Evaluation of the comet assay as a method for the detection of DNA damage in the cells of a marine invertebrate, Mytilus edulis L. (Mollusca: Pelecypoda)

Wilson, James T.; Pascoe, P. L.; Parry, James M.; Dixon, David R.

doi:10.1016/s0027-5107(97)00268-6

Cited by 152 publications

(93 citation statements)

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“…For in vivo studies in aquatic invertebrates such as oysters, mussels, clams and shrimp, cells from haemolymph, embryos, gills and digestive glands were used for Comet assay (Lee and Steinert, 2003). In vitro studies of genotoxicants were reported in haemocytes, gill cells and digestive gland cells from various mollusks namely Mytillus edulis, Nassarius tegula and Musculista senhousia (Sastre et al, 1997;Wilson et al, 1998). Studies of genotoxicants in shrimp cell cultures were not reported while Lee et al (2000) studied the effects of genotoxicants such as chromium (III) chloride, sodium chromate, mercuric chloride, and 2-methyl-1,2-naphthoquinone (MNQ) on grass shrimp, P. pugio, embryos employing Comet assay.…”

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Application of primary haemocyte culture of Penaeus monodon in the assessment of cytotoxicity and genotoxicity of heavy metals and pesticides

Jose

Puthumana

Mohandas

et al. 2011

Marine Environmental Research

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a b s t r a c tLack of shrimp cell lines has hindered the study of pollutants which adversely affects shrimp health and its export value. In this context a primary haemocyte culture developed from Penaeus monodon was employed for assessing the cytotoxicity and genotoxicity of two heavy metal compounds, cadmium chloride and mercuric chloride and two organophosphate insecticides, malathion and monocrotophos.Using MTT assay 12 h IC 50 values calculated were 31.09 AE 16.27 mM and 5.52 AE 1.16 mM for cadmium chloride and mercuric chloride and 59.94 AE 52.30 mg l À1 and 186.76 AE 77.00 mg l À1 for malathion and monocrotophos respectively. Employing Comet assay, DNA damage inflicted by these pollutants on haemocytes were evaluated and the pollutants induced DNA damage in >60% of the cells. The study suggested that haemocyte culture could be used as a tool for quantifying cytotoxicity and genotoxicity of aquaculture drugs, management chemicals and pollutants.

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confidence: 99%

Application of primary haemocyte culture of Penaeus monodon in the assessment of cytotoxicity and genotoxicity of heavy metals and pesticides

Jose

Puthumana

Mohandas

et al. 2011

Marine Environmental Research

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“…Decreased growth of C. tenuissimus may be due to combined effect of increased DNA damage (as observed by Wilson et al, 1998), while studying effect of different concentration of NDMA and H 2 O 2 on Mytilus) and competition among cells for the nutrients and space.…”

Section: ] Discussionmentioning

confidence: 97%

Genotoxicity of cadmium in marine diatom Chaetoceros tenuissimus using the alkaline Comet assay

et al. 2006

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Genotoxic effects of Cadmium on phytoplankton Chaetoceros tenuissimus have been evaluated using DNA damage by comet assay. Cadmium concentrations ranging from 2.4 to 10 mg/L were used to evaluate the effects. Results showed that as the concentration of Cd increased growth of the diatom decreased. Alkaline single-cell gel electrophoresis (Comet assay) method, which is highly sensitive in detection of DNA damage in eukaryotic cells, was used to observe genomic changes in marine diatom cells. DNA damage was measured as percent number of comets and normal cells. 65% cells were found to be damaged at 10 mg/L concentration of Cd as compared to 23% in 2.4 mg/L and only 5% in controls. More than 50% apoptic cells were observed on 8 th day at 10mg/L and 12 th day at 7.5 mg/L concentrations. At lower Cd concentrations (4.5 mg/L and below) the damage was below 30% till the last day. This suggested that higher Cd levels have early damaging effects on cell nuclear material and that % injury increases with advancement of exposure period. One advantage of use of C. tenuissimus is the ease with which it can be cultured in a defined medium. Chaetoceros tenuissimus diatom can be used as an in vivo model for ecogenotoxicity assessment using the Comet assay.

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“…El Ensayo Cometa Alcalino fue estandarizado utilizando tratamientos in vitro en tejido branquial de A. ater a partir del protocolo descrito por Wilson et al (1998). La extracción del tejido y aislamiento de células se realizó en CMFS a 10 °C, bajo luz amarilla para evitar el daño por ondas UV.…”

Section: Ensayo Cometaunclassified

“…Micronucleus Assay were modified in the obtaining of suspension cell, we used 0,9% Sodium citrate hipotonic solution, fixation in methanol for seconds after the spread and dyed with 2% Giemsa. The Alkaline Comet Assay protocol described by Wilson et al (1998) was standardized modifying the single-cell suspension embedded in LMP Agarose (1% in Kenny solution, pH 7,5), the exposure at lysing solution (pH 10 with DMSO and Triton) with changes from 1 to 16 h, electrophoresis in alkaline solution pH 13,7 and dyed with Ethidium bromide and Silver nitrate for agarose gels. Changes made at both techniques allowed us to obtain a high cell number with a defined morphology, recognizing macrolessions like the MN formation and nuclear aberrations, and the qualitative determination of microlessions observed by the fragmentation and migration of the DNA.…”

