3Rs and in vitro toxicology Flashcards
Describe replacement
Replace them with non-sentient alternatives
Limitations of animal testing methods
30% of effects are unique to humans
Humans will have different metabolic profiles and immune responses + anatomical differences.
Identifying toxic effects can be difficult, and trial clinicians need to know what the effects look like.
Some drugs are toxic in their therapeutic range (e.g., chemo, digoxin)
What are the 3Rs
Replacement, reduce, refine
Bringing about the 3Rs is done by the national centre 3Rs (NC3R’s)
Describe reduction and methods to do so
reduce to a minimum the number of animals required. Can be done by imaging for longitudinal studies, sharing data and resources, and improving experimental design and analysis
Describe refinement and methods to do so
refine the experiments to reduce the pain and distress. Done by improving housing and husbandry, while also incorporating non-invasive techniques. Also improves the data - stress can affect results. Finding better biomarkers can also be used for early toxicity testing, e.g., gene expression allaying RNA gene chip
This will generate a heat map that identifies alterations to gene expression that can indicate before side effects are noticeable the toxicity that will arise.
Ways that in vitro tests still utilise animals
Antibodies and nano bodies are derived from animals
cell cultures use foetal calf serum
Cell lines will still include human or animal cells.
What is partial replacement
Utilisation of animals that are not considered capable of experiencing suffering.
Can also include embryos or foetus.
Description of the Ames test
An in vitro test for the mutagenicity of substances.
uses salmonella with mutations for histidine synthesis (while they are dependent on histidine for growth).
When the salmonella is able to grow in histidine-free media it indicates that the test substance is mutagenic.
the assay can also be modified to include a microsomal activation system to account for metabolism in the activation of many mutagenic compounds.
This does fail to identify non-genotoxic carcinogens however.
Advantages and limitations of cell cultures
it is simple to set up a screening assay.
In these systems, the secondary messenger coupling is not necessarily the same as in the native receptor system.
Hepatocyte in vitro tests are limited by their non-proliferative nature, and the degeneration of CYP450 enzymes. Furthermore, the hepatocytes tend to undergo a metabolic switch (warburg effect), where they utilise glycolysis - lactate build up, which can effect the results.
Way to overcome hepatocyte in vitro issues
Addition of antioxidants reduce the degeneration of CYP450 enzymes, while also decreasing lactate synthesis. The mitochondrial membrane potential also increases.
Indicates the Warburg effect can be partially reversed.
Using a collagen EC matrix can also enhance the hepatocyte cell culturing.
Co-culturing of hepatocytes with endothelial and kupffer cells also enhances functionality.
Nervous tissue cell culturing techniques
New methods have arisen enabling sensory nerve culturing from dorsal root ganglion cells (from spine). Stem cells also offer this potential.
Limitations and future prospects of in vitro toxicology
In cell cultures, normally only one cell type is represented, whereas toxicological responses normally involve multiple cell types.
Toxicity also often occurs in multiple organs, and this is difficult to mirror in vitro. Furthermore, organ specific function (and its loss) is difficult to develop in vitro.
3D culture systems have improved modelling organ-specific function in vitro, and many cell types can be cultured together.
Stem cell technologies offers a wide range of future possibilities in in vitro research.
