Drug Toxicity and Testing - Conda Sheridan Slides PDF

Drug Toxicity and Testing Foundations of Molecules to Medicines (PHSI3210) Essentials of Drug Discovery and Development (PHSC802)  Toxicology The study of the adverse effects caused by chemical compounds on living systems. Mechanisms of action and exposu...

Drug Toxicity and Testing Foundations of Molecules to Medicines (PHSI3210) Essentials of Drug Discovery and Development (PHSC802)  Toxicology The study of the adverse effects caused by chemical compounds on living systems. Mechanisms of action and exposure, a cause of acute/chronic disease. Understanding physiology and/or pharmacology by using toxic molecules as chemical probes. Recognition, identification, and quantification of hazards caused by exposure to chemicals. Discovery of new drugs. Development of standards and regulations to protect humans, animals, and the environment from the adverse effects of chemical entities. https://hazwoper-osha.com/blog-post/what-is-toxicology  Definitions Cell viability: the number of healthy cells in a sample Cell toxicity: caused by exogenous toxic drug. It can damage cells, especially when the toxicant causes cell death and/or serious organ dysfunction Cell cytotoxicity and Cell proliferation assays: mainly used for drug screening purposes. Detect whether the studied compounds have effects on cell proliferation or display cytotoxic effects. https://scitovation.com/services/cell-based-assays/cell-cytotoxicity-and-viability/; DOI: 10.2174/1389201017666160808160513  Some Definitions Apoptosis: an active, programmed process of autonomous cellular dismantling that avoids eliciting inflammation. In other words, programmed cell death Necrosis: passive, accidental cell death resulting from environmental perturbations with uncontrolled release of inflammatory cellular contents. Death of cells due to diseases, injury, or limited blood supply. Autophagy: self-degradative process that is key for balancing sources of energy at critical times in response to nutrient stress https://www.ptglab.com/news/blog/what-is-the-difference-between-necrosis-and-apoptosis/; doi: 10.1002/path.2697; 10.1128/IAI.73.4.1907-1916.2005  Some Definitions Ferroptosis: a regulated form of iron-dependent, non-apoptotic oxidative cell death induced by erastin and RSL3 Pyroptosis: a highly inflammatory form of programmed cell death that occurs upon infection by pathogens and is likely to form part of the antimicrobial response (Wikipedia). Note, viability assays can be used to evaluate the response to internal or external stimuli. https://www.mdpi.com/1422-0067/23/15/8363  Dose-Response Curves A common way to portray data in both basic and clinical science For example, we could examine the effect of increasing amounts of an analgesic on pain threshold To show the data, the drug concentration would be plotted on the x- axis and the effect on pain threshold on the y-axis A plot of drug concentration ([D]) versus effect (E/Emax) is shown as a rectangular hyperbola Note how the drug effect reaches a plateau (maximum) This is because there is a finite number of receptors  Dose-Response Curves Hyperbolic plots are cumbersome graphs because drug concentrations can vary 1000-fold To overcome this problem, the log of the drug concentration is plotted versus the effect. A plot of the log of [D] versus E/Emax presents sigmoidal shape  Dose-Response Curves The position and shape of the log-dose response curve depends on the affinity of the ligand for the receptor and its intrinsic activity Affinity determines the position of the dose-response curve on the X-axis Intrinsic activity affects the magnitude of the response  Dose-Response Curves In most physiological systems, the relationship between receptor occupancy and response is not linear but some unknown function F of receptor occupancy. Also, all receptors do not have to be occupied to produce a full response Because of this hyperbolic relationship, maximal responses are elicited at less than maximal receptor occupancy  Therapeutic Index Effective dose (ED50) = dose at which 50% population shows response Lethal dose (LD50) =dose at which 50% population dies TI = LD50/ED50, an indication of safety of a drug (higher is better)  Efficacy Toxicity Consider a drug that requires ED50 LD50 a concentration of 15 mcg/mL to control depression but causes anemia at a concentration of 30 mcg/mL. This drug would be considered to have a narrow therapeutic index. It may be effective but it is too toxic. https://drawittoknowit.com/course/pharmacology/glossary/pharmacology/therapeutic-index-therapeutic- window#:~:text=MTC%20and%20MEC.-,Example,have%20a%20narrow%20therapeutic%20index.  Using TI to Compare Relative Safety 100 100 80 80 60 ED 60 ED 40 TD 40 TD % reponse % response 20 20 0 0 -5 -4 -3 -2 -1 0 10 10 10 10 10 10 10 10 10 10 1 2 3 4 10-5 10-4 10-3 10-2 10-1 100 101 102 103 104 drug A dose (mg/kg) drug B dose (mg/kg) Drug A: TI = TD50/ED50 = 100/0.01= 10000 Drug B: TI = TD50/ED50 = 1/0.01 = 100 Which drug is safer?  Disadvantages of Using TI 100 99 % response 80 ED LD A and B A 60 50 % response 40 LD B % response 20 1 % response 0 10-3 10-2 10-1 100 101 102 103 dose (mg/kg) Drug A: ED50 = 2 mg/kg; LD50= 100 mg/kg Drug B: ED50 = 2 mg/kg; LD50= 100 mg/kg Drugs A and B both have the same TI = 100/2 = 50 TI does not take into account the slope of the dose-response curves.  Cell Toxicity Cell viability/cytotoxicity assays measure cellular or metabolic changes associated with viable or nonviable cells. These assays detect structural changes such as loss of membrane integrity or physiological and biochemical activities indicative of living cells. Cytotoxicity assays may detect cytosolic proteins that should not be released by the cell, unless the cell has lost membrane integrity and the proteins have leaked out. A clear definitions: “Reduction of cell viability by more than 30% is considered a cytotoxic effect” (EN ISO 10993-5, section 8.5) https://www.cellandgene.com/doc/unravel-the-complexity-of-single-cell-natural-killer-cell-cytotoxicity-and-adcc-mediation-0001; https://www.promega.com/products/cell-health-assays/cell-viability-and-cytotoxicity-assays  Cell Toxicity Cell viability/cytotoxicity is essential in the drug discovery process, you can study: The toxicity towards normal cells The toxicity against cancer cells Transient vs permanent effects (membrane damage) Mechanism of action Parameters to take into account: Time of incubation Concentration (range and ammounts) Technical