Exam 1 Flashcards
Lecture 1
- List the five general steps of toxicological analysis
- List the basic steps for formulating a toxicological differential list
- Explain why additional information is needed before requesting toxicological analyses
- Describe what samples (and how much) to collect for antemortem and postmortem toxicological analyses
- Identify reliable sources of information about toxicants, including MTDs and MLDs
Definitions
What is
-Poison
-Poisonous
-Toxin
-Toxicant
-Venom
-Toxicosis
-Intoxication
-Poisoning
-Toxicity
-Poison: a substance that is capable of causing the illness or death of a living organism when introduced or absorbed
-Poisonous: a pathological condition
-Toxin: specific product of metabolic activities of a living organism, unstable and notably toxic when introduced into the tissues and typically capable of inducing antibody formation
-Toxicant: any toxic substance, can be poisonous and they may be man-made or naturally occurring, found in air, water, soil, or food.
-Toxic: containing or being poisonous material especially when capable of causing death or serious debilitation.
-Venom: toxic substance produced by some animals (as snakes, scorpion or bees) that is injected into prey by biting or stinging. It can be lethal, injurious, or broadly effect
-Toxicosis: a pathologic condition cause by a toxin or poison.
-Intoxication: The stage of being intoxicated, especially by alcohol or by any toxin.
-Poisoning: the action of administering a poison to a person or animal.
-Toxicity: the quality of being poisonous or toxic.
-Xenobiotic: general term referring to any chemical foreign to an organism, compound not occurring within the normal metabolic pathways of a biologic system.
**Depending on the compound and the level of exposure, interactions between xenobiotics and animals can be benign, therapeutic, or toxic in nature.
What is ADME? What is DAMNITV?
The disposition of a xenobiotic is what the animal’s body does to that compound following exposure and consists of ADME
Absorption
Distribution
Metabolism
Excretion
Degenerative, developmental
Anatomic
Metabolic
Neoplastic
Infectious, inflammatory, Immune-mediated, Idiopathic, Iatrogenic.
Traumatic, Toxic
Vascular.
Toxicokinetics refers to the quantification and determination of the time course of the disposition of ADME.
- Bioavailability
- Volume of Distribution
- Clearance
- Half-life
- One-compartment model
- First and zero order kinetics.
Toxicodynamics describes what a toxicant does physiologically.
What are the seven most deadly (to humans) chemical substances?
- Botulinum toxin
- Tetanus toxin
- Diphtheria toxin (Cornybacterium diphtheriae)
- Dioxin (manufactured)
- Muscarine (Amanita mushrooms)
- Bufotoxin ( from the common toad- Bufo)
- Sarin (manufactured).
- List the five general steps of toxicological analysis
What to do?
-Obtain a complete history
-Consult with a veterinary toxicologist or animal poison center
-Perform comple PE
-Perform an exposure assessment
-Perform an postmortem examination
-Collect specimens suitable for toxicologic testing
- List the toxicological differentials: need to know what we’re looking for and how to find it
a. Signalment: predisposing factors. Species, age, gender, additionally nutritional status, disease status, etc.
b. History: feeding, environmental, medical history. Potential hazards, such as plants, contaminated water, insecticides, herbicides, etc.
c. Clinical signs
d. Other diagnostics: use when you suspect something not just because you can
- Obtain appropriate samples
-Different toxins detectable in different matrices
-Everything can be a good sample
- Determine the appropriate extraction technique
-Making colorful solids into clear liquids
- Determining the appropriate analysis
-Complexometric Reactions: litmus paper, dipstick test.
-Microscopy
-Ultraviolet/visible spectroscopy
-Inductively coupled plasma: mass spectrometry, atomic emission.
-Ion exchange chromatography
-Gas chromatography
-Liquid chromatography
- Interpret the results
-Clinical significance vs. Quantitative
- List the basic steps for formulating a toxicological differential list
Case example DDx:
- History
-Testing for toxins without additional information is stumbling blindfolded through a dark room looking back for a black cat that may or not be there.
-Analysis is often confirmatory rather than diagnostic. Many toxicological diagnoses are based on history.
-Ask owner about medications in the household, new toys, cages, insecticides, herbicides, food including human food. The time of the year can also yield important information.
-The breed of the animal may generate information about sensitivity to a drug. For example: Collies, Australian shepherds, and other herding dogs are overly sensitive to anthelmintic IVERMECTIN.
-Signalment
-Signs, onset, progression
-Other animals affected or exposed
-Medical history
-Food, water, supplements
Do not assume that what you are told is necessarily true, that the owner will volunteer information necessary.
Do not jump into conclusions even if the case seems cut-and-dried
Ask important questions several times in several different ways
- Physical Exam
-Physical exam
-Postmortem
-Does the physical exam fit the history?
-Sometimes treatment is needed before PE, tremors, dehydration, etc.
Clinical signs
-Specific signs: example inducible seizures caused by rodenticide
-Nonspecific sigs: vomiting, drowsiness.
- Baseline diagnostics
Minimum database Antemortem
-CBC
-Serum chemistry (electrolytes, glucose, blood urea nitrogen, creatinine, and calcium).
-Urinalysis
-Feces
-Vomit, gastric lavage
-Hair in cases of topical exposure
-Coagulation profiles (PT, ACT)
-Hb SpO2
-Liver enzymes
-Radiographs, ultrasound, etc.
-Call the lab or check for test kits and appropriate collection technique.
Usually baseline diagnostics before specific toxicological testing
Postmortem Sampling
-Collect everything and lots of it!
-No, more!
-Every organ: liver, kidney, spleen, brain, heart, skeletal muscle, fat, etc.
-Fluids: Urine, heart blood, ocular fluid.
-GI contents
-Skin/hair
- Environmental exam in large animal cases.
- Explain why additional information is needed before requesting toxicological analyses
-To maximize the efficacy of laboratory tests, veterinarians must have knowledge of the use of each test, a basic grasp of specific laboratory procedures, how to obtain and proper handle specimens.
