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Toxicological Targets

Russell L. Carr
Department of Basic Sciences
College of Veterinary Medicine
Definitions
Ø Toxicology = study of poisons
Ø Poison = any agent capable of producing a deleterious
response in a biological system
Types of Poisons
1. Toxin = toxic substances which are produced naturally
Ø Animal venoms (snakes, spiders, scorpions, insects, bacteria,
etc…)
Ø Plant poisons (poison ivy, mushrooms, etc…)
2. Toxicant = toxic substances which are produced by or are a by-
product of man’s activity
(pesticides, fertilizers, metals, oil spills, industrial solvents, etc…)
Ø For simplicity, we will refer to a poisonous chemical as a
toxicant but remember some toxins may have similar effects.
The Target
Ø Poisons are toxic because they interfere with some sort of normal
physiological process resulting in problems.

Ø Poisons may interact at multiple sites within an organism but not


all of these interactions result in deleterious effects.

Ø Usually, a poison has a specific molecule with which it interacts


and it is that interaction that causes the toxicity.

Ø That specific molecule is what we call the


“Ultimate target.”

Ø If a poison interacts with other molecules but these do not result


in toxicity, we call these “non-target sites.”
What Makes a Good Target?
Three attributes of a target.
1. A target must be accessible to the toxicant.
The toxicant has to be able to reach the target at a high enough
concentration in order for toxicity to occur.
2. The target must be reactive or possess the appropriate
configuration that allows the toxicant to interact with it.
This relies on the affinity of the target for the toxicant.
If it cannot react with the target, no toxicity will occur.
3. The target must have a critical function related to the observed
toxicity.
For example, if a toxicant is neurotoxic, it must bind to some molecule in
the nervous system. If it also binds to some molecules elsewhere (say in
the blood), these binding sites are not considered the target because they
are not related to the ultimate toxicity. They are “non-target sites”.
All three attributes must be met in order to characterize a target.
What Can Be a Target?
Ø Generally, a target is a component of a cell.

Ø A toxicant interacting with its target can interfere with


the appropriate function(s) of the cell.
What are the Outcomes of Interactions
between the Toxicant and the Target?
1. Dysfunction of Target Molecules
a) Activation
Ø Many toxicants mimic endogenous ligands.
v endogenous = the body produces these molecules
v ligand = molecule which binds and elicit a response
Ø These toxicants activate protein target molecules.
Ø The target molecules can be such things as receptors or enzymes.
b) Inhibition
Ø Many toxicants inhibit the function of target molecules.
Ø The target molecules can be such things as receptors, enzymes,
ion channels, ion transporters, or the proteins that make up the cell
skeleton (the cellular infrastructure that all maintains the position
of all cellular molecules).
What are the Outcomes of Interactions
between the Toxicant and the Target?
1. Dysfunction of Target Molecules (continued)
c) Impaired Protein Function
Ø Many toxicants alter the conformation or structure of a protein.
Ø The efficient function of all proteins depends on their 3-dimensional
structure (conformation). Why?
v Proteins are composed of a chain of amino acids.
v However, this chain is bent and twisted such that certain amino
acids from different regions of the straight chain are clustered
together to form the “business” region of the protein.
v Anything that binds to a protein (even if not at the “business”
region of a protein) and alters this bending and twisting will
alter the ability of the protein to function efficiently.
What are the Outcomes of Interactions
between the Toxicant and the Target?
1. Dysfunction of Target Molecules (continued)
d) Interference with the Template Function of DNA
Ø DNA contains the information that codes for proteins.
Ø The information must be maintained in the correct sequence for
correct construction of proteins.
Ø Any toxicant which binds to DNA and disrupts this sequence can
alter the coding for proteins and thus, alters its conformation
(folding). What does wrong folding result in?
v Proteins may be non-functional.
v Proteins may be inefficient and not work to full capacity.
v Proteins may be enhanced and work too good. Some
proteins have degradation responsibilities and if are
hyperactive will result in negative effects.
What are the Outcomes of Interactions
between the Toxicant and the Target?
2. Destruction of Target Molecules
a) Cross-linking induced by a toxicant
Ø Cross-linking of the cell skeleton proteins disrupting the
position of the cellular molecules.
Ø Cross-linking of DNA to DNA or DNA to proteins such that DNA
doesn’t code correctly and the cell is unable to sustain life.
Ø In general, cross-linking imposes functional and structural constraints
on the linked molecules.
b) Degradation and fragmentation of proteins
Ø Some snake toxins are actually enzymes that degrade
cellular proteins.
Ø Some toxicants can induce DNA to fragment (literally fall to
pieces).
Ø These types of effects are not very common.
What are the Outcomes of Interactions
between the Toxicant and the Target?
3. Neoantigen Formation
Ø Some toxicants can actually act as antigens (the term for the bad
stuff that antibodies recognize and bind to in the body).
Ø These toxicants induce the body to make antibodies against the
toxicant.
Ø This is a problem if a portion of the toxicant is bound to the
membrane of immune cells (this is commonly done as part of the
clean up process of the immune system) and the immune system
attacks and destroys these cells.

