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Biomedical toxicology final review.docx

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TOX 2000
Aaron Witham

Biomedical toxicology final review Toxicant –any substance that causes an adverse biological effect, toxic response Toxicosis –chemically caused disease Drug- pharmacological effect (use toxic effect for benefit ex. chemotherapy) Human made chemicals perceived to be more dangerous but have lots of testing, well researched, many trials compared to natural products perceived to be safe Endogenous substances – normal components in body required or not Xenobiotics – chemicals not normally present in the body (exogenous) Toxicology principle #1-all substances natural or man-made our poisons (toxicants), the dose makes the poison Toxicology principle #2 –every toxicant has a threshold dose –dose where toxicosis occurs and below which no adverse effect occurs depends on response measured (acute vs. chronic), sensitivity of measurement (how low can we detect the level), number of individuals studied (more individuals greater accuracy) Toxicology principle #3 –everyone isn’t affected the same way due to biological variability ex. copper needed in enzymes has optimal range and below which deficiency and death, above which toxicosis and death with threshold on either level, biological variability –depends on genetics, might have different form of enzyme, may be able to excrete copper more readily Toxicology principle #4 – dose response increase with increased dose (a graded response) as dose increases a) in individuals: effected increase, symptoms more severe, death; b) in a population number affected increases Dose response group – exceptions are immunological reactions things system can handle Hormesis –stimulates biological systems at low doses, inhibit them high doses, non-nutritional substances have beneficial effects at low doses, implications of low dose extrapolation ex. radiation, alcohol, fluorine Measures of toxicity: toxicity –amount of chemical required to cause adverse effect under specific conditions, expressed as dosage in mg toxicant/kg body weight (equalizes size variability) Toxic endpoints –used as predictors a) death (lethal dosage) b) adverse biological effecttoxic dose “effect” most sensitive effect preferred Determine safety: nontoxic endpoints –use to determine safe levels a) LOAEL b) NOAEL safety margin – magnitude of the difference between the dose required to produce a maximum therapeutic effect and that which produces a toxic effect; don’t use just LOAEL, NOAEL because of statistical uncertainty Use of the chemical – interconnection with nature must protect all living organisms, data to regulate toxins, better recognition of ethical responsibilities, measures to regulate production, use and disposal; Chemical use: risk/benefit decision Chemical production Human exposure risk disposal Environmental exposure risk < Benefit determination subjective to change between people and society Risk – probability of toxicosis following exposure to hazardous substance determined by 1. Potential exposure Regulatory, 2. Inherent toxicity –experimental determination Exposure and toxicosis – dose required to cause toxicity may vary within individuals and between individuals –biological variability children vs. adults what determines if toxicosis occursToxicokinetic parametersconcentration of chemical at body target (site of action) Pb exposure – cognitive defects, low RBC count and anemia, visual toxicity, anticipated human carcinogen, children are more at risk- absorb more than adults, CNS still developing (few defense mechanisms), exposure through skin, risks with inner city children –poor nutrition not enough Ca and Fe which compete with Pb for binding, living near major roadways and freeways (Pb in gas), Pb pipes (older piping) lupinosis- anagyrine- plant lupine contains anagyrine- lipophilic, stable naturally occurring chemical teratogen, belongs to quinolizidine alkaloid group, affinity for nicotinic receptors (para- sympathetic nervous system), acute signs- lethargy, frequent urination, respiratory paralysis, deformities, thought to be caused by poor tendon muscle and ligament tension from lack of movement of fetus in womb, stimulation slowed breathing and increased blood flow to GI because lipophilic passing