Pharmaco/toxico

Give a few reasons to why we use animal models?

1\.similar physiology between animals and humans

2\.to help cure diseases (finding treatments, e.g vaccine)

3\.Lack of suitable alternative testing methods

Why do we use different strains of mice depending on the study?

Different mice strains have different phenotypes, for example some have high anxiety, some have low anxiety. Some have a tendency to be more alcoholic than others.

What type of breeding do we commonly use for mice models? why?

Inbred and F1 hybrid strains are frequently the mouse models of choice for research because of their unique and stable phenotypic traits, and hence, uniformity, and predictable experiment response

What is the lifespan of a mouse?

1-2.5 years

What is the pulse rate of a mouse?

120 beats per minute

What is the body temperature of a mouse?

37\.4 degres

What is the gestation period of mice?

19-21 days

what is the most common strain of mouse?

C57BL/6

What are the 3 R’s in animal models?

1\.Replace (use cells, tissues, organs, computer models)

2\.Reduce (fewer animals)

3\.Refinemenent (minimize pain)

Define animal model

Experimental stimulation in which a simple system represents another system which is more complex with a less accessibility to the experimentation.

\
(e.g mouse is more simple than human)

what is **creative** validity in animal models?

Model has the same symptoms as the ones of human illness

what is **theoretical** validity in animal models?

Same mechanism and pathology as humans

what is **predictive** validity in animal models?

The treatment used for the model should be the same as for humans.

What is the difference between transgenic and knockout mice?

transgenic=gain of gene

knockout=loss of gene

Explain briefly the microinjection method

use to transfer exogenous genes with unrestricted length and transfer of the embryo to pseudopregnant female. Test the offspring’s genes to check if method is successful using PCR.

How to prepare a construct?

1\.Gene of interest +antibiotic resistance

2\.Micropipette embryonic stem cells

3\.culture the cells

4\.transgenic DNA incorporation

5\.Insert stem cell in the blastocyst

What are the 3 ways in which we can measure blood pressure in rodents?

1\.**Catheterism** which directly measures the blood pressure, but the method is invasive and requires anesthesia, which naturally lowers the heart rate giving a bias

2\.**Tail cuff** is non-invasive, you put the mouse in the tube and measure the blood pressure from the tail

3\.**Radiotelemetry** uses an implant in the animal with a battery that is connected, to give continuous monitoring of blood pressure

How can we measure cardiac morphology and function?

Echocardiography is a non-invasive technique using ultra sounds. We can measure volume of blood ejected, thickness of walls etc.

\
We can measure stroke volume, ejection fraction and cardiac output.

Describe 2 rodent models for hypertension:

__1.genetic hypertension__

**SHR** (spontaneous hypertensive rat) Breeding from the WKY strain of mice, naturally has high blood pressure and high PVR (thickening of walls) with age.-> most commonly used

**SPSHR**(stroke prone SHR)

Has even higher blood pressure than SHR rats and very prone to have a stroke

control strain is WKY strain

\
__2.environment hypertension__

**Dahl-salt sensitive rat**

develop rapid high blood pressure under high salt diet.

we compare a 4% salt diet to 0.4% (control)

What are the 3 main symptoms of parkisons?

1 Resting tremor

2 Muscle stiffness

3\.Bradykenesia

What is the standard treatment of parkinson?

L-dopa +Carbidopa

What are the 3 common gene mutation observed in parkinsons?

PARK2 (codes for parkin)

PINK1

SNCA (formation of lewy bodies)

What does PARK2 do?

parkin is a protein responsible for the metabolism of ROS, mutation leads to ROS accumulation in the brain, leading to neuronal cell death (autophagy of mitochondria)

What does PINK1 do?

Also participates in mitochondria regulation. Responsible for the parkinsonian symptoms

What are the environmental factors that can affect/cause parkinsons?

MPTP induced parkinsons (heroine addicts, farmers from pesticides)

What are 2 main toxin models for parkinsons?

1. MPTP model
2. 6-OHDA model

Describe the MPTP model for parkinsons

MPTP crosses the BBB, is metabolized in the astrocytes by MAO-B-→MPP+ this is then transported through DAT (channel), into the dopaminergic neuron

Describe the 6-OHDA model for parkinsons

6-OHDA does not cross the BBB, it is injected into the median forebrain bundle MFB(only inject on one side **only** for control). The MFB is easier to access than the substantia nigra that is why we inject it there. It is transformed into H2O2, which is toxic, it then enters the brain via the DAT transporter

Why can we not use MPTP in rats?

