PHEO*610*600*TOXICOLOGY IN PUBLIC HEALTH*EXAM I (in class)

Xenobiotic (Slides Wk 1)

"a pharmacologically, endocrinologically, or toxicologically active substance not endogenously produced and therefore foreign to an organism" - Stedman's Medical Dictionary, 25th ed.

Ecotoxicology (Slides Wk 1)

"science of contaminants in the biosphere and their effects on constituents of the biosphere" (Casarett and Doull, 2008)

Presystemic Elimination (Slides Wk 2-3: Presystemic Elimination)

(First-pass effect or First-pass metabolism): •Chemicals absorbed via the GI tract must first pass through the GI mucosal cells and liver before reaching systemic circulation (and distribution to the rest of the body) •The liver and small intestine may metabolize a significant fraction of the toxicant, decreasing systemic availability - FIRST PASS EFFECT. •Extent of presystemic elimination differs between chemicals and between individuals Notes: - Presystemic or first-pass elimination = elimination of toxicants during process of transfer from exposure site to systemic circulation Ex: compounds absorbed from gastrointestinal (GI) tract must initially pass through GI mucosal cells, liver and lung before distribution to rest of body by systemic circulation

Toxicity due to alteration of biological microenvironment (Slides Wk 2-3)

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Metabolic activation may involve formation of these reactive metabolites (Slides Wk 2-3: Toxication vs. Detoxication)

1. Electrophile = molecule with electron-deficient atom with partial or full positive charge (common mechanism of toxication) 2. Free radical = molecule or fragment of molecule with one or more unpaired electrons in outer orbital, e.g., hydroxyl radical (HO.) 3. Nucleophile = molecule with electron-rich sites that may be negatively charged (less common mechanism of toxication) 4. Can also have formation of other redox-active reactants, e.g., ascorbic acid reduces Cr(VI) to Cr(V), and Cr(V) can then catalyze hydroxyl radical (HO.)formation Notes: - Free radicals are typically quite reactive. Free radical is denoted by this dot next to "HO". We don't need to dive into the chemistry, just understand the basics. - nucleophile - Know that redox reaction are oxidation and reduction

Parent compound (Slides Wk 2-3: Ultimate toxicant)

= toxicant to which organism was exposed

Lipid-soluble compounds (Slides Wk 2-3: Distribution to/away from target)

diffuse by passive diffusion through membrane into cells

Example: Acetaminophen (Slides Wk 1)

•Ex: metabolism of acetaminophen results in formation of toxic metabolite NAPQI (N-acetyl-p-benzoquinoneimine) •at regular therapeutic doses, NAPQI quickly conjugated with glutathione (intracellular antioxidant) à very low rate of liver toxicity •at high doses, glutathione in liver is depleted à NAPQI accumulates à liver toxicity that can be fatal Notes: - Know NAPQI on test! • Note! -Small difference between therapeutic dose and toxic dose (relatively narrow therapeutic ratio) - http://www.fda.gov/Drugs/EmergencyPreparedness/BioterrorismandDrugPreparedness/ucm133425.htm - http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/psn/transcript.cfm?show=87 (This link does not work anymore)

Nonvolatile, highly lipophilic chemicals (Slides Wk 2-3: Reabsorption vs. Excretion)

•Metabolism can increase a xenobiotic's water solubility (hydrophilicity) •Without metabolism: •no efficient mechanisms for excretion •These chemicals tend to accumulate with repeated exposure (e.g., DDT) Notes: - Volatile chem (eg Benzene) elimination

Local vs. Systemic effects (Slides Wk 1)

•Most compounds produce systemic effects, some produce both local and systemic effects •If systemic toxicity occurs, in most cases the major toxicity occurs in only one or two organs •Examples? - (Notes: Lead - brain and Acetaminophen - liver are examples of systemic effects)

Reactive oxygen or nitrogen species (Slides Wk 2-3: Ultimate toxicant)

(ROS, RNS) generated during metabolism of toxicant •ex: metabolism of diquat produces hydroxyl radical (HO )

Endogenous molecule (Slides Wk 2-3: Ultimate toxicant)

(ex: HO reacts with unsaturated fatty acids to produce other radicals (e.g., lipid peroxyl radicals)

Medications That Inhibit and Up-Regulate Cytochrome P450 2E1 and 3A4 Enzymes (Slides Wk 1: Look at Figure 4-5)

- Know the toxic and non-toxic routes - Glutathione is called a co-factor - Dr. C won't ask about drawing chemical structures. She cares about the concepts. For exams. Know like the the different routes of metabolism. - We'll revisit this slide. - Remember multiple routes of metabolism are common. Anything that switches multiple pathways can affect toxicity.

