In vitro testing of rug toxicity


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Testing of toxicity of environmental pollutants in cell culture

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  • Numerous end-points have been used by different investigators to measure toxicity. These include growth determined by protein analysis, plating efficiency, enzyme. The release of SlCr isanother index of lethality measuring membrane functions (Holden et at., 1973).release, exclusion or inclusion of dyes or radioactive markers and metabolic alterationssuch as oxygen consumption and ATP levels. Morphological changes in cells exposed to chemicals, observed by light or electronmicroscopy, have also been used to demonstrate basic cytotoxicity.effect commonly observed include 'cytoplasmic blobs suggesting injury of the cell membraneand vacuolization (Ekwall, 1983)
  • different cell lines are used to investigate the nature and the location of free radical damage at the cellular level . Liver slices orhepatocytes have been extensively used in the investigation of metabolic pathways,and in the identification of intermediates or secondary products. cell types derived from other organsor tissues such as lung (Baird et ai., 1980; Tell and Douglas, 1980), aorta (Baird etai., 1980) intestine (Schiller and Lucier, 1978), and ovary (Drake et ai., 1982) alsodisplay metabolic activities which may have a major significance in determiningtoxicity. Phase I and II metabolic reactions have been studied in vitro by cocultivatinghuman adult hepatocytes and rat liver epithelial cells for several weeks(Begueet ai., 1983).
  • End-points more commonly used as markers of toxic effects in specializedcells*
  • Qualitative (morphological estimation)Quantitative (e.g. NRU, MTT assay, LDH assay)
  • After preparation, the extracts are transferred onto a layer of cells and incubated. Prepare extract of a material Keep the material in solution Chemicals will leach intosolution Apply solution to cell-culture
  • In this method, a piece of test material is placed directly on a mammalian cell layer that is protected from mechanical damage by a layer of agar.
  • Reactivity of the test sample is indicated by malformation, degeneration and lysis of cells around the test material. induced by the test material. Cells may Change morphologyDieLose adherence to dish
  • Annex C of ISO 10993-5:2009 refers to the MTT cytotoxicity assay, which can accurately quantify as few as 950 cells.
  • Cell death can occur either by apoptosis or by necrosis.Necrosis is accompanied by mitochondrial swelling and increased plasma membrane permeability, whereas apoptosis involves an articulated breakdown of the cell into membrane-bound apoptotic bodies. LDH is rapidly released from the damaged cells.
  • LDH(lactate dehydrogenase) enzyme catalyzes the oxidation of lactate to pyruvate in the presence of co-enzyme, NAD+.
  • Three-dimensional cell culture offers a practical alternative to natural tissue models .These systems provide an cell types can be encouraged to form tissue-like constructs, often using natural or synthetic scaffolds such as collagen, alginate, polyethylene glycol hydrogels and nanofibres to provide structural support to the growing tissue.
  • Three-dimensional cell culture offers a practical alternative to natural tissue models .These systems provide an cell types can be encouraged to form tissue-like constructs, often using natural or synthetic scaffolds such as collagen, alginate, polyethylene glycol hydrogels and nanofibres to provide structural support to the growing tissue.
  • Colon cancer cells exposed to gemcitabine showed that 3-D multilayer cells were over 1000 times more resistant to gemcitabine than cells in 2-D cultures.These results coincided with the lack of in vivo efficacy of this drug for colon cancer in clinical trials.