Because the metabolism of MPTP is not possible in rats (enzyme MAO-B)

Which toxin model can we use in rats? mice? and monkey?

rat→ 6-OHDA only

mice→ 6-OHDA AND MPTP

monkey→MPTP only

What are the advantages of injecting MPTP in both the striatum and the MFB?

Injection to MFB→ observe parkinsonian symptom

injection to striatum→observe prodromal phase

What is the pros and cons of **MPTP** model?

What is the pros and cons of **6-OHDA** model?

\

What are some tests we can use to assess behaviour in mice (parkinsons disease)

\-Locomotor activity

\-Stepping test

\-Limb-Use

\-Elevated body swing

What is the limb-use test (parkinson)?

put the mice in a glass cylinder and measure the number of contacts of the impaired paw vs intact paw and the number of turns

What is the stepping test? (parkinson)

Hold the animal and slide it against a surface, then check the use of its paw to see if it steps.

Give a test that evaluates both antidepressive activity AND anxiolytic activity:

Novelty supressed feeding

Give a test that evaluates antidepressive activity:

Forced swim test

tail suspension test

Splash test

Give a test that evaluates anxiolytic activity:

Elevated plus maze

Open field

Light-dark case test

Explain the forced swim test

The forced swim test allows to measure the climbing behaviour and the immobile behaviour.

What is the tail suspension test?

We hold the animal by the tail, when it is stressed it will be more immobile. With antidepressants, the mouse tends to move more to escape the position.

What is the splash test?

To measure the grooming activity of the rodent, they usually groom less with depression.

What is the CORT model?

Chronic corticosterone (CORT) administration alone for 4 weeks and then administration of fluoxetine at week 4. Fluoxetine was able

What is the main function of biotransformation of xenobiotics?

To make lipophilic molecules water soluble for better elimination

Where does xenobiotic metabolism occur?

The liver

What is the enterophepatic cycle?

The liver excretes the drug through the biliary system, then the drug ends up in the intestinal lumen where it is reabsorbed.

What are the phases of xenobiotic metabolism?

phase 1 - CYP (functionalization)

phase 2-Glucuronidation through UGT (conjugation)

Explain phase 1 reaction

Creation of a functional group that is more hydrosoluble:

* oxidation (O2)
* reduction (-O2 or +H2)
* hydrolysis (+H20)
* hydrataion (+H20)

What is the most common enzymes involved in oxidation?

Cytochrome P450 (microsomes)

Xanthines oxidase

what is the most common enzyme for hydrolysis?

epoxide hydrolase (EH)

epoxy→dihydrothiol

How does CYP450 catalyze oxidation reactions?

Using oxygen and NADPH (monooxygenase activity)

Where is CYP450 located?

in the membranes of the smooth ER in hepatocytes

What is the reaction catalyzed by CYP450?

RH+O2+NADPH+H →ROH+H2O+NADP+

Draw and briefly explain the structure of CYP450 enzymes

There is a NADPH cytochrome reductase unit AND a CYP450 subunit carrying a heme

Explain phase 2 reactions

They are conjugation reaction such as glucorinadation (UGT), acetylation (acetyl COA), glucothione (GSH)

What are the 4 factors that can affect drug metabolism?

1\.Genetic (slow, fast metabolizer)

2\.Physiological (new borns and older people)

3\.Environmental (smoking, alcohol, food like grapefruit)

4\.Pathological (liver damage)

What is the CYP responsible for the metabolism of acetaminophen in the liver?

CYP2E1

What are the three main enzyme pathways of paracetamol, indicate which one is toxic?

Glucoconjugation (detoxification)

Sulfoconjugation (detoxification)

Oxidation into NAPQI (toxication)

What are the therapeutic consequences of different types of metabolizers

1\.Slow metabolizers→ TOXIC

2\.Fast metabolizers →Therapeutic efficacy

3\.Ultrafast metabolizers → TOXIC

What is rifampicin?

An inducer of CYP450 (3A4, 2C9)

What is isoniazid?

An inhibitor of CYP450 (3A4, 2C19)

An inducer of 2E1

What is ethanbutol?

An inhibitor of CYP450

Draw and label the phase 1, phase 2 pathway

is alcohol and tobacco and inducer or inhitor?

Inducer

What are the risks of rifampicin and oral contraception together?

Risk of unwanted pregancy due to the decrease in the effect of the contraceptiv because rifamicpin is a CYP inducer

Explain the metabolism of codeine

codeine is metabolized by CYP2D6 into morphine

codeine is metabolized by CYP3A4 into norcodeine

What are the 3 most abundant isoenzymes of CYP450

CYP3A4/5

CYP2D6

CYP2C8/9

what is the difference between central and peripheral tolerance?

