which may appear as an option in others, usually with 6 ECTS credits, which includes among its descriptors risk e stimation assessment toxicology, the use of alternative methods and the basis for regulatory toxicology. In relation to postgraduate studies, masters were identified with a variety of topics and approaches, which in some cases cover a very wide range of disciplines, including toxicology and other covering in depth the specific toxicologic areas. From the transversal review of the teaching programs at under graduate, graduate and doctoral degrees in Spain, it was deduced a gradual adaptation to the EHEA and the appropriate inclusion of subjetcs on experimental evaluation of toxicity , toxic hazard prevention and regulatory toxicology (AU)

Clinical features of chromosome 22q11.2 microdeletion syndrome in 208 Chilean patients.

Patients with chromosome 22q11 deletion syndrome exhibit significant phenotypic variability. Epidemiologic data suggest a higher incidence in Hispanics, but limited clinical information is available from Latin-American patients. We describe the clinical features of Chilean patients with 22q11 deletion syndrome and compare their findings with those reported in large European, Japanese and US series. Data were obtained from 208 patients from five medical centers. Mean age at diagnosis was 5.2 years, with a median of 2.3 years. Congenital heart defects were present in 59.6%, lower than other large series that averaged 75.8%. Palate abnormalities were present in 79%, higher than previous reports averaging 56%. Patients with congenital heart disease were diagnosed earlier (median 0.3 years of age) than those without heart defects (median 5.6 years) and had greater mortality attributable to the syndrome (9.8% vs 2.4%, respectively). The differences in frequencies of major anomalies may be due to growing awareness of more subtle manifestations of the syndrome, differences in clinical ascertainment or the presence of modifier factors. These observations provide additional data useful for patient counseling and for the proposal of health care guidelines.

Aquatic toxicity assessment of the additive 6-methylcoumarine using four experimental systems.

The toxicity assessment of chemicals is one of the main issues in the current policies in order to protect the health of the environment and human beings. Food and cosmetic additives have been extensively studied in relation to their toxicity to humans, but data about their ecotoxicological effects are scarce. The aim of this study was to evaluate the toxic effects of the additive 6-methylcoumarine in the aquatic milieu using a test battery comprising experimental model systems from different trophic levels. The inhibition of bioluminiscence was studied in the bacteria Vibrio fischeri (decomposer), the inhibition of growth was evaluated in the alga Chlorella vulgaris (producer) and immobilization was studied in the cladoceran Daphnia magna (first consumer). Finally, several end points were evaluated in the RTG-2 salmonid fish cell line, including neutral red uptake, protein content, methylthiazol tetrazolium salt metabolization, glucose-6-phosphate dehydrogenase activity, lactate dehydrogenase activity and leakage, and morphology. The sensitivity of the test systems employed was as follows: V. fischeri > D. magna > C. vulgaris > RTG-2 cell line. The results show that 6-methylcoumarine is not expected to produce acute toxic effects on the aquatic biota. However, chronic and synergistic effects with other chemicals cannot be excluded and should be further investigated.

La regulación de la protección frente al riesgo por agentes físicos / The regulation of the protection against the risk by physical agents

Aunque el toxicólogo se preocupa principalmente de los efectos nocivos de las sustancias químicas, no puede inhibirse de considerar las acciones y efectos de los agentes físicos sobre los seres vivos, ya que también son objeto de interés de la Toxicología, tanto por su interacción directa con las biomoléculas como por influir en las acciones de las sustancias químicas sobre los sistemas biológicos. Esta preocupación también llega al legislador que ha elaborado diferentes tipos de normativas que persiguen la protección de la salud humana y el medio ambiente. Los agentes físicos lesivos y tóxicos suelen agruparse según el tipo de energía en que se manifiesten: la energía mecánica, en forma de ruido y vibraciones; la energía calorífica; lapresión y la gravedad; y la energía electromagnética, en la que se distinguen radiaciones ionizantes y no ionizantes. Las radiaciones ionizantes son las más peligrosas, siendo sus principales ámbitos normalizados la protección sanitaria de la población o de los trabajadores, el control de las exposiciones en usos médicos, de los traslados de sustancias radiactivas, de las fuentes radiactivas selladas de actividad elevada y de las fuentes huérfanas y de la contaminación de aguas y alimentos, así como la conservación de alimentos por irradiación. Las regulaciones sobre campos electromagnéticos persiguen la protección del público en general y de los trabajadores frente al riesgo eléctrico y electromagnético, la armonización del espectro radioeléctrico, la protección del dominio público radioeléctrico y el control de los equipos. La radiación ultravioleta es la más peligrosa de las radiaciones ópticas, por lo que se controla tanto la exposición solar como la de fuentes artificiales, incluyendo los aparatos de bronceado. El ruido, de interés en ototoxicología y salud ambiental, se encuentra regulado tanto en el ámbito ocupacional como en el ambiental. Las vibraciones más peligrosas son las de alta frecuencia, por lo que también a ellas se refieren normativas específicas (AU)