Animal studies (Slides Wk 1: Duration and Frequency of Exposure)

-Acute: exposure less than 24 hrs -Subacute: repeated exposure for up to 1 month -Subchronic: repeated exposure for 1-3 months -Chronic: repeated exposure for > 3 months, often at least 1 yr

Human exposures (Slides Wk 1: Duration and Frequency of Exposure)

-Acute: resulting from single incident -Subchronic: repeated exposure over several weeks to months -Chronic: repeated exposure for many months or years

Toxicants (Slides Wk 1)

-Toxic compounds produced by human (anthropogenic) activities -Toxic compounds that are not produced by human activities or by biological organisms. Ex: polycyclic aromatic hydrocarbons produced by forest fires, or naturally-occurring arsenic in groundwater

Mechanisms that reduce distribution to target include: (Slides Wk 2-3: Distribution to/away from target)

1. Binding of toxicants to plasma proteins: this prevents most toxicants from leaving the blood and entering the cells 2. Specialized barriers (e.g., blood-brain barrier) restrict access of hydrophilic compounds but not lipophilic compounds 3. Accumulation of toxicant in storage sites away from the target (this can be reversible). Storage sites include: •Binding to plasma proteins, e.g., albumin •Liver, kidney •Fat (storage of highly lipophilic compounds) •Bone (storage of fluoride, lead, strontium, etc.) 4. Binding to non-target intracellular sites (can be reversible) 5. Transport from cells to extracellular space, e.g., ABC transporters (a superfamily of membrane proteins) can be overexpressed in tumor cells, making these cells resistant to antitumor drugs that are pumped by these transporters, through the cell membrane, out of the cells Notes: - Specialized barriers (Dr. C said this is "not perfect") - If lead is in bone, it's not in brain - Binding to non-target intracellular sites (Dr. C said this is "still goof since not target") - Rees et al,2009, Nature 10:218-227; Figure 1 | Molecular architecture of aBc transporters. a | A cartoon of the modular organization of ATP-binding cassette (ABC) transporters, which are composed of two transmembrane domains (TMDs) and two ABC domains (or nucleotide-binding domains). The binding protein component that is required by importers is also shown. Two conformational states of the ABC transporter — outward facing and inward facing, with the substrate-binding site orientated towards the periplasmic (extracellular) and cytoplasmic (intracellular) regions, respectively — are depicted to show the alternating access mechanism of transport (BOX 1). b | The Escherichia coli vitamin B12 importer BtuCDF22 (Protein Data Bank (PDB) code 2QI9). The core transporter consists of four subunits: the two TMD BtuC subunits (purple and red) and the two ABC BtuD subunits (green and blue). This complex also contains one copy of BtuF, the periplasmic binding protein (cyan). c | The Staphylococcus aureus Sav1866 multidrug exporter18 (PDB code 2ONJ). Sav1866 consists of two subunits (green and dark blue), which contain a fused TMD and ABC domain. The nucleotides that are bound in this structure are shown by yellow space-filling models. Molecular figures in this article were prepared with MolScript and Raster3D77,78 using coordinates from the PDB79. ICL, intracellular loop.