-Screening tests are maximally effective only if clinician has good reason (based on the history and clinical signs) to suspect a particular poison and request a specific lab procedure. “Fishing expeditions” should be avoided if they are to be successful to confirm or deny the presence of a particular poison, and the only way to confirm diagnosis.
- Describe what samples (and how much) to collect for antemortem and postmortem toxicological analyses
Minimum database Antemortem
-CBC
-Serum chemistry (electrolytes, glucose, blood urea nitrogen, creatinine, and calcium).
-Urinalysis
-Feces
-Vomit, gastric lavage
-Hair in cases of topical exposure
-Coagulation profiles (PT, ACT)
-Hb SpO2
-Liver enzymes
-Radiographs, ultrasound, etc.
-Call the lab or check for test kits and appropriate collection technique.
Usually baseline diagnostics before specific toxicological testing
Postmortem Sampling
-Collect everything and lots of it!
-No, more!
-Every organ: liver, kidney, spleen, brain, heart, skeletal muscle, fat, etc.
-Fluids: Urine, heart blood, ocular fluid.
-GI contents
-Skin/hair
Case example: Tito
Methyl (carbamate insecticide)
-Large amount in the stomach contents
-Cholinesterase inhibitor that causes muscarinic, nicotinic, and CNS signs = Blockage overstimulation.
- Identify reliable sources of information about toxicants, including MTDs and MLDs
ASPCA APCC mobile app
Product websites
as-capri.org
petpoisbononlinehelp.com/veterinarians
Lecture 2
Basic Principles of Toxicology
Basic principle #1
Dosage makes the poison or Everything is toxic at the right dose
-Dosage is the most important factor that determines response to poisons
-Toxicity is the quantitative amount of toxicant (dosage) required to produce a defined effect.
-Species specific values for toxicity
-Cats are not small dogs. Acetaminophen toxic to cats bc they don’t have the same coagulation pathways as dogs do
-Experimental conditions to determine toxicity measures can vary greatly from real-world cases.
Basic Principle # 2
Exposure does not equal intoxication
-The toxin must be absorbed and reach its site at a high enough concentration and from a sufficient period of time to cause a toxic effect (toxic-kinetics).
-Treatment involves decontamination
The disposition of a xenobiotic is what the animal’s body does to that compound following exposure and consists of ADME
- Absorption: how does it get into the body. Involves crossing cellular membranes, phospholipid bilayers with various sized pores and embedded proteins. Physiochemical properties of the toxicant (resemblance to endogenous compounds, molecular size, lipid and water solubilities, association constant, weak acid or weak base) determine how it is absorbed.
-Oral/ingestion
-Derma
-Injection
-IV
-IM, SQ, IM, IP
-Inhalation - Distribution
-Where does it go
-Fat soluble vs water soluble (Fat soluble vitamin D, E, K, A.)
-Protein binding
-pH of tissues and compartments
-Many other factors
-Molecular weight
-Vd: value of disassociation
-Crossing BBB or not - Metabolism
-What happens to it when it get there?
-Transported to liver, biotransformation, GI biotransformation, excretion.
-Biotransformation (metabolites more readily excreted)
-Often converted to a more water-soluble product.
-Often in the liver, but in many other organs.
Big differences in species can exists for various metabolic pathways
-Sometimes metabolites are less toxic or more toxic
-Cytochrome P450 enzymes catalyzes Oxidation reactions = phase I biotransformation. - Excretion
-How does it get our of the body?
a. Urinary
b. Biliary/Fecal
c. Milk, Sweat, Saliva.
d. Exhalation
-We can facilitate or speed up excretion: clearance rate.
Toxicokinetics refers to the quantification and determination of the time course of the disposition of ADME.
- Bioavailability
- Volume of Distribution
- Clearance
- Half-life
- One-compartment model
- First and zero order kinetics.
Toxicodynamics describes what a toxicant does physiologically.
Basic Principle # 3
The “typical” dose-response curve has important exceptions
Compare and contrast MTD, MLD, LD50
LD50: commonly used for comparison of toxicity differences among chemicals but does not define the nature of toxicosis produced or the safe dosage. The end point of and LD50 is death.
LC50: the lethal concentration for 50% of the animals exposed. Measured in mg/kg
NOAEL: No observed Adverse Effect Level
MTD: Minimum toxic dose
MLD/MLC: Minimum lethal dose. Minimum lethal concentration.
MTD is the value of most clinical use
Describe exceptions to the typical
Basic Principle # 3
The “typical” dose-response curve has important exceptions
Response vs. Dose (at target tissue)
Ex: beer
-No effect, Happy, Giddy, Asleep, Deep Sleep, Unconscious, Depressed breathing, Dead
Ex: Essential nutrients
Threshold of adverse effect
-Deficiency = death
Region of homeostasis
-Toxicity = death
Therapeutic Index
-Equal to the ratio of the dose of the drug that produces and unwanted (toxic) effect to that producing a wanted (therapeutic) effect.
Describe elements of toxixokinetics and the factors that influence toxicity
Basic Principle # 2
Exposure does not equal intoxication
-The toxin must be absorbed and reach its site at a high enough concentration and from a sufficient period of time to cause a toxic effect (toxic-kinetics).
-Treatment involves decontamination
The disposition of a xenobiotic is what the animal’s body does to that compound following exposure and consists of ADME
- Absorption: how does it get into the body. Involves crossing cellular membranes, phospholipid bilayers with various sized pores and embedded proteins. Physiochemical properties of the toxicant (resemblance to endogenous compounds, molecular size, lipid and water solubilities, association constant, weak acid or weak base) determine how it is absorbed.
-Oral/ingestion
-Derma
-Injection
-IV
-IM, SQ, IM, IP
-Inhalation - Distribution
-Where does it go
-Fat soluble vs water soluble (Fat soluble vitamin D, E, K, A.)
-Protein binding
-pH of tissues and compartments
-Many other factors
-Molecular weight
-Vd: value of disassociation
-Crossing BBB or not - Metabolism
-What happens to it when it get there?
-Transported to liver, biotransformation, GI biotransformation, excretion.
