Applications of CometChip for Environmental Health Studies

Abstract

Environmental exposures have long been known to impact public health and safety. For example, exposures to airborne particulates, heavy metals in water, or certain industrial chemicals can contribute to aging and to risk of developing cancer and other diseases. Environmental factors can impact health in a variety of ways, but a key concern is DNA damage, which can lead to mutations that cause cancer. Cancer can take years to develop following chemical exposure; however, one way to predict carcinogenicity in a more practical time frame is by studying the chemical's ability to induce DNA damage. The comet assay (or single-cell gel electrophoresis assay) has been used successfully for genotoxicity testing. The comet assay allows for the detection of DNA strand breaks via analysis of DNA migration during electrophoresis. Previously, the Engelward laboratory, in collaboration with the Bhatia laboratory, developed the CometChip for measurements of DNA damage and repair. The CometChip is a high-throughput comet assay that improves user reproducibility and significantly shortens total assay time. Here, we describe how the high-throughput CometChip platform can be used to measure DNA damage in established cell lines, animal models, and human samples. We also discuss technical challenges associated with these studies and provide recommendations on how to achieve optimal results for researchers interested in adopting this assay.

Figure 1.

DNA is damaged by both external and internal agents. The lower boxes are examples of some of the classes of damage that can result from the exposures listed in the upper boxes.

Figure 2.

Undamaged DNA remains highly supercoiled and does not migrate significantly during electrophoresis (left). DNA damage leads to release of DNA supercoiling and fragmentation, enabling migration and the formation of the comet tail.

Figure 3.

CometChip protocol involves loading cells into microwells, removing excess cells, and trapping cells with low melting point agarose. Shown here, cells are treated after being loaded into the CometChip. Cells can also be treated prior to loading into the CometChip (not shown).

Figure 4.

Base excision repair (BER) intermediates can be detected using the alkaline comet assay. During BER, DNA repair enzymes create abasic sites and single-strand breaks (boxed in green) that can be measured. Illustrated here is a simplified version of BER and is one of several pathways.

Figure 5.

At the base of each macrowell on a 96-well plate are ~300 microwells in a grid. Pictured here are microwells that are ~40 μm in diameter and spaced 240 μm apart. Cells are loaded into each macrowell, and they settle into the microwells over time. Loading efficiency can be checked using a microscope by visualizing a grid of loaded microwells among the off-grid/excess cells (examples of loaded wells are marked by the blue arrows). Following the wash step, excess cells are removed, leaving behind an array of patterned cells. For these images, 40,000 TK6 cells were added to each macrowell in a volume of 80 μL. Cells remain trapped in wells following 20 min of loading time. Note that loading time can be optimized for different cell types, but can be as low as 5 min.