Toxicological Targets Russell L. Carr Department of Basic Sciences College of Veterinary Medicine
Toxicology Definitions = study of poisons Poison = any agent capable of producing a deleterious response in a biological system Types of Poisons 1. Toxin = toxic substances which are produced naturally Animal venoms (snakes, spiders, scorpions, insects, bacteria, etc ) Plant poisons (poison ivy, mushrooms, etc ) 2. Toxicant= toxic substances which are produced by or are a byproduct of man s activity (pesticides, fertilizers, metals, oil spills, industrial solvents, etc ) For simplicity, we will refer to a poisonous chemical as a toxicant but remember some toxins may have similar effects.
The Target Poisons are toxic because they interfere with some sort of normal physiological process resulting in problems. Poisons may interact at multiple sites within an organism but not all of these interactions result in deleterious effects. Usually, a poison has a specific molecule with which it interacts and it is that interaction that causes the toxicity. That specific molecule is what we call the Ultimate target. 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 Many toxicants mimic endogenous ligands. endogenous= the body produces these molecules ligand = molecule which binds and elicit a response These toxicants activate protein target molecules. The target molecules can be such things as receptors or enzymes. b) Inhibition Many toxicants inhibit the function of target molecules. 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 Many toxicants alter the conformation or structure of a protein. The efficient function of all proteins depends on their 3-dimensional structure (conformation). Why? Proteins are composed of a chain of amino acids. 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. 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 DNA contains the information that codes for proteins. The information must be maintained in the correct sequence for correct construction of proteins. 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? Proteins may be non-functional. Proteins may be inefficient and not work to full capacity. 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 Cross-linking of the cell skeleton proteins disrupting the position of the cellular molecules. 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. In general, cross-linking imposes functional and structural constraints on the linked molecules. b) Degradation and fragmentation of proteins Some snake toxins are actually enzymes that degrade cellular proteins. Some toxicants can induce DNA to fragment (literally fall to pieces). These types of effects are not very common.
What are the Outcomes of Interactions between the Toxicant and the Target? 3. Neoantigen Formation Some toxicants can actually act as antigens (the term for the bad stuff that antibodies recognize and bind to in the body). These toxicants induce the body to make antibodies against the toxicant. 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? It all has to do with the Organization of the Body First, let us introduce the Scheme of Organization 1. Cellular Components Within one cell, the components work together to keep the cell running. 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 Alteration of a cellular component has the potential to alter the overall functioning of a cell. 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? Scheme of Organization 3. Tissue Cells of different types often work together within a tissue to perform a function. The inability of one type of cell to function may negatively effect the functioning of cells of other types in a tissue. 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. Some of these cells induce stimulation whereas others induce inhibition of that stimulation. 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. If the cells which inhibit stimulation are inappropriately stimulated, inhibition of the stimulatory response will occur and no output will occur. Thus, the overall performance of the tissue is disrupted.
Why is Interference With Cell Function Important? Scheme of Organization 4. Organ Organs (brain, heart, kidney) are composed of multiple type of tissue and each tissue plays a role in the function of the organ. For example, let us consider the kidney: Certain tissues of the kidney filter the blood and excrete fluid and macromolecules into the urine (glomerulus). Other tissues of the kidney selectively reabsorb water and other macromolecules that the body needs to keep (tubules). There are certain markers (i.e., inulin) which are filtered out into the urine are only reabsorbed when decreased filtration is required. 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. This negatively affects the function of the entire kidney.
Why is Interference With Cell Function Important? Scheme of Organization 4. Systems The different systems (nervous, circulatory, respiratory) are composed of multiple organs and the systems interact with each other to keep the body functioning. For example: The cardiovascular system feeds all of the other systems and blockage of or reduction in blood flow will negatively affect each system. The renal system (kidneys) filter the blood to remove wastes from the other systems. The respiratory system supplies oxygen and the digestive system supplies nutrients for use by other systems and these are carried by the blood. The nervous system controls the functions of the cardiovascular, respiratory, and digestive systems and is in turn fed by these systems. Thus, all the systems work together to help one another.
