Toxicology Flashcards
1
Q
Toxicology
A
- study of potential harmful effects of chemicals on living organisms, biological systems and human health
2
Q
Examples of toxic substances
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- Drugs and their metabolites
- Chemical substances, classified by their source of exposure: occupational, environmental, household
- Pesticides (certainly toxic to at least one life form)
- Natural toxins (e.g. snake venom)
- Food additives (some E-numbers) and contaminants (e.g. melamine contamination of baby formula)
3
Q
Thalidomide
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- Prescribed to relieve morning sickness during pregnancy – placental delivery unknown
- Introduced in 1956
- World-wide increase in malformations in new-borns
- Mainly phocomelia (shortening of limbs)
- Withdrawn in 1961 after link between the malformations and Thalidomide was proven
- Resulted in much more rigorous drug testing before licence is granted
- Now licensed for leprosy; multiple myeloma
4
Q
Occupational Toxicology: Examples
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- Chimney sweeps’ carcinoma: scrotal cancer due to prolonged exposure to polyaromatic hydrocarbons (PAHs) present in soot
- Nasal carcinomas in wood workers due to PAHs formed during sawing and sanding (incomplete combustion due to friction-based heat generation)
- Bladder cancer in dye workers due to use of naphtylamine, an azodye precursor (nowadays largely replaced by less carcinogenic alternatives)
- Neuropathy as result of long-term exposure to hexane (leather workers) or carbon disulphide (nylon workers)
5
Q
Lungs as a susceptible organ
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- Highly perfused, receive 100% of right side cardiac output
- Exposed to high concentration of molecular oxygen
- Metabolic activity (local or in liver) can lead to formation of reactive metabolites
6
Q
Liver as a susceptible organ
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- Receives 80% of blood supply from portal circulation, exposed to highest concentration of ingested xenobiotics
- Most important concentration of metabolic activity – inducers of CYPs and/or inhibitors can have significant effects on ‘capacity’ to deal with toxic substances
7
Q
Kidneys as a susceptible organ
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- Highly perfused, only 5% of body weight but receives 25% of right side cardiac output
- Glomerular filtrate is highly concentrated (to drive reabsorption) – non-toxic compounds in plasma may reach toxic concentrations in tubular fluid
- Ionic chemicals require active transport to be re-absorbed – non specific transport can lead to reabsorption of toxic chemicals
- Metabolic activity can lead to formation of reactive metabolites
8
Q
Central nervous system
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- Highly perfused
- Blood brain barrier not 100% effective
- Efflux transporters require ATP
- Neurons have high metabolic rate, high mitochondrial activity
9
Q
Acute toxic effects
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- Rapid development whilst chemicals or metabolites are still in body
- Short lived - may be lethal
- May result from single or short term exposure
- Cause and effect easily identified
10
Q
Chronic toxic effects
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- Delayed development - develops after chemical excreted
- Retrospective detection by epidemiology
- May result from single, multiple or chronic exposure
- May be cumulative effect from long term low level exposure e.g. cancer, neurodegenerative diseases
11
Q
Visible/detectable cause of toxic effects
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- exposure to toxic substance Absorption via: gastro-intestinal tract, respiratory system, skin contact - distribution - metabolism - elimination
12
Q
Visible/detectable effects of toxicity
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- primary target (biomolecular interaction/reaction) - cell damage - organ damage - adverse effect on organism (disease)
13
Q
ADME
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- Absorption: how chemical enters the body and systemic circulation
- Distribution: how chemical is distributed in the systemic circulation to the rest of the body
- Metabolism: what happens to chemical inside the body
- Elimination: how the parent chemical or metabolite leaves the body (urine, faeces, other)
14
Q
Absorption (pharmokinetics)
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- great majority of therapeutics are orally administered
- absorbed through buccal/oral mucosa, stomach and/or intestine
- pH differs in these compartments differences in ionisation state of drugs e.g. salicylate
- non-ionized forms are more readily absorbed – more easily cross cell membranes
- Most absorption occurs in intestine
15
Q
Absorption of Aspirin
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- uptake via the stomach
- near neutral pH of blood ensures that aspirin is ‘locked’
16
Q
Absorption of Morphine
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- Ionized in the stomach – no absorption
- pKa is within the intestinal pH range, resulting in absorption
- Many drugs contain an amine-based ionizable group with similar pKa to facilitate intestinal uptake
17
Q
Distribution (pharmokinetics)
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- Initial phase happens rapidly as a factor of blood flow to organs and tissues
- Some tissues are highly perfused (lungs, brain, liver, kidney)
