Catherine Shaffer holds a master's degree in biological chemistry and has worked as a research scientist. She is also an award-winning science fiction author and part-time reporter for local public radio.
In the context of drug development, toxicity is a double-edged sword. Toxicity is desirable when a drug is designed to kill cells or stop them from growing. On the other hand, undesirable toxic effects are a factor in the development of most drugs.
Key to successful development workflow and ultimately commercial release of a drug is being able to identify and address potential toxicity factors in the target validation process as early as possible.
Here we discuss some of the latest offerings available to researchers as well as improvements to existing toxicity assays.
Classic tox assay
Drug candidates are typically screened for chemical toxicity in cell culture during the early, preclinical phases of drug development. The most traditional assay for cell toxicity is the MTT or MTS assay. In healthy cells, activity of NAD(P)H-dependent oxidoreductase enzymes creates a reducing environment within the cell. The yellow MTT dye is reduced, resulting in a purple color. MTT assays are typically endpoint assays. The cells are destroyed by lysing to make the measurement. Recent improvements to the assay by various vendors have provided additional options for measuring toxicity of cells. For example, using cell-permeable dyes, the assay can be performed in real time. Promega's RealTime-Glo assays can be used to monitor toxicity in cell culture over time without interfering with the growth of the cells. Also, the basic colorimetric viability assay has been updated with newer dyes and reagents, offering a range of options for readouts, including fluorescent and bioluminescent signals. The CellTiter-Blue Cell Viability Assay from Promega is an example of a redox-based cell-viability assay updated to produce a fluorescent signal.
Toxicity assays encompass a diverse array of measurements, such as ATP consumption by the cell, apoptotic events through activity of caspase enzymes, monitoring membrane integrity, measuring mitochondrial membrane potential and more. Alternate assay modalities offer features such as real-time cell viability measurements, multiple microplate formats and flow cytometry readouts.
According to Jim Cali, director of research at Promega, that company's development efforts have been focused on multiplex assays, meaning the ability to get more than a single datapoint from a single assay well. Promega's MultiTox line of products enables measurement of live and dead cells in a single well. Simultaneous measurement of live and dead cells can minimize errors and correct for false-positive and false-negative results by providing an internal control. ThermoFisher Scientific also offers a widely used family of cell-viability and cytotoxicity assays—the Live/Dead product line.
“Early efforts that focused just on live and dead measurements eventually evolved to product offerings that not only tell you at a high level to what extent you've killed the cells, or if the cells are alive or expanding, but from a toxicity perspective, what's the mechanism,” says Cali. “Toxicity isn't always about killing cells. Fundamentally, all toxic input[s] have some mechanism.”
Cellular toxicity mechanisms typically involve the processes of apoptosis and necrosis. Apoptosis is also known as programmed cell death. In mammalian cells, the process is regulated by caspases, and the cell goes through a process of shrinkage, collapse and release of apoptotic bodies. Cell necrosis is a process in which cells die unexpectedly because of a trauma or some cellular event. Identifying the exact mechanism of cellular toxicity can yield clues as to how the drug candidate compound is acting on the cells.
Caspase activation
Promega's Caspase-Glo 3/7 measures activity of caspase-3 and -7 activity using a bioluminescent substrate as indicator. “The ‘Glo’ part of that name is indicative of a broad theme of Promega to focus on using bioluminescence as an optical approach to markers of cell health or cell function,” says Cali. The assay uses a proprietary firefly luciferase designed for extended stability and utility. For a broader determination of mechanism, Promega markets the ApoTox-Glo Triplex assay. ApoTox-Glo can distinguish between viable, apoptotic and necrotic cells in a single well.
Real-time analysis can be applied to apoptosis assays, as well. Biotium's NucView Caspase-3 substrates are designed to track caspase-3 activity in real time. The substrate is attached to a DNA-binding dye. When it enters a cell and is cleaved by caspase-3, it releases the fluorogenic DNA dye and migrates to the cell nucleus. As a result, the cell nucleus can be visualized for apoptotic morphology. Because NucView does not interfere with caspase activity, it enables monitoring of apoptosis in real time.
