Glioblastoma research on zebrafish xenograft models: a systematic review

Glioblastoma (GBM) constitutes the most common primary brain tumor in adults. The challenges in GBM therapeutics have shed light on zebrafish used as a promising animal model for preclinical GBM xenograft studies without a standardized methodology. This systematic review aims to summarize the advances in zebrafish GBM xenografting, compare research protocols to pinpoint advantages and underlying limitations, and designate the predominant xenografting parameters. Based on the PRISMA checklist, we systematically searched PubMed, Scopus, and ZFIN using the keywords “glioblastoma,” “xenotransplantation,” and “zebrafish” for papers published from 2005 to 2022, available in English. 46 articles meeting the review criteria were examined for the zebrafish strain, cancer cell line, cell labeling technique, injected cell number, time and site of injection, and maintenance temperature. Our review designated that AB wild-type zebrafish, Casper transparent mutants, transgenic Tg(fli1:EGFP), or crossbreeding of these predominate among the zebrafish strains. Orthotopic transplantation is more commonly employed. A number of 50–100 cells injected at 48 h post-fertilization in high density and low infusion volume is considered as an effective xenografting approach. U87 cells are used for GBM angiogenesis studies, U251 for GBM proliferation studies, and patient-derived xenograft (PDX) to achieve clinical relevance. Gradual acclimatization to 32–33 °C can partly address the temperature differential between the zebrafish and the GBM cells. Zebrafish xenograft models constitute valuable tools for preclinical studies with clinical relevance regarding PDX. The GBM xenografting research requires modification based on the objective of each research team. Automation and further optimization of the protocol parameters could scale up the anticancer drug trials (AU)

Histone acetyltransferases derived RW20 protects and promotes rapid clearance of Pseudomonas aeruginosa in zebrafish larvae / La histona acetiltransferasa derivada RW20 protege y promueve la rápida eliminación de Pseudomonas aeruginosa en larvas de pez cebra

Pseudomonas is a group of bacteria that can cause a wide range of infections, particularly in people with weakened immune systems, such as those with cystic fibrosis or who are hospitalized. It can also cause infections in the skin and soft tissue, including cellulitis, abscesses and wound infections. Antimicrobial peptides (AMPS) are the alternative strategy due to their broad spectrum of activity and act as effective treatment against multi-drug resistance pathogens. In this study, we have used an AMP, RW20 (1RPVKRKKGWPKGVKRGPPKW20). RW20 peptide is derived from the histone acetyltransferases (HATs) of the freshwater teleost, Channa striatus. The antimicrobial prediction tool has been utilized to identify the RW20 sequence from the HATs sequence. We synthesized the peptide to explore its mechanism of action. In an in vitro assay, RW20 was challenged against P. aeruginosa and we showed that RW20 displayed antibacterial properties and damaged the cell membrane. The mechanism of action of RW20 against P. aeruginosa has been established via field emission scanning electron microscopy (FESEM) as well as fluorescence assisted cell sorter (FACS) analysis. Both these experiments established that RW20 caused bacterial membrane disruption and cell death. Moreover, the impact of RW20, in-vivo, was tested against P. aeruginosa-infected zebrafish larvae. In the infected larvae, RW20 showed protective effect against P. aeruginosa by increasing the larval antioxidant enzymes, reducing the excess oxidative stress and apoptosis. Thus, it is possible that HATs-derived RW20 can be an efficient antimicrobial molecule against P. aeruginosa.(AU)

Lactobacillus rhamnosus GG treatment potentiates ethanol-induced behavioral changes through modulation of intestinal epithelium in Danio rerio / El tratamiento con Lactobacillus rhamnosus GG potencia los cambios de comportamiento inducidos por etanol a través de la modulación del epitelio intestinal en Danio rerio

