E2L2 Rodent Models in Toxicology

Question 1

what are the two main reasons to use animal models?

Answer 1

1. predictive use (regulatory)
2. mechanistic studies (academic)

Question 2

what are the three R’s of animal research?

Answer 2

1. replace: use alternatives instead of animals if you can
2. reduce: do enough for your data, but don’t be wasteful
3. refine: minimize pain and suffering

Question 3

what are the three study designs of animal/rodent models?

Answer 3

1. acute tox
2. sub-chronic
3. 2-year chronic bio assay

Question 4

what are acute tox studies?

Answer 4

single dose over a short period of time (1 day) administered in a way that is similar to humans

Question 4

what is the LD50 of Aflatoxin B1 (food contaminant/fungal toxin) in rats administered orally?

Answer 4

0.48 mg/kg

Question 4

what is the LD50 of APAP (acetaminophen) in rats administered orally?

Answer 4

2 g/kg

Question 4

what are acute tox studies used for?

Answer 4

• do the animals die? (does it cause toxicity)
• what doses do they die at?
• what organs seem to be affected?

Question 4

what is the LD50 of Chlorpyrifos (pesticide) in rats administered orally?

Answer 4

200 mg/kg

Question 5

what is the LD50 of TCDD (dioxin) in rats administered orally?

Answer 5

20 µg/kg

Question 6

what is the LD50 of Botulinum toxin in mice administered via IP?

Answer 6

0.02 ng/kg

Question 7

what is LD50?

Answer 7

the dose that causes death in 50% of the participating animals

Question 8

what is the margin of safety?

Answer 8

the ratio between dose that causes toxicity and the dose for efficacy (don’t want the margin of safety to be small..)

Question 9

what can you use the acute tox study results to design?

Answer 9

a sub-chronic study!

Question 10

what is a sub-chronic study?

Answer 10

usually 90 days; 3+ doses with a control group; uses LD50 from the acute tox study

Question 11

what are sub-chronic studies used for?

Answer 11

finding appropriate doses, determine NOAELs and LOAELs, etc.

Question 12

what is a two year chronic bio assay?

Answer 12

a 2-3 year long study to determine whether a drug/chemical increases tumor incidence after long term exposure

• usually done in 2 species so result isn’t just one species

Question 13

what are the three things we look for when doing a two-year chronic bio assay?

Answer 13

1. unique/unusual tumors
2. increased frequency/incidence of tumors
3. do they get the tumors sooner than usual?

Question 14

how much does a 2-year chronic bio assay cost for one compound to be tested?

Answer 14

about a million dollars

Question 15

what are the five NTP levels of evidence?

Answer 15

1. Clear Evidence (CE)
2. Some Evidence (SE)
3. Equivocal Evidence (EE)
4. No Evidence (NE)
5. Inadequate Study (IS)

Question 16

what NTP level of evidence score does TCDD (dioxin) have?

Answer 16

CE Clear Evidence

Question 17

what is sensitivity?

Answer 17

how often do you identify true positives?

Question 18

what is specificity?

Answer 18

how often do you identify true negatives?

Question 19

why are mice so commonly used in animal models?

Answer 19

• about 90-95% of the human genome is shared with mouse genome
• easy to work with, breed well, etc.

Question 20

why use mice as an experimental organism?

Answer 20

• mammalian species (high degree of conservation of genes and biological pathways)
• hardy
• require little space
• breed well and often
• many approaches for genetic engineering

Question 21

what is the inbred strain of mice called?

Answer 21

C57BL/6J

Question 22

what is the outbred strain of mice called?

Answer 22

CD-1

Question 23

what is inbred?

Answer 23

genetically identical; clones; "isogenic"

Question 24

what is outbred?

Answer 24

genome is not fixed; many alleles are segregating/varying

Question 25

what are hybrids?

Answer 25

cross between two inbred strains (B6C3F1)

Question 26

what are the pros and cons of outbred mice?

Answer 26

PROS: - cheap - easily available - breed well - "outbred like humans" CONS: - unknown genetics - subject to genetic change - lack of reliable background information about population

Question 27

what are the pros and cons of inbred mice?

Answer 27

PROS: - long-term stability - better for study reproducibility - know what you're working with CONS: - not much variance in genes - more expensive - brother/sister mating can produce mutations/problems

Question 28

what lowers your risk of false positive and false negatives?

Answer 28

multiple strain use

Question 29

what are the two engineered genetic models?

Answer 29

1. knockouts (KO) 2. transgenics

Question 30

what are the two approaches to humanize mice?

Answer 30

genetic (syntenic replacement) and tissue replacement

Question 31

what is the syntenic locus?

Answer 31

the corresponding locus in the mouse genome

Question 32

what is the genetic approach to humanization?

Answer 32

syntenic replacement of mouse DNA with orthologous human sequences - cut out the syntenic locus in mouse - paste the human genome segment you want into that area of the mouse that you cut out

Question 33

how is the ADME of arsenic different in mice vs humans?

Answer 33

mice are super efficient at forming the di-methylated species of arsenic

Question 34

how can we use syntenic replacement to study arsenic metabolism in mice?

Answer 34

cut out mice syntenic locus and put the human AS3MT gene in; now mouse has arsenic metabolism of humans

Question 35

what is the tissue replacement (chimeric) humanization approach?

Answer 35

1. get rid of the tissues/cells we don't want in the mouse 2. replace with human cells/tissues that we do want

Question 36

what is an example of tissue replacement in mice?

Answer 36

bone marrow: - use radiation to deplete the bone marrow in the mouse - put in hematopoietic stem cells from humans - over some months, the hematopoietic cells are human-derived

Question 37

how would we use tissue replacement in mice if we wanted a humanized liver?

Answer 37

1. get rid of mouse liver 2. put human hepatocytes in 3. human hepatocytes will ingraft in that space forming a human liver

Question 38

how do knockouts help with genetic humanization in mice?

Answer 38

if you just put a human cell in a mouse, their immune system would form a response to it; if you knock out a bunch of immune system genes, you can prevent that. on the downside, you now have a mouse that has a poor immune system

Question 39

what questions should we ask to determine if our tissue replacement model is working well?

Answer 39

- do they have human hepatocytes there? - do the livers look more human? - are the livers doing human liver things (measure enzyme levels) - have we corrected ADME?

Question 40

what are the pros and cons of tissue replacement humanization?

Answer 40

PROS: - all pathways in a tissue are humanized; efficient in this way CONS: - very labor intensive - only 1 organ system modified at a time - lack of control over donor genetics - immunocompromised because of knockout - variation in extent of humanization due to the rat itself (how much the rat becomes human is dependent on that specific rat itself)

Question 41

what are the pros and cons of the syntenic replacement genetic humanization approach?

Answer 41

PROS: - upfront labor large, but renewable resource afterwards - total control over the human genetics CONS: - fairly skill intensive - limited to a single gene or gene family - possible mismatch between TFs and gene regulatory elements could alter the ability of the human gene to be expressed