  • In vitro testing of rug toxicity

    1. 1. Submitted by: Dr. Vijayata (
    2. 2. • In vitro toxicity testing is the scientific analysis of the effects of toxic chemical substances on cultured bacteria or mammalian cells. • In vitro (literally 'in glass') testing methods are employed primarily : ̵ ̵ to identify potentially hazardous chemicals to confirm the lack of certain toxic properties in the early stages of the development of potentially useful new substances such as therapeutic drugs, agricultural chemicals and food additives. • In vitro toxicity testing methods can be more useful and cost-effective than toxicology studies in living animals (which are termed in vivo or "in life" methods). 2
    3. 3. • Cell culture can be used to screen for toxicity both - by estimation of the basal functions of the cell (i.e. those processes common to all types of cells) or - by tests on specialized cell functions. • For general toxicity studies, the more commonly used cell lines include the well characterized diploid human fibroblast lines, WI38 and tumour cell lines, HeLa. • The first and most readily observed effect following exposure of cells to toxicants is morphological alteration in the cell layer or cell shape in monolayer culture. 3
    4. 4. • The cytotoxic concentrations of chemicals determined in vitro have been shown to correlate well with lethal doses in laboratory animals and man for a range of selected drugs and chemicals (Ekwall, 1983). • Morphological changes in cells exposed to chemicals (blebbing - suggesting injury of the cell membrane or vacuolization) observed by light or electron microscopy, have also been used to demonstrate basic cytotoxicity. (Ekwall, 1983). • These observations may provide valuable information about the pathologic processes that occur as a consequence of exposure to a chemical substance. 4
    5. 5. End-points for the assessment of general cellular toxicity • Cell morphology - Blebbing, vacuolisation, fine ultrastructural modification • Cell viability - Trypan blue (enters dead cells), neutral red (actively taken up by living cells), Cr51 release • Cell growth - Cell count, plating efficiency, DNA or protein content, glucose consumption, lactate production, NR-test, MTT-test • Metabolic parameters - O2 consumption or ATP level, pool of DNA and RNA precursors, NADH-NAD conversion. 5 Ekwall et al (1990)
    6. 6. Organ of origin Primary cultures or isolated cells Cell lines Nervous system Chick embryo ganglia; chick embryo brain cells; mouse and rat cerebellum cells C 1300 (mouse); C 6 (rat) Lung Human, rabbit and rat alveoloar macrophages P 388Dl (mouse); A 549 (human) Reticuloendothelial system Human, mouse lymphocytes and erythrocytes; rat and mouse peritoneal macrophages ------ Liver Rat and chick embryo hepatocytes Chang (human); CC1144 (rat); ARL (rat); RLC-GA (rat) 6
    7. 7. End-points more commonly used as markers of toxic effects in specialized cells Synthesis or release of specific molecules: – Collagen mat, heme, haemoglobin, albumin, urea, lipoprotein, bile salts, metallothionein,glycosaminoglycans, proline and hydroxyproline, energy-dependent choline accumulation, and histamine release . Synthesis, activity or release of specific enzymes: – Glucuronidase, lactate-dehydrogenase, oubain-insensitive ATPase, G-6-P dehydrogenase, glycogen phosphorylase, glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase, acetylcholinesterase, and renin. 7
    8. 8. Interactions of compound with cells: – Phagocytosis, cytoplasmic inclusions, intracellular accumulation, uptake or binding of compound to cytosol and lipoproteins, mitogenic response. Alterations of metabolic pathways: – Methaemoglobin reduction, glucose-transport, 5-methyl tetra hydropholate accumulation, hormone-stimulated gluconeogenesis, lipid peroxidation, fat accumulation and glucosamine and galactose incorporation. 8
    9. 9. Cell surface activities: – Adhesiveness, antibody-mediated rosette formation, complement deposition on treated cell membrane, chemotactic migration, GABA-mediated postsynaptic inhibition, membrane polarization, fibre retraction or outgrowth, and electrophysiological alteration. Intracellular markers – Mitochondria, lysosomes, peroxisomes 9
    10. 10. Introduction • The quality of materials used (cell lines, media and other reagents) will affect the quality of the cultures and the subsequent scientific data and products derived from them. • The main areas of quality control that are of concern for tissue culture are: – The quality of the reagents and materials. – The provenance and integrity of the cell lines. – The avoidance of microbial contamination. 10
