Inter-species and Inter-individual differences in toxicology

Question 1

Study of adverse effects of chemicals on living systems, including -

Answer 1

• Mechanisms of action & exposure to chemicals as a cause of acute/chronic illness.
• Recognition, identification, quantification of hazards from exposure to chemicals.
• Discovery and safety of new medicines, personal care products, agrochemicals.
• Development of standards and regulations to protect humans and the environment from adverse effects of chemicals

Question 2

Preclincial toxicity screening highlight

Answer 2

Mutagenicity, Carcinogencity, Reproductive toxicity, acuet toxicty, subchronic, chronic

Question 3

how is mutagenicity and carcinogenicity

Answer 3

Mutagenicity = in vitro tests including Ames test
Carcinogenicity = repeated dose given throughout lifetime of animal (chronic exposure)

Question 4

how is reproductive toxicity determined

Answer 4

Effect on fertility, implantation, foetal growth, fetal abnormalities, neonatal growth

Question 5

Describe the ways to identify acute toxicity

Answer 5

Animal model: single dose given by proposed route for humans
Two mammalian species, Dose Ranging Finding study (lethal / limit dose)
Defines dose range associated with toxicity

Question 6

How is subchronic toxicity determined

Answer 6

repeated doses given for 14 or 28 days

Question 7

how is chronic toxicity determined

Answer 7

– Repeated doses given up to six months, Reveals target(s) for toxicity
– 2 species – rodent + non-rodent
– Define doses associated with adverse effects and “no observed adverse effect level” associated with “safe” dose

Question 8

what species are used in general toxicity studies

Answer 8

Rat/ Mouse/ Guinea Pig (Rodent)
Dog/ Minipig/ Monkey (Non-rodent)
Rabbit (reproductive toxicity)

Question 9

what are the reasons for chosing a specific species when examining general toxicity

Answer 9

• May require metabolism similar to humans
• At least one species should show pharmacological/ response activity
• Repeat dose studies normally in 2 species (Rat and dog are most common)
• Other species can be used e.g. mouse instead of rat, mini-pig rather than dog.
• Primates rarely used for NCEs unless there is a very compelling reason. (Generally need a strong justification if: Specific metabolism + Disease/therapy model)

Question 10

Explain the reasoning behind extrapolating animal models to humans from toxicology studies

Answer 10

Animal models = historically critical role in exploration & characterisation of novel therapeutic agents & treatments and toxicity
‘Ideal’ animal model should replicate human metabolism of the chemical = models should share with human same molecular pathway(s)/ enzymes involved in test chemical metabolism
If testing therapeutic drug, model should be a good model for a given human disease

Question 11

Thus our ability to perform informed extrapolation is intimately linked to _

Answer 11

the degree of our mechanistic understanding of the toxicity

Question 12

what are the factors which affect drug metabolism

Answer 12

Genetic background = animals (Inbred), Humans (outbred)
Age = A (usually same), H (foetus,child,adult,elderly)
Sex = A (usually same), H (influence of sex hormones)
Diet = A (controlled), H (starvation, malnutrition ect)
Pathological = A (usually none), H (Comorbidities, liver+ )
Chem-Chem Interac- = A (usually none) , H (potential)
Short vs Long = A (short term exposure), H (both long and short)

Question 13

What are the problems associated with variation in metabolism when extrapolating toxicology from animal studies

Answer 13

Between species
= change in proportion, specificity and distribution phase I/II enzymes between species/strains
= change in mechanisms regulating phase I/II enzymes expression(e.g PXR)
Between humans
= Population diversity (SNPs, age, body fat, gender, health
= Extreme condition = starvation, dieting excersising
= Exposure = short/long term, low/high dose, repeated/single

Question 14

what is a major difference between animal and human which effects difference in toxicity

Answer 14

Differences in response to DNA damage ( DNA repair; immune system) between animals and human

Question 15

What is the difference in CYP distribution between human and rat livers

Answer 15

Human = CYP 3A,2C,1A2,2A6,2E1
Rat = CYP 2C11,3A2,1A2 very low levels of 2E1

Question 16

What is the dis/advantages in difference in metabolism between lab animals and human

