Lecture 5 - In Vitro Toxicology Flashcards
What are the disadvantages of animal (in vivo) studies?
- high doses are required, need a lot of chemical
- expensive - cost for one rat is $$$
- low throughput
- time consuming
- large # of animals
- apical endpoints
- ethical issues and issues of accountability; pain and distress
What were the drivers of in vitro tox?
- Societal pressure; animal rights
- Knowledge: our need to know about more chemicals, at a more high throughput pace
- money: costs less, more rapid, easier to get a cell liens than to take care of animals
- Efficiency
- Ethical issues of using animals
What were the key events in in vitro tox?
1824: SPCA, and UK saw its first curelty to animals act
1931: Marshal Hall (physiologist) proposes 5 “rules” that should govern naimal testing
1959: Russel and Burch: The Princple of Humane Experimental Technique –< identified 3 R’s: refine, reduce, replace. This lay the foundation, groundwork for in vitro tox
1980s: henry spira leads campaign for cosmetic industry and use of toxic chemicals on animals
Soon after, led to the creation of Centre for Alternatives to Animal Testing at Johns Hopkins and European Centre for Validation of alternative methods (ECVAM)
-In Canada, no act was adopted but we have some legislation: the Canadian council on animal care credits all places that use animals
What were Marshal Hall’s 5 proposed guidelines on animal experiment?
- NO experiment should be carried out on animals if results can be determined by observation
- Use least sentient beings and minimize suffering
- Experiments ust be done under conditions that provide most accurate/clearest possible results
- Ifi t has been done, dont do it gain
- Need a clearly defined and obtainable objective
Surrogate Responsibility
The debate on whether we should be defending those who cannot defend themselves - and how far down the animal kingdom does this apply?
Ex: appropriate endpoints, minimizing suffering, treating wth care, and decreasing sentient being use ex: used to do studies on CNS with cats ans cvS WITH dogs, rarely done anymore
The 3 R’s
Refine: refine experiment techniques to lessen pain, distress
–> ex 1: guinea pig maximization test –> murine local lymph node assay test (reliable data, faster, # of tumours and age-related diseases is less, duration of exposure is less)
–> ex 2: use of transgenic animals has decreased need for # of animals, allows use of specialized targets
–> ex 3: use of non-invasive monitoring: MRI, PET, 3D X-rays, implantable monitor devices, optical imaging
Reduce: decrease # of animals
–> ex 1: limb bud culture: 1 female rat can birth 18 pups; yields ~70 limbs, then introduce a toxicant and study limb morphology.
–> ex 2: embryo culture system: collect the embryos from a pregnant rat and study the effects of a toxin on organogenesis
Replace: replace animals using in vitro methods such as cell cultures
–> ex 1: pregnancy tests used to be done by injecting rabbits with human urine and seeing if the rabbit mounted an immune response to human chorionic gonadotropin (hCG), today we use monoclonal antibodies against hCG in pregnancy kits (think: expression used to be; the rabbit died)
–> ex 2: in vitro skin irritancy tests have been developed; there are differnet in vitro skin tissue models (bc skin on vaginal epothelum, gingival, oral, etc. Are diff). You can take these models and apply any chemical you are interested in, in the tissue epithelium you wanna study
Pros: highly reproducible and can easily distinguish corrosive vs not corrosive
Cons: cannot distinguish mild or very irritable and cannot reproduce the entire skin but only one portion
Example of irritancy tests that are available: EPISKIN=use of reconstructed human skin model, approved by ECVAM, do not need to use animals
Example of another irritancy test: excised rat skin transcutaneous electrical resistance test
ECVAM concludes both are valid!
The Daize Test (replacement, but
For makeup and eyes - bad
Ecvam created another called the alternative eye irritancy test
1. If the chemical tested is strong acid or base; remove right away (measure pH)
2. If it is OK, try irritanc ytest on in vitro eye model
3. Then do structure-activity relationship - if it resembles a structure of irritant, is thee potential of toxicity
4. If 2 and 3 (structure and skin) are positive (yes), then use Draize test. Do it on oNLY ANIMAl. If it causes a problem, remove. If not, do it on 2 animals. (Today, this isnt even done)
5. If no problem, chemical is OK
Advantages and Limitations of in vitro Tox
Advantages:
- Controlled testing
- Cheaper
- Faster
- Higher throughput
- Reduction of variability between experiments
- Reduction of systemic effects (eliminates possibility of infection o stress in the animal manipulating teh results)
- Very small amount of test material is required
- Limited amount of toxic waste produced
- Human cells and tissues can be used
- Reduction of testing in animals
- Transgenic cells carrying human genes can be made and used
Limitations
- Cannot asses general side effects/off-taregt drug effects pr toxin effects
- Systemic effectsc(whole body) cannot be evaluated
- Cannotassess interactions between tissues/organs
- Cannot evaluate pharmacokinetics
Predictive Toxicology
- 21st century toxicology screening invovles a lot of predictive toxicity testing, using:
- -> mucroplate screenign systems
- -> high throughput screenign methods
- –defined as 100,000+ chemicals per day examining their biological activity, analyze many chemicals at once, try to assess toxicity of REPLACEMENT chemicals
TOX21
-human cell lines for toxicity assessment
–> hepatocytes, neurons, renal proximal tubule cells….
They have been cultured, evaluated using marker genes, and are in use to assess cytotoxicity
There is also concept of “human on a chip”
