5. 3R's and In Vitro Toxicology Flashcards
What is in vivo toxicology?
Using animal biology to test if something is toxic or not.
Test for acute or chronic toxicity.
How is toxicity evaluated in in Vivo toxicology? (what type of endpoints)
Apical endpoints: need the whole animal/organism to measure these endpoints
Ex: death, breeding behaviours, reproductive failure
What are some key challenges of in vivo toxicology?
High doses Low throughput Expensive Time consuming Large number of animals needed Little focus on mode of action
What is in vitro toxicology?
- word meaning: in the glass
- in practice: doing a study outside normal biological context
What are 3 examples of in vitro toxicology?
- Explant culture: in biology, explant culture is a technique to organotypically culture cells from a piece or pieces of tissue or organ removed from a plant or animal. The term explant can be applied to samples obtained from any part of the organism.
- Organ slice
- Cell lines: Immortalized cell lines originally came from a tissue and were adjusted so they could grow for a very long time under certain conditions. It’s beneficial because we can do studies on human derived tissue samples.
What were the drivers of in vitro toxicology?
- Societal concerns
- Ethics (is it okay?)
- Knowledge
- Speed
- Efficiency
- Money
What is important about the SPCA in relation to in Vitro toxicology?
The SPCA was established in the UK in 1824 and it represents the fact that people were starting to become more conscious about having respect towards animals and brought awareness to animal cruelty. It brought about ethical implications involving animals.
Name Hall’s principles of ethical animal experimentation? What are they for?
Hall proposed 5 principles that he thought should govern animal experimentation. Before this, there was no documentation for how animal experiments should be performed.
- Experiment should not be done if the information can be obtained by observation (ex: observing something in nature)
- Experiment must have a clearly defined and obtainable objective
- Prevent duplication (If the experiment has already been done, and conclusions were rigorously drawn, don’t do it again)
- Experiments should be done with least infliction of suffering and use of least sentient animals
- Do experiment to provide clearest possible results (no ambiguity)
Relating to Hall’s principles, what is a less sentient species? Explain it and give examples.
To be sentient means to experience emotions such as fear or pain. According to the scientific community, beings that are not sentient have no centralized nervous system. This includes: Bacteria, archaea, protists, fungi, plants, and certain animals (e.g. sponges). Therefore, “keyless sentient” means species lower on the phylogenetic tree.
Why do we do a lot of experiments on invertebrates?
Because they are low on the phylogenetic tree.
Who proposed the 3R’s and what are they? What’s the title?
Russell and Burch Proposed the 3 R’s and called it “the principles of humane experimental technique”:
- Refinement
- Reduction
- Replacement
What big event affected cosmetic companies and their testing methods?
Society started to become more against animal testing. A man named Henry Spira started a campaign against cosmetic companies for testing on animals. A very famous picture in the new york times depicts a rabbit getting its eyes tested on for the sake of cosmetics with the title “How many Rabbits does Revlon blind for beauty’s sake?”.
What did Johns Hopkins university do to contribute to the key events in in Vitro toxicology?
They developed the centre for alternatives to animal testing. This was a way to combine all of the different tests (in vitro or specialized tests) to evaluate compounds and their risks without the use of animals.
What is the health research extension act? How did it contribute to in vitro toxicology?
The health research extension act requires a certain protocol in order to do research on animals. This protocol is to ensure that you are working on animals ethically and it makes you have to justify the use of every animal. It has to get approved before you can proceed.
US institutions that use animals must have an Institutional Animal Care and Use Committee (IACUC)
In Canada it has to go through the CCAC (CCPA in french)
What 2 centers were created with the aim to validate alternative testing measures?
- ECVAM (European Centre for the Validation of Alternative Methods)
- ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods)
These centres group the different types of toxicity testing methods together AND validate the methods so we know certain in vitro methods are good at detecting toxicity and that they are good alternative tests to use.
Which event is considered a key event for in vitro toxicology?
A. Hall launched a campaign against cosmetic companies
B. Henri Spira proposed 5 principles of ethical animal experimentation
C. Creation of OECD
D. Russell and Burch Proposed the 3Rs
D. Russell and Burch Proposed the 3Rs
Define the 3R’s of Russell and Burch’s Principles of Humane Experimental Technique.