replicates (running the reaction on the same sample 3 times would be 3 technical replicates). Biological replicates (running the test on different samples treated the same way). https://visikol.com/blog/2023/01/25/cellular-toxicity/  Technical Replicates Repeated measurements of the same sample Technical replicates address the reproducibility of the assay or technique; however, they do not address the reproducibility of the effect you are studying. Rather, they indicate whether your measurements are robust Examples include loading multiple lanes with each sample on the same blot, running multiple blots in parallel, or repeating the blot with the same samples on different days. Technical replicates help identify variation in technique. For example, a lysate derived from a mouse is loaded thrice (A1, A2, A3), then run and measured independently to identify variations in the protocol. https://www.licor.com/bio/blog/technical-and-biological-replicates  Biological Replicates Parallel measurements of biologically distinct samples to capture random biological variation. Important because they address how widely your experimental results can be generalized. They indicate if an experimental effect is sustainable under a different set of biological variables. Include repeating a particular assay with independently generated samples or samples derived from various cells, tissues, or organisms to see if similar results are observed. Examples include analysis of samples from multiple mice rather than a single mouse, or from multiple batches of independently cultured and treated cells. Biological replicates derived from independent samples help capture random biological variation. For example, three biological replicates (A, B, and C) are collected from three idifferent mice. Each of these biological replicates was run in three technical replicates (A1, A2, A3; B1, B2, B3; C1, C2, C3) using a Western blot assay. https://www.licor.com/bio/blog/technical-and-biological-replicates  A Brief Overview The most common methods for estimating dead cells take advantage of (i) loss of membrane integrity and (ii) the ability of molecules to partition into a compartment not achievable if the cell membrane is intact. Test used to detect dead cells include measuring the leakage of a component (usually an enzyme) from the cytoplasm into the culture medium or the penetration of an otherwise non-permeable dye into cells with a compromised membrane. https://bpsbioscience.com/cell-based-services/cell-cytotoxicity-assay-services  A Brief Overview Dyes that selectively penetrate dead cells Trypan blue. One of the most common methods to determine the cell number and percent viability in a population of cells Fluorescent DNA Binding Dyes That Penetrate Dead Cells Large number of nucleic acid binding dyes that can be used to stain cells for microscopy or flow cytometry Propidium iodide (PI), SYTOX green, GelRed, CellTox Green, YOYO-1 Iodide, DRAQ7 far-red fluorescent dye Markers That Leak Out of the Cytoplasm The presence of dead cells that have lost membrane integrity can be detected by measuring markers that leak from the cytoplasm into the culture medium Examples include LDH, Calcein-AM, CytoTox 96, Vybrant Cytotoxicity Assay (Glucose-6-Phosphate Dehydrogenase Release Assay), In Vitro Toxicology Assay (Lactic Dehydrogenase based) HepG2 cells treated with Terfenadine. CellTox Green was added at time of dosing. Fluorescence was measured every hour over 3 d. Increasing fluorescence indicates increase in number of dead cells in the culture well. https://www.ncbi.nlm.nih.gov/books/NBK540958/  Studying Cell Toxicity As listed a main event that occurs after cell death is the loss of membrane integrity, which allows molecules to freely enter or exit the cell. Thus, membrane disruption can be used as proxy for cell toxicity Dyes That Selectively Penetrate Dead Cells Trypan blue: frequently used. Stains selectively dead tissues or cells with a blue color, use microscope Fluorescent DNA-Binding Dyes That Penetrate Dead Cells Nucleic acid binding dyes. Key to consider the emission wavelength spectrum Some common dyes include: Propidium iodide, Hoechst 33342, SYTOX Green https://adipogen.com/; https://www.bio-rad-antibodies.com/cell-health-nuclear-staining-cell-pureblu-hoechst-33342.html; https://www.ncbi.nlm.nih.gov/books/NBK540958/  Trypan Blue Exclusion Assay Used to determine the number of viable cells present in a cell suspension. Based on the notion that live cells possess intact cell membranes that exclude trypan blue whereas dead cells do not. Binds intracellular proteins In this test, a cell suspension is mixed with the dye and then visually examined to determine whether cells have taken up, or excluded, the dye. Rapid and simple assay and is good for analyzing membrane integrity Viability is determined indirectly from cell membrane integrity Concentration measurement of TB, PI, and AO/PI-stained room temperature- incubated Jurkat cells https://www.nexcelom.com/applications/cellometer/viability/comparing-trypan-blue-ao-pi-staining-methods/; https://pubmed.ncbi.nlm.nih.gov/18432654/  Trypan Blue Problems It is possible that cell’s viability have been compromised even though its membrane is fine. Conversely, membrane integrity may be abnormal yet the cell may repair itself and become fully viable. Dye uptake is assessed subjectively and small amounts of uptaken dye may go unnoticed. Considered to be carcinogenic and must be handled with care. Over time it forms aggregates and crystals. Samples that are lower than 70% viable show higher measured cell viability when compared to fluorescent based methods 10.5455/jrmds.2017526; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6716531/  Common Fluorescent Dyes Propidium Iodide (PI) is a fluorescent DNA-binding dye It penetrates cell membranes of dead or dying cells but is excluded from viable cells. These allows the use of PI for evaluation of (apoptotic) cell death or for cell cycle analysis. Hoechst 33342 and propidium iodide are used together for simultaneous flow cytometry and fluorescence analysis of apoptosis and cell-cycle distribution stages. Hoechst 33342 can be uptaken by live and dead cells They can be distinguished by their morphology: live cells have flat nuclei with noncondensed chromatin while dead cells have small nuclei with condensed and/or fragmented chromatin. Ideal assay for distinguishing apoptotic cells from healthy or necrotic cells Commonly used with 5-bromo-2'-deoxyuridine (BrdU) to distinguish