Measurement of Cell Death

The cellular responses in this table are listed in order of severity of stress [11, 36]. 

Stress For example, mitochondrial inhibitor, oxidant, substance accumulation (e.g., lipid, phospholipid, fluid) 

Stress signal For example, nrf-2, AP-1, NF-kB transcription factor translocation 

Induction For example, antioxidant enzymes, mitochondrial, membrane hyperpolarization, mitochondrial biogenesis 

Stress susceptibility Loss of functional reserve capacity 

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Sublethal Measurement as opposed to measurement of cell death is required. Measurement of the noxious substance or a signal transduction event may be too early in the pathogenesis if cell injury as opposed to measurement of adaptive and adverse effects... 

Loss of cytoplasmic membrane function occurs early in necrosis and late in apoptosis (so-called apoptotic necrosis ). If, in a given experimental system, the mode of death has been adequately characterized morphologically and the assay timepoint judiciously chosen, loss of dye exclusion can serve as a useful, easy to quantify measure of cell death in either apoptotic or necrotic model systems. Commonly used dyes for this type of analysis include propidium iodide, 7-AAD, Sytox green, and ethidium homodimer. 

Before further testing and to confirm that the compounds are cytotoxic rather than merely interfering with the Alamar blue indicator dye, they are re-bioassayed using two other indicator dyes. Calcein-AM is a fluorescent dye that measures changes in cell mem brane permeability, an indicator for one of the penultimate steps of cell death, uci e e measures the amount of adenosine triphosphate (ATP) synthesis in a chemilurmne assay. For some compounds, cell death was also confirmed by microscopic exami Papanicolaou-stained cell preparations.11... 

Jester, J.V, Ling, J., HarbeU, J. Measuring depth of injury (DOI) in an isolated rabbit eye irritation test (IRE) using biomarkers of cell death and viability. Toxicol In Vitro 24(2), 597-604 (2010). Epub 2009 Oct 24... 

AP-induced toxicity and measurement of cell survival/injury were performed as described in detail elsewhere.1011 Briefly, 6-day-old cells were exposed to fresh solutions of either Ap25 35 or Ap, 42 for 24 h, in the presence or absence of different drugs. Cell survival and extent of cell death were determined using MTT and Sytox green assays, respectively. Measurement of intracellular reactive oxygen species was determined by dichlorofluorescein (DCF) fluorescence assay, as described previously.23... 

Fig. 21 LDH (Lactate dehydrogenase, an enzyme released at cell death is used as a measure of cell viability) release from human cells which indicates that there is no significant difference between the DLC and control samples.2 3...

Information gained from assays and future potential So far, few field measurements have been made of caspase-like activities (Berman-Frank et al., 2004 Vardi et al., 1999), but the assay methods appear to be sensitive enough to allow use in natural communities. As work using cultures proceeds, and our understanding of cell death processes improves, assays of capase-like activity may offer an important means to distinguish different forms of cell mortality. Aside from bulk in vitro assays, the availability of ceU-permeable substrates, coupled with flow cytometry will provide improved resolution and specificity (e.g. Bidle and Bender, 2008). 

The extent of cell death is determined by measuring the cells unable to exclude the Trypan blue dye. In prolonged continuous culture or in unfavourable environments the additional phenomenon of cell lysis may also occur. The number of cells that have lysed can be determined by measuring the lactate dehydrogenase (LDH) concentration released into the medium (see Chapter 2, section 2.5). Adding the number of visible and lysed dead cells yields an evaluation of the total rate of cell death. 

It is often possible to address function more specifically in in vitro assays, where functional parameters are usually very sensitive readouts of adverse effects. For example trans-epithelial electrical resistance (TEER) is a very sensitive marker of epithelial disturbances. TEER measures the barrier function of the entire mono-layer and is utilized to study functional disturbances of many epithelial/endothelial cell types including blood-brain barrier, pulmonary, renal, and gastrointestinal cells. Its sensitivity lies in the fact that only a small proportion of cell death has a very large impact on barrier function. Additionally, cell stress can interfere with the arrangement and population of tight junction proteins [16] thus, TEER can in certain conditions measure functional disturbances in the absence of cell death [13]. Also since TEER can be measured noninvasively, it is nondestructive and can be used to monitor the effects of treatment over days and weeks [13, 17]. For excitable cells, electrical activity has also been proven to be an extremely sensitive parameter of adverse drug reactions and microelectrode arrays have been employed successfully to monitor neurotoxicity in vitro [18]. Also, for contractile cells, such as cardiomyocytes, the use of impedance measurements to measure the effects of compounds on spontaneous contraction has been demonstrated to be a very sensitive functional monitoring parameter in vitro [19, 20], Admittedly, none of the aforementioned techniques are true biomarkers per se however, such measurements illustrate the fact that in vitro techniques allow certain possibilities that are not practically tenable in the whole body. 

The above tests are all labor-intensive and time-intensive. Orth [19] has developed a test which may predict the behavior of preservatives in only 48 h. This test is based on measurements of the kinetics of cell death. In this test, the decimal reduction time (D value) [i.e., the time required for one log (90%) reduction in the bacterial population] is obtained by linear regression for different preservative concentrations. This gives a rate of killing of specific organisms and also allows comparisons between the effectiveness of different antimicrobials. Criteria of preservative acceptance based on the D value are discussed in detail elsewhere [20]. 

The choice of an assay in quantifying cell death is critical. The selection and timing of an appropriate end-point to measure is based on knowledge of the underlying mechanism and kinetics of cell death. The first decision to be made is whether to monitor the marker of dead cells or the marker for surviving (live) cells. Advancement in understanding and techniques to detect the biochemical markers typical of cell death led to the development of relatively simple and direct cell death detection assays, which in many cases represent a better choice of assay. 

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