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Assay Tutorials : Apr 1, 2009 (Vol. 29, No. 7)

High-Content Methods to Assess Toxicity

Extensions to Thermo Scientific Cellomics Platform Address Comet and Zebrafish Applications
  • Mark Collins

Assessing the toxicity of a sample for its risk to human health is key throughout the life sciences. This assessment is perhaps most visible in terms of the pharmaceutical industry, e.g., drug failures due to liver- or cardio-toxicity. There is also considerable toxicity testing that occurs for environmental toxicants as well as consumer product safety.

In addition, there are various regulatory initiatives (for example, REACH in Europe) to reduce the use of animals for toxicity testing, initially for nonpharmaceutical products. Couple this overwhelming rise in the volume of toxicity testing with the fact that many traditional methods of in vitro toxicity testing offer low predictivity, or use subjective, manual methods and there is now a perfect storm  that is driving innovation and the desire for both better tools and more relevant, predictive models.

High-content methods based on automated imaging are able to generate multiple endpoints of size, shape, texture, and intensity in individual cells in each well, thereby reducing losses in data integrity due to population averaging. Because each cell is measured independently, subtle changes in physiology can be detected and quantified robustly. Additionally, high content allows previously nonaddressable targets to be evaluated, particularly those where a morphological change occurs in cells.

Comet Assay

The Comet (more correctly known as single-cell gel electrophoresis) assay is a simple-to-perform, sensitive technique for the detection of DNA damage in single cells. In the assay, cells having been exposed to a potential agent of interest are embedded in a thin layer of agarose on a microscope slide. The DNA is allowed to unwind under neutral/alkaline conditions.

The sample then undergoes electrophoresis, allowing the broken DNA fragments or damaged DNA to migrate away from the nucleus. After staining, the resulting image looks like a comet with a distinctive head and tail (Figure 1). The image is then evaluated for the amount of fluorescence in the head and tail and the length of the tail. The length of the tail is directly proportional to the amount of DNA damage.

The comet assay is already being adopted by regulatory agencies as approved for regulatory submissions, and it is being widely adopted for genetic toxicology for screening and regulatory testing of industrial chemicals, pharmaceuticals, biocides, and cosmetics, as well as for ecogenotoxicology.

There is also a move to replace some traditional assays (e.g., liver UDS assay) with an in vivo Comet assay. Comet assays are therefore gaining popularity; they are  limited in scalability by the manual scoring, a subjective assessment. Automating and quantifying the assay would overcome the limitations and allow it to be used more widely.

To address the need for improved genotoxicity testing and the study of DNA damage using Comet, the Thermo Scientific Cellomics HCS Platform recently added the capability to measure Comet assays.

The high-content approach to evaluating Comet assays offers a number of advantages in terms of speed, robustness, reproducibility, and objectivity. The Comet BioApplication allows scoring of Comets in seconds, removing tedious manual scoring as well as providing a wealth of objective measurements such as olive tail moment, tail length, tail extent moment, and percent DNA in tail.

Comets can be classified into normal/ abnormal, increasing statistical robustness, and the available measurements provide much more information on DNA damage than manual scoring by eye. When combined with suitable assay systems (e.g., Trevigen Comet slide; dnadamage/cometslides.php) many compounds and many Comets can be analyzed, offering a step-change in productivity and the ability to easily scale the assessment of DNA damage.

Comet assays are seen as key in defining new reliable and validated in vitro assays to test for genotoxic and cytotoxic effects of chemicals (as in the REACH program) without resorting to animal experiments. The Thermo Scientific automated high-content approach will certainly go a long way to aiding that initiative.

The Cellomics Comet BioApplication also allows the analysis of Comet-FISH  assays, which extends its utility into determining sequence or gene-specific damage and repair as well as possible diagnostic use.

The focus on Comet addresses one aspect of the innovation need, in that it allows a tedious manual assay to be automated and made more robust. The search for more relevant, predictive models of toxicity is ongoing, however, and again high content has a role to play here.

Zebrafish

Over the past few years, zebrafish embryos have become an increasingly important tool for biologists as a model to study development. The zebrafish has become important because the embryo is highly transparent (easing analysis), genes are highly conserved (around 75%) with humans, it is a vertebrate, and the embryo develops quickly.

Large numbers of offspring make them amenable for large-scale small molecule or genetic screens. In addition hundreds of transgenic zebrafish lines exist, many with GFP tags that allow for detailed investigations into gene function. It is not surprising therefore that zebrafish are also seen as a suitable system for studying toxicity in an animal model that is cheaper, more scalable, and more humane than rodents for example. The fact that the developing embryo is highly sensitive to a toxic insult makes the zebrafish system a highly relevant in vivo model for use by drug discovery, environmental, and even consumer products toxicity testing.

Analysis of hundreds of embryos for the subtle morphological and other changes in response to a toxic insult represents a challenge. With the recent release of the ZebraTox BioApplication for the Cellomics Arrayscan HCS platform, however, researchers can now bring the power of high content to the assessment of toxicity in zebrafish.

The Zebrafish BioApplication allows analysis of either Brightfield (transmitted light, nonfluorescently labeled) embryos or those that are fluorescently labeled. The application measures a sophisticated range of morphological changes in response to a toxic insult, such as shape, curvature, and area.

Zebrafish tend to curl up in response to a toxicant (Figure 2), and this can be measured and a dose range defined. Figure 3  shows the results of an in-house study looking at four different compounds; the graph clearly quantifies the degree of toxicity based on the morphological measurements. The BioApplication coupled with the Arrayscan platform and utilizing 96- or 384-well plates can screen large numbers of compounds in minutes, yielding highly relevant data. In addition, using fluorescently labeled zebrafish the application can evaluate for angiogenesis, neuronal development and so on, allowing deeper insights into mechanisms of toxicity.

There is no doubt that there is pressure on many fronts to change the way toxicity testing is done—from the viewpoint of improving predictivity, through scaling relevant manual assays, to more relevant models of human toxicity. With recent extensions to Thermo Scientific high-content platform (Comet and Zebra fish toxicity assessment), coupled with the long standing multiparameter cytotoxicty evaluation, we have a real chance to improve human health in terms of safer drugs (for lowered costs), understanding the effect of environmental toxins, and as ensuring that the products we use, from shampoos to topical skin treatments, are safe.