Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Understanding the effects of small molecules, compounds and chemicals on cells is the very core of drug discovery, one in which the pharmaceutical industry continues to invest billions of dollars. Yet alongside the question of whether such entities have a desired effect looms that of whether they have a toxic effect on those cells—and ultimately the tissues and organisms the cells compose. This question has equal importance to those who protect our environment and assure that our food is safe to eat.
Testing chemical toxicity can take many forms, from looking for simple surrogates of death, such as the inability to exclude trypan blue, to sophisticated measures of changes in a specific cell type’s physiology. Various assays look at pathways leading to cell death, membrane integrity, depletion of energy, ability to proliferate and changes in differentiation. They are accomplished using instruments ranging from a hemocytometer and light microscope; to a Coulter counter, microplate reader or flow cytometer; to a high-content analysis solution found principally in screening cores at biotech and larger pharmaceutical companies.
Screens for loss of viability are often the first line of inquiry, and only after an entity is shown to cause a decrease in survival is it then subjected to more nuanced assays [1]. Here we look at the principal means by which entities are tested for their effects on viability.
Membrane integrity
One of the defining characteristics of a cell is its ability to compartmentalize the inside environment from the outside. When things such as hydrophilic dyes cross into the cytoplasm, for example, or conversely when certain enzymes that are supposed to be contained with the cell are found in the supernatant, this is prima facie evidence that something has gone wrong. Thus membrane integrity is a basis for many cytotoxicity assays.
Membrane-impermeant (exclusion) dyes such as propidium iodide (PI) can cross a leaky membrane and bind to a cell’s nucleic acids. Once bound, PI’s fluorescence shifts and increases about 25-fold, allowing for an assay that can be read by flow cytometry or by a plate reader. Other dyes are available that can be used similarly to label nucleic acids or other cellular components.
A twist on the concept uses substances that can freely traverse the cell membrane but become trapped after enzymatic conversion. For example, the lipophilic, nonfluorescent calcein acetoxymethylester (calcein-AM) is hydrolyzed to a fluorescent, hydrophilic product by endogenous esterases and thus retained by cells with intact membranes [2].
Enzymatic activities on the outside of the cell also are used as indicators of a leaky membrane. A host of vendors offer kits to measure the activity of lactate dehydrogenase (LDH) in the supernatant, for example, and kits looking at enzymes such as glucose 6-phosphate dehydrogenase (G6PD) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) also are marketed. These use a variety of formats and detection chemistries, including colorimetric-, fluorescence- and bioluminescence-based assays.
Assays that measure membrane integrity—including Lonza’s ToxiLight™, which looks at leakage of adenylate kinase (AK)—don’t measure cell death so much as cellular damage, says Jeffrey Bergerson, Lonza product manager for toxicity assays. Some damaged cells recover, he notes.
Metabolism
Several assays look at cellular metabolism as an inverse measure of toxicity. The first and perhaps best known of these is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which is very robust and works on most cell types [3]. Here, a pale yellow substrate is reduced by the cells into a blue precipitate that can be quantitated colorimetrically after cell lysis and detergent solubilization. Other tetrazolium derivatives, such as 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl] -2H- tetrazolium hydroxide (XTT), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) and water-soluble tetrazoliums (collectively known as WSTs), have since been developed whose reduced product is soluble and is released into the medium, thus bypassing several steps. But beware, some of these dyes reportedly may not work with all cell types.
Resazirin-based (commonly known as Alamar Blue) assays are also widely used and widely available. In this case, the reduced product is both colored and fluorescent and thus can be read in either colorimetric or fluorescence mode—with the latter being sensitive enough to detect as few as 80 cells, with intact reducing capacity.
The most widely used surrogate biochemical marker for cell viability is ATP (because a cell’s energy currency is rapidly depleted as the cell dies), and its detection has become the “gold standard” for high-throughput screening [4]. Assays such as Promega’s CellTiter-Glo® Luminescent Cell Viability Assay interrogate ATP by using the ability of luciferase to generate light from the catalysis of luciferin, a reaction that uses ATP as its energy source.
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Sometimes it’s important to understand not only whether cells are being killed, but how, and “there are a variety of assays that measure markers that indicate mechanisms leading to cytotoxicity,” notes Terry Riss, global strategic marketing manager for Promega. Distinguishing necrosis from apoptosis may be of importance in vetting cancer therapeutics, for example, while discerning which of several apoptotic pathways is affected could allow for more specific interventions. Assays ranging from querying of reactive oxygen species or glutathione production, to differential caspase activity, to examining whether phosphatidylserines are found on the outer leaflet of the plasma membrane, to staining for distinct DNA cleavage patterns, are all being used to mine for such information.
Sometimes toxins make cells sick rather than kill them, and this can be picked up by functional assays—some generic, and some cell-type-specific. For Molecular Devices’ EarlyTox Cardiotoxicity Kit, for example, which uses calcium influx as a proxy readout for muscle contraction, “the primary focus is not on cell death, it’s on the change of behavior,” notes Xin Jiang, the company’s product manager for reagent systems.
In fact, many measures of cytotoxicity look at proxies; therefore, caution must be exercised when interpreting the results. Many assays, for example, measure reversible events and so can overestimate cell death, or they look at cytostatic, rather than cytotoxic, phenomena. Assays for later events (such as membrane permeability) may underestimate the amount of toxicity, if they are read too early. It’s important to know what is being measured and to include the proper controls—and in some cases, to multiplex or follow up with a second assay to get at the nuanced information you’re seeking. So choose your platform, decide on throughput and sensitivity, pick your readout and find an assay that will sort the toxic from the benign—there are many to choose from.
References
[1] Cree, IA (ed.), “Cancer Cell Culture: Methods and Protocols,” second edition, Methods in Molecular Biology series, vol. 731, Springer Science+Business Media, pp. 219-236, 2011. [PMID: 21516411]
[2] Kepp, O, et al., “Cell death assays for drug discovery,” Nat Rev Drug Discov, 10(3):221-37, 2011. [PMID: 21358741]
[3] Stoddart, MJ (ed.), “Mammalian Cell Viability: Methods and Protocols,” Methods in Molecular Biology series, vol. 740, Springer Science+Business Media, pp. 1-6, 2011. [PMID: 21468961]
[4] Stoddart, MJ (ed.), “Mammalian Cell Viability: Methods and Protocols,” Methods in Molecular Biology series, vol. 740, Springer Science+Business Media, pp. 103-114, 2011. [PMID: 21468972]
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