** As you can tell, the majority of things that a toxicant can act
on are Cellular Components and interaction of the toxicant
with these components interferes with Cell Function **
Why Is Interference With Cell Function
Important?
Ø It all has to do with the Organization of the Body
Ø First, let us introduce the Scheme of Organization
1. Cellular Components
Ø Within one cell, the components work together to keep the cell running.
Ø Failure of one component to operate effectively or over-stimulation of one
component may interfere with the ability of the other components to do their
job.
2. Cell
Ø Alteration of a cellular component has the potential to alter the overall
functioning of a cell.
Ø If a toxicant targets a certain type of cells, all of the cells of that type can be
affected and their contribution to maintaining normal functioning is eliminated
or diminished.
Why is Interference With Cell Function
Important?
Ø Scheme of Organization
3. Tissue
Ø Cells of different types often work together within a tissue to perform a function.
Ø The inability of one type of cell to function may negatively effect the
functioning of cells of other types in a tissue.
Ø A good example of cooperation of different cell types within a tissue occurs in
the brain where there are multiple cell types can be found in the same region.
v Some of these cells induce stimulation whereas others induce inhibition of
that stimulation.
v If the cells which induce stimulation are inappropriately stimulated, the
normal influence of the inhibitory cells will be overridden and the overall
output from that region will be hyperexcitatory.
v If the cells which inhibit stimulation are inappropriately stimulated,
inhibition of the stimulatory response will occur and no output will occur.
v Thus, the overall performance of the tissue is disrupted.
Why is Interference With Cell Function
Important?
Ø Scheme of Organization
4. Organ
Ø Organs (brain, heart, kidney) are composed of multiple type of tissue and each
tissue plays a role in the function of the organ.
Ø For example, let us consider the kidney:
v Certain tissues of the kidney filter the blood and excrete fluid and
macromolecules into the urine (glomerulus).
v Other tissues of the kidney selectively reabsorb water and other
macromolecules that the body needs to keep (tubules).
v There are certain markers (i.e., inulin) which are filtered out into the urine
are only reabsorbed when decreased filtration is required.
v If a toxicant affects a target in the tubules and causes non-selective
resorption, the tubules become more permeable to inulin, then inulin
causes the glomerulus to decrease urine filtration.
v This negatively affects the function of the entire kidney.
Why is Interference With Cell Function
Important?
Ø Scheme of Organization
4. Systems
Ø The different systems (nervous, circulatory, respiratory) are composed of
multiple organs and the systems interact with each other to keep the body
functioning.
Ø For example:
v The cardiovascular system feeds all of the other systems and blockage of
or reduction in blood flow will negatively affect each system.
v The renal system (kidneys) filter the blood to remove wastes from the
other systems.
v The respiratory system supplies oxygen and the digestive system supplies
nutrients for use by other systems and these are carried by the blood.
v The nervous system controls the functions of the cardiovascular,
respiratory, and digestive systems and is in turn fed by these systems.
v Thus, all the systems work together to help one another.
Why is Interference With Cell Function
Important?
5. Organism