the compact chromatin of apoptotic nuclei, to identify replicating cells and to sort cells based on their DNA content https://www.creative-bioarray.com/support/double-staining-apoptosis-assay-hoechst33342-pi.htm; https://www.sciencedirect.com/topics/medicine-and-dentistry/hoe-  Recap TB assay is based on the ability of the membrane of viable cells to exclude the dye, while nonviable cells are stained blue TB assay can be difficult to interpret because of staining artifacts Alternative assays using acridine orange (AO) and propidium iodide (PI) have been used for simultaneous visualization of viable and nonviable cells AO is a membrane-permeable, cationic dye that binds to nucleic acids of viable (causes a green fluorescence) PI is impermeable to intact membranes but readily penetrates the membranes of nonviable cells and binds to DNA or RNA (orange fluorescence) When AO and PI are used simultaneously, viable cells fluoresce green and nonviable cells fluoresce orange under fluorescence microscopy. Acridine orange (AO, green) and propidium iodide (PI, red) double staining fluorescent micrographs of differentiated neuronal cells (a) 4 h H2O2 treated cells, (b) 72 h myrosinase pre-treated plus 4 h H2O2 exposed cells https://doi.org/10.1371/journal.pone.0196403; https://doi.org/10.1046/j.1537-2995.2000.40060693.x  CellTox Green CellTox Green is a fluorescent dye that binds DNA of cells with impaired membrane integrity. The fluorescence is enhanced upon binding generating a stable signal. Measures changes in membrane integrity that occur as a result of cell death. Thus, the cyanine dye is excluded from viable cells but stains the DNA from dead cells. CellTox Green Dye was mixed with K562 Cells. CellTiter-Glo® Cell Viability Assay Reagent was added CellTox Green can be used in kinetic mode to determine and luminescence (viability) measured. These two the onset of cytotoxicity followed by multiplexing with the inverse measures of cell health resulted in Caspase-Glo® 3/7 Assay in the same sample well to EC50 agreement. confirm caspase-3/7 activation at the appropriate time. https://www.fishersci.com/shop/products/promega-celltox-green-cytotoxicity-assay-5/p-4575193; https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/cytotoxicity.html  Important Notes Some people may argue your experiments is as important as your control. When studying DNA-Binding Dye, you may use the following controls Background control (without cells). To determine background fluorescence. Negative control (untreated cells). Measure fluorescence from samples containing cells in culture medium treated with the same amount of buffer used to deliver test compounds and containing same concentration of dye. Positive control (total cell number). To determine a value for the positive control representing all of the cells in the sample, a detergent is used to lyse the cells and enable staining of the entire population. (A) DRAQ5, a DNA-intercalating dye. (B) Hoechst, a groove-binding dye. https://doi.org/10.1042/BST20170301  Fluorescent Markers Dead cells that lost membrane integrity can be detected by measuring markers that leak from the cytoplasm into the culture medium. Some common reagents/assays include: Calcein-AM; CytoTox-Glo; LDH; Sytox https://www.creative-bioarray.com/support/calcein-am-cell-viability-assay.htm; https://www.ncbi.nlm.nih.gov/books/NBK540958/  Calcein AM It is a membrane-permeable cell marker that can permeate into intact cells Non-fluorescent acetomethoxy derivate of calcein (calcein AM, AM = acetoxymethyl) Once inside the cells, non-fluorescent calcein AM is hydrolyzed by esterases to calcein (green, fluorescent), which is highly negatively charged and is well- retained in cell cytoplasm Used to study cell membrane integrity and for long-term cell tracking due to its low cellular toxicity Used to quantify the number of viable cells. Limitations: it is a P-gp substrate, which limits its ability to enter the cells. for certain cell lines and experimental conditions, relatively high concentrations of Calcein, AM can be toxic to cells. FBS may cause hydrolysis outside of cells Dose-dependent cytotoxicity results at different time points The results showed increasing % cytotoxicity as the antibody concentration increased for each time point https://www.aatbio.com/resources/faq-frequently-asked-questions/Is-calcein-AM-toxic-to-cells; https://www.nexcelom.com/blogs/nk92-cell-mediated-adcc-using- calcein-am/; https://www.creative-bioarray.com/support/calcein-am-cell-viability-assay.htm  Lactate dehydrogenase (LDH) Assay Lactate dehydrogenase (LDH) is a cytoplasmic enzyme present in all cells that can be used as an indicator of cytotoxicity. When the plasma membrane is damaged, LDH is released into the cell culture supernatant LDH’s activity can be quantified by using the NADH produced during the oxidation of lactate to pyruvate The released NADH can be used to reduce a yellow tetrazolium salt, INT, into a red, water-soluble formazan-class dye by absorbance at 492 nm. The amount of formazan is directly proportional to the amount of LDH in the culture, which is, in turn, directly proportional to the number of dead or damaged cells. DOI: 10.1101/pdb.prot095497; https://en.wikipedia.org/wiki/INT_%28chemical%29; https://www.cephamls.com/ldh-cytotoxicity-assay-kit-colorimetric-2/  Lactate dehydrogenase (LDH) Assay HeLa cells were seeded into a 96-well plate at varying densities using media containing 10% FBS. HeLa cells were seeded in medium After overnight incubation, the cells were replaced supplemented with 1% FBS at density of with serum-free media and then treated with either 10,000 cells/well and then treated for 24 hr Assay Buffer (Spontaneous LDH Release) or 10% with either vehicle control or 1 μM Triton X-100 solution (Maximum LDH Release). The Staurosporine #9953. LDH released into the amount of LDH released into the medium was medium and percent cytotoxicity was determined using the LDH Cytotoxicity Assay Kit calculated using the LDH Cytotoxicity Assay protocol. Kit protocol. https://www.cellsignal.com/products/cellular-assay-kits/ldh-cytotoxicity-assay-kit/37291?