Although the toxicologist is concerned mainly with the adverse effects of chemicals, can not be inhibited to consider the actions and effects of physical agents on living organisms. It is also a subject of interest of Toxicology, both for their direct interaction with biomolecules as to influence the action of chemicals on biological systems. This concern has also come to the legislator that has developed different types of regulations to protect human health and the environment. Harmful and toxic physical agents are usually grouped by the type of energy: the mechanical energy in the form of noise and vibration; the heat energy; the pressure and gravity; and the electromagnetic energy, expressed in ionizing and non-ionis ingradiations. Ionizing radiation are the most dangerous, and their main regulated areas are the protection of the health of the public and workers, the control of the exposure on medical uses, the transportation of radioactive substances, the sources of high-activity sealed radioactive substances, and the contamination of water and food, as well as food preservation by irradiation. The regulations on electromagnetic fields pursue the protection of the general public and workers against electrical and electromagnetic risks, the harmonization of radio spectrum and the control of equipment. The ultraviolet radiation is the most dangerous optical radiation, so it should be monitored both solar exposure as artificial sources, including tanning devices. The noise, of interest in ototoxicology and environmental health is regulated at both occupational and environmental levels. As the most dangerous vibrations are those of high frequency, they also have specific regulation (AU)

Functional treatment of snoring using oral shields in conjunction with the tongue repositioning manoevre

The tongue repositioning manoeuvre has been demonstrated to lead to a closed rest position of orofacial structures with increased contact of the velum with the tongue and a contact position of the tongue at the hard palate. Within the multifactorial etiology of snoring, the tongue repositioning manoeuvre was used as training method in conjunction with pressure indicating oral shields to reduce symptoms of snoring by stabilisation of the orofacial system. Bed partner ranking of 128 snorers treated consecutively showed a score before treatment of 8.9 on a 10 cm visual analogue scale. After treatment the score decrease to 4.2 (p<0.01). No significant BMI , age or gender specific influence of the outcome could beobserved. The data give evidence, that dynamic stabilisation of the orofacial system with oral shields in conjunction with the tongue repositioning manoevre is a valuable instrument to reduce the snoring problem.

La maniobra de reposicionamiento lingual ha demostrado tener ventaja para mantener cerrada el resto de estructuras orofaciales, con un aumento del contacto del paladar blando con la lengua y también en posición de contacto el paladar duro con la lengua. Dentro de la etiología multifactorial del ronquido, la maniobra de reposicionamiento lingual ha sido usada como método de entrenamiento, en conjunto con protectores orales que indican la presión para reducir los síntomas del ronquido y estabilizar el sistema orofacial. Un total de 128 pacientes roncadores tratados consecutivamente mostraron una puntuación antes del tratamiento de 8,9 a 10 cm en una escala visual análoga. Después del tratamiento, el puntaje disminuyó a 4,2 cm (p< 0,01). El índice de masa corporal no fue significativo, y no pudo ser observado si la edad o el género tenían influencia. Los datos evidenciaron que la estabilización dinámica del sistema orofacial, en conjunto con la maniobra de reposicionamiento lingual resulta ser una valiosa herramienta para reducir el problema del ronquido.

Acute and subacute toxic effects produced by microcystin-YR on the fish cell lines RTG-2 and PLHC-1.

Approximately 80 microcystins (MCs) variants have been isolated in surface water worldwide. The toxicity of the most frequently MCs are encountered, MC-LR and MC-RR, has been extensively studied in humans and animals. However, studies dealing with MC-YR toxicity are still scarce. In this work, the toxic effects of MC-YR were investigated in the fish cell line PLHC-1, derived from a hepatocellular carcinoma of the topminnow Poeciliopsis lucida, and RTG-2 fibroblast-like cells derived from the gonads of rainbow trout Oncorhynchus mykiss. After 48 h, morphological and biochemical changes (total protein content, neutral red uptake and methylthiazol tetrazolium salt metabolization) were determined. The most sensitive endpoint for both cell lines was the reduction of total protein content, with EC(50) values of 35 microM for PLHC-1 cells and 67 microM for the RTG-2 cell line. Lysosomal function and methylthiazol tetrazolium salt metabolization were stimulated at low concentrations, while they decreased at high doses. Increase of piknotic cells, rounding effects, reduction in cell number and cell size, hydropic degeneration, and death mainly by necrosis but also by apoptosis were observed in the morphological study. Furthermore, PLHC-1 cells are more sensitive than RTG-2 cells to MC-YR exposure. These results were similar to those obtained when both cell lines were exposed for 24h to a Microcystis aeruginosa isolated strain extract containing MC-LR.