from animals to humans (milk), anagyrine built up in mothers and passed on to fetus; Investigation- human mothers were observed to consume goat’s milk and cheese lupin goat milk mother fetus Biological membranes- barriers to movement, compartmentalization (what to keep what to get rid of); bimolecular layer of phospholipids, primarily phophotidylcholine, and phosphotidylethanolamine; contains structural and functional proteins, signalling and cell adhesion molecules Principles of lipid/water solubility of chemicals in body, fluid, and tissues- body compartments have different pH’s, many toxicants are weak acids and bases, chemical movement related to ionization or lack there of Membrane barriers- organs ie. skin; tissue ie. epithelium; cells ie. cell membrane; cell organelles ie. nucleus; extracellular barriers- intercellular junctions, tight junctions, basement membrane (anchors epithelium) Barriers control fluid/chemical movement, cells form tissue membranes, kidney has greatest pore size (filter), then most cells; brain has intercellular junctions (extra filtration), endothelium has tight junctions that hinder movement (size varies controlled by hormone factors) Can cross the membranes: lipid soluble chemicals ie. DDT, steroids, PAHs (without energy requirement), small water soluble molecules through aqueous channels and pores, large water soluble molecules require special transport mechanisms Passive diffusion- lipid- transcellular difference, water soluble-pores, para-cellular rate depends on [ ] gradient (high to low), follow Fick’s law, surface area (larger area more diffusion), molecular size (smaller diffuse better), transport mechanisms- chemicals use endogenous 2+ 2+ substance carriers (Pb /Ca )- membrane protein carrier, structure specific, saturation and competition; Types: facilitated diffusion- moved with gradient, no energy required, active transport- energy dependent low high [ ] (paraquat in lungs), pinocytosis- least significant to absorption; filtration- energy dependent- water soluble chemical passing through pores and channels under hydrostatic pressure eg. Pressure of blood flow to a filter, small enough and size dependent; small molecules bulk flow, where bowel, kidney, capillaries Membrane damage- most chemicals are irritants that cause local damage to membranes/barriers via chemically altering membrane composition (hydrolysis, oxidation, reduction; physical damage or elicit immune response- inflammatory response increases blood flow, alters membrane) permeability responsible parties: cellular signaling proteins, WBC and immune cell release compounds that cause physical damage Membrane compromisedincreased absorption, pH of body compartment (weak acid/base) effects ionization, lipophilicity/hydrophilicity influencing chemical movement Xenobiotics- highly lipophilic, organic (more easily absorbed), act as weak acid/base, shift reactivity/solubility based on pH, stable, non-reactive (survive harsh conditions in gut), dangerous to human and environment- Equilibrium; partition coefficient measures lipid solubility increase ionization, increase water solubility, decrease ability to cross membrane Acidic toxins: Body compartment: increase pH, increase ionization of chemical; decrease pH, decrease ionization Basic toxins: body compartment: increase pH, decrease ionization of chemical; decrease pH, increase ionization Weak organic acids absorbed in stomach, weak organic bases absorbed in intestine but larger SA in gut overcomes lower [ ] Aspirin in blood pKa=3.5 pH blood ~7, if urine is pH=8 aspirin in ionized form and unable to cross membrane therefore trapped in urine compartment; 1 equilibrium of (non)ionized molecules created only non-ionized can leave which will create new equilibrium; if pH=3.5 in urine 50/50 ionized therefore recirculation and cycle repeats; changing pH of urine- usually around pH=6-6.5, acidify urine increase excretion of weak bases uses ascorbic acid, ammonium chloride used to get rid of PCP in drug users; basify urine- increase excretion of weak acids- use sodium bicarbonate used to treat phenobarbital toxicosis (pKa=7.2) or aspirin Routes of absorption: Integument structure (skin): keritinization- decreased water, increased lipid, increased thickness, layer of dead skin on top, don’t have intracellular spaces creates thick lipid