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Lecture 5

Biomedical Toxicology - Lecture 5 through 8

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TOX 2000
Aaron Witham

Lecture 5 – November 9, 2012 Review Questions - Excessive consumption of fat-soluble vitamins may be toxic because they are not easily eliminated (B) - toxicity associated with fluorosis involves brittle teeth and bones (D) Removal of Chemicals from the Body - excretion and biotransformation (metabolism) work in tandem to eliminate exogenous toxicants - main routes: o kidney  urine (aqueous)  deals with molecular weight chemicals o liver/bile  fecal (lipids and aqueous)  deals with molecular weight chemicals - characteristics favoring excretion: o lipid solubility o ionized and water soluble o free toxin = non-protein bound Mechanisms of Excretion - filtration o renal – urine - active secretion o kidney – urine and liver – bile - diffusion into glandular secretions o mammary, sweat, saliva - these mechanisms can fail: o lipid-soluble  soluble  bioaccumulation o reabsorbed  kidney (Hg-metalothionine)  GIT (bile salts)  active transport o binding  storage  bioaccumulation  lead The Kidney - nephrons (1 million) glomerulus - high blood flow - leaky podocytes - spaces 40 angstroms o 1A = 1/100000000 cm - hydrostatic filtration: hydrophilic chemicals are pushed through - water, sugars, and peptides are reabsorbed o transport mediated - some transporters contribute to secretion - balance between influx and efflux Liver Anatomy - receives oxygenated blood from the heart via the hepatic artery - receives blood from the stomach, SI, LI, pancreas, and spleen via the portal vein - tissue is divided into lobules and portal triads o lobules: hexagonal o portal triads: divisions on corners - hepatocytes lines sinusoids o hepatic capillaries - bile ducts are arranged to collect fat-soluble waste o drain into the gall bladder Liver Secretion - liver is the first target of the molecules absorbed from the GI tract - liver is a major site of metabolism o many xenobiotics transporters filter blood and import molecules - hepatocyte functions: o synthesize bile o secrete conjugates into bile  bile drains into the gall bladder and ends up back in the intestine Billiary Excretion - bile salts: compounds that emulsify fats by breaking down large globules for easier digestion o composition similar to detergents:  both lipophilic and hydrophilic characteristics o size is very important - compounds are divided into three categories based on the ratio of biliary to plasma secretion o ratios close to one  glucose, mercury, thallium, cesium, sodium o ratios greater than one  bile acids, bilirubin, lead, many xenobiotics o ratios less than one  insulin, albumin, zinc, iron, gold - MW affects ability to go through biliary secretion: o MW < 325Da are poorly excreted o MW > 325Da are easily excreted o example: glutathione and glucuronide conjugates generally excreted well in the bile - excretion depends on the concentration and the position of specialized transporters - biliary excretion can be increased by agents that induce enzymes and transporters Major Routes of Excretion - depends on size and lipophilicity o larger conjugates and molecules  bile o smaller conjugates and molecules  urine - depends on location and abundance of transporter proteins - from general circulation: o kidney-filtration trough pores and in PCT cells  both active transport and diffusion can reabsorb nutrients and toxins o pH of environment (bladder) will affect these processes o large conjugates will be actively transported in the bile to the SI to become fecal waste  applies to both hydrophilic and lipophilic substances Enterohepatic Circulation A. Liver - portal blood flow (from bowl)  high exposure - active uptake  large MW compounds – active secretion into bile - metabolism  conjugation – active secretion in bile - transferred by the bile to the gut B. Gut - lipid soluble chemicals reabsorbed into portal blood or bacteria remove conjugate and release lipid soluble chemicals o reabsorbed into the portal blood Glandular Secretions A. Mammary - diffusion mediated - pH is slightly acidic so basic toxicants can get trapped o protonated conjugate acid - 3 – 4% fat content o some lipophilic concentration  PAH, DDT  significant for accumulation in children B. Sweat and Salivary Glands - diffusion mediated - electrolytes, urea, water-soluble molecules (ethanol) DDT: An example of stable lipid soluble chemical toxicokinetics - Lactating Mother  Oral intake (0.0005 mg/kg/day DDT) - Mother’s Body Fat  1.0 ppm DDT - Milk (eggs) contains  0.08 ppm DDT - Infant Dosage  0.0112 mg/kg/day o Infant Dose 20X greater than mother - PCB and mehtlmercury also undergo enterohepatic circulation # Lecture 6 – November 12, 2012 Biotransformation - definition: transformation of chemicals (both endogenous and xenobiotics) by metabolic enzymes - purpose: change lipophilic, easily absorbed chemicals from GI into a suitable excretory form - organs: (most important to lest important) o liver o kidney o gut o blood o placenta (all cells) - result: o chemical structure altered  activation of deactivation (detocify) o ionization   H O so2ubility o lipid solubility o Process