Induction of EROD activity by 1-phenylimidazole and beta-naphthoflavone in rainbow trout cultured hepatocytes: a comparative study.

The classical pathway for induction of cytochrome P4501A (CYP1A) by xenobiotics is ligand binding to the aryl hydrocarbon receptor (AhR). However, several studies with mammalian cell systems point out a range of xenobiotics including imidazole derivatives, which are able to activate CYP1A through non-classical mechanisms. The objective of the present work is to compare induction of CYP1A (determined at the catalytic level as 7-ethoxyresorufin-O-deethylase, EROD) in rainbow trout (Oncorhynchus mykiss) hepatocytes by the prototypic AhR ligand, beta-naphthoflavone (betaNF), and by the imidazole derivative, 1-phenylimidazole (PIM). PIM was able to induce EROD activity although its potency was clearly lower than that of betaNF. In order to assess the relative importance of classical AhR ligand binding and alternative signaling pathways in CYP1A induction by PIM, co-exposure experiments with the partial AhR antagonist alpha-naphthoflavone (alphaNF) or with inhibitors of protein kinase C (staurosporine) and tyrosine kinases (genistein, herbimicine) were performed. alphaNF and herbimicin provoked a decrease of EROD induction both by betaNF and PIM, whereas staurosporine and genistein remained without effect. The overall similarities in the response of betaNF and PIM to the various inhibitors suggest that both compounds, in apparent contrast to the behaviour of some other imidazole derivatives, induce CYP1A following similar mechanisms.

Comparative cytotoxicity of alachlor on RTG-2 trout and SH-SY5Y human cells.

The cytotoxic effects of the herbicide alachlor were compared on rainbow trout gonadal RTG-2 and human neuroblastoma SH-SY5Y cell lines. The end points evaluated in both cells after 24, 48, and 72 h of exposure were total protein content (PC), lysosomal function, and mitochondrial's integrity by mitochondrial succinate dehydrogenase (SDH) activity. After 24 h, cytoplasmic membrane integrity by cytosolic lactate dehydrogenase (LDH) leakage and LDH intracellular activity were also studied. In addition, acetylcholinesterase activity (AChE) was quantified in SH-SY5Y cells. The possible biotransformation of alachlor by RTG-2 cells was investigated by analyzing the exposure culture medium by liquid chromatography-mass spectrometry. In RTG-2, EC50 values on PC, lysosomal function, and SDH activity after 24 h exposure ranged from 80 to 95 microM and decreased to approximately 40 microM for longer exposure time periods. SH-SY5Y cells were slightly more sensitive than RTG-2 cells, with EC50 values on PC and lysosomal function ranging from 87 to 75 microM at 24 h and decreasing to 47 microM and 34 microM at 72 h, respectively. AChE activity was increased, being the most sensitive marker for SH-SY5Y with an EC50 of 20 microM at 24 h. The metabolic enzyme SDH was stimulated in SH-SY5Y and reduced in RTG-2 cells. At the studied conditions, no metabolites of alachlor were detected in RTG-2 cultures. In conclusion, the proposed battery approach is an effective screening tool for the safety assessment of environmental contaminants as a complement to fish and animal toxicity procedures.

Toxic effects produced by microcystins from a natural cyanobacterial bloom and a Microcystis aeruginosa isolated strain on the fish cell lines RTG-2 and PLHC-1.

Toxic cyanobacterial blooms are a worldwide problem, causing serious water pollution and public health hazard to humans and livestock. The intact cells as well as the toxins released after cellular lysis can be responsible for toxic effects in both animals and humans and are actually associated with fish kills. Two fish cell lines-PLHC-1 derived from a hepatocellular carcinoma of the topminnow Poeciliopsis lucida and RTG-2 fibroblast-like cells derived from the gonads of rainbow trout Oncorhynchus mykiss were exposed to several concentrations of extracts from a natural cyanobacterial bloom and a Microcystis aeruginosa-isolated strain. After 24 hours, morphologic and biochemical changes (total protein content, lactate dehydrogenase leakage, neutral red uptake, methathiazole tetrazolium salt metabolization, lysosomal function, and succinate dehydrogenase [SDH] activity) were investigated. The most sensitive end point for both cyanobacterial extracts in PLHC-1 cells was SDH activity, with similar EC(50) values (6 microM for the cyanobacterial bloom and 7 microM for the isolated strain). RTG-2 cells were less susceptible according to SDH activity, with their most sensitive end point lysosomal function with an EC(50) of 4 microM for the M. aeruginosa-isolated strain and 72 microM for the cyanobacterial bloom. The lysosomal function was stimulated at low concentrations, although SDH activity increased at high doses, indicating lysosomal and energetic alterations. Increased secretion vesicles, rounding effects, decreased cell numbers and size, hydropic degeneration, esteatosis, and apoptosis were observed in the morphologic study. Similar sensitivity to the M. aeruginosa-isolated strain was observed in both cell lines, whereas the cyanobacterial bloom was more toxic to the PLHC-1 cell line.