Section: Introductionmentioning

confidence: 99%

“…El Test de Micronúcleo se modificó en la forma de obtener el disgregado celular, realizando la hipotonización en Citrato de sodio 0,9%, fijación en metanol por segundos posterior al frotis y la tinción con Giemsa 2%. La estandarización del Ensayo Cometa Alcalino, descrita por Wilson et al (1998), se realizó modificando la suspensión celular en agarosa LMP (1% en solución Kenny, pH 7,5), la exposición a solución de lisis (pH 10 con DMSO y Tritón) con variaciones en el tiempo de 1 a 16 h, y la electroforesis en solución alcalina pH 13,7 y tinción con Bromuro de etidio y Nitrato de plata para geles de agarosa. Los cambios realizados en ambas técnicas permitieron obtener un alto número celular con morfología definida, observándose macrolesiones como la formación de MN y aberraciones nucleares, y la determinación cualitativa de microlesiones observadas por fragmentación y migración del DNA.…”

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Aplicación de dos biomarcadores para el análisis de lesiones en el DNA de bivalvos marinos

et al. 2006

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ResumenEl presente trabajo describe la estandarización de las técnicas del Test de Micronúcleo (MN) y Ensayo Cometa para la evaluación del daño en el DNA de bivalvos marinos. La importancia de estas técnicas radica en que permiten evaluar la respuesta temprana al estrés ocasionado por agentes contaminantes y cambios ambientales. Tejidos branquiales de Semimytilus algosus y Aulacomya ater fueron utilizados para tratamientos in vivo e in vitro. Los tejidos fueron expuestos a los agentes mutagénicos Mitomicina C en concentraciones de 0,02; 0,04 y 0,06 x 10 -6 M y H 2 O 2 en una concentración de 100 μM con tiempos de evaluación de 1, 3, 6, 24 y 48 h. Diferentes soluciones para la colecta del tejido y aislamiento celular fueron evaluados obteniendo una mayor viabilidad celular en la solución salina CMFS, pH 7,3; en frío. El Test de Micronúcleo se modificó en la forma de obtener el disgregado celular, realizando la hipotonización en Citrato de sodio 0,9%, fijación en metanol por segundos posterior al frotis y la tinción con Giemsa 2%. La estandarización del Ensayo Cometa Alcalino, descrita por Wilson et al. (1998), se realizó modificando la suspensión celular en agarosa LMP (1% en solución Kenny, pH 7,5), la exposición a solución de lisis (pH 10 con DMSO y Tritón) con variaciones en el tiempo de 1 a 16 h, y la electroforesis en solución alcalina pH 13,7 y tinción con Bromuro de etidio y Nitrato de plata para geles de agarosa. Los cambios realizados en ambas técnicas permitieron obtener un alto número celular con morfología definida, observándose macrolesiones como la formación de MN y aberraciones nucleares, y la determinación cualitativa de microlesiones observadas por fragmentación y migración del DNA. Palabras claves: daño del DNA, micronúcleo, Ensayo Cometa alcalino, estrés, bivalvos AbstractThis work describes the standardization of Micronucleus Assay (MN) and Comet Assay techniques used for the evaluation of the DNA damage on marine bivalves. These techniques are important because they can evaluate the early stress response caused by pollutant agents and environmental changes. Branchial tissues of Semimytilus algosus and Aulacomya ater were used for in vivo and in vitro treatments. Tissues were exposed to the mutagenetic agents Mitomicine C in concentrations of 0,02; 0,04 and 0,06 x 10 -6 M and H 2 O 2 in concentration of 100 μM evaluated at 1, 3, 6, 24 and 48 h. Different solutions for the tissue collection and cell isolation were evaluated, obtaining more cell viability with cold CMFS pH 7,3 saline solution. Micronucleus Assay were modified in the obtaining of suspension cell, we used 0,9% Sodium citrate hipotonic solution, fixation in methanol for seconds after the spread and dyed with 2% Giemsa. The Alkaline Comet Assay protocol described by Wilson et al. (1998) was standardized modifying the single-cell suspension embedded in LMP Agarose (1% in Kenny solution, pH 7,5), the exposure at lysing solution (pH 10 with DMSO and Triton) with changes from 1 to 16 h, electrophoresis in alkaline solution pH 13,7 and d...

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Evaluation of the comet assay as a method for the detection of DNA damage in the cells of a marine invertebrate, Mytilus edulis L. (Mollusca: Pelecypoda)

Cited by 152 publications

References 23 publications

Application of primary haemocyte culture of Penaeus monodon in the assessment of cytotoxicity and genotoxicity of heavy metals and pesticides

Application of primary haemocyte culture of Penaeus monodon in the assessment of cytotoxicity and genotoxicity of heavy metals and pesticides

Genotoxicity of cadmium in marine diatom Chaetoceros tenuissimus using the alkaline Comet assay

Aplicación de dos biomarcadores para el análisis de lesiones en el DNA de bivalvos marinos

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