“Most assays require lysing the cell,” says Lori Roberts, director of bioscience for Biotium. “Ours can be used for time lapse in live cell culture or as a homogenous endpoint assay for flow cytometry or fluorescence microplate reader.”
Mitochondrial stress
A breakdown in the functioning of oxidoreductive enzymes in the cell results in production of reactive oxygen species as the cell loses its ability to reduce those intermediates. The presence of those oxygen species—superoxides and other free radicals—can be used as indicators of toxicity via mitochondrial stress.
The FlowCellect MitoStress kit from MilliporeSigma measures oxidative stress and apoptosis in a single assay. The kit can detect multiple populations of cells. These include: live cells with little or no superoxide; cells with superoxide radicals; cells with both superoxide radicals and early apoptosis; and apoptotic cells without superoxide.
MilliporeSigma's FlowCellect kits (sold in combination with Guava easyCyte flow cytometers) are designed to detect multiple cell-health parameters and pinpoint mechanisms.
According to Kamala Tyagarajan, R&D director for flow cytometry assays and applications at MilliporeSigma, the use of flow cytometry for detection provides an “in-cell answer” rather than average impacts. “This is very important in understanding cellular impacts. It also allows for more statistically relevant results by rapidly providing measurement of a large number of cells in parallel,” says Tyagarajan. “Our solutions are multiparametric and typically allow for the study of at least two parameters in parallel, thus providing more data from a single experiment.”
Enzo offers a line of kits, ROS-ID, which includes assays for total reactive oxygen species (ROS) as well as other specific species, like nitric oxide and superoxide. Promega also provides its homogenous ROS-Glo kit, which measures ROS levels in an add-and-read format.
Membrane integrity
Another simple, classic method for looking at chemical toxicity is to assess the membrane integrity of cells. Certain substrates can't cross the membrane of a healthy cell, but they will enter and give a signal when the membrane begins to break down during necrosis.
The CELLESTIAL line of products from Enzo Life Sciences features an apoptosis/necrosis assay that solves a common problem in fluorescence-based assays. Many apoptosis assay kits use a fluorescent dye that occupies the same channel as green fluorescent protein (GFP), making them difficult to use with a GFP-transfected cell line. Wini Luty, senior product manager for Enzo, says, “Our kit allows you to use it in multiplex with any GFP-transfected or BFP-transfected cell line.”
Lactate dehydrogenase (LDH) activity can be used as another signal for loss of membrane integrity. LDH is a cytosolic enzyme released into the culture medium when cells lose membrane integrity because of cytotoxicity. Enzo also offers the LDH Cytotoxicity WST (water-soluble tetrazolium) Assay for detection of LDH activity. “It's a simple colorimetric assay that doesn't require fluorescence,” says Luty.
Cell viability
Although specific markers of cell death are commonly used in chemical toxicity testing, it is often helpful to capture a snapshot of cellular health and robustness. For example, Promega’s CellTiter-Glo assay uses bioluminescent technology to track the presence of ATP in cells. Healthy cells produce ATP through oxidative phosphorylation and glycolysis. The assay picks up the ATP to activate the luciferin, generating luminescence, for a simple, direct read on the cells’ metabolic health.
Options for researchers
Tool providers have created many variations on the traditional MTT/MTS chemical toxicity assay, using a range of colorimetric, fluorescent and bioluminescent dyes. Those dyes in many cases have been engineered for greater stability and higher performance when compared with previously-released versions. In addition to offering the ability to determine whether cells are alive or dead, many assays pinpoint the mechanism of toxicity or track markers of viability. Assay formats range from small-scale individual assays to assays for flow cytometry-based analysis and high-throughput microplate screening. Researchers have an abundance of assay options available to measure and assess pharmaceutical compounds and their potential toxicity effects on cells.
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