The gut-brain axis directly regulates the brain homeostatic environment; an imbalance in gut microbial composition following ethanol exposure is maleficent. In this context, involvement of probiotics as prophylactic intervention against ethanol-induced neurotoxicity is elusive in the literature. Therefore, the present study was aimed to determine the impact of chronic ethanol exposure on the neurobehavioral response of zebrafish and possible neuroprotection through co-supplementation of probiotic Lactobacillus rhamnosus GG (LGG). Zebrafish were divided into naive, control, ethanol (0.01% v/v), LGG, and ethanol co-supplemented with LGG groups. Neurobehavioral assessment was performed after 7 days of chronic waterborne exposure to ethanol with LGG co-supplementation followed by histopathological studies. The findings indicated that there was a clear alteration in locomotor activity and habitat preference, with animals preferentially migrating toward altered zones on exposure to ethanol. However, co-supplementation of LGG showed restoration against ethanol-induced neurobehavioral and cognitive dysfunction. Brain tissue pyknosis and intestinal epithelial disruption were significantly mitigated on LGG co-supplementation against ethanol in zebrafish. The present study provides a novel approach toward supplementation of probiotics such as LGG in modulation of gut commensal microbiota influencing zebrafish behavior. Moreover, the findings delineate the possible role of probiotics as a curative administration to counter ethanol-persuaded neurological outcomes.(AU)

Actividad embriotóxica de los extractos acuosos de Allophylus edulis y Matricaria recutita en pez cebra / Embryotoxic activity of aqueous extracts of Allophylus edulis and Matricaria recutita in zebrafish

La planta de uso medicinal Allophylus edulis se utiliza para el tratamiento de enfriamiento, catarro, diabetes y la planta Matricaria recutita (manzanilla) se utiliza como antiinflamatorio, descongestivo, tratar conjuntivitis, cólicos. Ambas especies son de amplio uso a nivel nacional, en Paraguay, e internacional, pero no se conocen bien sus posibles efectos secundarios sobre embriones de vertebrados. El objetivo de este trabajo fue evaluar si los extractos acuosos de, Allophylus edulis y Matricaria recutita tienen efecto tóxico en embriones de Danio rerio. Se trataron embriones de 6 horas pos fecundación con diferentes concentraciones de cada extracto para determinar la toxicidad de los extractos acuosos de Allophylus edulis y Matricaria recutita. En los resultados obtenidos, ambos extractos presentaron efectos tóxicos significativos en los siguientes parámetros: ausencia de formación de somitas, de latidos cardiacos, de circulación sanguínea y de pigmentación. No se observó un efecto teratogénico significativo. Se concluye que los extractos acuosos de Allophylus edulis y Matricaria recutita resultaron tóxicos para embriones de Danio rerio, de manera dosis dependiente. (AU)

Medicinal plant Allophylus edulis is used to treat colds, catarrh, diabetes and the Matricaria recutita plant (chamomile) is used as an anti-inflammatory, decongestant, conjunctivitis treatment, and colic. Both species are widely used nationally in Paraguay, and internationally, but their possible side effects on vertebrate embryos are not well known. The objectives of this work were to evaluate whether the aqueous extracts of Allophylus edulis and Matricaria recutita have a toxic effect on Danio rerio embryos. Embryos 6 hours after its fertilization were treated with different concentrations of each extract to determine the toxicity of the aqueous extracts of Allophylus edulis and Matricaria recutita. In the results obtained, both extracts presented significant toxic effects on the following parameters: absence of somite formation, heartbeat, blood circulation and pigmentation. No significant teratogenic effect was observed. It is concluded that the aqueous extracts of Allophylus edulis and Matricaria recutita were toxic for Danio rerio embryos, in a dose dependent way. (AU)

Un analisis de imagen por segmentación de la histopatología producida por la acrilamida en el pez cebra adulto (Danio rerio) / An image analysis by segmentation of histopathology produced by acrylamide in the adult zebrafish (Danio rerio)

