Primary Cell and Micromass Culture in Assessing Developmental Toxicity

Pratten, Margaret K.; Ahir, Bhavesh K.; Smith-Hurst, H.; Memon, Shabana; Mutch, P.; Cumberland, Peter F. T.

doi:10.1007/978-1-61779-867-2_9

Cited by 20 publications

(13 citation statements)

References 77 publications

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“…Although animal tests are intrinsically capable of capturing complex biological processes, and often allow direct observation of adverse effects, there is widespread agreement that replacing animal tests with in silico (e.g., quantitative structure-activity relationship [QSAR] models, structural alerts), in chemico (e.g., receptor binding assays), in vitro (e.g., stem cell assays) and in vivo tests using non-protected taxa or life stages (e.g., fish embryo tests) is high on the priority list of 21 st century toxicity assessment [1]. Many non-animal-based assays, or alternatives to current whole animal test designs that require fewer animals [2], focused on reproductive and developmental toxicity have already been developed (e.g. mouse whole embryo culture, limb micromass assay, mouse embryonic stem cell assay, fish embryo test, preantral follicle culture).…”

Section: Introductionmentioning

confidence: 99%

The potential of AOP networks for reproductive and developmental toxicity assay development

Knapen

Vergauwen

Villeneuve

et al. 2015

Reproductive Toxicology

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Historically, the prediction of reproductive and developmental toxicity has largely relied on the use of animals. The adverse outcome pathway (AOP) framework forms a basis for the development of new non-animal test methods. It also provides biological context for mechanistic information from existing assays. However, a single AOP may not capture all events that contribute to any relevant toxic effect, even in single chemical exposure scenarios. AOP networks, defined as sets of AOPs sharing at least one common element, are capable of more realistically representing potential chemical effects. They provide information on interactions between AOPs and have the potential to reveal previously unknown links between biological pathways. Analysis of these AOP networks can aid the prioritization of assay development, whether the goal is to develop a single assay with predictive utility of multiple outcomes, or development of assays that are highly specific for a particular mode of action. This paper provides a brief overview of the AOPs related to reproductive and developmental toxicity currently available in the AOP Wiki (http://aopwiki.org), and gives an example of an AOP network based on five reproductive and developmental toxicity-related AOPs for fish to illustrate how AOP networks can be used for assay development and refinement.

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Section: Introductionmentioning

confidence: 99%

The potential of AOP networks for reproductive and developmental toxicity assay development

Knapen

Vergauwen

Villeneuve

et al. 2015

Reproductive Toxicology

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“…To obtain primary heart cells for MM culture, White Leghorn Chicken eggs (Henry Stewart & Co., Louth, Lincs, UK) were placed on to the auto egg turner and incubated at 38 °C in 100% relative humidity for 5 days, day 0 being defined as the day when the eggs were set in the incubator. In the chick cardiomyocyte MM culture system the 5‐day‐old chick embryo (HH stages 25–26) heart cells were cultured and treated with chemicals as previously described (Ahir and Pratten, , ; Pratten et al , ). In brief, the embryonic chick hearts were trypsinized and 20 µl of single cell suspension (3 × 10 6 cells ml –1 ) in a defined culture medium [Dulbecco minimal Eagle's medium (DMEM) supplemented with 10% heat inactivated fetal calf serum, 2 m m l ‐glutamine and 50 U ml –1 penicillin with 50 µg ml –1 streptomycin] was pipetted on to the centre of each 24‐well tissue culture plate.…”

Section: Methodsmentioning

confidence: 99%

“…There is not much evidence of work being performed on embryonic chick heart cells (cardiomyocytes) as a MM culture system. However, the system may be useful for studying embryonic developmental mechanisms and the perturbation of heart development (Ahir and Pratten, , ; Hurst et al , ; Memon and Pratten, ; Pratten et al , ), while limb buds and neural tissue (midbrain MM culture) may be used for effects on chondrogenesis and neurogenesis by teratogens (Atterwill et al , ; Wiger et al , ).…”