A. Phase I Metabolism - produces primary metabolites with a functional group - increased reactivity (chemical handle) o OH, NH , S2, COOH B. Phase II Metabolism - adds (conjugates) a large molecule (i.e. sugar) to functional group o water soluble non-reactive product o easily excreted Exceptions A. Germfibrozil - goes through phase II before it is oxidized by its phase I product o already has a carboxyl group (chemical handle) B. Acetaminophen - conjugated directly with glucronic acid o does not undergo phase I metabolism o already has a reactive functional group Summary Phase I Reactions ―Oxidation‖ Reduction and Hydrolysis - primary enzymes: cytochrome P-450 - location: smooth endoplasmic reticulum (SER) - characteristics: o broad substrate specificity  binds lip3+ soluble2+hemicals o contain iron (Fe and Fe ) in the binding site  can bind O 2  can accept e - o split O a2d add it to substrates  ―oxidative reactions‖  results in the addition of ―chemical tags‖ o R-OH, R-NH , R-S2, R-COOH - recall that electrophilic molecules have greater attraction for electrons o stronger bonding A. CYP 450 - hydroxylation: addition of OH group to a carbon chain - epoxidation: addition of OH group to non-carbon atoms - heteroatom hydroxylation C. Mixed Function Oxidase Reactions - products are primary metabolites - advantages: o potentially less reactive with target  detoxified o more readily excreted o ready for conjugation (phase II)  have reactive sites - disadvantages o more reactive ―functional group‖  bioactivation  toxicity o biotransformation error  electrion transfer reaction  leads to free radicals  toxicity o enzyme ―induction‖  activity and biotransformation o enzyme ―inhibition‖  activity and biotransformation - consider effects of these results Inducible Enzymes - induction mediated by ligan-activated receptors (xenosensors) - DNA binding proteins, transcription factors, up-regulate transcription of P450 o get more protein in the cells o also can up-regulate by stabilizing proteins at promoter site - receptors: o AHR o CAR o PXR o ARNT Updated Summary Phase II Reactions Conjugation - synthetic reactions add large water soluble compounds (cellular metabolites or by- products) to functional groups - makes phase I products hydrophilic o reactivity - location: smooth endoplasmic reticulum, cytoplasm, mitochondria - conjugating agents  products o glucuronic acid  glucuronides o acetyl coenzyme A  acetylated o PAPS  sulfonated o gluthione  mercapturic acid  undergoes further metabolism - conjugating agents are lost in the elimination process o possible use as biomarkers Glucuronic Acid Phase II Metabolism - substrate is UDP-glucuronic acid o site of attack marked by a star - enzyme is UDP-glucuronosyltransferase - low affinity but high capacity system - - a nucleophile will attack the anomeric carbon of the sugar (e weak area) o usually OH, NH , SH 2esponsible o UDP is leaving group - phosphate = stable anion = good leaving group o anomeric carbon = attack site - UDP glucuronic acid presence in body o lots of substrate Acetylation - substrate: Acetyl CoA o only really concerned with the boxed group o e deficient site  nucleophile attacks here  either uses N-acetyltransferase (NAT) or O- acetyltransferase (OAT) enzymes depending on the nucleophile (OH NH ) 2 - already has acetyl bound to it but can still undergo PI with CYP-450 to neutralize that reactive group Sulfonation - catalyzed by sulfotransferase enzyme (SULT) - xenobiotics enzymes are not membrane bound - uses 3=-phosphoadenosine-5’-phosphosulfate (PAPS) o [PAPS] because synthesis requires concentration cytosine (limited) - high affinity, low capacity conjugation - involves nucleophilic attack on electrophilic sulfur  cleavage of phosphosulfate bond o much wider substrate specificity  contrast to glucuronic conjugation o all 4 methods discussed involve nucleophilic attack Glutathione Conjugation - substrate: glutathione o tripeptide (glycine, cytosine, glutamic acid) - enzyme: glutathione-S-transferase (GST) - process: electrophilic attack by nucleophilic thiol on electron deficient sites o creates electrophilic sites  both carbons very e -  not nucleophiles o creates diol nucleophile to attack conjugation agent at one of the e deficient sites - must be synthesized in the cell o amino acids required  nutritional status matters - for the exam… know reactivity Phase II Reactions: Summary - characteristics of conjugated chemicals: o highly water soluble o non-reactive and non-toxic o easily excreted  urine  bile (large molecules) Updated Summary Free Radicals and Lipid Peroxidation - systems are not perfect o ETC can form free radicals and/or superoxide metabolism products o UV light can form free radicals o phase I metabolism areas can form free radicals in the boxed area - radical species are damaging to the cell in various ways o can participate in lipid peroxidation and DNA cleavage o can deplete cellular stores of reducing agents - lipid peroxidation: o lipid missing electrons  holes pokes in cell membranes  can react with DNA  abstract to H4and mess up DNA (at the sugar) Free Radical Detoxification - occurs in all cells (cytoplasm) and blood - for the exam… know enzyme and reaction A. Superoxide (O) Dismutase (contains