5. 3R's and In Vitro Toxicology

Question 1

What is in vivo toxicology?

Answer 1

Using animal biology to test if something is toxic or not.

Test for acute or chronic toxicity.

Question 2

How is toxicity evaluated in in Vivo toxicology? (what type of endpoints)

Answer 2

Apical endpoints: need the whole animal/organism to measure these endpoints
Ex: death, breeding behaviours, reproductive failure

Question 3

What are some key challenges of in vivo toxicology?

Answer 3

High doses
Low throughput
Expensive
Time consuming
Large number of animals needed
Little focus on mode of action

Question 4

What is in vitro toxicology?

Answer 4

• word meaning: in the glass

- in practice: doing a study outside normal biological context

Question 5

What are 3 examples of in vitro toxicology?

Answer 5

1. 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.
2. Organ slice
3. 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.

Question 6

What were the drivers of in vitro toxicology?

Answer 6

1. Societal concerns
2. Ethics (is it okay?)
3. Knowledge
4. Speed
5. Efficiency
6. Money

Question 7

What is important about the SPCA in relation to in Vitro toxicology?

Answer 7

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.

Question 8

Name Hall’s principles of ethical animal experimentation? What are they for?

Answer 8

Hall proposed 5 principles that he thought should govern animal experimentation. Before this, there was no documentation for how animal experiments should be performed.

1. Experiment should not be done if the information can be obtained by observation (ex: observing something in nature)
2. Experiment must have a clearly defined and obtainable objective
3. Prevent duplication (If the experiment has already been done, and conclusions were rigorously drawn, don’t do it again)
4. Experiments should be done with least infliction of suffering and use of least sentient animals
5. Do experiment to provide clearest possible results (no ambiguity)

Question 9

Relating to Hall’s principles, what is a less sentient species? Explain it and give examples.

Answer 9

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.

Question 10

Why do we do a lot of experiments on invertebrates?

Answer 10

Because they are low on the phylogenetic tree.

Question 11

Who proposed the 3R’s and what are they? What’s the title?

Answer 11

Russell and Burch Proposed the 3 R’s and called it “the principles of humane experimental technique”:

1. Refinement
2. Reduction
3. Replacement

Question 12

What big event affected cosmetic companies and their testing methods?

Answer 12

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?”.

Question 13

What did Johns Hopkins university do to contribute to the key events in in Vitro toxicology?

Answer 13

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.

Question 14

What is the health research extension act? How did it contribute to in vitro toxicology?

Answer 14

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)

Question 15

What 2 centers were created with the aim to validate alternative testing measures?

Answer 15

1. ECVAM (European Centre for the Validation of Alternative Methods)
2. 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.

Question 16

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

Answer 16

D. Russell and Burch Proposed the 3Rs

Question 17

Define the 3R’s of Russell and Burch’s Principles of Humane Experimental Technique.

Answer 17

1. Refine experimental techniques to lessen pain or distress
2. Reduce the number of animals necessary for a particular test
3. Replace animals with other models

Question 18

What can be done to satisfy the refinement category of the 3 R’s? Give examples of methods AND tests.

Answer 18

Modification of a procedure that decreases potential pain or distress OR Procedure that uses animals lower in a phylogenetic category.

Examples of methods are:

1. Anaesthesia, Analgesics, sedatives
2. Non-invasive techniques
3. Habituate animals to study procedure
4. Careful choice of animal model
5. 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

Question 19

What old test is the Mouse local lymph node assay (LLNA) an alternative for? Describe the old method.

Answer 19

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.

Question 20

Describe the alternative test for the guinea pig maximization test.

Answer 20

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.

Question 21

What are the advantages of LLNA compared to the guinea pig maximization test?

Answer 21

1. It takes less time to perform: minimizes pain and stress animal has to go through. (REFINE)
2. It requires less animals: this applies to the “reduction” part of the 3 R’s
3. There is no adjuvant required so there are less problems for the animal to go through. (REFINE)

Question 22

What are the different types of transgenic animals? What are the pros and cons?