_requestid=1201242  Lactate dehydrogenase (LDH) Assay Typically, cells are seeded into 96- well plates and maintained in culture for 24 h. Cells are then exposed to eight compound concentrations. After 24 h exposure, NADH consumption in the supernatant is determined for each treatment concentration and compared to control. Negative controls (blank, untreated and solvent) and concentrations of positive control (sodium lauryl Toxicity evaluation using LDH measurements. Different batches (assayed in triplicates) of cardiomyocytes exposed sulfate or H2O2) are tested. to doxorubicin at various concentrations. Bar graph shows A solution 1% Triton is used to the mean values of the fold change related to the baseline and the error bars illustrate the SD (* = p < 0.05, generate the reference value for the ** = p < 0.01). total amount of LDH. https://doi.org/10.1016/j.tox.2014.12.018; https://www.nucro-technics.com/services/genetic-toxicology/ldhcytotoxicity/  SYTOX SYTOX dead cell stains are cell-impermeant dyes with increased fluorescence upon binding to dsDNA Dead cells have bright fluorescence and live cells have dim fluorescence. Can be used in cells without an additional wash step and visualized with minimal background staining since they are nonfluorescent in aqueous media. Useful to study gram-positive and gram-negative bacteria, where an exceptionally bright signal is required May be used in conjunction with DAPI Bovine pulmonary artery endothelial cells (BPAEC) incubated with the fixable, mitochondrion-selective MitoTracker Red CMXRos. After staining, the cells were formaldehyde-fixed, acetone-permeabilized, treated with DNase-free RNase and counterstained using SYTOX Green nucleic acid stain. https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/cell-viability.html  SYTOX The advantage of Sytox® is that it provides a very high signal But it does not give you any insight into the molecular causes of the cell death Recommended to confirm the results with some other assay to determine the signal is true (false negatives). (A) Live embryo stained with SYTOX green. Only a few cells are stained (fluorescence shown in green), which have been wounded during the dissection of the embryo (arrows). (B) Transmission image of the embryo shown in (A). (C) Fixed embryo stained with SYTOX green. (D) CLSM section through the root of a fixed embryo stained with SYTOX green. (E) Higher magnification of (C) showing that predominantly the nuclei are stained in the embryonic cells. (F) Fixed embryo stained with SYTOX orange. (G) Fixed embryo stained with SYTOX blue. All images with the exception of (D) are overlay projections of CLSM images. Scalebars: 10 μm in (D) and (E), otherwise 200 μm. https://plantmethods.biomedcentral.com/articles/10.1186/1746-4811-4-15  Metabolic Activity Assays Metabolic activity viability assays are live-cell, membrane permeant reagents that detect cellular indicators such as the reducing environment or cellular redox potential. Most common assays are Alamar blue, MTT, and XTT assays https://www.abpbio.com/product/cell-viability-assay-based-on-metabolic-activity/; Dose response curve of ellipticine in HepG2 cell https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/cell-viability.html line using the AlamarBlue and MTT assays  CytoTox-Fluor Cytotoxicity Assay Single-reagent-addition, homogeneous, fluorescent assay that measures the relative number of dead cells in cell populations. The assay measures protease activity associated with cytotoxicity It uses a fluorescent peptide substrate (bis-alanyl-alanyl-phenylalanyl-rhodamine 110; bis-AAF-R110) to measure "dead- cell activity," from cells that have lost membrane integrity. Bis-AAF-R110 cannot cross the membrane of live cells and gives no signal. Rhodamine 110 https://www.promega.com/products/cell-health-assays/cell-viability-and-cytotoxicity-assays/cytotox_fluor-cytotoxicity-assay/?catNum=G9260  Alamar Blue Assay Cell viability assay that contains the cell permeable, non-toxic, and weakly fluorescent blue indicator resazurin Useful and non-toxic alternative to MTT Quantitatively measures proliferation in human, animal, and bacterial cells Useful for cytokine bioassays, cell viability assays, and in vitro cytotoxicity. Growing cells reduce the alamarBlue dye from non-fluorescent blue to red fluorescent. The continuous growth of viable cells maintains a reducing environment (fluorescent, red). The inhibition of growth maintains an oxidized environment (non-fluorescent, blue), which can be detected using a fluorescence detector. https://www.gbiosciences.com/Bioassays/Alamar-Blue-Cell-Viability-Assay; https://www.bio-rad-antibodies.com/alamarblue-cell-viability-assay-resazurin.html  MTT Assay Colorimetric assay that detects the color change from yellow of the tetrazolium dye to purple due to the formation of formazan in the presence of viable cells with active metabolism So, it is a test that measures cellular metabolic activity The MTT reagent can pass through the cell membrane as well as the mitochondrial inner membrane of viable cells presumably due to its positive charge and lipophilic structure https://www.integra-biosciences.com/denmark/en/applications/mtt-assay-assist-plus-pipetting-robot  MTT Limitations Number of seeded cells Concentration of MTT reagent added Amount of MTT that enters the cells Time of incubation with MTT Type of culture media Suboptimal supernatant removal Wavelength at which optical density is measured Cannot be used with compounds that may interfere with the reduction reaction such as polyphenols, ascorbic acid, vitamin A, coenzyme A, and DTT. https://www.aatbio.com/; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8657538/  MTT Results OD as a function of cell seeding number and how it varies for varying concentrations of MTT and incubation time. As the data shows, regardless of [MTT] or incubation time, increasing of # cells increases OD Increasing the number of cells increases the amount of produced formazan by the cell population and, as a result, the OD OD depends on cell seeding number/density, MTT concentration, and incubation time: OD changes with increasing cell seeding number/density. PC-3 cells were allowed to grow for 26 h before MTT addition. Absorbance was measured following 2 h (a), 3 h (b), and 4 h (c) of incubating cells with different concentrations of MTT. Data shown as mean OD of triplicate wells and error bars represent standard deviation (SD).  