Answer 16

• Advantage: can help to deduce mechanisms of toxicity
• Disadvantage: limit experimental approach - implies same metabolic pathways shared by model and human

Question 17

how is nicotine metabolised in both human and animals

Answer 17

humans = metabolsied by CYP2A6 (highly expressed in human, nicotinic dependence has been seen in humans
Rats - No CYP2A6 but nicotine metabolised by CYP2B enzymes

Question 18

How is CYP1A2 different between humans & rats

Answer 18

Humans: Polycyclic Aromatic Hydrocarbons induce CYP1A2 expression
Lower expression in rodents compared with humans - No CYP1A2 in monkeys –> so not a good model

Question 19

Looking at Cat, Human, Rat, Rabbit and then Pig
phenol conjugated to glucuronide _ and sulfate _

Answer 19

increases
decrease

Question 20

Variation in rate of phenol conjugation can lead to

Answer 20

significant variations in metabolism between species

Question 21

How does gender difference in different species affect metabolic

Answer 21

Humans = generally no major sex differences in metabolic enzymes in humans
Rodents = some P450s (CYP 3A,2A,2B,2C) higher in male

Question 22

What are the inter-species differences in hydrolysis of esters/ esterases

Answer 22

Cholinesterases (greater in human)
Carboxylesterases (greater in rodents)

Question 23

How does interspecies difference in Metabolsim-Age affect toxicology testing

Answer 23

Human = Children (many changes in P450 in first 4 years), Adults (between 20-70 specific activity of metabolising enzymes doesnt decrease), Elderly (reduced liver size and blood flow reduced clearance in metabolism.
Rodents have short life span, rats mature at 6 weeks so hard to study toxicology in children

Question 24

Cut of point in terms of molecular weight for biliary versus renal excretion varies between _

Answer 24

species
- Rat MW>400Da for anions excreted in the bile
- Dog >400Da for anions
- Human-approx 500Da for anions

Question 25

Inter-species variation in biliary excretion may reflect what ?

Answer 25

differences in specificity for transporter ABCC2 (main transporter for glucoronidated compounds) between human and other species. Human ABCC2 more restrictive in what it transports across the bile canaliculus

Question 26

Why might strain differences effect drug metabolism

Answer 26

• genetic deficiency
  Dark Agouti Rat = Females lack CYP2D1(hCYP2D6) and unable to hydroxylate debrisoquine, Males express a spesific member of CYP2D isoform that hydroxylate debrisoquine. Females = good models to study debrisoquine toxicity
  Gunn Rat = Unable to synthesise certain phenol glucuronides e.g. deficiency of O-glucuronide
  of 2-aminophenol. Good model for paracetamol (N-acetyl-4-aminophenol) induced hepatotoxicity in
  individuals with SNP in UDP-Glucuronosyltransferase (UGT) 1A reducing glucuronidation

Question 27

What is the link between sex and tissue difference

Answer 27

Genotoxic Carcinogens: aan cause cancer in any tissue, multispecies and show a linear risk
Non-Genotoxic Carcinogens: Maybe tissue specific, restricted species and show sex differences
(e.g Unleaded Petrol :-causes kidney cancer in male rats)

Question 28

what is the proposed mechanism of carcinogenicity of unlead petrol in male rats

Answer 28

Normal renal cells take up a2-microglobulin-TMP complex. This causes a lysosomal overload which causes cell death
Regenerative hyperplasia eventually leads to renal tumours

Question 29

why do female rats experience carcinogenicity of unlead petrol

Answer 29

Synthesis of α2-microglobulin under hormonal control by androgens. Females make 1% compared to males:
So in female rats there is:
no complex, no uptake, no hyperplasia, no tumours

Question 30

Using the example PPAR-a agonists, why do tumours appear in rodent who received fibrate drugs but not humans

Answer 30

PPAR-a = Nuclear receptor which is a transcription factor, major physiological regulator of lipid metabolism. Directly induces transcription of CYP3A4 & 4A
Agonist used as hypolipidaemic drugs
Mouse/Rat = highly responsive to PPAR-a agonist therefore liver tumour
Guinea Pig = No peroxisome proliferation -> hypolipidaemia however no evidence of liver tumours
Marmoset/ dog =No response, no evidence of liver tumours
Humans = believed to be unresponsive however there is evidence of hypolipideamia

Question 31

Chemicals bind to human PPARα, but DO NOT cause liver hyperplasia/hypertrophy
Why difference between rodent and humans?