Los sistemas de análisis de imágenes han permitido la implementación de técnicas de histomorfometría para cuantificar con precisión las irregularidades arquitectónicas y la complejidad de las estructuras histológicas en condiciones normales, así como en situaciones de daño tisular inducido. El objetivo del trabajo fue cuantificar los efectos histológicos producidos por la acrilamida en el modelo de vertebrado Danio rerio adulto expuesto durante 8 días a la concentración de 0.85 mM de un alqueno como la acrilamida. Se aplico un proceso de segmentación de imagenes a través del programa de dominio público ImageJ. Fiji, sobre preparaciones histológicas de tejido renal, intestinal y hepático, aplicando umbrales de intensidad y diseñando contornos alrededor de las áreas de interés ubicadas en imágenes mascaras binarias de 8-bit. Esta fue obtenida a partir de la transformación de la imagen original contrastada con hematoxilina y eosina. Los datos obtenidos fueron incorporados al programa estadístico GraphPad Prims. 7 para su cuantificación y representación gráfica. Los espacios internos de las Capsulas de Bowman en el tejido renal registraron un mayor tamaño promedio en comparación con el tejido control, mientras que en el tejido intestinal se registraron vacuolas citoplasmáticas de mayor tamaño promedio en comparación a las vacuolas registradas en el tejido hepático, con un valor de ****p < 0,0001, altamente significativo en contraste con los respectivos controles. Concluimos que con la segmentación fue posible caracterizar a la histopatología inducida por la acrilamida como un efecto tejido-dependiente, resultando más sensible el tejido renal y el intestinal en comparación con el tejido hepático

Image analysis systems have allowed the implementation of histomorphometry techniques to precisely quantify architectural irregularities and the complexity of histological structures in normal conditions, as well as in situations of induced tissue damage. The aim of this work was to quantify the histological effects produced by acrylamide in the vertebrate model Danio rerio adults exposed for 8 days to the concentration of 0.85 mM of an alkene such as acrylamide. Segmentation was performed on histological preparations of renal, intestinal and hepatic tissue, through the use of the public domain program ImageJ Fiji, applying intensity thresholds and designing contours around the areas of interest located in binary 8-bit mask images. This was obtained from the transformation of the original image contrasted with hematoxylin and eosin. The data obtained were incorporated into the GraphPad Prims statistical program 7 for quantification and graphic representation. The internal spaces of the Bowman Capsules in the renal tissue registered a larger average size compared to the control tissue, while in the intestinal tissue cytoplasmic vacuoles of larger average size were recorded compared to the vacuoles recorded in the liver tissue , with a value of ****p <0,0001, highly significant in contrast to the respective controls. We conclude that with segmentation it was possible to characterize the histopathology acrylamide-induced as a tissue-dependent effect, with renal and intestinal tissue being more sensitive compared to liver tissue

Efecto del neonicotinoide - tiametoxam en el desarrollo embrionario del pez cebra (Danio rerio) / Effect of neonicotinoid - thiamethoxam in the zebrafish (Danio rerio) embryonic development

El pez cebra ha surgido como la mejor opción para ensayos toxicológicos, ofrece ventajas marcadas como un modelo de desarrollo de vertebrados y permite comprender las dinámicas de diversas sustancias tóxicas. Actualmente se ha encontrado una creciente preocupación por el uso de plaguicidas neonicotinoides y los posibles efectos ecológicos y ecotoxicológicos sobre los polinizadores y los invertebrados, y los efectos indirectos en ecosistemas acuáticos. El objetivo principal fue evaluar las alteraciones fenotípicas desde huevo hasta el periodo larval del pez cebra (Danio rerio) (6 días aproximadamente) relacionadas con tres dosis (0,48µg/ml, 0,90µg/ml, 1,30µg/ml) de plaguicida Neonicotinoide (tiametoxam). Para esto se usaron cuatro repeticiones, cada repetición con veinte huevos. Se realizó un diseño de bloques al azar evaluando la tasa de mortalidad y una descripción macroscópica de las alteraciones provocadas por este plaguicida, se encontraron alteraciones como: cifosis, lordosis, edema pericárdico, edema del saco vitelino, hipertrofia en las aletas y una ralentización en el desarrollo, la mayor tasa la presentó el tratamiento 3 (1,30µg/ml), demostrando que el neonicotinoide tiametoxam es tóxico y letal para el pez cebra