    11. 11. • Cell viability and Cytotoxicity assays are used for drug screening and cytotoxicity tests of chemicals. • This in vitro test evaluates the potential of the materials or their extracts to cause damage to cells in culture.(useful in evaluating the toxicity or irritancy potential of materials and chemicals. ) • Cell cultures are suitable test systems for the determination of cytotoxic reactions such as changes in cell cycle, inhibition of cell division, and cell death. (caused by eluates or extracts of products either natural or processed.) ISO 10993-5:2009 Part 5: Tests for in vitro cytotoxicity 11
    12. 12. Cytotoxicity is evaluated by qualitative and quantitative means. Qualitative Quantitative A. Qualitative Cytotoxicity Tests Three different qualitative cytotoxicity tests are commonly used : 1. Extraction method/ MEM Elution 2. Agar Diffusion or Agarose overlay assay 3. Direct contact method In general, in these tests, toxicity is verified after a period of exposure (typically 24–72 hours) of the cells to the extract or device. 12
    13. 13. 1. MEM Elution - Test on Extracts (ISO 10993-5) • The test material is extracted for 24 hours in Minimum Essential Medium (MEM). • An extract is prepared from the test material which is then placed over the cultured cells. (L-929 mouse fibroblast cells) • Following incubation, the cells are examined microscopically for morphological changes, degeneration and lysis of the cells. • Potentially cytotoxic substances are uniformly distributed throughout the cell culture cell line L929 13
    14. 14. 14
    15. 15. 2. Agar Diffusion or Agarose overlay assay - (ISO 10993-5) • In this method, a thin layer of nutrient-supplemented agar is placed over the cultured cells.(L-929 mouse fibroblast cells ) • The test material (or an extract of the test material dried on filter paper/sample) is placed on top of the agar layer, and the cells are incubated for 24 hours. • Cytotoxic leachates diffuse into the cell layer via the agar, and a zone of malformed, degenerative or lysed cells under and around the test material indicates cytotoxicity. • MRC-5 Human Embryonic Lung Cells Material Agar layer Agar overlay: positive control 15
    16. 16. 3. Direct Contact (ISO 10993-5) • In this method, a piece of test material is placed directly onto cells growing on culture medium.(without the agar layer) • Cell cultures are grown to a standard monolayer. • The cells are then incubated for 24 hours at 37 C. • During incubation, leachable chemicals in the test material can diffuse into the culture medium and contact the cell layer. • Subsequently, the monolayers are examined microscopically for the presence of morphological changes, reduction in cell density or lysis of cells around the test material. 16
    17. 17. B. Quantitative cytotoxic assays: • Evaluation of cell number and viability by measuring of metabolic activity, membrane permeability. MTT assay Neutral Red assay Calcein assay LDH assay 17
    18. 18. 1. MTT assay:- Mitochondrial activity [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenylformazan bromide ] • MTT assay is used often in determining cell viability (detects viable cells). • The MTT is a colorimetric method that measures the reduction of yellow 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide into an insoluble purple formazan product by mitochondrial succinate dehydrogenase. • MTT being water soluble can penetrate through cell membrane, water insoluble formazan is trapped inside the cell. • Dead cells do not have active mitochondrial reductases (as the cellular reduction is only catalyzed by living cells), MTT is not reduced and the purple formazan is not formed. 18
    19. 19. Calcein – AM (non – fluorescent compound) Water soluble (Colourless substrate) Esterases Calcein Nucleus Viable cell NADH,NADH Dehydrogenase Mitochondrial Miochondria succinate dehydrogenase. Formazan dye MTT Formazan dyes MTT 19
    20. 20. • Samples are read using an ELISA plate reader at a wavelength of 570 nm. • The amount of color produced is directly proportional to the number of viable cells. • The MTT can be used to evaluate the cytotoxicity of: ‐ ‐ ‐ ‐ Toxic compounds Toxins and environmental pollutants Potential anti-cancer drugs Antibodies to examine growth inhibiting potential 20