Reasons detoxication may not be adequate (Slides Wk 2-3: Toxication vs. Detoxication)

1. Detoxication processes may be overwhelmed, with accumulation of ultimate toxicant (case of acetaminophen toxicity - depletion of glutathione) 2. Toxicant may inactivate a detoxicating enzyme 3. Conjugation reactions may be reversible (in conjugation reactions, cofactors react with xenobiotic functional groups. Conjugation reactions often greatly increase xenobiotic hydrophilicity, thereby facilitating excretion.) 4. Detoxication reactions may produce toxic by-products Noes: - Enzymes are catalyzing a reaction and not consuming a reaction. Things that end in "-ase" are enzymes. - Methylation, acetylation conjugation reactions don't greatly increase xenobiotic hydrophilicity - Detoxication reactions may produce by-products that can serve as toxic agents, e.g., production of glutathione thiyl radicals during detoxication of free radicals

Mechanisms of reabsorption include (Slides Wk 2-3: Reabsorption vs. Excretion)

1. Reabsorption from renal tubules 2. Reabsorption by diffusion across intestinal mucosa Notes: Mechanisms of reabsorption include: 1.After delivery to renal tubules: can have reabsorption by diffusion across renal tubular cells into peritubular capillaries 2.After delivery to GI tract (e.g., by biliary, gastric and intestinal excretion): can have reabsorption of relatively lipophilic molecules by diffusion across intestinal mucosa

Mechanisms that enhance distribution to target include: (Slides Wk 2-3: Distribution to/away from target)

1.Porosity of capillary endothelium, e.g., xenobiotics can accumulate more easily in liver and kidneys because pores ("fenestrae") are larger in some parts of liver and kidney. This allows protein-bound toxicants to pass through 2.Specialized membrane transporters and ion channels can help deliver toxicants to targets in cell 3. Accumulation in certain cell organelles depending on toxicant properties •E.g.: following overdose, highly lipophilic local anesthetics (e.g., tetracaine) can accumulate in cardiac mitochondria, interfering with energy production, resulting in cardiac failure 4. Reversible intracellular binding of toxicants leading to accumulation of toxicant in cells Notes: - Endothelium - layer of flat cells lining especially blood and lymphatic vessels and heart Plural = Fenestrae ( "FeNEStree")- anatomical opening, often closed by a membrane Singular = sounds like "fenestruh" - Sinusoidal capillary; a thin-walled terminal blood vessel having an irregular and larger caliber than an ordinary capillary; its endothelial cells have large gaps and the basal lamina is either discontinuous or absent. - From Braet and Wisse,2002, Comp Hepatol 2—2 1:1, Structural and functional aspects of liver sinusoidal endothelial cell fenestrae: a review: - "The liver sinusoids can be regarded as unique capillaries which differ from other capillaries in the body, because of the presence of open pores or fenestrae lacking a diaphragm and a basal lamina underneath the endothelium." - Cutaway view of blood vessel: Innermost layer of blood vessels is a type of epithelium, called endothelium (a smooth surface that keeps blood flowing freely) - Porosity of capillary endothelium (Dr. said this is "pores equilibrium") - Specialized membrane transporters and ion channels (Dr. said this is "specific") - Accumulation in certain cell organelles (Dr. C said this is "stuck")

Ultimate toxicant (Slides Wk 2-3: Ultimate toxicant)

= chemical species that •reacts with target molecule, OR •critically alters the biological microenvironment Ultimate toxicant may be: • parent compound = toxicant to which organism was exposed • Metabolite of parent compound - ex: acetaminophen metabolized to reactive toxic compound • Reactive oxygen or nitrogen species (ROS, RNS) generated during metabolism of toxicant - ex: metabolism of diquat produces hydroxyl radical (HO ) • Endogenous molecule - (ex: HO reacts with unsaturated fatty acids to produce other radicals (e.g., lipid peroxyl radicals) Notes: - Brought up the image of "Medications That Inhibit and Up-Regulate Cytochrome P450 2E1 and 3A4 Enzymes". Medications like Acetaminophen. - toxication = the body's system making something more toxic

Absorption (Slides Wk 2-3: Absorption)

= transfer of compound from exposure site into systemic circulation •Most toxicants cross epithelial barriers and enter blood capillaries by diffusing through cells Notes: - Will look at the blood brain barrier - Lipid solubility is a major factor in determining what can cross the epithelium

The process of toxicant delivery is the first step in the development of toxicity (Slides Wk 2-3: Ultimate toxicant)