Answer 22

Types:

1. Gene knockdown/specific mutations
2. Knock in (human gene; reporter gene)

Pros:

1. Specific target yields decrease in number of animals needed
2. Decreases necessity for companion species

Cons:
Most transgenics require additional care because they’re more sensitive.

Question 23

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?

Answer 23

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.

1. 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.
2. 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

Question 24

What are types of non-invasive monitoring (refinement category)?

Answer 24

1. 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
2. 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
3. Transgenic animals for fluorescent imaging
   - Engineering the animals to express fluorescent fusion proteins
   - Image a protein’s distribution, dynamics
   - EGFP = enhanced green fluorescent protein
4. Magnetic resonance imaging (MRI)
5. Ultrasound imaging

Question 25

Give an example of an experiment done using transgenic animals for fluorescent imaging. Which of the 3Rs?

Answer 25

Looking at zebrafish to see if there is damage to the lining of capillaries due to MeHg. Tagged GFP to Fli-1 (protein in endothelial cells) so they can see the impact on the capillaries of zebra fish of exposing methyl mercury to them. In the imaging you can see that the endothelial cells are getting destroyed because of the treatment.
Refinement: using less sentient animals (zebrafish) and you don’t have to kill them to see.

Question 26

What can be done to satisfy the Reduction category of the 3 R’s?

Answer 26

Use the fewest animals appropriate to provide valid, statistically significant information

• Careful experimental design
• Appropriate statistical analysis
• Use existing published data (don’t duplicate for no reason)
• Pilot studies (smaller studies)
• In animal protocols you must justify the number of animals used

Question 27

Name and explain an alternative assay that can be used to satisfy the reduction condition.

Answer 27

The assay is called “Murine Limb Bud”. It is a limb bud culture that isolates the fore limbs in a day 12 embryo of a pregnant mouse in order to measure their ability to grow. In this assay, one mouse embryo has more than 1 fore limb so you need to use less mice. Once the fore limbs are isolated, they are cultured for 3 days with the substance being tested and then for 3 more days without it. Finally, you fix the limbs, stain them, and then score the differentiation.

Question 28

What can be done to satisfy the Replacement category of the 3 R’s?

Answer 28

Animals should be used only if the researcher’s best efforts to find an alternative have failed

• Non-animal models (in vitro, etc.)
• Less sentient species (e.g., invertebrates)
• Computer modelling
• In animal protocols you must justify the animal species

Question 29

What are examples of replacement alternatives?

Answer 29

Replacement alternatives:

1. Skin irritancy tests (in vitro)
2. Stem cells

Question 30

What is the in vitro skin irritancy test?

Answer 30

It is a replacement alternative because you don’t need animals at all. It uses layers of reconstructed skin (either EpiSkin or SkinEthic) that you can add your product on and then incubate it at 37 ˚C. After incubation you can measure cytotoxicity, look at histology (imaging to see skin damage), and see the release of cells such as cytokines to see if there is an immune response ex: inflammation response. You can also perform multiple endpoint analysis, for example you can check for tissue viability.

Question 31

What replacement alternative in vitro skin was validated? Why? Who validated it?

Answer 31

EpiSkin

• Reproducible (within and among laboratories)
• Distinguishes corrosive and non-corrosive chemicals
• ECVAM concluded that the EpiSkin is scientifically validated as a replacement for the animal test to distinguish chemicals

Question 32

What are stem cells?

Answer 32

Stem cells are immature cells that eventually get programmed into all of the different cells that we have in our bodies. They are the non differentiated cells.

Question 33

What are the benefits of using stem cells as a replacement alternative. When was it validated and by who?

Answer 33

• They can be used to study embryonic and developmental toxicity
• Allows access to all cell types which may be difficult to obtain (and therefore study functional toxicity, reproductive toxicity, cytotoxicity)
  In 2008: European Center for the Validation of Alternative Methods (ECVAM) successfully validated stem cells.

Question 34

What are the 2 different ways to derive stem cells?