Section: Introductionmentioning

confidence: 99%

The impact of caffeine on connexin expression in the embryonic chick cardiomyocyte micromass culture system

Ahir

Pratten

2015

J of Applied Toxicology

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Cardiomyocytes are electrically coupled by gap junctions, defined as clusters of low-resistance multisubunit transmembrane channels composed of connexins (Cxs). The expression of Cx40, Cx43 and Cx45, which are present in cardiomyocytes, is known to be developmentally regulated. This study investigates the premise that alterations in gap junction proteins are one of the mechanisms by which teratogens may act. Specifically, those molecules known to be teratogenic in humans could cause their effects via disruption of cell-to-cell communication pathways, resulting in an inability to co-ordinate tissue development. Caffeine significantly inhibited contractile activity at concentrations above and including 1500 μm (P < 0.05), while not affecting cell viability and total protein, in the embryonic chick cardiomyocyte micromass culture system. The effects of caffeine on key cardiac gap junction protein (Cx40, Cx43 and Cx45) expression were analysed using immunocytochemistry and in-cell Western blotting. The results indicated that caffeine altered the expression pattern of Cx40, Cx43 and Cx45 at non-cytotoxic concentrations (≥2000 μm), i.e., at concentrations that did not affect total cell protein and cell viability. In addition the effects of caffeine on cardiomyocyte formation and function (contractile activity score) were correlated with modulation of Cxs (Cx40, Cx43 and Cx45) expression, at above and including 2000 μm caffeine concentrations (P < 0.05). These experiments provide evidence that embryonic chick cardiomyocyte micromass culture may be a useful in vitro method for mechanistic studies of perturbation of embryonic heart development. Copyright © 2015 John Wiley & Sons, Ltd.

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“…Briefl y, dissociated single cells from the secondary neurospheres were plated at a density of 1.5 × 10 4 cells/well in three 96 well plates [ 23 ] and fed with a DMEM/F12/N2 culture medium for 48-72 h. At the end of the incubation time, the medium was replaced with a diff erent concentration of gabapentin (0.25, 0.5, 1, 2, 4, 8, 16, 32, 64 and 128 μg/ml) dissolved in DMEM/F12/N2 as a vehicle. At indicated time points (24, 48 and 72 h), for each 96 well plate the medium was aspirated and the cells were gently washed twice with 200 μl pre-warmed PBS.…”

Section: Cytotoxicity Assaymentioning

confidence: 99%

Gabapentin Enhances Neurogenesis in E14 Rat Embryonic Neocortex Stem Cells

et al. 2014

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Many anticonvulsant drugs have been studied for their non conventional therapeutic effects on neurodegerative diseases but merely a few demonstrated potential neurogenic characteristic. Gabapentin as a well-known mood stabilizer was studied for its potential capability to promote neurogenesis in embryonic rat cortical stem cells. Rat E14 cortical stem cells were exposed to gabapentin during differentiation for 7 days and subjected to immunocytochemistry. The phenotypic changes were evaluated in the ultimately survived and differentiated cells. Gabapentin (16 µg/ml) exposure significantly increased the number and percentage of MAP2 immunopositive neurons with no significant alterations in nestin or GFAP immunopositivity in neural or glial progenitors. The enhanced number of neurons by therapeutic doses of gabapentin via augmentation of the neuronal differentiation in neural stem cells may participate to the therapeutic properties of gabapentin in the treatment of mood disorder.

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Primary Cell and Micromass Culture in Assessing Developmental Toxicity

Cited by 20 publications

References 77 publications

The potential of AOP networks for reproductive and developmental toxicity assay development

The potential of AOP networks for reproductive and developmental toxicity assay development

The impact of caffeine on connexin expression in the embryonic chick cardiomyocyte micromass culture system

Gabapentin Enhances Neurogenesis in E14 Rat Embryonic Neocortex Stem Cells

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