How does delivery influence toxicity? Figure from textbook for this class but it's a 3rd edition: - Exposure site: Skin, GI tract, respiratory tract, injection/bite site, placenta -> Toxicant) -> DELIVERY -> Absorption -> Presystemic elimination -> Distribution toward target -> Distribution away from target -> Reabsorption -> Excretion -> Toxication -> Detoxication -> Ultimate toxicant -> Target molecule (Protein, lipid, nucleic acid macromolecular complex) -> Target site Notes: - Transporters also help with the elimination

Individual Dose-Response Relationship (Slides Wk 1: Individual Dose-Response Relationship)

Notes: Individual dose-response relationships are characterized by a dose-related increase in the severity of the response. The dose relatedness of the response often results from an alteration of a specific biochemical process. In panel B: has been plotted using the base 10 log of the dose on x-axis In contrast to the "graded" or continuous-scale dose-response relationship that occurs in individuals, the dose-response relationships in a population are by defnintion quantal - or "all or none" - in nature, that is, at any given dose, an individual in the population is classiifed as either a "responder" or a "non-responder".

Tolerance (Slides Wk 1: Tolerance)

Tolerance: reduced responsiveness to toxic effect of a chemical due to prior exposure to that compound or a structurally related compound. • Two major mechanisms: 1.Tissue becomes less responsive to the compound (less well understood) 2.Dispositional tolerance: reduced amount of toxicant reaches site of toxic effect •Example of dispositional tolerance: - -Exposure to cadmium induces metallothionein, a metal binding protein -cadmium toxicity is reduced when cadmium binds to metallothionein rather than to important macromolecules Notes: Cadmium mechanism of action Induction = increase in expression (upregulation), usually in response to exposure to high concentrations of xenobiotics

Potential stages in the development of toxicity after chemical exposure (Slides Wk 2-3)

Toxicant -> 1) Delivery -> 2a) Interaction with target molecule or 2b) Alteration of biological environment -> 3) Cellular dysfunction, injury, and 4) Inappropriate repair and adaptation. There is TOXICITY in 3 and 4. - Molecular level -> cellular level -> etc. Notes: - "Target" is site where toxicity occurs. - "Target molecule" is the molecular site where toxicity occurs - Nitrogen is an example of 2b. Alteration of biological environment - She might have a question with empty boxes above and we fill in the blank and explain this flow chart - Know the definitions on the slides

Chemical allergy (Slides Wk 1)

adverse reaction to a chemical that is immunologically mediated: - occurs following previous sensitization to that chemical or one with similar structure -can occur with exposure to very low doses of the chemical -For a particular person - allergic reactions are dose-related (Notes: see p. 15-16)

Detoxication (Slides Wk 2-3: Toxication vs. Detoxication)

biotransformation of a molecule that prevents the formation of an ultimate toxicant or eliminates it

Metabolic activation or toxication (Slides Wk 2-3: Toxication vs. Detoxication)

biotransformation to harmful metabolites •Compounds may be directly toxic or may be toxic following metabolic activation •With toxication: xenobiotics are altered so they are very reactive toward endogenous molecules with vulnerable functional groups

"Selective toxicity" (Slides Wk 1: Variations in Toxic Responses)

chemical injures one type of biological organism but not another: -Case where chemical equally toxic to both but accumulated more by susceptible species -Case where chemical interacts with a cellular or biochemical component that is not present in resistant species •Can have large differences in toxic response even with similar species (i.e., mice and rats)

Chemical anatagonism (inactivation) (Slides Wk 1: Types of Antagonism)

chemical reaction between two substances to generate a product that is less toxic

Additive effect (Slides Wk 1: Chemical Interactions)

combined effect equals sum of effects of each compound alone (1+2 = 3) (most common) •Ex: cholinesterase inhibition by two organophosphate insecticides is generally additive •Image: see https://depts.washington.edu/opchild/acute.html

Synergistic effect (Slides Wk 1: Chemical Interactions)

combined effects much greater than sum of effects of each compound alone (2+2 = 20) Ex: Ethanol (hepatotoxic) + Carbon tetrachloride (hepatotoxic) -> Greater than additive hepatotoxicity