Zebrafish has emerged as the best choice for toxicological testing, present marked advantages as a model of vertebrate development and allows to understand the dynamics of various toxic substances. There has been growing concern over the use of neonicotinoid pesticides and possible ecological and ecotoxicological effects on pollinators and invertebrates, and indirect effects on aquatic ecosystems. The objective was to evaluate phenotypic alterations from egg to larval period of zebrafish (Danio rerio) (6 days approximately) related to three doses (0,48µg/ml, 0,90µg/ml, 1,30µg/ml) of the pesticide Neonicotinoid (Thiamethoxam), for this, four replicates were used, each repetition with twenty eggs. A randomized block design was performed evaluating the mortality rate and a macroscopic description of the alterations caused by this pesticide, alterations were found such as: kyphosis, lordosis, pericardial edema, yolk sac edema, fins hypertrophy and a slowdown in development, demonstrating a higher mortality rate in the treatment with higher concentration (1,30µg/ml). The neonicotinoid thiamethoxam that proved to be toxic and lethal to the zebrafish

Retinal development and photoreceptor synaptic ultrastructure are altered by abnormal rearing light regimes

The effects of light rearing regimen on distal retinal development and photoreceptor ultrastructure were investigated using light and electron microscopy. Zebrafish larvae were reared in constant light, control/cyclic light (14 hr light/10 hr dark), or constant dark conditions until 4 or 8 days postfertilization (dpf). Gross retinal morphology was not altered by light rearing conditions; however, ultrastructural differences were noted both within and between age groups. Significant differences were seen in photoreceptor outer segments (OS) and synaptic ribbons, the size of cone photoreceptor mitochondria, and postsynaptic horizontal cell spinules. Larvae reared in constant dark displayed reduced pigment dispersion; OS development was delayed and cone mitochondria were smaller at 4dpf, two results that reversed by 8dpf. Photoreceptor terminals of larvae reared in all treatment conditions displayed anchored synaptic ribbons with arciform densities and no significant differences in ribbon number. Ribbons were 30- 40% longer in photoreceptor terminals within the constant light treatment. The number of horizontal cell spinules invaginating into cone terminals varied and the spinule-to-ribbon ratio was higher in control and constant light-reared tissue by more than 2x at 4dpf. By 8dpf, this ratio was significantly highest in retinas reared in control/cyclic light conditions. Taken together, these results show that abnormal light rearing conditions affect synaptic structure in distal retina. These changes suggest a mechanism for the physiological and behavioral deficits reported in zebrafish larvae grown under constant light and/or dark conditions

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Evaluación de la citotoxicidad y bioseguridad de un extracto de polifenoles de huesos de aceitunas / Assessment of cytotoxicity and biosafety of polyphenolic extracts from olive pits

El olivo constituye una fuente de compuestos bioactivos, tanto en su fruto, como en sus subproductos. Algunos de sus compuestos han mostrado beneficios para la salud, siendo objetivo de este trabajo la evaluación de la bioseguridad in vitro e in vivo de extractos de huesos de aceituna ricos en polifenoles. Material y métodos: Se ha evaluado la citotoxicidad mediante adición de extracto de hueso de olivas disuelto en PBS (0-400 mg/l) a un cultivo de la línea celular THP1- XBlue-CD14 y evaluación de la viabilidad celular mediante la reacción de reducción de la resazurina por las células vivas. La bioseguridad se ha evaluado en pez cebra, incubando huevos fecundados en extracto de 0 a 100 mg/l durante 24 a 72 horas y midiendo los parámetros: a) letales (embriones muertos, huevos coagulados), b) subletales (movimientos espontáneos, pigmentación, edemas) y c) teratogénicos (malformaciones, retraso desarrollo). Resultados: La citotoxicidad (efecto tóxico cuando viabilidad inferior al 75%) del extracto de huesos de oliva en la línea celular THP1-XBlue-CD14, está en concentraciones superiores a 50 mg/l de extracto (viabilidad 77,5%), calculando una LD50 (dosis de letalidad 50%) superior a 800 mg/l. La bioseguridad in vivo con los embriones de pez cebra expuestos a concentraciones de extracto de 0- 100 mg/l mostró total viabilidad a 24, 48 y 72 horas post fecundación (hpf), no observándose mortalidad ni se apreciaron embriones con efectos subletales, teratógenos, ni adelanto o retraso en la eclosión. Se puede concluir que el extracto de huesos de olivas es altamente bioseguro hasta al menos concentraciones de 100 mg/l (AU)