Interference with the normal function of a cell can


cause dysfunction at the cellular level which can
have resounding effects all the way up to the level of
the organism.
The Action of a Toxicant on its Target
can Affect Many Different Systems
Ø The interaction between systems allows the effects of a toxicant to
be widespread.
Ø For example, take the neurotransmitter acetylcholine (in nervous system).
vThe receptors for acetylcholine are present in many different
tissues/organs and there are many toxicants/drugs which can cause the
stimulation of these receptors.
vThe response of stimulation will vary according to the
tissue/organ involved:
ü Stimulation of a heart cell results in a decrease in frequency of
contraction.
ü Stimulation of the salivary gland results in excessive secretion.
ü Stimulation of a muscle cell results in excessive contraction.
v So basically, if a toxicant mimics acetylcholine,its toxic effects will not
only in occur in the nervous system and affect other systems through
that route but it can have direct effects on the other systems as well.
Whether or Not a Toxicant Will Have an
Effect Is Determined at the Cellular Level
Cellular No Response?
Component

Toxicant Cell

Why no response?
v Signal not strong enough to elicit a response.
v Cell compensates such that inappropriate response does not occur.
v The damage in minimal and the cell repairs the damage.
v The damage is so severe and the cell undergoes spontaneous death
(apoptosis) and is replaced.
Whether or not a Toxicant will have an
Effect is Determined at the Cellular Level
Cellular No Response?
Component

Toxicant Cell
Response

To get that response:


v Signal must be strong enough to elicit a response.
v Signal so strong the cell cannot compensate. Tissue
v The damage is to a point where the cell cannot
repair the damage but does not die.
v The damage is so severe and the cell undergoes spontaneous death
(apoptosis). Either too many cells are affected to be effectively replaced
or the tissue is such that cells cannot be replaced (such as the brain).
Toxicant-induced Cellular Dysfunction will
depend on the Role of the Target Molecule
Which is Affected
•Cancer
Cell Division •Birth defects

Dysregulation of •Tissue degradation


Cell Death
gene expression •Missing tissues
Protein Synthesis •Non-functional proteins
•Proteins have negative actions
Cell
Regulation

•Excessive Excitation
Excitable Cells •Depression of Activity
Dysregulation of
Ongoing cell activity
Other Cells •Metabolic Alteration
•Increase Glandular Secretion

Role of
Target
Molecule
Energy Production

Ion Regulation •Cell Injury


Impaired
internal maintenance •Cell Death
Protein Synthesis

Membrane Function
Cell
Maintenance

•Liver damage – build up of


Impaired
Impaired toxic substances
Functioning
external maintenance •Kidney damage – build up of
between Systems
wastes in blood
What are Some Good Targets?
Ø A common target of toxicants are Enzymes.
Ø What are Enzymes?
Ø Enzymes are proteins that function to catalyze a reaction.
Ø By catalyze, we mean that they take a substance (Substrate) and change
it to something else (Product).

S
P
S
E S E E
+ S +
P
S

E+S ES E+P
Enzymes
Ø Normally enzymes have two functions:
1. Activation
v The enzyme reaction can results in a chemical product which
induces some sort of physiological or biochemical response.
v The enzyme can also react with a cellular protein (change its
conformation) such that it is now free to react with chemicals
already present and thereby, induce a physiological response.
2. Inactivation
v The enzyme reaction destroys a substrate which was inducing
some sort of physiological or biochemical response.
v The enzyme can deactivate a cellular protein (change its
conformation) such that is insensitive to the effects of the
chemicals it would normally react with to induce a response.
Enzymes
Ø Many enzymes have two sites to which things can bind to:
1. Active Site
v This is the “business” portion of the enzyme which binds to the
substrate and produces the product.
2. Allosteric Site
v This is a site to which compounds can bind to that will regulate the
function of the enzyme (make it faster or slower).