-Biotransformation (metabolites more readily excreted)
-Often converted to a more water-soluble product.
-Often in the liver, but in many other organs.
Big differences in species can exists for various metabolic pathways
-Sometimes metabolites are less toxic or more toxic
-Cytochrome P450 enzymes catalyzes Oxidation reactions = phase I biotransformation. - Excretion
-How does it get our of the body?
a. Urinary
b. Biliary/Fecal
c. Milk, Sweat, Saliva.
d. Exhalation
-We can facilitate or speed up excretion: clearance rate.
Toxicokinetics refers to the quantification and determination of the time course of the disposition of ADME.
- Bioavailability
- Volume of Distribution
- Clearance
- Half-life
- One-compartment model
- First and zero order kinetics.
Basic Principle #4
Many factors influence toxicity
-Characteristics of the animal/animals exposed
-Route of exposure
-Frequency of exposure
-Characteristics of the toxicant
-Environmental conditions
Characteristics of the animal
-Species
-Genetic differences (polymorphisms)
-Age
-Sex
-Reproductive status
-Concurrent disease
-Concurrent exposure to other drugs or chemicals
-Nutritional status
Characteristics of the chemical or toxicant
-Formulation, vehicle
-Valence state of metals
-Ionization
-Decomposition
-Impurities
-Strain/subspecies
Frequency of Exposure
-One time exposure
-Repeated exposure
-Cumulative exposure
Environmental Conditions
-Drought
-Time of the year
-Growth stage
-Temperature
-Photo period (daylight time)
-Winds
Factors that may alter Response to Toxicants
Impurities or contaminants
-Melamine and cyanic acid contaminants in cat food caused renal failure. For dogs, aflatoxin contaminated corn in food caused bleeding and liver failure.
Changes in chemical composition or salts of inorganic agents
-Toxicity of metals relates to the valance state.
-Trivalent arsenicals are more toxic than pentavalance arsenic.
Ionization
-Dependent on pH and aka
-Compounds that are highly ionized in the stomach are poorly absorbed thus less toxic.
Vehicle effects
-Non-polar and lipid-soluble vehicles usually increase toxicity by promoting absorption and membrane penetration. Ex; petroleum products and highly volatile hydrocarbons.
Protein binding effects
-Binding to serum albumin is common for many drugs and toxicants, limiting the bioavailability and reducing toxicity. Agents displaced from protein binding (e.g., vitamin K responsive anticoagulants) enhance toxicity.
Chemical or drug interactions
-Barbiturate drugs stimulate metabolic activation of many toxicants to a more toxic metabolite. May directly bind, inactivate, or potentiate one another or induce microsomal enzymes to influence metabolism of the other.
Biotransformation
-Prior exposure may induced increased metabolic activity. Activated Microsomal MFOs = increased toxicity. Otherwise, excreted. Altered ability of MFOs to begin metabolism can be influence by liver disease, breed, age. Phase II compromised and less detoxification.
Liver disease
-Reduced synthesis of glutathione, metallothionein, and coagulation factors may alter response to acetaminophen, cadmium, anticoagulant rodenticides, etc.
Diet and Nutritional status
-Calcium, zinc may affect absorption and response to lead. Vitamin C and E can aid in scavenging of free radicals and repair of cellular protective mechanisms.
Calculations and Conversions
1 once = 28 g
1 pound = 0.454 kg
2.2 lb = 1 kg
1 ton (short) = 0.9 metric tons
1 ton metric = 1000 kg = 2200 lb
1 fl ounce = 30 ml
1 teaspoon = 5 ml
1 table spoon = 15 ml
1 cup = 0.24 L = 240 ml
1 quart = approx 920/950 ml = 0.95 L
1 gallon = 3.8 L
Explain why you would calculate dose of exposure
To determine if the dose is high enough to pose a risk of intoxication or death
Why not just treat?
Risks and expense of Tx may outweigh risk or consequence of intoxication. Aggressiveness of treatment will be determined by severity of intoxication
Always: At what dose? In what species? Under what conditions?
Calculate and interpret does of exposure given the necessary information
Sample case
4 blocks x 14g/block = 56g
56g x 0.075 (percent) cholecalciferol/100g of bait = 0.042g of cholecalciferol
20lb puppy = 9.1 kg
0.042 g = 42 mg, 42mg/9.1kg BW = 4.62 mg/kg
MLD: 2mg/kg
MTD: 0.1 mg/kg
Puppy potentially ingested twice the MLD, so need to tx ASAP, gastric lavage, blood work, control the situation.
Lecture 3
Basic Mechanisms of Toxicants
- Describe how the types of exposure and types of effects contribute to toxicity
- Describe and compare how the attributes of the target contribute to toxicity
- Describe and compare the different reaction types of the toxicant with the target
- Describe the possible outcomes of the toxicant-target interaction
- Give examples of toxicants that interact directly with ion channels and explain their mechanism of toxicity
- Describe how the types of exposure and types of effects contribute to toxicity
Types of Exposures
- Acute toxicity is a term usually reserved to mean the effects of a single dose or multiple doses measured during a 24-hour period. If toxic effects become apparent over a period of up to 7 days, it may be considered ACUTE EFFECT.
- Subacute toxicity may refer to effects seen between 1 week and 1 month of exposure. 30-90 days interval.
- Chronic toxicity: effects produced by prolonged exposure of 3 months or longer. Essentially for the lifetime of the species.
Types of Effects
- Local: the site of action takes places at the point of contact
-The site: Skin, mucous membranes of eyes, nose, mouth, throat, or anywhere along the respiratory or GI system. - Systemic: The toxic substance has been absorbed and distributed throughout the body
- Cumulative: Over a period of time, the material is only partially excreted and the remaining quantities are gradually collected. The retained toxic compound accumulates and becomes great enough to cause pathological response.
Factors affecting toxicity
-Rate of entry and route of exposure
-Age
-State of health
-Previous exposure
-Environmental factors
-Host factors
-Specie
-Breed: Labrador and Golden Retriever top the list. Young adult dogs are most inclined to be exposed to potentially toxic agents.