The olive tree is a source of bioactive compounds, both its fruit and its by-products. Some of its compounds have shown health benefits, being objective of this work the evaluation of biosafety in-vitro and in vivo of extracts of olive stones rich in polyphenols. Material and methods: He has been evaluated for cytotoxicity by addition of lyophilized extract dissolved in PBS (0-400 mg/l) to a culture of the cell line THP1-XBlueCD14 and evaluation of cell viability by the reaction of reduction of resazurin by living cells. Biosecurity has been evaluated in zebrafish, incubating eggs fertilized in 0 to 100 mg/l extract for 24 to 72 hours and measuring parameters: a) lethal (dead embryos, coagulated eggs), b) sublethal (spontaneous movements, pigmentation, edemas) and c) teratogenic (malformations, retardation development). Results: Cytotoxicity (toxic effect when less than 75% viability) extract bones of olive in the cell line THP1- XBlue-CD14, is in concentrations higher than 50 mg/l, calculating a LD50 (dose lethality 50) more than 800 mg/l. The biosafety of zebrafish embryos exposed to concentrations of extract from 0-100 mg/l showed total viability at 24, 48 and 72 hours post fertilization (hpf), not observed mortality or appreciated embryos with sublethal effects, teratogenic, or advancement or delay in hatching. It can be concluded that the bones of olive extract is highly biosecured until at least 100 mg/l concentrations (AU)

Relación de los estadíos de maduración del ovario y el efecto del BPA a nivel hipofisario en pez cebra (Danio rerio) / Relationship of ovarian maturation stages and the effect of BPA on pituitary level in zebrafish (Danio rerio)

El bisphenol A (BPA), que es utilizado en la fabricación de envases y contenedores de alimentos, puede actuar como disruptor endocrino uniéndose a los receptores estrogénicos. Dado que la hipófisis esencialmente juega su papel en la gametogénesis y esteroidogénesis, el objetivo fue evaluar la acción del BPA sobre la hipófisis y estudiar la posible implicación sobre la maduración folicular mediante estudio histopatológico y morfométrico. Se utilizaron 60 pez cebra (Danio rerio) de 16 semanas, distribuidos al azar en un grupo control y cuatro grupos expuestos a BPA (1, 10, 100 y 1000 μg/L) en el agua durante 14 días). Se cuantificaron los niveles de BPA en peces mediante LC-MS/MS y se realizó el estudio histopatológico de adenohipófisis y ovario. Estructuralmente se reveló un proceso degenerativo de las células gonadotropas de todos los grupos tratados. Ultraestructuralmente, se observó en el grupo control y tratado con 1 μg/L una activación de las células, aumento de gránulos de secreción en el complejo de Golgi, y fundamentalmente, del retículo endoplásmico rugoso, mientras que en el resto de grupos estas células mostraron signos de degeneración. En los grupos tratados con 100 y 1000 μg/L, se observaron células de castración. En ovario pudo determinarse morfométricamente un incremento de atresia folicular a medida que se incrementó la concentración de exposición al BPA. Nuestros resultados indican que el BPA produce una degeneración de células gonadotropas a nivel hipofisario y un aumento de la atresia folicular en las gónadas probablemente debido a una inhibición en la liberación de las gonadotropinas (AU)