Sulforhodamine B The test is based on the stoichiometric binding of the SRB dye to the basic amino acids of proteins (based in cellular protein content) under mild acidic conditions followed by extraction under basic conditions. The amount of dye extracted relates to protein concentration, which is a proxy for cell mass and the number of cells in a sample. It is used for analysis of cell viability, cytotoxicity and cell proliferation. Very popular due to the following: High sensitivity, Accurate and reproducible, Low signal-to-noise ratio, High resolution (1000–2000 cells/well), Linear results ranging from 1 to 200% of confluence, colorimetric, nondestructive, and indefinitely stable end point, Rapid and low-cost, Suitable for high-throughput screening https://www.cephamls.com/srb-cytotoxicity-assay-kit-colorimetric/; https://www.sciencedirect.com/topics/agricultural-and-biological- sciences/sulforhodamine-b; https://www.nature.com/articles/nprot.2006.179#article-info  CellTiter-Glo® Luminescent Cell Viability Assay Very sensitive cell viability assay, is the CellTiter-Glo® Luminescent Cell Viability Assay Homogeneous method of determining the number of viable cells by quantitation of ATP present, an indicator of metabolically active cells. It measures intracellular ATP. The system detects as few as 15 cells/well in a 384-well format in 10 minutes after adding reagent and mixing. https://www.promega.com/products/cell-health-assays/cell-viability-and-cytotoxicity-assays/celltiter_glo- luminescent-cell-viability-assay/?catNum=G7570  CellTiter-Glo® Assay Light production is proportional to the number of viable cells. Luciferase acts on luciferin in the presence of Mg2+ and ATP to produce oxyluciferin and to release luminescence. Since the luciferase reaction requires ATP, the luminescence produced is proportional to the Cisplatin mediated reduction of viable cells. The luminescence generated by activated caspase‐3/7 was measured after incubation for amount of ATP present, an 2 h, 3 h, and overnight. Cell viability (% of control) was compared with the absolute caspase‐3/7 activity. The increase of caspase‐3/7 activity indicator of cellular metabolic after exposure of cells to 5, 10, 15, 20 and 40 µM CP was statistically activity. significant (**P < 0.01). Statistical significance was valuated with the ANOVA test. https://doi.org/10.1002/jcb.20531  Comparing Assays Alamar Blue MTT XTT LDH Assay type Fluorescent Colorimetric Colorimetric Colorimetric (also fluorescent versión) Use For viability studies or Well-established, end- Quick and continuous Quantitate lactate when using high cell point assay for assay for quantitatively dehydrogenase (LDH) density in microplates quantitatively determining viability release from damaged determining viability and proliferation cells and proliferation Measurement Detects metabolic Detecting metabolic activity using the redox Detection of activity on cells. Only potential in active, live mammalian cells only extracellular LDH from live cells are detected damaged cells Only damaged cells detected Mechanism of Resazurin converted to Water-soluble MTT Water-soluble XTT Tetrazolium salt (INT) detection fluorescent resorufin converted into converted into water- is converted to a red insoluble purple soluble orange formazan formazan formazan https://www.thermofisher.com/us/en/home/life-science/cell-analysis/fluorescence-microplate-assays/microplate-assays-cell-viability.html  Advantages/disadvantages of the methods Trypan blue MTT XTT LDH Type Dye exclusion Colorimetric Colorimetric Colorimetric Pros Simple, cheap, and a good Far superior to dye exclusion. speed, sensitive, easy to use, Reliability, speed, indicator of membrane integrity. Easy to use, high and safe method. It has high and simple Dead cells are colored blue reproducibility, and used to sensitivity and accuracy evaluation. within seconds of exposure to determine cell viability and the dye cytotoxicity Cons Cell counting is generally done MTT formazan is insoluble in Assay performance depends Serum and some using a hemacytometer, water, prior to measuring the on reductive capacity of viable other compounds counting errors (~10%) could absorbance, an organic is cells with mitochondrial have inherent LDH occur. difficult to process large required to solubilize the dehydrogenase activity. activity. Llimited to number of samples crystals. Difficult to remove cell Changes of reductive capacity serum-free or low- concurrently, cannot be used to culture media from the wells resulting from enzymatic serum conditions distinguish between the healthy due to floating cells with MTT regulation, pH, cellular ion cells and the cells that are alive formazan needles, giving concentration, cell cycle but losing cell functions significant well-to-well error variation, etc may affect the final reading https://www.intechopen.com/chapters/57717 Advantages/disadvantages of the methods SRB (Sulforhodamine B) Alamar Blue ATP Type Colorimetric Fluorometric Luminometric Pros Simple, fast, and sensitive. It Relatively inexpensive and Fastest cell viability assay to use, the most provided good linearity with cell more sensitive than sensitive, and is less prone to artifacts than number, permits the use of tetrazolium assays. It can be other viability assays. The luminescent saturating dye concentrations, multiplexed with other signal reaches steady state and stabilizes less sensitive to environmental methods such as measuring within 10 min after addition of reagent. It changes, independent of caspase activity to obtain does not have an incubation step for intermediary metabolism. more info regarding the conversion of substrate into colored Reproducibility is high. cytotoxicity mechanism. compound. Cons Important to obtain and maintain Fluorescent interference Sensitivity is usually limited by a homogeneous cell suspension. from test compounds and the reproducibility of pipetting replicate Cellular clumps/aggregates often overlooked direct toxic samples rather than a result of the assay should be avoided for high assay effects on the cells are chemistry performance. possible https://www.intechopen.com/chapters/57717  Apoptosis Assays As mentioned, apoptosis (programmed cell death) is a highly regulated process that allows growth and development by removing unneeded cells It also minimizes threats to the organism by destroying surplus cells Unlike necrotic cells, apoptotic cells present compaction of the nuclear chromatin, shrinkage of the cytoplasm, and production of membrane-bound apoptotic bodies “Identifying cells that are going through apoptosis can be challenging because many of the assays used as indicators are detecting structural and functional changes that occur in other processes as well” https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/apoptosis.html  M30 CytoDeath M30 CytoDeath ELISA (enzyme-linked immunosorbent assay) is a plate-based assay designed used to detect and