Routes of entry
-Oral
-Dermal
-Nasal
-Percutaneous
-Inhalation
-Ocular
-Intraperitoneal
-Intravenous
-Subcutaneous
The most potent routes of exposure are usually intravenous, intrapulmonary, and intraperitoneal. Oral and dermal routes are the most common, these routes generally delay the absorption and diffuse exposure over a longer period.
Retention in GI tract and enterohepatic cycling can prolong exposure. Concurrent organ damage can accentuate the toxic effects.
Species and breed differences are important influences. For example, the cat and intolerance to phenolic compounds results directly from their limited glucuronyl transferase activity, which is necessary to produce glucuronides for the excretion of phenolic metabolites. A common example is Acetaminophen is toxic to cats, partially as a result of ineffective excretion of the toxic metabolite. Also, the relative lack of methemoglobin reductase in erythrocytes makes it more susceptible to oxidant damage.
- Describe and compare how the attributes of the target contribute to toxicity
Attributes of Target
Cassarett
-Appropriate reactivity and/or steric configuration to allow the toxicant to enter into covalent or noncovalent reactions.
-First target is usually the enzyme that catalyzes their production or the adjacent intracellular structures
-Example: Thyroperoxidase (Thyroid hormone synthesis) converts methimazole, amitrole, and resorcinol into reactive free radicals that inactivate thyroperoxidase. This is the basis for antithyroid as well as thyroid tumor-inducing effect of this chemicals.
-Example: Carbon tetrachloride activated by cytochrome P450, destroys this enzyme as well as the neighboring chromosomal membranes.
-DNA targets: Electrophiles metabolites react with nucleophilic atoms in nucleic acids. Example: Vinyl chloride epoxide, formed in the hepatocytes reaches DNA in neighboring endothelial cells, which are more sensitive than liver cells, initiating hepatic hemangiosarcoma.
-Some are not harmful. Example: Covalent binding of organophosphate insecticides to plasma chlolinesterase is a protective mechanism counteracting phosphorylation of acetylcholinesterase, the target molecule.
-Accessibility: usually the enzyme that catalyzes their production or the adjacent intracellular structures
-Critical function: to conclusively identify a target molecule as being responsible for toxicity, the toxicant must react with it and adversely affect its function, reach an effective concentration at the target site and alter the target in a way that is mechanistically related to the observed toxicity.
Mechanisms of Toxicity
1. Delivery: site of exposure to the target
The first step in the development of a toxicosis is the delivery of the “ultimate toxicant” (the parent xenobiotic, its metabolite, or even a generated reactive oxygen species that actually causes cellular damage) to its site of action or “target” (molecule that interacts with the ultimate toxicant resulting in an adverse effect). Targets can also be be referring to a cell type, organ, or tissue that is susceptible.
The distribution and biotransformation of a xenobiotic often limit the delivery of the ultimate toxicant.
-Transport of the chemical and cellular uptake: rate of transfer determined by physiochemical properties (lipid soluble, molecular weight), blood flow to the organ or tissue, rate of diffusion across endothelial walls of the capillary beds into cells.
-Storage depots can be protective and limit adverse effects. Plasma proteins represent a storage site for many xenobiotics and vitamins, steroid hormones, essential minerals. Displacement from plasma proteins can increase amount of unbound xenobiotics distributed to target tissues.
-Presystemic elimination or the firs-pass effect prevents toxic xenobiotics from ever reaching the general circulation. Metabolism produces mostly water soluble metabolites, thus more readily eliminated from the body. Hepatic biotransformation affects bioavailability of xenobiotics, rapid hepatic degradation = first pass effect. In contrast, biliary excretion cycle can enhance bioavailability and enterohepatic recirculation.
-Excretion: Renal is the most common. Feces, biliary, saliva, sweat, cerebrospinal fluid, or even milk. Exhalation too.
Excretion is the removal from blood to the external environment.
-Detoxification
- Describe and compare the different reaction types of the toxicant with the target
Which reaction blocks protein synthesis?
Warfarin falls into what category?
Proteins and nucleic acids toxicant reactions are?
Which involves Hb?
Which converts proteins into carbonyls?
- Reaction of the Ultimate toxicant with the target molecule
The ultimate toxicant may bind to the target molecules and alter it by hydrogen abstraction, electron transfer, or enzymatically.
Casarett - Noncovalent binding
-Apolar interactions
-Involves membrane receptors, intracellular receptors, ion channels, and some enzymes.
-Example: Warfarin to Vitamin K 2,3-epoxide reductase.
-Key fit to lock mechanism that is usually reversible due to low bonding energy. - Covalent binding
-Practically irreversible
-Permanent and alters endogenous molecules.
-Electrophilic toxicants such as nonionic and cationic electrophiles and radical reactions.
-Involves proteins and nucleic acids. - Hydrogen abstraction
-Neutral free radicals abstract H atoms from endogenous compounds converting those compounds into free radicals
-Radicals can remove hydrogen from CH2 groups of free amino acids or residues in proteins and convert them to carbonyls. These react covalently with amines forming cross-links with DNA or other proteins.
-Abstraction of H from fatty acids produce lipid radicals and initiates lipid peroxidation. - Electron transfer
-Chemicals can oxidize Fe (II) in hemoglobin to Fe (III) producing methemoglobinemia.
-Nitrite can oxidize hemoglobin. - Enzymatic reactions
-Example: plant toxins ricin and abrin are N-glycosidases that hydrolyze a specific glycosidic bond in Ribosomal RNA, blocking protein synthesis.
Lipophilic (lipid-soluble) properties of xenobiotics that favor absorption are biotransformed into hydrophilic/water-soluble compounds that favor excretion in urine and feces.
Phase I
-Oxidation, hydrolysis, or reduction reactions to convert into polar molecules.
-Hydroxyl, amino, carboxyl, or thiol moeities are exposed or added to increase water solubility.
-Oxidation catalyzed by P450 enzyme, many xenobiotics induce it.