Dispositional antagonism (Slides Wk 1: Types of Antagonism)

disposition (absorption, distribution, metabolism or excretion) of compound changed so duration or concentration of chemical at target organ is reduced •Ex: preventing absorption of chemical by administering charcoal, or administering compounds to alter drug metabolism to decrease toxicity •Ex: Adsorption of the mycotoxin aflatoxin B1 by calcium montmorillonite clay (HSCAS clay) •Aflatoxin is a common contaminant of corn, peanuts, dairy products, breakfast cereals, animal feeds •Heat stable •HSCAS clay can be ingested in diet à rapid chemisorption of aflatoxins in GI tract à reduced bioavailability and altered movement to blood, target organs

Threshold (Slide Wk 1: Changes in slope of dose-response)

dose below which there is a negligible risk of adverse effects Thresholds exist for many compounds Identification of threshold depends on adverse effect being considered, number of subjects studied, sensitivity of the measurement www.epa.gov/iris: see RfD, RfC values •However: -Toxic responses may not have a threshold, e.g. slope is linear at low doses and there is no dose without risk -Ex: development of cancer following exposure to genotoxic carcinogens Notes: Look at NOAEL vs LOAEL for determining threshold in IRIS database. Remember doing this stuff when you took Risk Assessment.

Metabolite of parent compound (Slides Wk 2-3: Ultimate toxicant)

ex: acetaminophen metabolized to reactive toxic compound

Volatile, nonreactive compounds (Slides Wk 2-3: Reabsorption vs. Excretion)

exhaled following diffusion from pulmonary capillaries into alveoli Notes: - Volatile chem (eg Benzene) elimination

Larger ions (molecular weight ≥50) and very polar toxicants (Slides Wk 2-3: Distribution to/away from target)

have a hydration shell that makes them too large to enter cell easily, unless moved across membrane by a special transport system

Biosphere (Slides Wk 1)

living organisms and their environment

Small ions and small water-soluble molecules (Slides Wk 2-3: Distribution to/away from target)

may diffuse through pores or aqueous channels in membrane •E.g., through aquaporins

Potentiation (Slides Wk 1: Chemical Interactions)

one substance not toxic for a certain organ or system, but when administered with a second compound, makes the second chemical much more toxic (0+2 = 10) Ex: Isopropanol (not hepatotoxic alone) + Carbon tetrachloride (CCl4) (hepatotoxic) -> Hepatotoxicity greater than expected with CCl4

Adduct (Slides Wk 2-3: Benzo(a)pyrene diol epoxide reacts with DNA to form (+)-trans-anti-[benzo[a]pyrene]-N 2 -dG adduct)

product of covalent bonding between a chemical (e.g., a carcinogen) and a biological macromolecule (DNA, protein)

Excretion (Slides Wk 2-3: Reabsorption vs. Excretion)

removal from the blood and elimination of toxicant (or metabolites) from body • Route, rate of excretion depend mostly on physicochemical properties of chemical • highly hydrophilic (usually ionized) compounds: can be removed by kidney or liver (major excretory organs) Notes: - Volatile chem (eg Benzene) elimination

Target organ(s) (Slides Wk 1)

site(s) where the major toxicity occurs following exposure to a compound •Target organ may not be location with the highest concentration of the compound -E.g., lead concentrates in bone but toxic effects occur in soft tissues, especially the brain

Toxicology (Slides Wk 1)

study of the adverse effects of chemicals; now includes study of molecular biology using these compounds as tools

Environmental toxicology (Slides Wk 1)

study of the impacts of environmental contaminants on biological organisms

Toxin (Slides Wk 1)

toxic compounds produced by biological organisms (e.g., bacteria, fungi, plants, animals)

Systemic effects (Slides Wk 1)

toxic effects are distant from entry point into body -e.g., ingestion of lead by young children and reduction of IQ scores, increased aggression & reduced attention span

Local effects (Slides Wk 1)

toxicity occurs at point of contact -e.g., acid irritation of skin

Functional antagonism (Slides Wk 1: Types of Antagonism)

two chemicals produce opposite effects on same physiologic function

Antagonism (Slides Wk 1: Chemical Interactions)

two compounds given together interfere with the actions of one or both compounds. •One or both compounds may be toxic if administered individually •Can serve as basis of antidotes Ex: Acetaminophen overdose + N-Acetylcysteine (within 10 h) -> Prevents massive hepatic necrosis