High concentrations of pralidoxime are needed for the adequate reactivation of human erythrocyte acetylcholinesterase inhibited by dimethoate in vitro.

Due to the current controversy about the real effectiveness of the oximes in the treatment of organophosphate poisoning, the reactivation capacity of pralidoxime has been evaluated in vitro on human erythrocyte acetylcholinesterase inhibited by dimethoate. In the in vitro model, a partial recovery of acetylcholinesterase activity was observed with concentrations from 0.066 mM pralidoxime, probably useful enough to prevent death in most cases in vivo. However, much more effectiveness was observed with concentrations up to 0.70 mM pralidoxime. Although pralidoxime should be applied as soon as possible after organophosphate exposure, the application of the antagonist can be useful even 24h after, particularly for organophosphates with biological half-life longer than one day. The protective capacity of pralidoxime after the application was reduced up to 50% in 6h and disappeared almost completely in 24h. Furthermore, the pesticide and its metabolites remained active and were able to inhibit the enzyme as soon as pralidoxime reduced its antagonist capacity. Our results in conjunction with the short half-life of pralidoxime suggest that the maintenance of higher plasmatic concentrations than the currently used should be considered in the management of severe poisoned patients, although adverse effects could be expected.

The use of the fish cell lines RTG-2 and PLHC-1 to compare the toxic effects produced by microcystins LR and RR.

Cyanobacterial toxins, especially microcystins (MC), are found in eutrophied waters through the world. Acute poisonings of animals and humans has been reported following MC exposure. In the present study, two fish cell lines, PLHC-1 and RTG-2, were evaluated after exposure to the cyanobacterial toxins MC-LR and MC-RR. The effects of different concentrations of the toxins were investigated in both cell lines at morphological and biochemical levels (total protein content, lactate dehydrogenase leakage, lysosomal activity and succinate dehydrogenase activity). The results obtained showed a decrease in protein content and no relevant increase in cell disruption, except for MC-LR in PLHC-1 cells. Morphological changes produced by microcystins were cellular swelling, blebbling, rounding, reduction in the cell number and increase in the number and size of lysosomal bodies. In addition, steatosis was produced in hepatoma PLHC-1 cells, particularly with MC-RR. Furthermore, the fish PLHC-1 cell line was more sensitive than RTG-2 cells to the cyanobacterial toxins compared, being the stimulation of the lysosomal function and the induction of steatosis the most specific changes detected.

Ecotoxicological evaluation of diethanolamine using a battery of microbiotests.

In order to investigate the potential ecotoxicity of diethanolamine (DEA), a battery of model systems was developed. DEA is widely used as a chemical intermediate and as a surface-active agent in cosmetic formulations, pharmaceuticals and agricultural products. DEA was studied using ecotoxicological model systems, representing four trophic levels, with several bioindicators evaluated at different exposure time periods. The battery included bioluminescence inhibition of the bacterium Vibrio fischeri, growth inhibition of the alga Chlorella vulgaris and immobilization of the cladoceran Daphnia magna. Cell morphology, total protein content, neutral red uptake, MTS metabolization, lysosomal function, succinate dehydrogenase activity, G6PDH activity, metallothionein levels and EROD activity were studied in the hepatoma fish cell line PLHC-1, derived from Poeciliopsis lucida. The systems most sensitive to DEA were both D. magna and V. fischeri, followed by C. vulgaris and the fish cell line PLHC-1. The most prominent morphological effect observed in PLHC-1 cultures exposed to DEA was the induction of a marked steatosis, followed by death at high concentrations, in some cases by apoptosis. The main biochemical modification was a nearly three-fold increase in metallothionein levels, followed by the stimulations of lysosomal function and succinate dehydrogenase and G6PDH activities. Judging by the EC(50) values in the assay systems, DEA is not expected to produce acute toxic effects in the aquatic biota. However, chronic and synergistic effects with other chemicals cannot be excluded.