- Liver receives 80% of blood via the portal system
- Subsequent uptake by tissues/organs is largely dependent on “affinity”
- Ability to cross biological membranes
- Specific transporters (influx and efflux)
- Protein binding
18
Q
Blood-brain barrier
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- Not an absolute barrier
- Interstitial fluid has a lower protein content than plasma
- Capillary endothelial cells tightly joined
- Transmembrane transporters: efflux
- Endothelial cells surrounded by glial cells: lipid membranes of glial cell processes a significant barrier
- “Gaps in BBB” e.g. pituitary gland, choroid plexus, olfactory bulb
19
Q
Phase I of mechanism of xenobiotics
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- Modification: Oxidation (e.g. P450 monooxygenases), Reductions (e.g. alcohol/aldehyde dehydrogenase), Hydrolysis (e.g. esterase, epoxide hydrolase)
- Can result in activated or deactivated xenobiotic
- Interaction with/modification of biomolecular target –> toxic effects
20
Q
Phase II of mechanism of antibiotics
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- Covalent modification of xenobiotics (and other ‘waste’ molecules e.g. bilirubin) with high molecular weight, polar, highly water soluble groups: Glucuronide (from UDP glucuronic acid), Glutathione (from reduced glutathione), Sulphate (from 3’-phosphoadenosine-5-phosphosulphate), Acetate (from acetyl CoA)
- Greatly increases efficiency of excretion, especially in bile, but also urine
21
Q
Cytochrome P450 (CYP450)
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- multigene superfamily: 37 different multigene families, 10 are known in mammals, 8 in humans
- More than 300 isoenzymes, relatively low substrate specificity, so broad range of reactions possible
- Inhibition can have severe pharmacological consequences: Deactivation (clearance rate decreases, increasing risk of adverse effects or even resulting in overdoses), Activation (drugs lose their therapeutic action)
- Four enzyme families play role in metabolism – CYP1-4
- contain Haem: characterised by absorbance at 450 nm when CO is bound
- CYP450 enzymes require a reductase for providing electrons
- Microsomal enzymes: associated to the ER membrane via a membrane anchor
22
Q
Mono-oxygenation of CYP450
A
- Oxidation of a broad range of compounds in order to (ultimately) functionalize them
- Generic reaction scheme:
R + O2 + NADPH + H+ –> RO + H2O + NADP+ - example reactions: aliphatic hydroxylation, N-hydroxylation, sulfoxidation
23
Q
Epoxidation of CYP450
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- Mono-oxygenation of a carbon-carbon double bond
- Epoxides are typically more reactive and can cause severe damage to biomolecules
- Hydrolysis of epoxides into vicinal diols by microsomal epoxide hydrolase (mEH) crucial to prevent damage by reactive epoxides
24
Q
Phosphotriesterase activity by paraoxonase 1 (PON1)
A
- Phase I enzyme
- Detoxifies organophosphates (e.g. pesticides, nerve gases)
- PON1 is also an anti-atherosclerotic component of high-density lipoprotein (HDL)
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Oxidoreductives
- Alcohol dehydrogenase (ADH)
- Aldehyde dehydrogenase (ALDH)
- Flavin mononucleotide oxygenases
- NADP(H) Quinone reductases
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Glucuronidation
- Modification of amine (-N) and hydroxyl (-O) groups in compounds to improve solubility
- Responsible enzymes: UDP-glucuronosyltransferases (UGTs)
- donor substrate - GlcA-UDP
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Glutathionylation
- Nucleophilic attack of sulphur group on electrophilic centers in a variety of substrates
- Responsible enzymes: glutathione-S-transferases (GSTs)
- donor substrate - GSH
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Sulphation
- Modification of amine (-N) and hydroxyl (-O) groups to improve solubility
- Similar purpose as glucuronidation
- Responsible enzymes: sulfotransferases (STs)
- donor substrate - PAPS
- Generic reactions: sulfation of alcohols and amines
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Acetylation
- Modification of arylamino compounds
- Responsible enzymes: N-acetyltransferases (NATs)
- donor substrate – Acetyl-CoA
- Generic reaction: N-acetylation of aniline
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Metabolism
- Main organ for detoxification: the liver
- Most xenobiotics enter body via oral ingestion and enter circulation via intestines, i.e. enter circulation in portal vein
- Contains high levels of several phase I enzymes and conjugating enzymes
- Some other organs (need to) possess their own metabolic capacity (may differ from liver in specificity and activity) due to high perfusion and:
- Lungs and skin: part of bodies ‘defence’, can be ‘attacked’ without liver being able to ‘intervene’
- Kidney: mechanism of action can cause increases in concentration of xenobiotics, reaching toxic levels
- Some enzymes (CYPs and transferases) exhibit polymorphisms: ideally, drugs should be metabolised by a variety of CYP isoforms
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Elimination
- Conjugated metabolites can be excreted via two routes: urine via the kidneys (the major route), biliary route - avoids exposure of other organs via systemic circulation, i.e. liver acts as a gatekeeper
- Certain metabolites may de-conjugate in bile (result of enzymatic activity of gut microflora) and hence be available for re-absorption – enterohepatic recirculation
- De-conjugation may also occur in the bladder.