Why is Interference With Cell Function 5. Organism Important? 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 The interaction between systems allows the effects of a toxicant to be widespread. For example, take the neurotransmitter acetylcholine (in nervous system). The receptors for acetylcholine are present in many different tissues/organs and there are many toxicants/drugs which can cause the stimulation of these receptors. The response of stimulation will vary according to the tissue/organ involved: Stimulation of a heart cell results in a decrease in frequency of contraction. Stimulation of the salivary gland results in excessive secretion. Stimulation of a muscle cell results in excessive contraction. 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 Component No Response? Toxicant Cell Why no response? Signal not strong enough to elicit a response. Cell compensates such that inappropriate response does not occur. The damage in minimal and the cell repairs the damage. 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 Component No Response? Toxicant Cell Response To get that response: Signal must be strong enough to elicit a response. Signal so strong the cell cannot compensate. Tissue The damage is to a point where the cell cannot repair the damage but does not die. 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
Dysregulation of gene expression Cell Division Cell Death Cancer Birth defects Tissue degradation Missing tissues Cell Regulation Protein Synthesis Non-functional proteins Proteins have negative actions Dysregulation of Ongoing cell activity Excitable Cells Other Cells Excessive Excitation Depression of Activity Metabolic Alteration Increase Glandular Secretion Role of Target Molecule Energy Production Impaired internal maintenance Ion Regulation Protein Synthesis Cell Injury Cell Death Cell Maintenance Membrane Function Impaired external maintenance Impaired Functioning between Systems Liver damage build up of toxic substances Kidney damage build up of wastes in blood
What are Some Good Targets? A common target of toxicants are Enzymes. What are Enzymes? Enzymes are proteins that function to catalyze a reaction. By catalyze, we mean that they take a substance (Substrate) and change it to something else (Product). E + S S S S S E E + P P E + S ES E + P
Enzymes Normally enzymes have two functions: 1. Activation The enzyme reaction can results in a chemical product which induces some sort of physiological or biochemical response. 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 The enzyme reaction destroys a substrate which was inducing some sort of physiological or biochemical response. 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 Many enzymes have two sites to which things can bind to: 1. Active Site This is the business portion of the enzyme which binds to the substrate and produces the product. 2. Allosteric Site 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 E Active Site
Toxicants and Enzymes The interaction of a toxicant with an enzyme can produce several outcomes with respect to functioning of the enzyme. 1. Inhibition The toxicant can bind to the active site and block the production of product. The toxicant which does this is called an inhibitor (I) I E + S S S S 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. This is the most common type of toxicant-enzyme interaction. I E
2. Reduced Activity E Toxicants and Enzymes 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 S S S E E + P 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. S
Toxicants and Enzymes 3. Enhanced Activity or Activation The toxicant can bind to the allosteric site and cause the enzyme to speed up and work more efficiently than normal. E + S S S S S In this case, substrate is eliminated faster and more product is produced. E P P + P P P 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. E
Toxicants and Enzymes How can you model the effects of toxicants on enzymes? Start Simple and Work Your Way Up First, find out a little about the reaction itself. E + S ES E + P When working normally, all enzymes have: A specific affinity for their substrate (K m ) A specific rate of reaction (V max ) E These can be determined experimentally. When measuring the effects of toxicants which bind to the allosteric site, either K m or V max or both will be affected. These rates can also be determined experimentally. 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 If dealing with an inhibitor of the enzyme, you can determine the rates of inhibition. Where: E + I [EI] R EI k 1 = rate of association of inhibitor and enzyme k -1 = rate of disassociation of inhibitor and enzyme k 1 k -1 [EI] R = reversible enzyme-inhibitor complex K p = rate of covalent binding with inhibitor The entire reaction has a rate as well: K p k 1 = rate of inhibition E + I k i EI
Toxicants and Enzymes 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. This might involve using the entire cell and measuring the response at the cellular level once the toxicant is introduced. This might include: measuring the response directly affected by the toxicant; measuring responses indirectly affected by the toxicant-enzyme interaction; measuring the effects on the overall health of the cell. 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. However, with every step that is taken towards looking at the effect on the whole organism, the complexity probably increases exponentially.
Another Good Target Another common target of toxicants are Receptors. What are Receptors? Receptors are proteins that endogenous chemicals bind to (ligand) in order to elicit a response of some sort. The response physically associated with the receptor can include: activation/deactivation of a protein opening a channel to let something in or out The overall response can include: increased/decreased synthesis of a protein; increased/decreased secretion from a gland; contraction/relaxation of a muscle; constriction/dilation of a blood vessel; and so on. Thus, the interaction of a ligand can induce a local response that through various mechanisms elicits a much greater response throughout the body.