barrier, movement by diffusion, no transporters, lipophilic rate of diffusion is very slow outer most layer- stratum corneun; Function- protect against external environment, thick lipoidal barrier; statum corneum- keratinized, thickness varies, no pores, tight intercellular junction, no transport systems , low blood flow to epidermis, more to dermis, lipids can be absorbed slowly, not hydrophilic compounds; access through dermal appendages - <1% total SA, more rapid passage, hydrophilic substances can use this route; dermis has various access points- glands, hair follicles, break continuous outer layer, high blood flow, take away absorbed toxins favourable equilibrium, fat stores (like dissolves like) Gastrointestinal route: (oral)- function- absorption of nutrients, characteristics favouring absorption- single cell lipoidal membrane (small diffusion, high blood flow and lymph flow, variable pH (3-8) range of organic acids, many specialized transport proteins (not diffusion dependent) absorption can occur from mouth to colon pH 2 in stomach, pH 6 in SI, 8 in colon, contents absorbed from small intestine go to liver, from colon bypasses liver, in GI blood supply keeps gradient in order, transport proteins for many xenobiotics expressed on brush border, transport proteins bring in large hydrophilic molecules protein families like organic anion transporters (OAT) or organic cation transporter (OCT) transport molecules with similar physical or chemical properties, competition for limited sites, equilibrium determines final blood [ ] Respiratory system- pulmonary route- lower airways (alveoli) allow for gas exchange (passive diffusion), high blood flow, thin membrane, depends on solubility (small particle can travel down, dissolve in fluid) protective mechanism- macrophages/phagocytosis of particulate matter/debris, susceptible to very small particles (penetration inversely proportional to particle size, gases diffuse across membrane depend on blood-gas partition constant, equilibrium dependent, high blood flow maintains favourable equilibrium, transporters present; Characteristics favouring absorption- single cell lipoidal membrane type 1 pneumocytes very thin, large SA- expansion of lungs, many alveoli, large blood supply, phagocytosis and migration (asbestos carcinogenesis) Asbestos toxicity- small silicate fibres, good insulator, fire resistant, chemical resistant, good tensile strength, different forms have different metals in their composition iron on some can produce toxicity through ROS production (creates radicals), gets ingested by phagocytic macrophages break membrane and proteins, causes local inflammation, radicals, degradative enzymes, prolonged inflammatory response, cell repair (collagen) and proliferation, asbestosis and possibly cancer Distribution of chemicals- transport in blood initially fast, dominated by blood flow, then based on affinity for free chemical receptors at tissue site; free chemical diffuses passively or through transporters at tissue- accumulation can occur, dependent on specific transporter larger ions have hydration shell not easy to transport in equilibrium between bound and unbound chemical Binding sites- albumin- major blood binding protein (barbituates, Ca , vit. C), erythrocytes (RBCs carry O , 2O  negative effect), lipoproteins (LDL, HDL, steroid hormones, fatty vitamins), globulins- lower [ ]s (alpha, beta carry acid and basic toxicants), blood binding proteins- various non-covalent forces in binding pocket will determine selectivity and binding affinity (hydrophobic interactions, van der Waal forces, H-bonding) based on electronic and hydrophobic state not ionic; Binding site competition- displaced toxin free form toxic; effect- ex. <99% bound decrease toxic effects, decrease elimination (storage), increase binding, decrease toxicity, decrease elimination only free toxicants eliminated Bilirubin- toxic to brain, jaundice, liver failure, sulfonamide displaced bound bilirubin on protein carrier albumin (had higher affinity BBB (undeveloped in premature babies), deposited in grey matter Highly protected areas barrier of protection- tight intracellular junction, no pores, thick lipoidal barriers, degrading enzymes (placenta), slow rate of distribution allows for increase time of elimination BBB- between blood and cerebral spinal fluid, selectivity- specific