Phase II
-Xenobiotic or its metabolite are conjugated with functional groups to increase water solubility.
-Not all mammals have ability for phase II, especially glucuronidate.
-Acetaminophen metabolites from phase I are more toxic than the parent xenobiotic.
Intoxication
-Chemical species such as electrophiles, free radicals, nucleophiles, and redox-active compuond metabolites.
-Indiscriminately reactive with endogenous molecules.
General Mechanism of Action
-A toxic xenobiotic’s mode or mechanism of action is the activity the compound or its metabolites at the molecular or cellular level that results in and adverse effect.
- Cellular damage
-Altered cellular maintenance, internally and externally
-Altering membrane integrity and ability to regulate volume and energy metabolism
-Cellular injury or death results from impaired cellular synthesis of ATP, oxidative phosphorylation and calcium regulation alterations.
-Ability to synthesis proteins and gene expression can also altered - Changing microenvironment through alterations of pH or receptor sites
-Some mimic the actions of normal nutrients and endogenous hormones or neurotransmitters.
-Specific receptors can be stimulated or blocked.
-Enzymes inactivated or inhibited.
- Describe the possible outcomes of the toxicant-target interaction
- Cellular dysfunction, injury
2.Inappropriate repair and adaptation
- Dysfunction of target molecules
-In the case of proteins it may render them foreign to the immune system
-Some toxicants activate protein targets mimicking endogenous ligands.
-Example: morphine activates opioid receptors, clofibrate is an agonist on the peroxisome proliferator-activated receptor, and phorbol esters and lead ions stimulate PKC.
-More commonly function is inhibited
-Example: atropine, curate, and sstrychnine (block neurotransmitter receptors by attaching to the ligand-binding sites, whereas others interfere with the function of ion channels.
-Example: DDT and insecticides inhibit closure of Na channels. - Inappropriate repair
-Thiol groups in proteins are critical for catalytic activity or assembly to macromolecular complexes. They may be innactivated by Thiol-reactive chemicals causing impaired maintenance.
-Binding can also lead to initiation of signal. Example: corrosive gases (chlorine) and excitation of neurons = lacrimation, pain, bronchial secretion.
-Template function of DNA interference. Example: Aflatoxin to guanine results in pairing of the adduct-bearing guanine with adenine rather than cytosine. Leading to p53 tumor repression due to mutation.
- Destruction
-Altered structure by means of cross-linking and fragmentation
-Converting proteins to reactive electrophiles, radicals, or subject to spontaneous degradation
-Lethally affect the cell
General Mechanism of Action
-A toxic xenobiotic’s mode or mechanism of action is the activity the compound or its metabolites at the molecular or cellular level that results in and adverse effect.
- Cellular damage
-Altered cellular maintenance, internally and externally
-Altering membrane integrity and ability to regulate volume and energy metabolism
-Cellular injury or death results from impaired cellular synthesis of ATP, oxidative phosphorylation and calcium regulation alterations.
-Ability to synthesis proteins and gene expression can also altered - Changing microenvironment through alterations of pH or receptor sites
-Some mimic the actions of normal nutrients and endogenous hormones or neurotransmitters.
-Specific receptors can be stimulated or blocked.
-Enzymes inactivated or inhibited.
- Give examples of toxicants that interact directly with ion channels and explain their mechanism of toxicity
Chemicals that specifically interact with protein targets
- Chemicals that activate or inactivate ion channels can cause widespread cellular dysfunction and cause cell death and many physiological symptoms.
Na, K, Ca, levels are extremely important in neurotransmission, muscle contraction and nearly every cellular function.
-Examples: - Tetrodotoxin from Puffer Fish closes/blocks voltage-gated Na channels, blocks action potentials
- alpha-bungarotoxin blocks nicotinic AChR from Kraits (elapid snakes)
- Dendrotoxins block K channels from Green mamba snakes
- w-agatoxin blocks Cav2.1 Calcium channels from Funnel web spider
- w-conotoxin blocks Cav2.1 Calcium channels from coneshell
- SNX-482 blocks Cav2.1 Calcium channels from Tarantula spider
- Alkaloids batrachontoxin Na channel inhibitor Nicotinic antagonist from Frog (Dendrobates) skin
Molecular mechanisms of Toxicology
Enzyme mediated Animal and Plant Toxins
-Parathion and Sarin (organophosphate)(insecticides): Irreversible anticholinesterase.
Interfere with metabolism and breakdown of Ach at the synaptic junction. AChR enzyme responsible for breakdown of Ach is inhibited resulting in accumulation of it which at first incites and then paralyzes transmission in these synapses giving “nerve gas” signs =central respiratory depression as with Atropine.
-Pralidoxime reactivation of acetylcholinesterase is defense against biological nerve gases.
-Oximes are strong nucleophiles that reactivate AChR.
Lecture 4
Basic Mechanisms of Toxicants 2
Interplay between the primary metabolic disorders spells cellular disaster
- Depletion of cellular ATP reserves deprives the endoplasmic and plasma membrane Ca pumps of fuel, causing elevation of Ca in the cytoplasm. Influx of Ca mitochondrial membrane potential decreases
- Intracellular hypercalcemia facilitates formation of ROS and RNS, which oxidatively inactivates the Thiol-dependent Ca pump, which in turn aggregates hypercalcemia.
- The ROS and RSN can also drain the ATP reserves.
- PARP (poly-ADP-ribose polymerase) activated trying to repair, consuming NAD(P)H
Mitochondrial electron transport increases, ATP synthesis decreases.
Xanthine oxidase increases.
Chemicals that interfere with ATP synthesis
Class A
-Interfere with delivery of H to electron chain transport.
-Fluoroacetate inhibits citric acid cycle and the production of reduced cofactors.
Class B
-Inhibit Cytochrome oxidase
-Inhibit the transfer of electrons along the electron transport chain to oxygen.
-Cyanide and Retonone (complex I, insecticide, Parkinson’s disease)
-Paraquat inhibits complex I, herbicide, lung hemorrhaging in humans.