apoptosis-inducing molecules, including kinetic and dose response relationships Detects caspase-cleaved cytokeratin 18; caspase activity is one of the earliest and most common markers for apoptosis Uses M30 CytoDEATH monoclonal antibodies Cells can be detected in situ by Typical M30 CytoDEATH stains of immunofluorescence microscopy and quantified apoptotic cells, exemplarily shown by flow cytometric analysis. for a mouse of the control cohort. Apoptotic cells were very scarce in the periperal zone of the tumors https://www.oncotarget.com/article/19263/text/; https://diapharma.com/product/biomarkers/liver_biomarkers/other-livbio/ck18-other-livbio/m30-cytodeath-elisa/  M30 CytoDeath Activation of caspase-3 in apoptotic HeLa cells after treatment with cisplatin (CP). Exponentially growing HeLaS3 were treated with 40 µM CP for indicated periods of time. The cells were fixed according to the manufacturer's recommendations and stained using antibodies recognizing specifically caspase-3 cleaved cytokeratine 18 (CytoDEATH). In cells fixed on slides the chromatin was visualized by sequential staining with DAPI. Cells present in the field were visualized by Hoffman modulation contrast (HMC). The right image was prepared by simultaneous detection of the CytoDEATH staining and HMC. https://doi.org/10.1002/jcb.20531  Annexin V Common method for detecting apoptotic cells. Annexin V is a 35–36 kDa, Ca2+-dependent phospholipid-binding protein that has a high affinity for the anionic phospholipid phosphatidylserine (PS). In normal cells, PS is located on the cytoplasmic surface of the plasma membrane. During apoptosis, the plasma membrane undergoes structural changes including the translocation of PS from the inner to the outer leaflet (extracellular side). The translocated PS marks the cell for recognition and phagocytosis by macrophages The Annexin V (modified with luciferase) proteins bind to PS during early apoptosis and are detected with a simple luminescence signal https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/apoptosis/annexin-v-staining.html  Annexin V Limitations Risk of false positives when staining with annexin V conjugates. Compromised plasma membranes of dead cells allow annexin V protein to pass through to the interior of the cell where it can bind PS in the inner leaflet. It can only be done on live cells and tissue Binding is limited by slow diffusion of this 36 kDa protein and by slow formation of its high-affinity complexes Jurkat cells (T cell leukemia, human) treated with 10 μM camptothecin (CPT) for 4 hs (right panel) or untreated (as control, left panel). Cells were then treated with Annexin V-Alexa Fluor 488 conjugate to identify apoptotic cells and with PI to identify dead cells, followed by flow cytometric analysis. The CPT-treated cells (right panel) have higher # of apoptotic cells (labelled “A”) than the basal level seen in the control cells (left panel). V = viable cells, D = dead cells. https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/apoptosis/annexin-v-staining.html  Mitochondrial Function Assay A feature of the early stages of programmed cell death is the disruption of mitochondrial function Mitochondrial disruption includes changes in the membrane potential - a central feature of mitochondrial health, and alterations to the oxidation– reduction potential of the mitochondria The inner mitochondrial membrane potential needs Ca2+, reactive oxygen species generation, detoxification, and the synthesis of ATP by oxidative phosphorylation Therefore, the membrane’s depolarization is a good indicator of mitochondrial dysfunction, which is increasingly implicated in drug toxicity https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/apoptosis/mitochondria-function.html  Mitochondrial Function Assay The disruption of mitochondria function can be detected using a variety of fluorescence-based assays including measurements of mitochondrial calcium, superoxide, mitochondrial permeability transition, and membrane potential Mitochondria function assays Membrane potential and Superoxide production Calcium detection Mitochondrial permeability Metabolism transition What can be Cells with healthy, Irreversible detection of Irreversible detection of Irreversible detection of the identified? metabolically active mitochondrial superoxide mitochondrial calcium in mitochondrial permeability mitochondria. production in live cells. live cells. transition (MPT) subsequent to dysregulation of the MPT pore. What is the Measure fluorescence of Measure the increasing Measure the increasing Loss of mitochondrial calcein basis of reagents that accumulate in fluorescence of a superoxide fluorescence of a calcium fluorescence due to assay? active (healthy) mitochondria detection reagent that indicator with increasing mitochondrial permeability with intact membrane accumulates in mitochondria calcium concentration in the transition. potentials. in a membrane potential- mitochondria. dependent fashion where it is selectively oxidized and subsequently binds nucleic https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/apoptosis/mitochondria-function.html acids.  DNA Damage Assays The genome is exposed to potentially genotoxic events during every cell division cycle due to cellular metabolism or routine errors in DNA replication. Exposure to ionizing radiation, UV light, free radical, and mutagenic compounds may modify nucleotides or produce DNA strand breaks. Several assays are available to assess the mechanisms underlying DNA damage and repair”. PAR & PARP Assays, detects Poly ADP-ribose (PAR) and PAR Polymerase (PARP). CometAssay. A simple method for evaluating DNA damage in single cells. Superoxide Dismutase Assay Kits. Fast plate-based method for assaying Superoxide Dismutase activity. HT 8-oxo-dG ELISA Kits. A plate-based immunoassay designed to detect and quantify 8-hydroxy-2’-deoxyguanosine (8-oxo-dG). https://blogs.bmj.com/bjsm/2022/01/07/dna-damage-and-repair-capacity-related-to-diet-and-exercise-a-new-dimension-in-cancer-treatment/; https://www.rndsystems.com/products/dna-damage-and-repair-kits-reagents  COMET Assay Also known as Single-cell gel electrophoresis assay Simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/research-and-disease-areas/cancer-research/comet-assay; https://link.springer.com/article/10.1385/MB:26:3:249  COMET Assay The most widely used method for measuring DNA damage in eukaryotic cells The Comet Assay can be used to detect DNA damage caused by double strand breaks, single