transporters, lipophilic substances (anaesthetics), endothelial cells form tight junctions, glial cells, astrocytes secrete chemical factors (hormonal influence), control permeability, structural role in maintaining integrity of BBB, no absorption of water soluble chemicals, regulate xenobiotic transporters that keep lipid soluble chemical out, low protein content in CSF limits transport of bound toxins BPB- nourishment, protection, gas exchange, get rid of waste, contains many cell layers to differentiate between mother and conceptus blood, active transport of essential molecules, diffusion of toxins, efflux transporters, metabolizing enzymes limit toxicant movement, not as robust as BBB, specific transporters bring in nutrients, pump toxins and toxic metabolites out, increase elimination (efflux>influx); diesthylstilbestrol- next generation carcinogen- pregnant mothers can have daughters who will develop vaginal cancer, able to diffuse across BPB BTB- few testicular toxins, sertoli cells provide barrier to nurture and develop growing spermatocytes, tight junctions between cells (stop diffusion) = escape barrier as entrance autoimmunity to sperm cells toxins: phthalate esters, glycol ethers, antiandrogenic agents Storage- concentrate and hold chemicals in tissue factors favouring storage: lipid nature (adipose, neural), pH, specific transport mechanisms, binding or incorporation of chemical into tissue, adipose tissue (PCBs), very fatty (like dissolves like), stay in favourable environment, decrease acute toxicity increase when fat stores mobilized (used for energy), decrease BFI Ex. THC takes about a month to eliminate Bone- Fluorine incorporated into matrix instead of Ca (F similar charge and size easily mistaken), hydroxyapatite (normal bone crystal), not permanent regular bone turnover - Teeth- discolouration (antibiotics), fluoride- replaces natural OH in tooth enamel crystals (protective) Liver and kidney- proteins (metallothionein) bind and sequester toxins many transporters (get bound to proteins and can’t leave), [ ] x50 > in liver than blood after 30 min Lung (paraquat)- specific one-way transport mechanism energy dependent storage effect on toxicity, decrease acute toxicity, can’t reach site of action, will alter dosage with pharmaceuticals, increase chronic toxicity because of chemical residues still present in body not metabolized or changed, will be released when fat stores mobilized, decreased elimination bound molecules too big for filtration, sequestered in organs outside of bloodstream, important for drug testing Paraquat- common industrial herbicide- low dose safe, ingested or inhaled= toxic, LD 50 oral=25-100mg/kg, dermal >200mg/kg (poor ability to cross epidermis), inhalation on very low toxicity, absorbed by specific organic cation transfer- accumulates in lung, kidney, liver; Pathogenesis- acute phase (1-3 days)- local GIT irritation and generalized herbicide, depression, edema, ulcers in GIT and brain, damages dopaminergic neurons (system of wellbeing and happiness) Mechanism of toxicity- free radical generation *OH , O , paraquat , O (singlet), o- 2 lipid peroxidation, NADH, free radical damage; delayed phase (3-8 day) type 1 alveolar cells damaged congestion and edema, hyline membrane formation; chronic phase (8 days)- diffuse fibrosis healing- concentrating in cells of lungs, damage-cell leakage, immune cells come to repair cause fibrosis which decreases function of cells by changing membrane creating scar tissue, epithelialization recruitment of more cells to the site (cause death 4-5 days); treatment- fuller’s earth bind cations acute: vit. E, C, selenium (antioxidants), steroids, mucomyst; chronic- supportive, grave prognosis, lung transplant Elimination: excretion, biotransformation- work in tandem to eliminate toxicants, characteristics favouring excretion: low lipid solubility, ionized and water soluble, non-protein bound, low molecular weight (kidney), high molecular weight (liver); mechanisms- filtration (renal), active secretion (urine/bile), diffusion into glandular secretions (mammary, sweat, saliva), excretion failure- lipid storage bioaccumulation, reabsorbed (Hg-metalothionine, bile salts), involves active transport, binding storage (lead) Kidney: high blood flow around loop of Henle, little xenobiotic reabsorption, hydrostatic filtration