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Metabolic/Cellular consequences in mechanism of toxicity
- Dysregulation of cellular homeostasis, mitochondrial function, cell cycle and others
- Cytotoxicity: necrosis and/or apoptosis
- Genotoxicity (direct or indirect)
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Molecular Mechanisms in toxicity
- Oxidative stress and generation of reactive oxygen species
| - Reactive parent chemicals or metabolites which bind to macromolecules such as proteins and/or DNA
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Un-stressed cell
- High levels of reduced glutathione - reduced environment within the cell
- Reduced thiols on proteins
- Anti-oxidants and enzymes available to protect cell
- Oxidatively damaged DNA bases are repaired
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Oxidative stress
- imbalance between cellular production of reactive oxygen species (ROS) and the ability to detoxify ROS/ability to repair damage
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Formation of ROS
- Molecular oxygen can accept electrons to give ROS such as hydrogen peroxide, superoxide and the hydroxyl radical
- Fenton reaction generates most toxic ROS: the hydroxyl radical
- Factors that drive the Fenton reaction: Directly (high levels of hydrogen peroxide and freely available Fe2+), Indirectly (superoxide)
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Remove hydrogen peroxide and superoxide (defence against ROS)
- Superoxide dismutase (SOD)
- This enzyme in isolation would not be enough
- In combination with catalase both superoxide and hydrogen peroxide are neutralized
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Alternative to catalase: glutathione peroxidase (GP)
| defence against ROS
- Like catalase turns hydrogen peroxide into harmless water
| - Oxidized glutathione (GSSG) regenerated to reduced glutathione (GSH) by glutathione reductase (GR)
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Antioxidants (defence against ROS)
- Vitamins: can accept single electrons from free radicals (R.) neutralizing them
- result is another free radical that is less reactive i.e. less damaging
- Lowered reactivity in free radical due to the possibility of mesomeric resonance structures
- Example: ascorbic acid (Vitamin C)
40
Glutathione
- tripeptide: g-glutamyl-cysteinyl-glycine
- Dual purpose in toxicology: conjugation of electrophiles followed by conversion of the conjugates into mercapturic acids which are excreted, protection against oxidative stress
- Conjugation results in depletion of reduced glutathione: both roles at odds with each other
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Toxicology of Carcinogenesis
Genotoxicants
- direct: toxic compounds interacts directly with DNA, causing mutations
- indirect: damage to proteins involved in e.g. DNA replication and repair
Other causes (mostly of a regulatory nature):
- endocrine disruptors (disrupted endocrine regulation)
- epigenetic changes (disrupted regulation of gene expression)
- peroxisome proliferators (a type of transcription factor --> disrupted regulation of gene expression)
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Necrosis
- Accidental
- Cell damage
- Organelle swelling/disruption
- ATP levels fall
- Loss of plasma membrane integrity: release of intracellular contents
- Digestion by lysosomal enzymes
- Local inflammation (phagocytes migrate to site)
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Apoptosis
- Programmed cell death = “natural” process
- Organelles remain intact for much of the time: cell often shrinks
- ATP levels high
- Plasma membrane remains intact: no release of cell contents
- Digestion by caspase enzymes
- No inflammation (neighbouring cells ingest apoptotic bodies)
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Examples of Metabolism of Xenobiotics/Drugs
- Benzo[a]pyrene metabolism and toxicity
- Aspirin overdose
- Paracetamol overdose
