Mechanism Of Toxicology - kenny ritchie Flashcards
Name 4 consequences of metabolism
- Biological half life decreased
- Duration of exposure reduced
- Accumulation of the compound in the body is avoided
- Duration of biological activity may be affected
Define xenobiotics
A foreign substance that does not naturally occur in humans and can be absorbed through various routes
What does cytochrome p450 require
Oxygen
NADPH
NADPH cytochrome p450 reductase
What is cytochrome p450
Is heme-containing enzyme system that is involved in the metabolism of xenobiotics.
It’s most abundant in the liver and is located in the smooth endoplasmic reticulum
Factors controlling xenobiotic metabolism
- Availability of cofactors eg NADPH- particularly important for phase 1 reactions
- Availability of co-substrates eg oxygen glutathione
- The level of particular enzymes
Two types of ROS
Superoxide anion radical
Hydroxyl radical
Explain redox cycling
Xenobiotics can accept an electron from the electron transport chain or from NADPH via reductase enzymes
The xenobiotic is reduced as it gains an electron and then oxidised as it gives the electron away to oxygen
The xenobiotic is then free to be reduced again
This process results in a lot of oxygen based radicals moi
What is the Fenton reaction
Where h202 is readily broken down to produce a superoxide anion radical and a hydroxyl radical
Catalysed by iron, UV light or active transition metals
3 methods of detoxification of ROS
- Glutathione
Is a strong antioxidant which detoxifies in 3 ways
By conjugation catalysed by GSH transferase
Chemical reaction with a reactive metabolite to form a conjugate
Donation of a proton to reactive metabolites eg ROS
The thiol group means it’s easily reduced and the superoxide anion radical can attach and be detoxifies
If GSH is depleted it leaves cells other thiol groups vulnerable to toxic compounds eg ones on important proteins
Could effect enzymes which control ion concentration and other important cellular activities
- Superoxide dismutase
Dismutates 2 molecules of superoxide into one of h202 and one 02
the h202 is then removed by catalase or GSH peroxidase action - producing water, oxygen and glutathione disulfide - Isothiocyanate
Also shows antioxidant activity
Works by inducing phase 2 enzymes eg GSTs (glutathione-s-transferases) which catalyse GSH dependent conjugations and redox reactions which prevent cells from DNA damage by ROS
Name 4 major primary events in toxicity
Lipid peroxidation
Covalent bonding to macromolecules
Changes in thiol status
Enzyme inhibition and ischemia
Name 3 important secondary events underlying cellular injury
Calcium release
Changes in cytoskeleton
Mitochondrial damage
Explain the 2 phases of biotransformstion (metabolism)
Phase 1;
Functionalists the xenobiotic by chemical conversion to allow phase 2 to occur. This changes the structure if the chemical ingredient. It can also increase the toxicity of the ingredient.
This is carried out by the cytochrome P450 monoxygenase system and is found in SER mostly in liver cells.
These enzymes are produced by supply and demand so if a certain chemical is absorbed by the body- more enzyme will be produced
Electrons are transferred from NADPH to P450 by NADPH cytochrome P450 reductase
Phase 2;
This is the true detox pathway where hydrophilic compounds are produced and excreted from the body. Therefore this phase takes the changed structure and excretes it.
It does this by adding a molecule eg a sulphate onto the changed structure to make it more water soluble so it can be excreted
Name 4 phase 2 enzymes;
UGTs
Responsible for the process of glucuronidation eg the metabolism of things such as retinoids and substances which involve glycosidic bonds
Sulfotransferase
Transfers the sulphate to the xenobiotic to make it more water soluble
N-acetytransferase
Transfers acetate to the xenobiotic
Glutathione-s-transferase
Catalyses the conjugation or glutathione with a sulfhydryl group to make it more water soluble
What controls xenobiotic metabolism?
- Availability of cofactors eg NADPH- particularly for the phase 1 reactions
- The availability of co - substrates eg oxygen and glucothione
- The levels of particular enzymes
Talk about free radicals
Free radicals are a consequence of living in an oxygen rich environment.
The most common reactive metabolite is an electrophile, these are produced by P450s and attracted to DNA (neg charged)
Nucleophiles
ROS eg superoxide anion radical and hydroxyl radical (most reactive)
H202 occurs naturally within us and also in cosmetics and is readily broken down via the Fenton reaction to produce a hydroxyl radical and a superoxide anion radical
Fenton reaction
The breakdown of h202 into the superoxide anion radical and the hydroxyl radical
Catalysed by iron, UV light or active transition metals
What do ROS do
Cause oxidative stress which results in ageing. Cells are constantly damaged as a consequence of living in an oxygen rich environment and part of general biochemistry where oxygen is taken in and ATP is produced
ROS are produced as a by-product in the electron transport chain
These can also be produced from redox cycling
Explain primary secondary and tertiary events in toxicity
Primary -
Initial damage to cellular constituent by toxicant
Secondary-
Changes to large cellular structures eg cytoskeleton or mitochondria
Tertiary -
Changes observable to the eye eg blebbing or apoptosis
Give 3 examples of a primary event
- Lipid peroxidation -
Free radicals attack the structure and damage the cell membrane
As a result of the attack other toxic chemicals eg MDA are produced - Covalent bonding to macromolecules
Chemicals physically bonding to cells eg electrophiles forming a covalent bonds with DNA
This bonding can cause a protein eg an enzyme to become inactivated and cause it to stop what it’s producing = INHIBITION - Thiol status
A functional group that contains a sulphur and hydrogen bond- these are very easily oxidised which allows the thiol group to be oxidised rather than other important cell molecules. When these run out, toxic substances can then bind to important molecules causing toxicity to be seen.
4 examples of secondary event
- Changes in membrane structure and permeability
Can result in influx / enflux of ions eg k+
Calcium entry
- Changes in cytoskeleton
Can cause calcium to rush in
Can cause calcium release from mitochondria
Compromises cell membrane - Mitochondrial damage
Interferes with ATP production
Can cause calcium release - Intracellular calium homeostasis
Damage to calcium pumps and sponges interferes with the mechanisms keeping the calcium conc at the correct levels
Give three tertiary events
Steatosis
Due to increased lipid excretion and synthesis and decreased metabolism
Accumulation of fats that the liver stores and blisters on the membrane
Blebbing
On plasma membrane
Rupture of these leads to cell death
Define necrosis and apoptosis
Necrosis is messy inflammatory cell death
Apoptosis is non inflammatory and requires atp is is efficient and regulated
Consequences of intracellular calcium
Alterations in cytoskeleton
Leads to membrane blebs
Calcium activated phospholipases
Enzymes which digest fat therefore the membranes and therefore starts to digest the cell
Calcium activated proteases
‘Calpains’ digest proteins and fat and protein eating enzymes which are activated by high calcium find means the cell is being digested
Nucleases also digest dna
Decreased ATP production
Because ca2+ destroys the potential differences across the membrane in the mitochondria and inhibits ATP production
ROS generation
Increase in calcium means more superoxide and h202 generates therefore more damage to proteins and more cell death
Calcium activated signalling pathways
Stimulates apoptosis by initiating cell death mechanisms