hydrophilic pushed through water, sugars, peptides reabsorbed (transport mediated) some transporters for secretion balance between influx and efflux Liver: receives oxygenated blood from heart (hepatic artery), receives blood from stomach, SI, LI, pancreas, spleen (portal vein) hepatocytes line hepatic capillaries, bile ducts collect fat soluble waste, drains to gall bladder, liver major site of metabolism secreted to bile released back to intestine Biliary excretion (active transport)- bile salts emulsify fats (break down for easier digestion) lipophilic and hydrophilic character, accommodates lipid soluble toxicants depends on size 3 categories: ratio around 1- glucose, Hg, thallium, cesium, Na; ratio >1- bile acids, bilirubin, lead, many xenobiotics; ratio <1- insulin, albumin, zinc, iron, gold, glutathione and glucaronide conjugates generally excreted well in bile, ultimately depends on concentration and position of specialized transporters, excretion increased by agents induced enzymes and transporters From general circulation- kidney- filtered through pores in PCT cells (active transport and diffusion), can reabsorb nutrients and toxins, pH dependent Enterohepatic circulation- A. liver- portal blood flow (from bowel) high exposure, active uptake (large MW compounds) active secretion, metabolism- conjugation active secretion; B- Gut- lipid soluble chemicals reabsorbed into portal blood or bacteria remove conjugate and release lipid (persistence), soluble chemical then reabsorbed into portal blood Glandular secretion: mammary- diffusion mediated, slightly acidic (traps basic toxicants), 3- 4% fat content some lipophilic [ ] like PAH- significant accumulation in child; sweat and salivary glands (not major route)- diffusion mediated, electrolytes, urea, water soluble <1% heavy metal excretion Biotransformation (metabolism): endogenous, xenobiotics, purpose- change lipophilicity, easily absorbed chemicals from GIT into suitable excretory form; organs: liver kidney > gut> blood > placenta; Result- alter chemical structure- activation and deactivation detoxify, increase ionization increase water solubility or decrease lipid solubility; Phase I- produces primary metabolites with functional group- increase reactivity (OH, COOH, SH, NH ); Phase II- adds 2 conjugates (large molecules ie. sugar) to functional group to increase water solubility creating non-reactive product, easily excreted; exceptions- gemfibrodil conjugated before oxidized, acetaminophen conjugated directly with glucoronic acid Phase I phase II Xenobiotic primary metabolite (functional group) final product Phase I: reactions- oxidation reduction and hydrolysis, Cytochrome P 450s located in SER, characteristics of CYP- 450s- lack substrate specificity- broad range of substrates, bind lipid soluble, hemoprotein- binds O a2d accepts electron, splits O – 2reaks chemical bonds of O  2 oxidative reactions, CYP 450 rxns- hydroxylation (Adding on OH group to C not with a double bond), heteroatom hydroxylation (adding OH to a non C atom), epoxidation; characteristics of phase I (mixed oxidase rxns MFO) products are primary metabolites; advantages- potentially less reactive (detoxified), more readily excreted, ready for conjugation if necessary; disadvantages- more reactive bioactivation toxicity, biotransformation error electron transfer reaction (free radicals), enzyme induction increases activity, increases biotransformation, enzyme inhibition decreases activity, decreases biotransformation Inducible enzymes- induction mediated by ligand- activated receptors (xenosensors), DNA binding proteins, transcription factors, up regulate transcription of P 450 get more protein in cells, can also up regulate by stabilizing proteins at promoter site toxicity after phase I if phase II overwhelmed Phase II conjugations- synthetic reactions that add large water soluble compounds to functional groups occurs in SER, cytoplasm, mitochondria conjugating agents- glucuronic acid glucuronides, acetyl Co-A acetyled, PAPS sulfonated, glutathione mercapturic acid, conjugating agents are lost in elimination process possibly used for biomarkers Glucuronic acid- substrate: UDP- glucuronic acid, enzyme: UDP-glucuronosyltransferase, low affinity but high capacity system, nucleophilic attacks at anomeric carbon of sugar usually OH, NH ,2SH responsible, UDP