Allosteric Site

Active Site
Toxicants and Enzymes
Ø The interaction of a toxicant with an enzyme can produce
several outcomes with respect to functioning of the enzyme.
1. Inhibition
v The toxicant can bind to the active site and block the production of
product. The toxicant which does this is called an inhibitor (I)
S
S
I E + I E
S
S
v The enzyme is inhibited and cannot function so either:
• The response being mediated by its substrate will continue.
• The response that needs to be mediated by the product will not occur.
v This is the most common type of toxicant-enzyme interaction.
Toxicants and Enzymes
2. Reduced Activity
v The toxicant can bind to the allosteric site and cause the enzyme to
slow down and not work as efficiently as before.
S S
S
E S E E
+ S + P

S S
v In this case, more substrate remains and less product is produced.
• If you need the product to mediate a response, the response
will not be as strong because less product is available
• If you need to stop a response being mediated by the
substrate, the time to stop the response will be delayed
because substrate is not broken down.
Toxicants and Enzymes
3. Enhanced Activity or Activation
v The toxicant can bind to the allosteric site and cause the enzyme to
speed up and work more efficiently than normal.
S P
S P
E S E E
+ S + P
P
S P

v In this case, substrate is eliminated faster and more product is produced.


• If you need the product to mediate a response but don’t need that
much of it, this may cause the response to be too strong.
• If you need the substrate to accomplish a response, activation of
the enzyme may eliminate the required substrate and the response
will not occur.
Toxicants and Enzymes
Ø How can you model the effects of toxicants on enzymes?
Ø Start Simple and Work Your Way Up
Ø First, find out a little about the reaction itself.
E+S ES E+P
Ø When working normally, all enzymes have:
v A specific affinity for their substrate (Km)
v A specific rate of reaction (Vmax) E
v These can be determined experimentally.
Ø When measuring the effects of toxicants which bind to the allosteric site,
either Km or Vmax or both will be affected.
v These rates can also be determined experimentally.
Ø Using other kinetic formulas, you can also determine the affinity of the
toxicant that binds to the allosteric site as well as the rate of association and
dissociation of the toxicant with the allosteric site.
Toxicants and Enzymes
Ø If dealing with an inhibitor of the enzyme, you can determine the rates of
inhibition.
k1 Kp
E+I [EI]R EI
k-1
Ø Where:
k1 = rate of association of inhibitor and enzyme
k-1= rate of disassociation of inhibitor and enzyme
[EI]R = reversible enzyme-inhibitor complex
Kp= rate of covalent binding with inhibitor
Ø The entire reaction has a rate as well:
ki
E+I EI
k1 = rate of inhibition
Toxicants and Enzymes
Ø After determining the effect of different levels of the toxicant at the
enzyme level, you can then move up and measure the effect of those
changes in enzyme rate on the response that the enzyme’s substrate or
product induces.
Ø This might involve using the entire cell and measuring the response at the
cellular level once the toxicant is introduced.
Ø This might include:
v measuring the response directly affected by the toxicant;
v measuring responses indirectly affected by the toxicant-enzyme interaction;
v measuring the effects on the overall health of the cell.
Ø After this, you could progress to measuring the toxicant effects on cell to
cell interactions, cell to tissue interactions, tissue to tissue interactions,
tissue to organ interactions, and so on….
Ø However, with every step that is taken towards looking at the effect on
the whole organism, the complexity probably increases exponentially.
Another Good Target
Ø Another common target of toxicants are Receptors.
Ø What are Receptors?
Ø Receptors are proteins that endogenous chemicals bind to
(ligand) in order to elicit a response of some sort.
Ø The response physically associated with the receptor can include:
v activation/deactivation of a protein
v opening a channel to let something in or out
Ø The overall response can include:
v increased/decreased synthesis of a protein;
v increased/decreased secretion from a gland;
v contraction/relaxation of a muscle;
v constriction/dilation of a blood vessel;
v and so on….
Ø Thus, the interaction of a ligand can induce a local response that through
various mechanisms elicits a much greater response throughout the body.
Receptor Locations
Ø Receptors can be located:

