Class Notes (835,495)
Canada (509,212)
Toxicology (14)
TOX 2000 (13)
Lecture 5

Biomedical Toxicology - Lecture 5 through 8

44 Pages
124 Views
Unlock Document

Department
Toxicology
Course
TOX 2000
Professor
Aaron Witham
Semester
Fall

Description
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-
More Less

Related notes for TOX 2000

Log In


OR

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


OR

By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.


Submit