The document discusses cytotoxicity, defined as the ability of drugs and chemicals to destroy living cells, influenced by various stimuli and environmental factors. It covers in-vitro assays for testing cytotoxicity, including endpoint assessments, such as dye exclusion and colorimetric assays, along with the study of nanotoxicology, which examines the toxicity of nanomaterials. It highlights the effects of nanomaterials on cellular functions, potential health risks, and the implications for various diseases.

1. CYTOTOXICITY
Shabnam Ameenudeen
M. Tech Biotechnology
Shabnam.jamaliyath@gmail.com

2. Introduction
• Cytotoxicity- ‘cyto’ means cell and ‘toxicity’ means poison.
• The ability of drugs or chemical molecules or mediator cells to
destroy the living cells.
• This is mediated by
i. Chemical stimuli (drugs)
ii. Targeted by other cells (immune cells- NK and T-cells)
iii. Environmental condition- radiation, temperature, etc.

3. Cellular Fates
• Cytotoxicity can result in one of the possible cellular fates
i. Necrosis- it results in rapid loss of membrane integrity and
cell lysis
ii. Apoptosis- slower, orderly and genetically controlled
iii. Cytostasis- cells remain avail but the growth is inhibited

4. In-vitro Assay
• It has helped with the traditional study of whole animal/plant
model toxicity study.
• It is done to exclude cytotoxicity in specific cells or to test for
specialized function.
• They are suitable test systems to determine the cytotoxicity
changes that affects the cell cycle.
• To look for adequate compound/condition for therapeutic
purpose.

5. Possible Cellular Alterations
• Cell morphology- membrane blebbing, vacuolization, etc
• Cell viability- the ability to take up or exclude cells
• Cell growth- cell count, plating efficiency, DNA or protein
content
• Metabolic parameters- O2 consumption, NADH-NAD
conversion and pool of DNA or RNA precursors

6. End- Point Assessment

7. Dye Exclusion
• Simplest and widely used method.
• Dyes like trypan blue, eosin, Congo red are used.
• Live cells exclude dyes dead cells does not exclude them.
• Evaluation of dead/ viable cells are made using hemocytometer.

8. Colorimetric Assay
• MTT assay is the most commonly used.
• It measures biochemical markers.
• Reagents create response to the viable cells.
• Measured using spectrophotometer.
• Determines mitochondrial function of cell.
• Based on conversion of MTT to purple formazan.
• Fluorometric assay is much more sensitive and uses fluorescence
microscope or flow cytometer.

9. Luminometric Assay
• ATP assay is the most sensitive cell viability measurement.
• It is based on luciferase enzyme activity.
• Graph is plotted as luminescent signal
intensity against ATP conc./ Cell number

10. Nanotoxicology
• Nanotoxicology is a branch of bioscience which deals with the
study and application of toxicity of nanomaterials.
• It determines the extent at which the nanomaterial becomes
threat to the environment and human being.
• Nanotoxicity is the effect.
• Smaller the size higher the surface area to volume ratio and
quantum effects possess threat.
• Nanomaterials made of inert materials like gold, silver are also
toxic.

11. Nanomaterials Classification
• Dimensionality- zero, one , two and three dimensions
• Morphology- nanowires, nanospheres, nanotubes, etc
• Composition- single material or composite based
• Uniformity/ Agglomeration state- dispersed and aggregated
• Strongly bound uncontrolled agglomerates influences the cell-
particle interaction.
• It hinders the actual cytotoxicity study.

14. Nano-cytotoxicity
• Aluminium oxide NPs used in fuels and paints alters mitochondrial
function, creates oxidative stress and damages the blood brain barrier.
• It is also associated with genotoxicity.
• Silver NPs are associated with blood diseases and in colon cancer.
• Metallic NPs can easily pass through the circulation and can cause
blood clotting.
• Fe, Cu, Zn NPs are associated with neurodegenerative diseases.
• Iron oxide NPs are targeted against adenocarcinoma cells.

15. Nanoparticle Inhalation
• Small concentration of NPs- distributed to brain, heart and
kidney
• High concentration of NPs- aggregates >100nm gets
phagocytosed
• Very high concentration of NPs- results in lung overload