Bisphenol-A (BPA) is widely used as part of packaging and food containers, and acts as an endocrine disruptor by binding to estrogen receptors. The importance of the pituitary lies in its role in gametogenesis and gonadal steroidogenesis level. The objective was to evaluate the effects of BPA on the pituitary and its possible relationship with follicular maturation by histopathology. 60 female 16 weeks of age zebrafish were used, randomly divided into a control group and four groups exposed to BPA (1, 10, 100 and 1000 μg/L) in water for 14 days. After the sacrifice, samples were taken to quantify BPA levels in fish using LC-MS/MS, in addition to the histopathological study of the anterior pituitary and the ovary. Structural studies revealed a degenerative process of the gonadotrope cells of the pituitary in all treated groups, being these cells positive to PAS reaction. The ultrastructure presented cell activation in the group treated with 1 μg/L, with increased secretion granules at the Golgi complex, and essentially the rough endoplasmic reticulum, whereas in the other groups, these cells appeared degenerated. In the groups treated with 100 and 1000 μg/L, castration cells were observed with a rough endoplasmic vacuole. Increased ovarian follicular atresia as BPA concentration increased was also determined. The results indicate that BPA produces a degeneration of the gonadotrope cells at pituitary level and an increase of atretic follicles in gonads probably due to an inhibition in the release of the gonadotropins (AU)

Efectos letales y subletales de nanopartículas y material soluble de oro en el desarrollo de embriones de pez cebra / The lethal and sub-lethal effects of nanoparticulated and soluble gold on the development of zebrafish embryos

La toxicidad de las nanopartículas de oro (AuNP) en el desarrollo de pez cebra resulta un tanto controvertida, ya que algunos autores demuestran su inocuidad, mientras que otros autores advierten de la presencia de efectos tóxicos. Por otro lado, la toxicidad del material base de la AuNP en solución aún no se ha investigado, aunque generalmente se requiera. Por consiguiente, el objetivo de este estudio fue investigar los efectos de la exposición de AuNP y el oro soluble en embriones de pez cebra, aportando reflexiones sobre la toxicidad de AuNP. Para ello, se llevaron a cabo ensayos toxicológicos en embriones de pez cebra con AuNP sin recubrimiento y recubiertas con ácido hialurónico (12,8 ± 1,6 nm, TEM), así como el material base (HAuCl4) de dichas AuNP. Los resultados demuestran que las AuNP no fueron tóxicas durante las primeras fases del desarrollo de los embriones (0-48 h). Por otro lado, el HAuCl4 resultó ser letal a concentraciones > 64,1 mg/L. A concentraciones subletales (48,1-0,7 mg/L), el HAuCl4 no alteró el desarrollo ni la actividad motora o cardiaca, aunque redujo notablemente la capacidad de eclosión a > 5.7 mg/L, situándose el NOAEC en 2,7 mg/L. Teniendo en cuenta nuestros resultados y los encontrados por otros autores, se puede concluir que la superficie de carga y en menor medida el tamaño de las AuNP resultaron ser los factores clave para explicar la toxicidad de la AuNP. Nuestros resultados también confirmaron que este modelo, con embriones de pez cebra, es adecuado para el estudio de toxicidad de las nanopartículas, siempre y cuando éstas estén completamente caracterizadas antes y durante el ensayo de toxicidad (AU)

Toxicity of gold nanoparticles (AuNPs) on zebrafish development is a rather controversial aspect in literature, and the investigation of the bulk material has not been considered yet, even though it is generally required. Therefore, we investigated the potential adverse effects of AuNPs and soluble gold on zebrafish embryos, while trying to find a rationale for gold nanoparticle toxicity. For such a purpose, uncoated and coated with hyaluronic acid AuNPs (12.8 ± 1.6 nm, TEM) and the bulk material (HAuCl4) were tested for their toxicity in zebrafish embryos. Results showed that the AuNPs were non-toxic for the zebrafish development during exposure (0-48h). Soluble gold (HAuCl4) was lethal at > 64.1 mg/L. At sublethal concentrations (48.1-0.7 mg/L) HAuCl4 did not alter embryo development, spontaneous movements or heartbeat rate, but the hatching ability was affected at > 5.7 mg/L, resulting in a NOAEC of 2.7 mg/L. According to our data and others found in scientific literature, it can be concluded that surface charge and, to a lesser extent, the size of the gold nanoparticles are key factors to explain toxicity of gold nanoparticles. Our results also confirmed that this model is suitable for studying nanoparticle toxicity, although the nanoparticles themselves should be fully characterised before and during the toxicity test (AU)