On the cell membrane

On the membrane
of organelles or
in the nucleus
Cell

Floating in the cytoplasm


Receptors on the Cell Membrane
1. Associated with ion channels.
Ø Ligand binds to receptor.
Ø Ion channel opens
+ Ø Ions (sodium, potassium, chloride, calcium,
+ etc…) on outside of membrane flow inward.
Cell Ø The influx of these ions can:
Membrane +
v Alter the electrical properties of the
+ membrane which can induce a cellular
response or inhibit a cellular response
(this involves ions that are charged).
v Bind to cellular components which
turns on cell signaling and gene
expression.
Toxicant-Mediated Effects on Receptor-Ion
Channel Complexes

+
+
Cell
Membrane +

Toxicant binds to the receptor causing a


stimulatory response (acting as an agonist).
Toxicant-Mediated Effects on Receptor-Ion
Channel Complexes

Cell
Membrane

Toxicant binds to the receptor but blocks the action of the


normal ligand so no response (acting as an antagonist).
Toxicant-Mediated Effects on Receptor-Ion
Channel Complexes

+ +
+
Cell
Membrane

Toxicant blocks the ion channel so even though the normal


ligand can bind to the receptor, there is no response.
vIn addition, certain toxicants can also bind to the channel and alter:
the rate at which the channel opens
or
the rate at which it closes.
Receptors on the Cell Membrane
2.Associated with proteins and enzyme
cascades and cell signaling.
Ø Ligand binds to receptor.
Ø Activates protein (G-protein).
Ø G-protein activates initial Enzyme Complex.
Cell G Ø Activated enzyme complex reacts with its
Membrane EC substrate which activates next enzyme and
so on down the enzyme cascade.

Ca++
release
Initiates Cellular
or uptake Machinery

Protein Synthesis
Receptors on Organelles or in the Nucleus
3. Associated with organelles (may be
associated with an ion channel or a protein
on the organelle) or in the nucleus
(associated with DNA).
Ø Activate cellular machinery (short term
Cell G response).
Membrane EC Ø Activate gene expression and protein synthesis
(long term response).

Ca++
release
Initiates Cellular
or uptake Machinery

Protein Synthesis
Toxicant-Mediated Effects on
Receptor-Enzyme Cascade Complexes

The toxicant binds to the receptor causing a


stimulatory response.

Cell G
Membrane EC

Ca++
release
Initiates Cellular
or uptake Machinery

Protein Synthesis
Toxicant-Mediated Effects on
Receptor-Enzyme Cascade Complexes

The toxicant binds to the receptor but blocks


the action of the normal ligand so no
response.
Cell G vAs with other receptors, certain toxicants can
Membrane EC also bind to the channel and alter the rate at
which it activates the G-protein.
Toxicant-Mediated Effects on
Receptor-Enzyme Cascade Complexes
The toxicant can interact at other sites in the
enzyme cascade (this goes back to enzyme-
toxicant interactions.

Cell G Ø Toxicants acting on the G-protein


Membrane EC

Cell G Ø Toxicants acting on the initial Enzyme Complex


Membrane EC
Toxicant-Mediated Effects on
Receptor-Enzyme Cascade Complexes
Ø Toxicants acting on the enzymes of the
Enzyme Cascade
Ø Toxicants acting on internal processes required for
the intracellular machinery.
Cell G Ø Toxicants acting on the processes involved in gene
Membrane EC expression and protein synthesis.