strand breaks, alkali labile sites, oxidative base damage, and DNA cross-linking with DNA or protein Also used to monitor DNA repair by living cells Treat cells with a DNA-damaging agent and then incubate them to allow repair to proceed, measuring the amount of damage remaining at intervals. a A high-throughput comet assay produces an output image with multiple comets. b Comets are classified into different types according to their shape https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7688437; https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/research-and-disease- areas/cancer-research/comet-assay  COMET Pros and Cons Pros Cons Possibility to be Measurement of transient DNA lesion instead of irreversible integrated with other gene and chromosomal mutations. In other words, it does not complementary short directly measure the number of specific DNA lesions, but term genotoxicity rather the migration of DNA in agarose gels as a result of the assays in the same relaxation produced by strand breaks under alkaline animals conditions No specific rodent Relative increase of readouts vs. controls instead of absolute strain requirement numbers of mutations Applicable to multiple Limited standardization of assay procedures organs Applicable to virtually Sensitive to indirect genotoxicity mechanisms linked to toxicity all animal models and cellular stress Insensitive to some genotoxicity modes of action Low cost (aneugenicity, DNA crosslinking) Variability is another serious issue https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885189/  Gamma-H2AX Enables the quatification of double strand DNA breaks through the detection of gamma-H2AX in peripheral blood mononuclear cells, cultured cells, and tissue biopsies. The phosphorylation of histone H2AX in Serine 139 (gamma-H2AX) marks regions of DNA double strand breaks and contributes to the recruitment of DNA repair factors to the site of DNA damage. Gamma-H2AX is used widely as DNA damage marker in vitro Quick read-out of DNA damage, can be performed on primary untransformed cells Efficient DSB determination with the γ-H2AX assay. Immunofluorescence staining with γ-H2AX antibodies enables the visualization of DSB-related nuclear foci which can be read out with the automated AKLIDES® system https://pubs.rsc.org/en/content/articlelanding/2019/NR/C9NR01021A; https://onlinelibrary.wiley.com/doi/full/10.1002/em.22238 ; https://www.rndsystems.com/products/ht-gamma-h2ax-pharmacodynamic-assay_4418-09 k#:~:text=HT%20gamma%20%2DH2AX%20Pharmacodynamic%20Assay%20Summary,cultured%20cells%2C%20and%20tissue%20biopsies.  Gamma-H2AX Whole blood was irradiated with 0, 200, 400, 800 or 1600 mGy, and incubated (37 °C) for 10 min. WBCs were isolated and the γ-H2AX analysis was performed by confocal microscopy and by epifluorescence microscope. a Fields of approx. 20 nuclei were selected based on DAPI staining only to exclude bias. 3D reconstruction and counting of Foci were done as described. b Shows dose response curves of WBCs analyzed by confocal microscopy plus 3D reconstruction red circles and by epifluorescence microscope triangle. Curves were obtained by non-linear, sigmoid curve fitting (dose response curve) https://link.springer.com/article/10.1007/s00411-019-00778-1  TUNEL Assay Detects the DNA breaks formed when DNA fragmentation occurs in the last phase of apoptosis by labeling the free 3ʹ-hydroxyl termini. It uses terminal deoxynucleotidyl transferase (TdT). TdT enzyme is able to recognize and bind to the ends of DNA strands that have been cleaved by caspases. Once TdT binds to the DNA, it catalyzes the addition of a nucleotide to the end of the DNA strand. – This is known as nick translation. The addition of a nucleotide creates a new DNA end that can then be detected with a fluorescent dye. https://www.assaygenie.com/blog/tunel-staining  TUNEL Assay TUNEL staining is highly sensitive, specific, and easy to use, when compared to flow cytometry or immunohistochemistry. Can be used to detect apoptosis at earlier stages It can be used on fixed cells (no need to perform live-cell imaging) Key advantage over Annexin V assay, which can only be used on live cells. TUNEL staining is resistant to photobleaching Can be combined with dapi or PI, to create a more complete picture of the cell. Useful when investigating the early stages of apoptosis, difficult to detect DNA damage using dapi or PIalone. Problems: background staining decreases the efficiency of this method (require additional blocking steps to neutralize endogenous biotin). False positive due to DNA fragments during DNA reactions, hard to quantify when DNA fragmentation is too high, low DNase I activity in some cell type may prevent good measurements https://www.mdpi.com/1422-0067/22/1/412; https://www.assaygenie.com/blog/tunel-staining  TUNEL Assay Variations of TUNEL images in kidneys of mice subjected to cisplatin injury (20 mg/kg, 4 days after exposure). (A) TUNEL image in kidney at low magnification; note poor TUNEL staining of glomeruli (shown by arrows) vs. tubules. (B) TUNEL-IHC combination; red staining indicates heme oxygenase 1 (HO-1) induced in two cells, one of which is TUNEL-positive (dead). (C) Commonly seen universal TUNEL signal indicative of any kind of cell death. (D) Late universal TUNEL signal with low DAPI staining of completely degraded DNA. (E) Apoptotic TUNEL signal; TUNEL staining of apoptotic bodies characteristic of apoptosis. (F) Cytoplasmic TUNEL signal that illustrates at least partial necrosis. (G) Necrotic dispersed TUNEL signal characterized by nuclear and plasma membrane lysis and irregular leakage of TUNEL-positive material from nucleus and cytoplasm https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7795088/  Autophagy “The segregation and delivery of cytoplasmic cargo, including proteins and organelles, for degradation by hydrolytic enzymes through the lysosomal machinery. It is critical to the healthy functioning of cells, and the failure of autophagy is a primary driver of the accumulation of cell damage and aging.” There are several test to study Autophagy including cell transfection with the fluorescent LC3 reporter (flow cytometry) or tracking p62 https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/autophagy.html  Tracking Autophagy Tracking Autophagy With LC3B: LC3B is recruited from the cytosol and associates with the phagophore early in autophagy. This localization serves as a general marker for autophagic membranes and for monitoring the process as it