    21. 21. 2. Calcein assay :- • The acetomethoxy derivate of calcein (calcein AM) is used in testing of cell viability as it can be transported through the cellular membrane into live cells. • After transport into the cells, intracellular esterases remove the acetomethoxy group, the molecule gets trapped inside and gives out strong green fluorescence. • As dead cells lack active esterases, only live cells are labeled and counted by flow cytometry. 21
    22. 22. Calcein – AM (non – fluorescent compound) Water soluble (Colourless substrate) Esterase Calcein Nucleus Viable cell NADH,NADH Dehydrogenase Miochondria Formazan dye MTT Formazan dyes MTT 22
    23. 23. 3. Neutral Red (NR) assay:- Lysosomal activity - Membrane permiability • The neutral red (NR) assay is a cell survival chemo sensitivity assay. • This assay is based on the incorporation of NR into the lysosomes of viable cells after being incubation with test agents. • NR (3-amino-7-dimethyl-2-methylphenazine hydrochloride) is a weak cationic dye that readily penetrates cell membranes by nonionic diffusion, accumulating intracelluarly in lysosomes, where it binds with anionic sites in the lysosmal matrix. 23
    24. 24. • Therefore, it is possible to distinguish between viable, damaged or dead cells as viable cells take up the NR dye, damaged or dead cells do not. • When a reduced cytotoxic effect was determined by neutral red (NR-test), which shows the activity of lysosomal enzymes, microscopically multiplication or enlargement of lysosomes was observed. 24
    25. 25. 4. Lactate dehydrogenase (LDH) leakage assay : (membrane integrity) • Lactate dehydrogenase (LDH), which is a soluble cytosolic enzyme present in most eukaryotic cells, releases into culture medium upon cell death due to damage of plasma membrane. • The increase of the LDH activity in culture supernatant is proportional to the number of lysed cells. • LDH activity, therefore, can be used as an indicator of cell membrane integrity and serves as a general means to assess cytotoxicity resulting from chemical compounds or environmental toxic factors. 25
    26. 26. • LDH Assay measures LDH activity present in the culture medium using a coupled two-step reaction. • In the first step, LDH catalyzes the reduction of NAD+ to NADH and H+ by oxidation of lactate to pyruvate. • In the second step of the reaction, diaphorase (NADPH dehydrogenase) uses the newly-formed NADH and H+ to catalyze the reduction of a tetrazolium salt to highly-colored formazan which absorbs strongly at 490-520 nm. 26
    27. 27. Calcein – AM (non – fluorescent compound) Water soluble tetrazolium (Colourless substrate) Esterase Calcein Nucleus Viable cell NADH,NADH Dehydrogenase Miochondria Formazan dye MTT Formazan dyes MTT 27
    28. 28. • Currently, pharmaceutical firms spend a large amount of money on the compound efficacy and cytotoxicity test. • There is still a 78% failure rate for all drugs, which may be devastating to developing companies. • Effective compounds in vitro may be non-effective in vivo for many reasons, including differences between in vitro and in vivo target biology, interrelated biochemical mechanism, metabolism, poor penetration into solid tissues, etc. 28
    29. 29. • Currently, almost all cell-based assays or biosensors are developed in 2-D culture systems, although conventional 2-D cultures usually suffer from contact inhibition and a loss of native cell morphology and functionality. • In comparison with 2-D cultures, 3-D cell models create a more realistic representation of real human tissues, which is critical to many important cell functions, including morphogenesis, cell metabolism, gene expression, differentiation and cell-cell interactions. 29
    30. 30. • 3D cell culture models can provide a platform for high throughput and systematic experimentation, reducing the need for animals, and permitting a more straightforward understanding of cause and effect in drug safety and efficacy studies. • Fibrous materials are used, such as non-woven polyethylene terephthalate (PET), to maintain tissue native morphology, because they can provide high specific surface areas, mechanical properties and void volumes. 30
    31. 31. • In studying cytotoxicity and drug testing, maintaining cells in their native functional state in a proper 3-D environment would improve predictions and have the potential to reduce clinical trial failures. • Therefore, although designing 3-D models is much more complicated than designing the 2-D counterparts, cell- and tissue-based assays with a 3-D model are superior and are the assays of choice for HTS (High-throughput screening) of drug cytotoxicity. 31
    32. 32. 32