Class C
-Interfere with delivery of oxygen to cytochrome oxidase (electron transporter)
-All chemicals that cause hypoxia.
-Ischemic agents such as Ergot alkaloids, cocaine
-Carbon monoxide displaces oxygen from Hb.
Class D
-Inhibit the activity of ATP synthase, key enzyme for phosphorylation.
-DDT, DIM, phytochemicals
Class E
-Cause mitochondrial DNA injury, impairing synthesis of specific proteins.
-Antivirals
Sustained Raise of Intracellular Ca++
Intracellular Calcium levels are highly regulated
-10,000-fold difference between extracellular and cytosolic Ca concentration is maintained by
a. Impermeability of plasma membrane to Ca
b. Transport mechanisms that remove Ca from cytoplasm (0.1 uM vs. 1000 uM).
-Calcium sources are from outside the cell or Ca stores in ER or mitochondria (as calcium phosphate).
Four mechanisms
1. Extracellular Ca/ATPase
2. ER Ca/ATPase
3. Extracellular Na/Ca exchanger
4. Mitochondrial Ca uniporter.
-Toxicants induce elevation of cytoplasmic Ca by promoting influx or inhibiting efflux.
-Opening ligand-gated channels or damage to plasma membrane causes Ca to move down its concentration gradient from extracellular to intracellular.
Harmful
- Depletion of energy reserves. Decreases mitochondrial ATP production and increases loss of ATP by activation of Ca/ATPase.
- Dysfunction of microfilaments. Impaired cell motility, disruption of cell morphology, cellular functions
- Activation of hydrolytic enzymes. Disentigration of membranes, proteins, DNA, etc.
- Generation of ROS and RNS. Disintegration of membranes, proteins, DNA, etc.
- Describe the role of reactive molecules in generating oxidative stress and cell injury
- Describe the imbalance of control of mitosis and apoptosis that can lead to carcinogenesis
- Define teratogen and describe at what phases of pregnancy different teratogens can have effects
Oxidative Stress: imbalance of cellular oxidants and antioxidants in favor of oxidants
A. Direct generation of ROS/RNS
a. Xenobiotic bioactivation = homolytic bond fission
-Carbon tetrachloride: free radical formed by electron transfer from Cytochrome P450 or hte mitochondrial transport chain. The trichloromethyl free radical reacts with oxygen to form even more reactive trichloromethylperoxy radical.
-Benzene
b. Redox cycling = one electron acceptor xenobiotic molecule can generate many O2- (radical) molecules. Superoxide radical.
-Paraquat: enters into pneumocytes via unspecified transporters and an MPTP metabolite which is MPP+ taken up into extrapyramidal dopaminergic neurons by the dopamine transporter, and internalization of ammino glycosides by renal tubular cells expose those cells to toxic concentrations.
Paraquat accepts an electron from reductase to give rise to a free radical which transfer the extra electron to molecular oxygen, forming a superoxide anion radical and generation the parent xenobiotic, which is ready to gain a new electron.
-MPP+: metabolite that electrophoretically accumulates in the mitochondria of dopaminergic neurons, causing mitochondrial dysfunction and cell death.
c. Transition metals = Hydrogen peroxide is a Hydroxyl radical generated by homolytic fission. Catalyzed by transition metals. Fenton reaction.
-Fe++
-Cu++
d. Inhibition of mitochondrial electron transport
-Many phytochemicals
Two pathways for toxication of superoxide anion radical via nonradical products (ONOO- peroxynitrite and HOOH hydrogen peroxide).
Spontaneous reaction of ONO2- with Carbon dioxide yields nitrosoperoxy carbonate that is hemolytically cleaved to Nitrogen dioxide. Carbonate ion radical, Nitrogen dioxide.
-NO- can readily diffuse and where the electron transport chain and the citric acid cycle produce O2- and CO2.
Lipid Peroxidation
Some target molecules are susceptible to spontaneous degradation after chemical attack.
1. Free radicals such as Cl3COO* and HO* can initiate peroxidative degradation of lipids by hydrogen abstraction from fatty acids.
2. The lipid radical (L) formed is converted successively to lipid peroxyl (LOO) radical by oxygen fixation (LOOH).
3. Lipid hydroxperoxide by hydrogen abstraction and lipid alkoxyl radical (LO) by the Fe++ catalyzed Fenton reaction.
-Subsequent fragmentation gives rise to hydrocarbons such as ethane and reactive aldehydes.
-Thus lipid peroxidation not only destroys lipids in cellular membranes but generates endogenous toxicants, both free radicals (LOO, LO). These substances can readily react with adjacent molecules such as membrane proteins, or diffuse to DNA.
-Lipid peroxidation is also a potent pulmonary and renal vasoconstrictor
Mutagenesis and Carcinogenesis
Mutagens cause changes to cell DNA that are passed on when cell divides, if this produces a neoplastic cell agent then it is termed Carcinogen.
Two major classes of gene are involved in carcinogenesis
- Proto-oncogenes: promote cell cycle progression
-Example: constituitive activity of growth factor tyrosine-kinase receptors can cause neoplastic transformation - Tumor-supressor genes: inhibit cell cycle progression
-Example: mutations in tumor suppression gene product p53
Genotoxic and Nongenotoxic Carcinogens
Genotoxic
Interact with DNA to damage or change structure
a. DNA damage
b. DNA replication
c. Mutation = Activation of oncogenic proteins, neoplastic cell transformation = clonal expansion = tumor
-Inactivation of tumor suppressor proteins = neoplastic cell transformation = clonal expansion = tumor
Cell Survival = repair
-Mutagenic
-Can be complete carcinomas
-Tumorigenicity is dose responsive
-No theoretical threshold
-Low dose linear patterns
-Lack reversibility
Nongenotoxic
Do not directly damage or interact with nuclear DNA
a. DNA replication (+)
b. Cell death (-)
-May change gene expression, modify cell function, bind or modify cellular receptors, and increase cell growth.
-Nonmutagenic
-Threshold reversible
-Tumorigenicity is dose responsive
-May function at tumor promotion stage
-No direct DNA damage
-Species, strain, tissue specificity.