Answer 31

• Human liver - insufficinet levels of PPARa to induce cell proliferation
• Rat/mouse liver - igher PPARα levels induce cell proliferation at equivalent doses
  Suggests PPARα has different role in human liver compared in control of homeostasis, oxidative stress response, lipid metabolism

Question 32

What is the cellular difference between rodents and humans

Answer 32

Different PPARα binding sequence on DNA in rat/mice compared to humans
Expression of PPARα protein significantly higher in rat/mice than humans

Question 33

What is tamoxifen

Answer 33

Non-steroidal anti-oestrogenic treatment of breast cancer
– Acts by blocking oestrogen binding to the receptor
– Tamoxifen is metabolised into compounds that also
bind to the oestrogen receptor but do not activate it

Question 34

What is the species specific genotoxic effect of tamoxifen

Answer 34

– Male rat: associated with hepatocellular carcinomas
– Female rat: hepatocellular and endometrial carcinomas
– Mice: Rare hepatocellular carcinoma
– Human: no hepatocellular carcinoma

Question 35

What is the difference in metabolism of tamoxifen between rats and human

Answer 35

After undergoing phase I metabolism (Tamoxifen -CYP3A4-> 4OH-Tamoxifen)
Humans detoxifies by glucuronidation (glucuronidation enzymes is 100 fold high in humans compared to rats)
In rats - 4OH-Tamoxifen undergoing sulfation into a genotoxin

Question 36

What is PON1

Answer 36

synthesised in the liver, secreted in plasma where it associates with HDL.
* PON1 detoxifies organophophate molecules (insecticides, herbicides, nerve agents)

Question 37

What is the effect of the SNP Q192R in paraoxonase (PON1)

Answer 37

• SNP Q192R induces AA change at position 192 and changes substrate specificity
• Glutamine (Q isoform) = rapid metabolisers of Diazinon (insecticide) but also rapid metabolisers of nerve agents
• Arginine (R isoform) = rapid metabolisers of Paraoxon (insecticides)

Question 38

What genotype (SNP) of PON1 makes workers who are in contact with insectides at dangerous doses (sheep dip)

Answer 38

Position 192 Glu/Arg Affects PON1 protein levels and substrate specificity
Position 55 Leu / Met SNP affects PON1 protein levels (not activity)
192QQ= Reference genotype, ORqq=1
ORqr or rr >1 ↑ disease risk in farmers because of a lower ability to detoxify diazinon

Question 39

How does absorption different in elderly people

Answer 39

There is decrease inhalation capacity, increase dermal absorption (thinner skin) and decreased gut absorption
Distribution = Altered blood flow and decreased clearance. There is changes in volume and distribution (fat/water ratio) changes as we age hydrophobic chemicals stored in fat tissue
There is a decrease expression of metabolic enzymes and plasma protein transporting drugs

Question 40

why are neonates more susceptibile to toxicity

Answer 40

• Increased dermal absorption (undeveloped skin not as protective)
• decrease gut absorption
• different fat/water ratio (distribution issues)
• reduced excretion due to immature renal function
• Metabolic enzymes expression: underdeveloped for
  some metabolic enzymes; e.g. P450s, UDP-glucuronosyltransferases

Question 41

Why are animal models not useless in identifying toxicity despite numerous differences

Answer 41

• Pretty much every drug in the pharmacopoeia is an example of where animal models have been a success
• However it is necessary precaution to avoid major adverse effects