Cu, Zn, or Mn) - H 2 i2 reactive oxygen species o less so than superoxide catalase anion - deficiency in any nutrients cause metal and ability for body to deal with superoxide anions - enzymes uses metal cofactor  gets reduced (gains electrons) to detoxify superoxide radical - outcompetes native reactions  protects cell from damage B. Catalase - has iron inside a heme porphyrin ring o can be inhibited by heavy metals - substrate: hydrogen peroxide - - not as important, but secondary detoxifying agent can be inhibited by CN and some heavy metals (Cu ) 2+ - iron is oxidized in the first step, and produces two water molecules in addition to oxygen C. Glutathione Peroxidase - very important o several reaction to detoxify several species o needed for both Phase I and Phase II, so needs to be regenerated  glutathione is not lost but can be regenerated by glutathione synthase and glutathione-disulfide reductase - contains Selenium and requires reduced glutathione – GSH - detoxifies radical species and hydrogen peroxide - produces oxidized glutathione dimer which undergoes redox reaction to regenerate reduced starting material D. Natural Antioxidants - group of molecules that help take care of reactive oxidative species (ROS) - direct chemical interaction o examples: Vitamin C, Vitamin E, Beta-Carotene - reducing cofactors are used in the redox cycle o examples: NADH, NADPH, flavin coenzymes - passing Ofrom reactive to less reactive, easier to deal with Oxidative Stress - occurs when concentrations of harmful pro-oxidant species overwhelms anti- oxidative species o harmful: radicals (superoxide, alkyl, hydroxyl), H O , H2Cl2 ONOO o anti-oxidant defence mechanism: glutathione peroxidase, SOD, Vit C - if oxidant concentration I shigh enough, it will exhaust the supply of anti-oxidants o examples: GSH, cytosine - poor diet can lead to decreased levels of cellular anti-oxidants - oxidative stress  Alxheimer’s, Parkinson’s, Cancer Phenols, Pro vs. Anti-Oxidants - behave as anti-oxidants o vitamin E o protect against lipid peroxidation o quench free radicals o electron releasing alkyl and methoxy groups - behave as pro-oxidants o PCP o electron withdrawing substituents Acetaminophen and Alcohol - ethanol induces CYP2E1 - will push metabolic pathway towards more production of NAPQ1 - ethanol is metabolized to acetaldehyde which is very reactive, forms free radicals, detox by viamin C, vitamin B12 and other antioxidants - can deplete anti-oxidant stores, glutathione needed to detox NAPQ1 - alcohol consumption will upregulate the CYP2E1 enzyme and deplete glutathione stores - both will produce more NAPQ1 which can react with proteins and cause hepatotoxicosis - chronic alcoholics develop cirrhosis but NAPQ1 can also cause liver failure # Lecture 7 – November 14, 2012 Toxicosis - toxicosis: occurs when the chemical reaches the target in sufficient concentrations to cause adverse effects - toxicodynamics: occurs if all parameters fail and high concentration of chemical reaches the target The Target - characteristics: o often large functional molecule  examples: lipid, protein, DNA  called body macromolecules A. High Specificity o chemical and target interact like ―locak and key‖ o specific receptor  example: estrogen/testosterone receptor o pharmacologic receptor  example: cholinergic/adrenergic  taken advantage of for drugs such as the birth control pill o low dose required B. Low Specificity - target ubiquitous within the body o located in many tissues and macromolecules - target has reactive functional groups o examples: -SH, -NH, -OH  exist on many protein enxymes and DNA; can be cross-linked by chemicals such as aflatoxin - reactions occur by nucleophilic or electrophilic attack - effects are non-specific o depends on what it interacts with - high does required Chemical-Target Interactions A. Competitive Binding - weak (ionic) interaction with target - H-bonding - Van der Waals forces - hydrophobic interactions - temporary o may compete with a natural substrate - examples: most drugs and some pesticides o carbamates (ache) o CO (competes with O on 2 hemoglobin) o natural toxins: mycotoxins, zeralenone B. Covalent Binding (Arylation, Alkylation) - permanent - example: organophosphate pesticides o permanently bind acetylcholine esterase (Ache) enzyme  arrests function - example: aflatoxin o binds permanently to DNA  can be removed enzymatically C. Incorporation - occurs when a chemical acts as a substitute for an endogenous compound o substrate can be incorporated into the target - examples: o fluoride  teeth and bones  brittleness o selenium  cytosine residues impacts S-S bonds  improper protein o folding; lead  bone  brittleness D. Peroxidation - free radicals  unstable target due to abstraction of electrons - example: lipid peroxidation o unsaturated fatty acids contain many double bonds, which are sites of electrophilic attack by free radicals - increase oxidative stress o associated with Alzheimers and Parkinsons E. Antigenic (Allergic) Interactions - antigen characteristics: o large o bind tissue  hpatenize tissue, making it antigenic as well - examples: penicillin, metals such as mercury, glomerulus Consequences of Chemical-
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