leaving group Acetylation- substrate: acetyl Co-A, enzyme: N-actyltransferase (NAT), or O-acetyltransferase (OAT), depending on nucleophile (NH , OH2, Sulfonation- enzyme: sulfonotransferase (SULT), xenobiotic enzymes are not membrane bound, 3’phosphoadenosine-5’phosphosulfate (PAPS), low [PAPS] (synthesis requires cysteine which is limited), high affinity, low capacity conjugation, involves nucleophilic attack on electrophilic sulfur, cleavage of phosphosulfate bond Glutathione- substrate: glutathione (tripeptide) enzyme: glutathione S-transferase (GST) electrophilic attack by nucleophilic thiol on electron deficient sites P450 epoxide  conjugate Free radicals/lipid peroxidation- radical species are damaging to cell in various ways lipid peroxidation- DNA cleavage as well as deplete cellular stores of reducing agents Free radical detoxification (occurs in all cells and blood)- superoxide (O•) dismutase, uses metal; cofactor gets reduced (gain electrons) to detoxify superoxide radical, outcompetes native reaction, protects cells from damage (nutritional status dictates ability to catalyse reactive species) Catalase- detoxifies hydrogen peroxide- not important in secondary detoxifying agent inhibited - st by heavy metals and CN , iron oxidized in 1 step, glutathione peroxidase- detoxifies radical species and hydrogen peroxide produces glutathione dimer which undergoes redox reaction to regenerate reduced starting material not lost but regenerated by glutathione synthase, glutathione-disulphide reductase Natural antioxidants- direct chemical reaction Vit. C, E, beta carotene, uses NADH, NADPH, flavin coenzymes (reducing coenzymes) passing of radical from more reactive to less reactive molecule; oxidative stress- occurs when [ ] of harmful pro-oxidant species- radicals (superoxide, alkyl, hydroxyl), overwhelm anti-oxidant defense mechanism (glutathione, peroxidase, SOD, Vit. E, C) also if [oxidant] high enough, will exhaust supply of antioxidants (GSH, cysteine) decreased diet decreased level of antioxidants Phenols pro vs. anti oxidants behave as antioxidants protect against lipid peroxidation, quench free radicals, electron releasing alkyl and methoxy groups, behave ass pro-oxidants- PCP, electron withdrawing substituents Toxiodynamics- target organ toxicity if all parameters fail and high [ ] of chemical reaches the target toxicosis occurs Alcohol + acetaminophen- direct phase II glucoronidation, sulphonation; phase I- CYP 450 monoxygenase hydroxylation rearrangement (NAPQI)  glutathione conjugation or toxicity (reacts with proteins and nucleic acids) alcohol role- ethanol induces CYP 2E1 (important for phase 1 tylenol), push metabolic pathway towards more production of NAPQI, ethanol metabolized to acetaldehyde which is very reactive forms free radicals can deplete antioxidant stores glutathione needed to detox NAPQI, alcohol consumption up regulates CYP 2E1 depletes gluatathione both produce more NAPQI hepatotoxicosiscirrhosis liver failure Toxicosis- when the chemical reaches the target in sufficient concentration to cause adverse effects;The target often large functional molecule: lipid, protein, or DNA called body macromolecules; high specificity- chemical and target interaction like lock and key, specific receptor, pharmacologic receptor, natural toxins, low dose required, high specificity- receptor is selective; low specificity- target located in many tissues/ macromolecules, macromolecules with reactive functional groups (DNA can be cross-linked), nucleophilic/electrophilic attack, exhibit nonspecific clinical effects, high dose required; competitive binding- weak (ionic) interactions with target, hydrogen bonds, van der Waals forces, hydrophobic interactions (non-covalent), not permanent (can be reversed), may compete with a natural substrate, most drugs, some pesticides (carbamates, CO, natural toxins), Covalent binding- target molecules permanently bound, sometimes called “arylation or alkylation”, organophosphate pesticides- permanently bind acetylcholine esterase enzyme stops function, aflatoxin-DNA binds permanently must be removed enzymatically; incorporation- chemical may act as substitute for endogenous compound/substrate and can be incorporated into target- fluoride, selenium; peroxidation- free radicals can
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