Central tolerance eliminate self-reactive lymphocytes during their initial development in the bone marrow and thymus. Peripheral tolerance (treg) eliminate self-reactive lymphocytes that escape the central mechanisms

What are haptens?

xenobiotic that is not immunogenic by themselves but have to be processed by antibodies in order to elicit immune response.

What are the 4 types of hypersensitivty?

1: **A**llergic **A**naphylaxis and **A**topy

2:Anti**B**ody

3\.Immune **C**omplex

4\.**D**elayed

Which types of hypersensitivity are often chemically induced?

type 1 and type 4

Type 4 is also known as …………

allergic contact dermatitis

What are the key steps of sensitization during allergic contact dermatisis?

1. Covalent binding of the allergen to the skin components

2\.Hapten induced activation of APC cells (cytokine production)

3\.Recognition of allergen modified by specific T cells and dentritic cells

4\.Proliferation of specific t cells in draining lymph nodes (C4+ and CD8+ are expressed)

what are the methods used to measure the sensitization phase?

LLNA (mouse local lymph node assay)

Explain the LLNA evaluation method

By introducing the allergen to the animal every day and then prepare a cell suspension from the lymph nodes of the animal and then you measure the lymphocyte proliferation using a radio label. →Quantitative evaluation

What are three alternative tests for skin **sensitization** phase?

1\. Direct Peptide Reactivity Assay (DPRA)

2\.Human Cell Line activation Test (h-CLAT)

3\.KeratinoSens assay

Explain the way DPRA works.

First they add chemicals to the peptides then wait for 24 hrs → Check the bound peptides compared to the free peptides. Using an **HPLC**, they measure the reactivity of the chemicals.

Explain the way h-CLAT works.

* In-vitro test
* Measure dendritic cell activation by measuring cell surface marker expression.

First they use t helper cell lines + chemicals then wait for 24hrs →add antibodies → stain antibodies → observe binding by flow cytometry.

Explain the way KeratinoSens assay works.

* In-vitro test (second AOP event)
* Study **keratinocyte activation** via the Keap1-Nrf2 pathway known to regulate the response to oxidative stress.
* Under oxidative stress, the transcription factor Nrf2 will bind and lead to the expression of genes such as luciferase.
* We measure the luciferase expression as a measure for the activation of the pathway
* MORE luciferase=chemical more toxic

\

What is an adverse outcome pathways (AOP)

It is a model that identifies a sequence of events required to produce a toxic effect, the adverse outcome after xenobiotic exposure

\

What are the key steps of an AOP ?

1\.Covalent bonding with cell’s protein (DRPA)

2\.Inflammatory response of keratinocytes (cytokines, IL-18) (*KeratinoSens*)

3\.Activation of dendritic cells

4\.T cell proliferation in the lymph nodes (LLNA)

What are the methods to evaluate immunosupression?

1\.sheep red blood cell T-dependent antibody response

2\.t-dependent antigen

3\.PFC and ELISA

What is the principle of plaque forming assay to evaluate immunosupression?

Immunize mice with sheep RBC, collect the spleen and isolate the lymphocytes, plate the lymphocytes and sheep RBC in an agar with complement. Look at the formation of white plaques as a consequence

How are chemical carcinogens classified?

1: carcinogenic to humans (e.g tabacco)

2A:Probably carcinogenic to humans

2B:Possibly carcinogenic to humans

3: Not classifiable

4:Probably not carcinogenic to humans

What is the difference between mutagens and carcinogens?

Mutagens affect the DNA in a non specific way to cancer

Carcinogen affect the DNA to make cancer more likely

What are 2 types of carcinogens?

genetic (alters DNA sequence)

epigenetic (alters cell growth, gene expression)

What are the 3 main stages of carcinogenesis?

1\.INITIATION

2\.PROMOTION

3\.PROGRESSION

Specify in each step of carcinogenesis which one is irreversible, and reversible

Initiation -irreversible

promotion-reversible

progression-irreversible

true or false: All Carcinogens can affect each stage of the carcinongenesis phase

False

What is the importance of the dose-effect relationship in carcinogenesis?

Each carcinogen has a threshold for which it causes damage, the severity of the effect is proportional to the dose absorbed (NOAEL)

What are oncogens?

Genes that promote cancer when they are mutated

Proto-oncogene→oncogene

How do non-genetic carcinogens work?

They act as promotors AKA peroxisome proliferators, they lead to the increased production of ROS and increased peroxisome metabolism leading to DNA damage→cell proliferation. An example of a non-genetic carcinogen is TCDD

What tests can we do to evaluate carcinogenic effects of a chemical?

EMT

soft agar and clonogenic test

tumour development in nude mice

SHE test

mutagenesis studies (genotoxic)

carcinogenic studies (non genotoxic)