Reabsorption (Slides Wk 2-3: Reabsorption vs. Excretion)

when a toxic molecule is in the process of being removed from the body, and then gets reabsorbed

Receptor antagonism (Slides Wk 1: Dispositional Anatagonism)

when two compounds that bind to same receptor produce less of an effect when given together than when given separately (4+6 = 8), or when one chemical antagonizes (blocks) the effect of the second chemical (0+4 = 1) • Example: Naloxone - binds to opioid receptors and reverses and blocks the effects of other opioids (see https://www.drugabuse.gov/publications/drugfacts/naloxone and https://medlineplus.gov/medlineplus-videos/how-naloxone-saves-lives-in-opioid-overdose/) Notes: e.g., competitive binding to same receptor https://www.youtube.com/watch?v=RcAaZQQqd50

Attributes of Target Molecules (Slides Wk 2-3)

• Target molecule = site of molecular damage • Target molecules include DNA, proteins, membrane lipids • Targets may be adjacent to locations where enzymes catalyze the formation of toxic metabolites • Toxicant can diffuse (e.g., from cytoplasm into nucleus) until appropriate "reaction partner" is encountered • If reaction with endogenous reaction partner does not cause toxicity, this can be protective by sparing toxicologically relevant targets Notes: - Binding to DNA may be worse than binding to proteins. Neither type of binding is good.

Teratogen (Slides Wk 1)

• compound that causes defects during development between the time of conception and birth • Carcinogenic and teratogenic effects - generally irreversible (Notes: Teratogen - compound that produces defects during development between conception and birth)

Mechanism of presystemic elimination (Slides Wk 2-3)

• drug enters the cells that line the gut •It can undergo metabolism, excretion back into the intestinal lumen, or transport into the portal vein •The portal vein takes it to the liver •From the liver, it may be excreted in bile or may enter systemic circulation. Notes: - Enterocyte () A type of epithelial cell that lines the gut. [entero- + -cyte] - biliary canaliculus. one of the intercellular channels, about 1 mcm or less in diameter, that occur between liver cells forming the first portion of the bile system. SYN: bile capillary.

Duration and Frequency of Exposure (Slides Wk 1: Duration and Frequency of Exposure)

•Duration & frequency of exposure are important in determining the toxic response that occurs •Toxic effects following single exposure are often different from effects following chronic exposure •Chronic exposure may result in acute effects after each administration as well as chronic effects •To evaluate the likelihood of toxic effects, exposure frequency should be evaluated in relation to: -elimination rate -concentration of compound at target site needed to produce toxicity -whether interval between doses allows complete tissue repair -whether toxic effects are reversible Notes: - Eg, benzene - single exposure - primary acute toxic manifestation is CNS depression, but after repeated exposure you can see bone marrow toxicity and an increased risk for leukemia - Half-life

Effects of Toxicants on Target Molecules (Slides Wk 2-3)

•Effects on target molecules can include: •Inhibition of target molecule function or destruction of target molecules, e.g.: •change in protein structure that impairs function •interference with template function of DNA leading to mutations •covalent crosslinking between bases in DNA

Characteristics of Exposure (Slides Wk 1: Characteristics of Exposure)

•Route of administration can influence toxicity -Ingested compounds undergo "first-pass metabolism" in liver -Compare metabolism of various routes of exposure (next slide)

Distribution to/away from target (Slides Wk 2-3: Distribution to/away from target)

•The rate of distribution to organs or tissues depends mostly on: • blood flow to that organ or tissue •rate of diffusion out of capillary bed into cells of that organ or tissue •Final distribution depends mostly on affinity of the chemical for particular tissues Notes: - capillary bed = the capillaries considered collectively and their volume capacity for blood. - Partition coefficients - Equillibrium - Dr. C said there is no definitive end and beginning because there is overlap with the "Distribution to/away from target"

Genetic polymorphisms (Slides Wk 1: Variations in Toxic Responses)

•When evaluating human variability in toxic responses, consider: -Genetic polymorphisms: hereditary difference in an individual gene that is present in >1% of the population •may cause differences between individuals in toxic response -gene-environment interactions (e.g., lifestyle, diet, occupational/environmental exposures) Notes: Start here top p 28 col 1 Ex with tumor suppressor gene or oncogene