- Refine experimental techniques to lessen pain or distress
- Reduce the number of animals necessary for a particular test
- Replace animals with other models
What can be done to satisfy the refinement category of the 3 R’s? Give examples of methods AND tests.
Modification of a procedure that decreases potential pain or distress OR Procedure that uses animals lower in a phylogenetic category.
Examples of methods are:
- Anaesthesia, Analgesics, sedatives
- Non-invasive techniques
- Habituate animals to study procedure
- Careful choice of animal model
- Modern imaging methods
Examples of tests are:
i. Mouse local lymph node assay (LLNA) – review from in vivo toxicology
ii. Transgenic animals
iii. Non-invasive monitoring
What old test is the Mouse local lymph node assay (LLNA) an alternative for? Describe the old method.
The Guinea pig maximization test. It was used to screen for substances that cause human skin sensitization (contact allergy).
The method:
1. Intradermal injection of substance + immunological adjuvant. In this step, the adjuvant is used to stimulate the immune system to ensure the animal can develop an allergy to it.
2. Animal is exposed to lower concentration and tested for allergic reaction. If they have developed an adverse response, they will visibly get skin irritations.
Describe the alternative test for the guinea pig maximization test.
The Mouse local lymph node assay (LLNA).
Scientific principle of test: sensitizers induce growth of lymphocytes
Lymphocyte proliferation can be measured by radiolabeling (quantifying tritiaded thymidine) or other methods.
This method allows you to measure the immune response directly without having to measure the apical effect of skin irritation.
What are the advantages of LLNA compared to the guinea pig maximization test?
- It takes less time to perform: minimizes pain and stress animal has to go through. (REFINE)
- It requires less animals: this applies to the “reduction” part of the 3 R’s
- There is no adjuvant required so there are less problems for the animal to go through. (REFINE)
What are the different types of transgenic animals? What are the pros and cons?
Types:
- Gene knockdown/specific mutations
- Knock in (human gene; reporter gene)
Pros:
- Specific target yields decrease in number of animals needed
- Decreases necessity for companion species
Cons:
Most transgenics require additional care because they’re more sensitive.
What are transgenic mice often used for? Give examples of transgenes used and compare it to regular mice. Which of the 3 R’s do they satisfy?
Transgenic animals are often used for carcinogenicity testing.
Original test: Using a lab strain of black mice called black6: the mice would need a duration of exposure of 24 months, you would need 200 mice (need a lot to compensate for the fact that 24 months is enough time to get spontaneous mutations), and there are spontaneous tumours that can form.
- Using a P53 (tumour suppressor gene) heterozygous knockdown mouse (+/-): the gene is mutated so that the mouse can’t suppress tumours as well. Therefore if it is exposed to a carcinogen, we should see a tumour much faster. Needs duration of exposure of 6 months, requires 60 animals, no spontaneous tumours are formed.
- Adding in a Ha-ras transgene (oncogene) so it behaves as a genetically initiated model for skin carcinogenesis. This oncogene is more likely to cause skin cancer.
The mouse is already initiated so if there is a carcinogen you will see signs of cancer very fast. Needs duration of exposure of 6 months, requires 60 animals, no spontaneous tumours are formed.
1&2 are examples of refining and reducing!
Refine: decrease duration of exposure therefore less pain and stress
Reduce: decrease number of animals
What are types of non-invasive monitoring (refinement category)?
- Computer Assisted Tomography (CT) - anatomic information (CAT scans):
- 3D X-ray technique
- Contrast generated by difference in tissue
absorption
- 3D reconstruction and computer analysis
- 3D images with a resolution of 100 x 100 x 100 microns obtained in a few minutes
- You can see whats happening inside without causing added stress to the animals - Positron Emission Tomography (PET) - metabolic information:
-gamma rays emitted by a positron-emitting radionuclide
(commonly fluorine-18 -> can be picked up by gamma radiation)
- introduced into the body on a biologically active molecule (usually fludeoxyglucose (18F)) -> Modify glucose with the fluorine atom and see how it is metabolized in tissues over time - Transgenic animals for fluorescent imaging
- Engineering the animals to express fluorescent fusion proteins
- Image a protein’s distribution, dynamics
- EGFP = enhanced green fluorescent protein - Magnetic resonance imaging (MRI)
- Ultrasound imaging