develops. Tracking Selective Autophagy With p62: p62 is a receptor for cargo destined to be degraded by autophagy, including ubiquitinated protein aggregates destined for clearance. The p62 protein is able to bind ubiquitin and also to LC3, thereby targeting the autophagosome and facilitating clearance of ubiquitinated proteins. Premo Autophagy Sensor Tandem RFP- GFP-LC3B can be used to discriminate Imaging motility of p62-positive vesicles along between acidic and neutral LC3B- microtubules using Premo Autophagy Sensor positive vesicles. GFP-p62 Kit. https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/autophagy/autophagy-lc3b-p62.html  Lysosome Markers Lysosomes are membrane-bound organelles that have enzymes that can degrade carbohydrates, lipids, nucleic acids, and peptides The intracellular molecules are digested through autophagy The lysosomal cell death, (LCD), pathway is a caspase-independent, cell death pathway LysoTrackers are cell-permeable markers that target acidic organelles in live cells. LysoTracker markers are made of a fluorophore linked to weakly basic amines. “These lysosomal markers accumulate in organelles that have a low pH and are only partially protonated at neutral pH.” Multi-color imaging of lysosomes and endocytosis. A549 cells were labeled with Hoechst 33342 and 50 nM LysoTracker Deep Red for 15 minutes in complete media. Cells were then washed with warm DPBS and incubated in DPBS containing 40 µg/mL pHrodo Green 10k-dextran for 90 minutes at 37°C. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611321/; https://www.thermofisher.com/us/en/home/life-science/cell- analysis/cell-structure/lysosomes.html  Detecting Free Radicals Generation of reactive oxygen species (ROS) happens in aerobic organisms In healthy cells, their production occurs at a controlled rate During oxidative stress conditions, the production of ROS is dramatically increased, resulting in subsequent alteration of membrane lipids, proteins, and nucleic acids. Oxidative damage of biomolecules is associated with aging as well as a various pathological conditions such as atherosclerosis, carcinogenesis, ischemia, and neurodegenerative disorders. https://solesence.com/what-are-free-radicals-anyway/; https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/oxidative-stress.html  Detecting Free Radicals CellROX Reagents are fluorogenic probes Lipid peroxidation is the oxidative that measure oxidative stress in cells using degradation of lipids. Lipid peroxidation is conventional fluorescence microscopy, high implicated in many human diseases content screening, microplate fluorometry, or including diabetes and cardiovascular flow cytometry. The dyes are nonfluorescent disease. in a reduced state and fluoresce bright green, orange, or deep red upon oxidation. General oxidative stress detection in U2OS Detection of lipid peroxidation with the Invitrogen cells with CellROX Green Reagent. Image-iT Lipid Peroxidation Kit. https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/oxidative-stress.html  How About Bacteria? Membrane can be used to study the integrity of bacterial membranes. The fluorescence of the lipophilic dye N-phenyl-1-napthylamine (NPN) can inform about the permeability of the outer membrane of Gram-negative bacteria NPN emits weak fluorescence in water but is highly fluorescent in hydrophobic environments. NPN cannot insert into intact bacteria membranes Bacteria membranes are polarized (they have a negative transmembrane potential); some molecules kill bacteria by depolarizing their membranes The dye 3,3′-Dipropylthiadicarbocyanine iodide [DiSC3(5)] is cationic, membrane-permeable, fluorescent in water and can be used to depolarization of bacterial membranes. DiSC3(5) has low fluorescence when bound to bacteria with polarized membranes; if the membrane depolarizes, the dye is released, and its fluorescence intensity increases https://www.news-medical.net/health/Technology-Advances-in-Bacteria-Detection.aspx; https://www.frontiersin.org/journals/medical-technology/articles/10.3389/fmedt.2020.610997/full  How About Bacteria? Flow cytometry analyzes the physical properties and fluorescence of individual cells within a population. Bacteria with permeabilized membranes can be distinguished from viable bacterial cells using propidium iodide (PI) and SYTO 9 using flow cytometry. PI and SYTO 9 become fluorescent when intercalating with DNA. PI is a red-fluorescent dye, non-permeable to intact membranes and cannot enter viable cells SYTO 9 is a green-fluorescent dye that can enter both live and dead bacterial cells PI has stronger affinity for nucleic acids than SYTO 9; therefore, when both dyes have access to nucleic acids inside bacteria, PI displaces SYTO 9 There are some factors that might interfere with readings: photobleaching of SYTO 9, binding affinities of SYTO 9 to live and dead cells, background fluorescence. Some bacteria strains have efflux pumps that can remove PI. SYTOX Green is a high-affinity nucleic acid stain used to investigate membrane integrity in bacteria using flow cytometry. This dye only enters cells with compromised plasma membrane. Binding of SYTOX green to nucleic acids results in >500-fold increase in fluorescence intensity This dye has a higher quantum yield compared to that of PI These might explain why many studies used SYTOX green to investigate bacterial membrane integrity instead of the combination of SYTO 9 and PI. https://www.news-medical.net/health/Technology-Advances-in-Bacteria-Detection.aspx  How About Bacteria? Schematic of a Gram- negative bacterium with inner membrane (gray), peptidoglycan layer (orange), outer membrane (green), intracellular content (purple spheres) and nucleic acids (helix) and the effect of fluorescent dyes before (left) and after (right) treatment with a membrane- active molecules. (C) NPN becoming highly fluorescent when in hydrophobic environment such as lipid membranes (D) [DiSC3(5)] packed within inner membrane. DiSC3(5) is released intoaqueous environment and becomes fluorescent once membrane is depolarized (E) Bacterial cells incubated with PI and SYTO 9. Left side shows PI unable to go through intact membrane, unlike SYTO 9 that penetrates intact membrane and binds to DNA. After damage, PI enters the cell and displaces SYTO 9 (right). (F) SYTOX Green is highly fluorescent if bound to DNA, however, unable to go through bacterial membrane unless membrane is damaged. https://www.news-medical.net/health/Technology-Advances-in-Bacteria-Detection.aspx