Cytotoxicity

1. What is Cytotoxicity?

             Cytotoxicity is the quality of being toxic to cells. Examples of toxic agents are an immune cell or some types of venom, e.g. from the puff adder (Bitis arietans) or brown recluse spider (Loxosceles reclusa).

             Cytotoxicity is the quality of being toxic to cells. Cells exposed to a cytotoxic compound can respond in a number of ways. The cells may undergo necrosis, in which they lose membrane integrity and die rapidly as a result of cell lysis; they can stop growing and dividing; or they can activate a genetic program of controlled cell death, termed apoptosis.

MEASURING CYTOTOXICITY 

 

               Cytotoxicity assays are widely used by the pharmaceutical industry to screen for cytotoxicity in compound libraries. Researchers can either look for cytotoxic compounds, if they are interested in developing a therapeutic that targets rapidly dividing cancer cells, for instance; or they can screen "hits" from initial high-throughput drug screens for unwanted cytotoxic effects before investing in their development as a pharmaceutical.

              Assessing cell membrane integrity is one of the most common ways to measure cell viability and cytotoxic effects. Compounds that have cytotoxic effects often compromise cell membrane integrity. Vital dyes, such as trypan blue or propidium iodide are normally excluded from the inside of healthy cells; however, if the cell membrane has been compromised, they freely cross the membrane and stain intracellular components.Alternatively, membrane integrity can be assessed by monitoring the passage of substances that are normally sequestered inside cells to the outside. One molecule, lactate dehydrogenase (LDH), is commonly measured using LDH assay. Protease biomarkers have been identified that allow researchers to measure relative numbers of live and dead cells within the same cell population. The live-cell protease is only active in cells that have a healthy cell membrane, and loses activity once the cell is compromised and the protease is exposed to the external environment. The dead-cell protease cannot cross the cell membrane, and can only be measured in culture media after cells have lost their membrane integrity.

                Cytotoxicity can also be monitored using the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) or MTS assay. This assay measures the reducing potential of the cell using a colorimetric reaction. Viable cells will reduce the MTS reagent to a colored formazan product. A similar redox-based assay has also been developed using the fluorescent dye, resazurin. In addition to using dyes to indicate the redox potential of cells in order to monitor their viability, researchers have developed assays that use ATPcontent as a marker of viability.Such ATP-based assays include bioluminescent assays in which ATP is the limiting reagent for the luciferase reaction.

             Cytotoxicity can also be measured by the sulforhodamine B (SRB) assay, WST assay and clonogenic assay.

             A label-free approach to follow the cytotoxic response of adherent animal cells in real-time is based on electric impedance measurements when the cells are grown on gold-film electrodes. This technology is referred to as electric cell-substrate impedance sensing (ECIS). Label-free real-time techniques provide the kinetics of the cytotoxic response rather than just a snapshot like many colorimetric endpoint assays.

  

1. What is Atomic absorption spectroscopy (AAS) ?
               
                Atomic absorption spectroscopy (AAS) is a spectroanalytical procedure for the quantitative determination of chemical elements using the absorption of optical radiation (light) by free atoms in the gaseous state.
                In analytical chemistry the technique is used for determining the concentration of a particular element (the analyte) in a sample to be analyzed. AAS can be used to determine over 70 different elements in solution or directly in solid samples used in pharmacology, biophysics and toxicology research.
Atomic absorption spectroscopy was first used as an analytical technique, and the underlying principles were established in the second half of the 19th century by Robert Wilhelm Bunsen and Gustav Robert Kirchhoff, both professors at the University of Heidelberg, Germany.
              Atomic absorption spectrometry has many uses in different areas of chemistry such as:

• Clinical analysis: Analyzing metals in biological fluids and tissues such as whole blood, plasma, urine, saliva, brain tissue, liver, muscle tissue, semen
• Pharmaceuticals: In some pharmaceutical manufacturing processes, minute quantities of a catalyst that remain in the final drug product
• Water analysis: Analyzing water for its metal content.                                                                          How it works
  Atoms of different elements absorb characteristic wavelengths of light. Analysing a sample to see if it contains a particular element means using light from that element. For example with lead, a lamp
  containing lead emits light from excited lead atoms that produce the right mix of wavelengths to be
  absorbed by any lead atoms from the sample. In AAS, the sample is atomised – ie converted into
  ground state free atoms in the vapour state – and a beam of electromagnetic radiation emitted from
  excited lead atoms is passed through the vaporised sample. Some of the radiation is absorbed by the lead atoms in the sample. The greater the number of between 1 Nm–2 and 5 Nm–2. The ionisation of some gas atoms occurs by applying a potential difference of about 300–400 V between the anode and the cathode. These gaseous ions bombard the cathode and eject metal atoms from the cathode in a process called sputtering. Some sputtered atoms are in excited states and emit radiation characteristic of the metal as they fall back to the ground state – eg Pb* → Pb + h (Fig. 2). The shape of the cathode concentrates the radiation into a beam which passes through a quartz window, and the shape of the lamp is such that most of the sputtered atoms are redeposited on the cathode.
   

  Atomic Absorption Theory

  Atomic absorption spectroscopy relies on the Beer-Lambert law to determine the concentration of a particular analyte in a sample. The absorption spectrum and molar absorbance of the desired sample element are known, a known amount of energy is passed through the atomized sample, and by then measuring the quantity of light it is possible to determine the concentration of the element being measured. Aurora’s TRACE AI1200 and TRACE 1300 Atomic Absorption Spectrometers are available with flame, graphite furnace and vapor hydride generation atomizers. These atomizers aspirate the sample into the light path where it is illuminated by a hollow-cathode lamp (HCL), which emits light at the wavelength characteristic of the desired elements. A built-in detector measures the light emissions both in presence and absence of sample, and the ratio of the absorbances are used to determine the analyte concentration.