Ca++
release
or uptake
Receptors in the Cytoplasm
Ø The receptors in the cytoplasm are designed to bind to endogenous ligands that can
cross the cell membrane.
Ø Once they bind to the ligand,
they move to the nucleus.
Ø In the nucleus, they bind to
DNA and stimulate gene
transcription leading to protein
synthesis.
Ø The resulting proteins that
are synthesized will elicit a
Response response.
Ø Steroid hormones
(estrogen and testosterone)
Protein Synthesis
are good examples of
ligands for these receptors.
Receptors in the Cytoplasm
The toxicant enters the cell, binds to the receptor, and causes a
response.

Response

Protein Synthesis
Receptors in the Cytoplasm
The toxicant binds to the receptor but blocks the action of the
normal ligand so no response.
Receptors in the Cytoplasm
Some toxicants can interfere with the binding of the ligand-
receptor complex to specific response elements on the DNA.
Allosteric Sites on Receptors
ØAs with enzymes, some receptors have allosteric binding sites.
ØAs with enzymes, binding of a ligand to this site will alter the
response of the receptor.
Toxicants and Receptors
Ø How can you model the effects of toxicants on receptor?
Ø The same principle we introduced for enzymes applies here:
Start Simple and Work Your Way Up
Ø The reaction is similar to that of enzymes but slightly different.
E+R ER
Ø As with enzymes, receptors have values associated with them:
v A specific affinity for their endogenous ligand (Kd)
• When measuring the effects of toxicants which bind to the allosteric
site, the Kd will be affected.
• So you can also have measures of affinity for the allosteric site.
• The receptor will also have a certain affinity for the toxicant that
binds to its active site.
v A specific number of receptors (Bmax)
Toxicants and Receptors
v A specific number of receptors (Bmax)
• Prolonged exposure to toxicants which active receptors will cause the
body to decrease the Bmax to reduce the activation (down-regulation).
• Prolonged exposure to toxicants which block receptors will cause
the body to increase the Bmax to improve the chance for activation
(up-regulation).
v These values can be determined experimentally.
Ø After determining the basic interactions of the toxicant with the receptor,
you can do the same types of things as with enzymes and move up and
measure the effect of those interactions on the response that the receptor
mediates.
Ø You can also progress to measuring the toxicant effects on cell to cell
interactions, cell to tissue interactions, tissue to tissue interactions, tissue
to organ interactions, and so on….
Ø Remember the further you move up, the more complex the situation
becomes.
Reactive Oxygen Species
Ø Other common toxicants are Reactive Oxygen Species.
Ø These are produced naturally by the body but can be produced
following exposure to environmental chemicals.
Ø These are gotten rid of by Anti-oxidants.
Ø The body has anti-oxidants to scavenge these compounds
when you produce them naturally.
Ø Dietary intake can add anti-oxidants. This is why we have the
big push in the “natural supplements” industry to get you to
buy their anti-oxidant products
Ø When you are exposed to an environmental chemical, the
breakdown of that chemical causes these Reactive Oxygen
Species to be produced above the normal levels of production
by your body.
Reactive Oxygen Species
Ø The Reactive Oxygen Species are highly reactive and will bind
to almost anything including enzymes, receptors, and DNA.
Ø This binding can damage proteins (enzymes and receptors) and
cause a 3-D conformational change in protein structure such
that they no longer function correctly.
Ø Binding to DNA by certain species will cause the
fragmentation of DNA we mentioned earlier (this is one of the
few things that causes fragmentation of DNA).
Ø So, in essence, we can have alteration of cell function by
changing the structure of the cellular components through the
action of non-specific toxicants as well as specific toxicants.
Conclusion
Ø The toxic effects of every poison is directly related to cell
dysfunction.
Ø The cell dysfunction induced by a toxicant is the result of its
interaction with a target molecule.
Ø This interaction can occur at:
v the “business” region (ligand or substrate binding site)
v other regions (allosteric site or other sites which induce a
conformational change in structure)
Ø This interaction can cause effects not only on the target
molecule itself but on other molecules which require the target
molecule to be fully functional .
Ø This chain of effects can cause dysfunction at various levels
including the cell, tissue, organ, system, and finally whole
organism.