Receptors can be located: Receptor Locations 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. Ligand binds to receptor. Ion channel opens + + Ions (sodium, potassium, chloride, calcium, etc ) on outside of membrane flow inward. Cell Membrane + + The influx of these ions can: Alter the electrical properties of the membrane which can induce a cellular response or inhibit a cellular response (this involves ions that are charged). 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. In 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 Cell Membrane G EC 2.Associated with proteins and enzyme cascades and cell signaling. Ligand binds to receptor. Activates protein (G-protein). G-protein activates initial Enzyme Complex. Activated enzyme complex reacts with its substrate which activates next enzyme and so on down the enzyme cascade. Ca ++ release or uptake Initiates Cellular Machinery Protein Synthesis
Receptors on Organelles or in the Nucleus Cell Membrane G EC 3. Associated with organelles (may be associated with an ion channel or a protein on the organelle) or in the nucleus (associated with DNA). Activate cellular machinery (short term response). Activate gene expression and protein synthesis (long term response). Ca ++ release or uptake Initiates Cellular Machinery Protein Synthesis
Toxicant-Mediated Effects on Receptor-Enzyme Cascade Complexes The toxicant binds to the receptor causing a stimulatory response. Cell Membrane G EC Ca ++ release or uptake Initiates Cellular Machinery Protein Synthesis
Toxicant-Mediated Effects on Receptor-Enzyme Cascade Complexes Cell Membrane G EC The toxicant binds to the receptor but blocks the action of the normal ligand so no response. As with other receptors, certain toxicants can 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 enzymetoxicant interactions. Cell Membrane G EC Toxicants acting on the G-protein Cell Membrane G EC Toxicants acting on the initial Enzyme Complex
Toxicant-Mediated Effects on Receptor-Enzyme Cascade Complexes Cell Membrane G EC Toxicants acting on the enzymes of the Enzyme Cascade Toxicants acting on internal processes required for the intracellular machinery. Toxicants acting on the processes involved in gene expression and protein synthesis. Ca ++ release or uptake
Receptors in the Cytoplasm The receptors in the cytoplasm are designed to bind to endogenous ligands that can cross the cell membrane. Once they bind to the ligand, they move to the nucleus. Response Protein Synthesis In the nucleus, they bind to DNA and stimulate gene transcription leading to protein synthesis. The resulting proteins that are synthesized will elicit a response. Steroid hormones (estrogen and testosterone) 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 ligandreceptor complex to specific response elements on the DNA.
Allosteric Sites on Receptors As with enzymes, some receptors have allosteric binding sites. As with enzymes, binding of a ligand to this site will alter the response of the receptor.
Toxicants and Receptors How can you model the effects of toxicants on receptor? The same principle we introduced for enzymes applies here: Start Simple and Work Your Way Up The reaction is similar to that of enzymes but slightly different. E + R ER As with enzymes, receptors have values associated with them: A specific affinity for their endogenous ligand (K d ) When measuring the effects of toxicants which bind to the allosteric site, the K d 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. A specific number of receptors (B max )
Toxicants and Receptors A specific number of receptors (B max ) Prolonged exposure to toxicants which active receptors will cause the body to decrease the B max to reduce the activation (down-regulation). Prolonged exposure to toxicants which block receptors will cause the body to increase the B max to improve the chance for activation (up-regulation). These values can be determined experimentally. 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. 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. Remember the further you move up, the more complex the situation becomes.
Reactive Oxygen Species Other common toxicants are Reactive Oxygen Species. These are produced naturally by the body but can be produced following exposure to environmental chemicals. These are gotten rid of by Anti-oxidants. The body has anti-oxidants to scavenge these compounds when you produce them naturally. 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 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 The Reactive Oxygen Species are highly reactive and will bind to almost anything including enzymes, receptors, and DNA. 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. 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). 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 The toxic effects of every poison is directly related to cell dysfunction. The cell dysfunction induced by a toxicant is the result of its interaction with a target molecule. This interaction can occur at: the business region (ligand or substrate binding site) other regions (allosteric site or other sites which induce a conformational change in structure) 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. This chain of effects can cause dysfunction at various levels including the cell, tissue, organ, system, and finally whole organism.