Toxicidad y teratogénesis por arsénico en aguas en el pez cebra (Danio rerio) / Toxicity and teratogenesis by arsenic in water in the zebra fish (Danio rerio)

El objetivo del trabajo fue estudiar los daños teratogénicos y la inducción de micronúcleos en células branquiales de peces cebra (Danio rerio) por presencia de arsénico en las aguas. Fueron mantenidos en aguas bicarbonatadas cálcicas magnésicas de un pozo de referencia y del pozo 'Zimapán 5', del Municipio Zimapán, Estado de Hidalgo, México. Este último, con un contenido de arsénico que varía de 0,395-0,630 mg/L. Para el estudio de genotoxi-cidad se evaluaron durante 180 días en 3 tratamientos: agua del pozo de referencia (control negativo, sin As), agua del pozo de referencia adicionada con 5,0 mg As (V)/L (control positivo), y en agua del pozo 'Zimapán 5', colocándose 65 especimenes por tratamiento. Después de 30 días hubo una disminución de As en el agua del control positivo de 1092,65 ppb (36,42 ppb/día) mientras en pescados hubo un incremento de 523,81 ppb (17,46 ppb/día). Para el agua del pozo 'Zimapán 5' hubo una disminución de 211,40 ppb (7,04 ppb/día), y en pescados hubo un incremento de 74,73 ppb (2,49 ppb/día). Este resultado pone de manifiesto el alto grado de bioacumulación de As en el pez, que en relación al control negativo muestra que es 2,54 veces mayor. En relación a la frecuencia de inducción de micronú-cleos en células branquiales, al final de los 180 días en el control negativo hubo una generación espontánea de 0,8 micronúcleos/1000 células, en el control positivo hubo una frecuencia de inducción de micronúcleos 163,5 veces mayor que en el control negativo, mientras que en los peces expuestos al agua del pozo 'Zimapán 5' fue 56,25 veces mayor con respecto al mismo. Estos resultados demuestran la genotoxicidad del As en Danio rerio. Para el estudio de teratogénesis, se colocó una hembra y un macho en apareamiento en las mismas condiciones de los tratamientos, obteniendo que a mayor concentra-ción de As en el agua mayor porcentaje de huevos no viables, menor porcentaje de huevos viables y de eclosión, mayor porcentaje de alevines recién eclosionados y juveniles con malformaciones, y menor porcentaje de juveniles sobrevivientes (AU)

The objective of this work was study the teratogenic damages and the induction of micronuclei in gill cells of zebra fish (Danio rerio). They was maintained in calcium-magnesium bicarbonated waters from a reference well and 'Zimapán 5' well, the latter with an arsenic (As) content ranging from 0.395 to 0.630 m/L. For the genotoxicity study the specimens were studied during 180 days in 3 separated lots; in reference well water (negative control), in reference water to which was added 5 mg/L As (V) (positive control); and in water from 'Zimapán 5' well, with specimens by treatment. In waters an As concentration diminution was observed with time, whereas in fish there was an increase. After 30 days there was an As diminution in water from positive control of 1092.65 ppb (36.42 ppb/day) whereas in fish it had increased to 523.81 ppb (17.46 ppb/day). For the water from 'Zimapán 5' well there was a diminution of 211.40 ppb (7.04 ppb/day), and in fish there was an increase of 74.73 ppb (2.49 ppb/day). This result indices the grade high of bioaccumulation of As in fish, in relation with negative control is 2.54 major. In relation to micronucleus frequency in gill cells, at the end of 180 days in the negative control there was a spontaneous generation of 0.8 micronuclei/1000 cells, in the positive control there was a micronucleus frequency 163.5 times greater than in negative control, whereas for the fish exposed to 'Zimapán 5' well water the micronucleus frequency was 56.25 times greater than with the negative control. Taken together these results demonstrate the genotoxicity to Danio rerio of As in the well water (AU)