The process of carcinogenesis is a multistep and multistage process involving modification and mutation to a number of normal cellular processes
DNA repair
Following the formation of a carcinogen-DNA adduct (covalently bond piece to a chemical) the persistence of the adduct is a major determination of the outcome.
-Ability of cell to repair the altered DNA
-Repair must occur before cell division, otherwise the presence of the adduct can give rise to mispairing of bases, rearrangements and translocations of segments.
Mechanisms of Repair
-Mutations in an oncogene, tumor suppressor gene, or gene that controls the cell cycle, can result in clonal cell population with proliferative or survival advantages.
What is the function of Proto-Oncogenes and Tumor-Suppressor Genes?
Proto-oncogenes and tumor suppressor genes play a key role in cancer induction.
These genes encode an wide variety of proteins that function to control cell growth and prolliferation.
Teratogenicity
What is Teratogenesis? Teragones?
Teratogenesis: The creation of birth defects during fetal development
Teratogens: substances that induce birth defects
Teratogenic alkaloids
Example: Arthrogyposis “crooked calf disase” caused by Lupines
Poisonous Species include
-Silky lupine, silvery lupine, velvet lupine, tailcup, lunara and yellow lupine.
-Tend to bloom in years of relative high moisture on the prairies and may attract cattle to graze them heavily.
What are some examples of Teratogenic alkaloids?
-Lupines
-Poison Hemlock: (Conium maculatam) is highly poisonous biennial herbaceous flowering plant in the carrot family Apiaceae. Regurgitation, dyspnea, tachycardia, and weakness.
-Nicotiana glauca: wild tabacco or tree tabaco. Annualy growing. C/S: regurgitation, tremors, tachycardia, seizures.
-Veratrum californicum or corn lilly contains CYCLOPAMINE.
Cyclopamine
-inhibits the action of the hedgehog signaling pathway which is involved in the formation of the neural system. Now investigated as anti-cancer potential therapy. SINGLE EYE.
Thalidomide (R) vs. (S) enantiomer
-(R) sedative, antemitic drug for pregnant women
-(S) teratogen
-1950’2 in Europe and Germany
-Problems when ingested between 3-6 wks gestation
-Today: tx for leprosy, multiple myeloma
- Describe the anatomical and physiological features that create different sensitivities to toxicity in the nervous system
- Doxorubicin (suicide transport)
- ddl, ddC, Thalium
- Diphtheria toxin
- Hexachlorophene
- Marine toxins (Ciguatera, Tetrodoxtin, Saxitoxin) Pyrethrins
- Vinca Alkaloids, taxoids, colchicine, Pyriminil
- Botulinum toxin, B-W spider venom, Elapid vemons.
- Organophosphates, carbamates
- alpha-bungarotoxin, d-tubocurarine, Marine toxins
- Tetanus toxin
- Statins, Zidovudine, Ethanol.
- Phencyclidine (PCP), Ketamine.
Manifestations of Structural neurotoxicity
- Neuronopathies
-Injury or death to neurons
-Target cell bodies
-Irreversible loss
-Initial injury followed by apoptosis or necrosis
- Axonopathies
-Primary site of toxicity is axon
-Degeneration of axon, also known as Wallerian degeneration
-Loss of axon distal to lesion
-Loss of surrounding myelin, and cell body remains intact
-Chromatolysis and margination of Nissl substance.
-IRREVERSIBLE in CNS, but reversible in PNS possible.
-Manifestation: chemical transection, Ca++ influx, Proteolysis, Axonal swelling, fragmentation of distal axon, macrophages influx, Schwann cell phagocytosis.
- Myelinopathies
Manifestation of functional neurotoxicity
-Intramyelinic edema
-Demyelination by affecting myelin or myelin-producing cells
-Remyelination in CNS occurs but limited extent - Oligodendrocytes (glial cells)
-Remyelination in PNS done by Schwann cells.
-Sultatory conduction
- Neurotransmission-associated abnormalities.
-Interruption of impulse transmission
-Blockage of trans-synaptic communication
-Inhibition of neurotransmitter uptake
-Interference with second-messenger systems
Neuropathy other causes
Buckeye, Horse Chesnut a.k.a
Glycoside AESCULIN and FRAXIN and possible a narcotic alkaloid, present in the young growing sprouts, leaves and seeds.
C/S: weakness, muscle twitching, may produce colic in horses
What toxicity does anemia, anisocystosis, poikilocytosis, polychromasia, basophilic stippling, metarubricytosis, and hypochromia indicate?
Metal intoxication - Lead
- Describe the mechanism of toxicity of tetanus
Second deadliest
LD50 = 1ng/kg
Clostridium tetani
-Anaerobic, gram positive
-Metabolizes and reproduces via spores, which are common mode of transmission.
C/S:
-Rigidity of masseter muscles, facial musces, straightening of the upper lip causing a grimacing posture to the face.
-Localized stiffness near the site of a penetrating wound followed by the axial muscles involving the neck, back muscles (opisthotonus) and abdomen.
-Spams can break long bones, horses susceptible.
-Larynx, diaphragm and intercostal muscles lead to respiratory failure.
-Cardiac arrhythmias, tachycardia, and hypertension.
WOUND contamination
-Toxin target and cleaves Synaptobrevin = SNARE component (on VAMP).
-Transported to the spinal cord from the neuromuscular junction and then transported by transcytosis to the inhibitory Renshaw cell to the upper motor neurons.
GABBA
-Blockage of glycine and GABBA from inhibitory neurons, leading to spastic paralysis.
-No relaxation of the antagonist muscle during normal contraction
- Describe the mechanism of toxicity of botulism
Deadliest toxin in the world
C/S:
-Flacid paralysis, tongue
-Waterfowl limberneck
-Disturbed vision
-Difficulty chewing and swallowing
-Generalized progressive paralysis.
-Death usually de to respiratory paralysis
Sources
-Rotten hay, forage, carcasses, etc.
