M7 In Vitro Toxicity Assays

Cellular Cytotoxicity Assays

• Assess the effect of the lead compound on:

  • Cell viability

  • Cell growth

  • Apoptosis

  • Oxidative stress

  • Drug-drug interaction

Measuring Cellular Toxicity

• Cellular toxicity is measured by assessing the effect of lead compounds on:

  • Cell viability

  • Cell growth and survival

  • Apoptosis

  • Oxidative stress

• Can be assessed in organ-specific systems (one or multiple cell types from the same organ) or multi-organ specific tests (cells from multiple organs).

• Outcomes determine the effect of the lead compound on one or multiple organs.

Cell Viability Assay (ATP Assay)

• Involves the use of ATP and luciferin (substrate for luciferase).

• Measures the effect of the lead compound on cell viability/survival.

• Procedure:

  • Liver cells (hepatocytes), kidney tubule cells, or lung epithelial cells are treated with the lead compound.

  • Cells are incubated with ATP, luciferin, and luciferase (or cells synthesize ATP).

  • Luciferin is converted to a light-emitting molecule by luciferase in the presence of ATP.

  • The emitted light is captured on a microplate.

  • The light is directly proportional to the number of viable cells.

MTT Assay (Cell Growth and Survival)

• Measures the effect of a lead compound on cell growth and survival.

• Procedure:

  • Cells are incubated with MTT substrate.

  • Living cells generate reducing factors such as NADH.

  • NADH oxidizes, and MTT is reduced to Formazan (purple color).

  • The color is measured; color intensity is directly proportional to cell growth and survival.

• Both the ATP and MTT assays can be adapted for high-throughput screening.

Programmed Cell Death (Apoptosis) Assay

• Measures the level of pro-apoptotic proteins (caspases).

• During apoptosis, caspase levels increase, especially caspase 8 and caspase 3 (terminal caspases).

• The level of apoptotic proteins is directly proportional to the degree of cell death programming.

• This assay can be adapted for high-throughput screening.

Oxidative Stress Assay

• Measures the effect of a lead compound on oxidative stress in cells.

• Oxidative Stress:

  • Refers to the balance between oxidative and antioxidative capacity of the cell.

  • Oxidative stress occurs when the oxidative capacity exceeds the antioxidative capacity.

  • Characterized by the presence of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), peroxyl radical, superoxide anion, etc.

  • ROS can cause oxidative damage to lipids, proteins, and DNA, leading to cell death.

• The cell uses glutathione as an antioxidant to counteract oxidative stress.

  • Glutathione helps convert hydrogen peroxide to water using glutathione peroxidase.

  • With glutathione reductase, it prevents peroxyl radicals from causing oxidative damage.

• Measuring Glutathione Levels:

  • Uses an oxidized substrate (DTNB).

  • Glutathione reduces DTNB to its reduced form.

  • The reduced form of DTNB has a yellow color, which is measured in a microplate reader.

  • Yellow coloration is proportional to glutathione levels, indicating the oxidative stress condition within the cells.

• This assay can be adapted for high-throughput screening.

Drug-Drug Interaction

• Measures the effect of a lead compound on drug-drug interactions in liver hepatocytes or microsomes.

• Involves CYP450 isoforms (enzymes in phase one metabolism of drugs).

  • Phase one metabolism involves oxidation, reduction, and hydrolysis to make compounds more hydrophilic for excretion through the kidneys.

• Procedure:

  • Microsomes containing the CYP450 enzyme system are incubated with/without the lead compound.

  • Known substrates of different CYP450 isoforms are added.

  • If metabolites are increased or reduced in the presence of the lead compound, it indicates drug-drug interaction.

  • The lead compound either induces or reduces the metabolism of other drugs.

  • Inducing metabolism: reduces the drug's effect.

  • Reducing metabolism: increases drug toxicity.