Answer 34

1. Embryonic stem cells (ESC): isolation of stem cells from an animal embryo
2. Induced pluripotent stem cells (iPSC): Take stem cells that are already differentiated (ex: a somatic cell such as fibroblasts or adipose cells) and reprogram them to go backwards until they are un-differentiated=back to stem cells.

Question 35

What results in a 1994 study showed that stem cells were important for testing developmental toxicity?

Answer 35

The IC50 (the inhibitory concentration in 50%) of 25 teratogens was lower in stem cells that were differentiating (developing from stem cells into a different cell type) vs proliferating cells (just dividing cells). This shows that differentiating stem cells are more sensitive to teratogens.

Question 36

Which assays are alternatives that help achieve “reduction”?
A. MRI, Guinea pig maximization test, PET,
B. LLNA, limb bud assay, Guinea pig maximization test, PET
C. limb bud assay, transgenic animals, LLNA
D. limb bud assay, EpiSkin, transgenic animals
E. LLNA, limb bud assay, iPSCs

Answer 36

C. limb bud assay, transgenic animals, LLNA

Question 37

What are advantages of in vitro toxicology?

Answer 37

• Controlled testing conditions
• Reduction of variability between experiments
• Reduction of systemic effects
• Testing is fast and cheap
• Time-dependent studies can be done & samples taken
• Same dose range can be tested in many test systems
• Very small amount of test material is required
• Limited amount of toxic waste is produced
• Human cells and tissues can be used
• Transgenic cells carrying human genes can be easily made
• Reduction of testing in animals

Question 38

What are some limitations of in vitro toxicology?

Answer 38

• General side effects cannot be assessed, e.g., weight reduction
• Systemic effects cannot be evaluated
• Interactions between tissues/organs cannot be tested
• Pharmacokinetic effects cannot be evaluated
• Chronic effects cannot be tested

Question 39

What are some successes of in vitro toxicology?

Answer 39

• The personal care/household good industries minimally use animals
• In the last 20 years, almost every Nobel Prize winner used in vitro methods and alternative approaches (i.e., these methods are mainstream)
• NCI (National Cancer Institute) 􏰁 screening for cancer drugs is done in vitro
• Several alternative methods formally validated

Question 40

How is toxicity evaluated in IN VIVO toxicology? (what type of endpoints)

Answer 40

Apical and pathology endpoints (e.g., death, breeding behaviours, reproductive failure).

Question 41

What endpoints are used for IN VITRO tests?

Answer 41

1. General cytotoxicity (cell death) or cell proliferation:
   - Breakdown of cellular permeability barrier
   - Changes in cell replication (ex: if they are stalled in a cell cycle)
   - Reduced mitochondrial function
   - Changes in protein synthesis
2. Cellular responses:
   - Genotoxicity (DNA damage and mutation)
   - Oxidative stress (ex: measuring an increase in ROS)
   - Heat shock proteins
   - Stress-activated protein kinases

Question 42

What is a challenge we are experiencing in toxicology?

Answer 42

There is an urgent need for faster and better methods of testing because we are pumping out more chemicals than ever and it is very difficult to catch up on looking at the potential harm or risks they pose for humans.

Question 43

What is the goal of toxicology in the 21st century and how will this be achieved?

Answer 43

Goal: To gain a better mechanistic understanding of the toxicity of different chemicals in order to predict what effects similar chemicals might have.
How:
- High throughput screening: Look at many samples very quickly. You have the same cells or genes in each well of a 1536-well plate, in each well you put a different chemical. The robots, laser detectors, and computers then identify which chemicals affect the cell’s function and what function is being affected.
- High-content cellular response (e.g., genomics, proteomics- next class!)
- Organ-on-a-chip: reconstructing parts of each organ on a chip where things flow through different parts. Goal is to connect all of these things together to get an entire human on a chip
- Bioinformatics and machine learning: computationally extrapolating and and predicting

Question 44

What is QSAR?

Answer 44

Quantitative structure-activity relationship models which takes all of the different results and putting them together in a way that we can predict the impact of other chemicals.