Clostridium botulinum
-Gram positive rod
-Anaerobic
-Reproduces via spores
-survives in soil, intestines and feces of animals
-Spores invades the body through seemingly insignificant wounds and subsequently multiplies.
-Most cases are intoxication not an infection, toxin in food.
Mechanism
-Toxins prevent release of Ach at motor endplates (neuromuscular junction), autonomic ganglia, postganglionic parasympathetic nerves, Post ganglionic sympathetic nerves (w/Ach).
-SNARE proteins complex does not form, membranes do not fuse.
-No release of Ach, no muscle contraction
- List the two plants associated with neurotoxicity and their mechanisms of action
- Locoweed (Oxytropis, Astralagus)
-Cattle, horses, sheep, elk, deer.
-Always toxic even when dry
-SWAINSONINE an indolizide alkaloid found in all parts of the plant inhibits the enzymes alpha-mannosidase I & II.
C/S: sudden changes in temperament
-Ataxia
-Aggressiveness
-Horses more severe
- Larkspur (Delphinium spp)
->60 ornamental in western US
-Toxic DITRERPENE ALKALOIDS
-Readily absorbed in GI tract and not degraded in rumen
-Compete with acetylcholine binding to the nicotinic receptors
-Muscle weakness and paralysis
-Reversible blockade
Monkshood (Aconitum spp)
-Tetrodotoxin
-Na channel blocking
-Polycyclic diterpenoid alkaloids.
-Flacid paralysis, interference with excitable tissue
Toxic dose: 0.1-0.5% of BW
C/S: arrhtythmias, paraesthesia, vomiting, diarrhea, muscle paralysis.
Lecture 8
What are the Muscarinic signs?
Ach everywhere
-Somatic: Motorneuron ganglionic to Nicotinic receptor on Skeletal muscle, neuromuscular junction
-Sympathetic: pre-ganglionic to Nicotinic ganglionic receptor for postganglionic NE receptors (alpha 1 & 2, Beta 1 & 2) on smooth muscle galnds.
-Sympathetic: preganglionic to nicotinic receptors then also postganglionic to Muscarinic receptors on sweat glands
-Parasympathetic: preganglionic to nicotinic receptors to postganglionic agains Ach to muscarinic receptors on smooth muscle glands.
-Adrenal medulla: preganglionic to nicotinic receptors on medulla then to circulation the gland makes Epi (80%) and NE (20%).
Cholinesterase Inhibitors Signs
Muscarinic Signs
SLUDGE or DUMBELS
-Salivation
-Lacrimation
-Urination
-Defecation
-GI pain
-Emesis
- Describe the acute toxicity associated with pesticide exposure (organophosphates (OP), carbamate, organochlorine)
- Describe the use of pralidoxime and atropine as treatment for organophosphate and carbamate toxicity
- Describe the mechanism of action of organochlorine pesticides
- Explain the mechanism of delayed neurotoxicity and why this is only associated with certain organophosphates
Organophosphates
Uses
-Insecticides, fuel additives, hydraulic fluids, and lubricants
MOA: AchE inhibitors, Inactivated Ach
-Results in overstimulation of Muscarinic and nicotinic receptors
-Absorbed through GI, Skin or repiratory tract
-PO4 group
Organophosphate Poisoning
- Inhibition of neuropathic esterase (NTE): Wallerian-type degeneration in distal CNS and PNS
-OPIDN: Organophosphate INduced Delayed Neuropathy, Axon degeneration - Inhibition of AchE
-Accumulation of Ach at muscarinic, nicotinic and CNS receptors. Acute cholinergic syndrome
-Excess Ach at NMJ leading to down regulation of Ach receptors. Intermediate Syndrome
C/S:
-Activation of the autonomic and CNS at the nicotinic receptors on skeletal muscle
Examples
-Melathion
-Methamidophos
-Parathion-methyl
-Chlorpyrifos
Antidote for AchE inhibition
2-PAM = Pralidoxime
Atropine
No antidote for NTE = OPIDN (neurotoxic esterase)
Carbamates
Examples
DDT, DDE, Endrin (older type)
DDT (Duchloro-Diphenyl-Trichloro ethane) banned in 1970s
-Endosulfan
-Lindane etc.
- Insecticides: R1 is a methyl group
- Herbicides: R1 is an aromatic moiety
- Fungicides: R1 is a benzimidazole moiety
MOA:
-Interferance with Na and K (excitatory blocking) conductance gating
-Mainly at nerve axon
-HCH groups (lindane) inhibiting the CNS GABA receptors
C/S:
-Hyperexcitability
-Seizures
-Tremors
-Paresthesias
-Ataxia
-Other neurological signs
ATROPINE treatment
-Drying secretions
-Mydriasis
-Arrhythmias
-Flushing
-Confusion
-Hallucinations
-Ileus:stasis of GI
Use low dose at first to determine if carbamates present or not
Extreme caution with horses Ileus = colic, death*
- Describe the mechanism of toxicity of nicotine products, methylxanthines, permethrins, strychnine, and tick paralysis
Pyrethrin/Pyrethroids
-Derived from Chrysanthemum plants
MOA: Na channel blocking
Clearance: metabolized through glucoronidation pathway. CATS lack this pathway.
CATS
-Flea collars
-Profuse drooling, vomiting, tremoring, hyperexcitability, agitation, seizures, weakness, difficulty breathing.
-Nicotinic and Pyrethrin signs
Coyote bait case
Strychnine (in bait)
-Seeds from Strychnos nux-vomica tree
-Dogs and cats are more susceptible
-Horses tolerate large amounts
MOA: Antagonist of glycine (inhibitory neurotransmitter)
C/S:
-Muscle convulsions
-Death through asphyxia
Methylxanthines = Phosphodiesterase Inhibitors
Caffeine, Paraxantine, Theophyline, Theobromine
-Beta agonists = Increase HR
-Chocolate
-Mulch
-Caffeinated products
Tick Paralysis
-Neurotoxin produced in the salivary gland of female ticks
-Lower Motor neuron paralysis
Rhipicephalus, Haemaphysalis, Otobius, and Argas
