1 Biotransformation and Elimination of Toxicants Principles of Environmental Toxicology Instructor: Gregory Möller, Ph.D. University of Idaho Principles of Environmental Toxicology 2 Learning Objectives • Explain the role of biotransformation in toxicokinetics. • Describe how biotransformation facilitates elimination of toxicants. • Distinguish between Phase I and Phase II reactions. • Define bioactivation or toxication. Principles of Environmental Toxicology 3 Learning Objectives, 2 • Identify tissues and factors involved in biotransformation. • Summarize the role of elimination in toxicokinetics. • Describe processes occurring in the kidney, liver and lung related to the elimination of toxicants. Principles of Environmental Toxicology 4 Metabolism • Sum of biochemical rxns occurring to a molecule within the body. – Anabolism - “build-up” – Catabolism - “break-down” • Occurs in the cytoplasm or at specific organelles within the cell. • Storage affects the body’s ability to biotransform and eliminate. – Bone, lipid. Principles of Environmental Toxicology 5 Biotransformation • Process that changes substances from hydrophobic to hydrophilic to aid in elimination (grease to salt). – Hydrophilic molecules are less able to cross cellular membranes, hence fluid filterable (kidneys). – Major elimination routes are feces (biliary) and urine. – Biological half-life, T ½ allows comparison of rates of removal. Principles of Environmental Toxicology 6 Biotransformation Reactions • Grouped as Phase I (functional group modification) and Phase II (conjugation). • Goals – Produce water soluble metabolites. – Activate natural/endogenous compounds for normal function. • Some compounds undergo bioactivation . – The biotransformed metabolite is more toxic than the original compound. 2 Principles of Environmental Toxicology 7 Results of Biotransformation • Increase toxicity via a toxic metabolite. • Decrease toxicity via metabolism of a toxic parent compound. • No effect on toxicity. • Present to metabolize endogenous compounds. Principles of Environmental Toxicology 8 Major Categories/Reactions Phase I Phase II Elimination oxidation reduction hydrolysis conjugation synthesis polar very polar Principles of Environmental Toxicology 9 Enzymes of Biotransformation • Oxidation (most important). – Add O, remove H, increase valence. – Cytochrome P-450, MFO, alcohol dehydrogenase, oxidases, others. • Reduction (less important). – Remove O, add H, decrease valence. – Reductases. • Hydrolysis. – Add water. – Esterases, phosphtases, others. Phase I Enzymes Principles of Environmental Toxicology 10 Phase I Reactions NH2R S R 2 R 1 R 1 R 2 C O R 2 R 1 C-O O R 1 R 2 CS RCH2OH R 1 R 2 CH OH SO R 2 R 1 NHOHR R 1 CH2OOH RCHO R 1 R 2 CO R 2 HO N-oxidation S-oxidation Carbonyl reduction Ester Hydrolysis Desulfuration Dehydrogenation + Hughes Principles of Environmental Toxicology 11 Enzymes of Biotransformation, 2 • Conjugation reactions. • Enzymes (tranferases) + cofactor. – Enzyme catalyzes. – Cofactor donates group. – Glucuronic acid, glutathione, sulfate, acetyl group, methyl group. – Tends to increase molecular size and polarity for excretion. Phase II Enzymes Principles of Environmental Toxicology 12 PII Cofactors: GSH H N O HS O N H NH 2 HO O OH O Glutathione 3 Principles of Environmental Toxicology 13 PII Cofactors: Acetyl-CoA Acetyl Coenzyme A N N N NH 2 N O O P OH O O O P O HO HO O NH NH O S HO O OH P HO O O Principles of Environmental Toxicology 14 PII Cofactors: PAPS OH O O N N N N NH 2 OSO O OH PO O OH P OH OOH 3’-Phosphoadenosine 5”-phosphosulfate Principles of Environmental Toxicology 15 PII Cofactors: UDPGA O HO HO H H H H 2 C H N H N O O OHP O O HO P O O O H HO H HO H OH H H - O O Uridine-5’- diphosphoglucuronic acid Principles of Environmental Toxicology 16 Benzene Metabolism OH ST O OH P450 PAPS O OSO 3 OH OH Glutathione Epoxidation G SH GST Toxic Epoxide Phenol Glucuronide UDP-GT Epoxide Hydratase Dihydrodiol Principles of Environmental Toxicology 17 Aniline NH 2 P450 H N OH Phase II Amine N-hydroxylation Principles of Environmental Toxicology 18 De-Alkylation N P450 H N HC O + Phase II Dimethyl-propyl-amine Methyl-propyl-amine Acetaldehyde 4 Principles of Environmental Toxicology 19 Free Radical Generation C Cl Cl Cl Cl N A DH P450 Reducatase C Cl Cl Cl To x ic F r ee Radical GSH Tet r achlo r o-methane Principles of Environmental Toxicology 20 Case Study: Fluorocitrate and Kangaroos • Fluorocitrate found in legume pasture plants of Western Australia. – Gastrolobium and Oxylobium. • Highly lethal (TD 1 mg/1080 kg). – Leaf concentrations can be 2.6 g/kg. • The rat and gray kangaroo of Western Australia have evolved resistance. – In vivo defluorination w/ glutathione. – Other kangaroos from areas w/o these plants are not tolerant. WACALM Harborne Principles of Environmental Toxicology 21 Rodenticide: Fluoroacetic Acid OH O F Co A SH FCCOSCoA H H Fluoroacetate Fluoroacetyl CoA Sodium Fluoroacetate Compound 1080 rodenticide predator control Principles of Environmental Toxicology 22 Fluorocitrate Metabolite HO O OOH O AcCoA FAcCoA OH HO O OH O O HO F Principles of Environmental Toxicology 23 Krebs Cycle OH HO O OH O O HO F AcCoA FAcCoA H 2 O HO O OOH O HO O OH O O HO Aconitase (Fluo r o)Cit r ate Oxaloacetate Cis-aconitate Mitochondrial energy production Principles of Environmental Toxicology 24 Deoxynivalenol, Vomitoxin O HO HO O O O HO HO OH O CH 2 OH Fusarium trichothecene mycotoxin found on corn and barley 5 Principles of Environmental Toxicology 25 Aflatoxin B 1 O O O O O H H O O O O O O H H O OH B 1 Q 1 = hepatic metabolite Aspergillus mycotoxin found on corn, peanuts and cottonseed Principles of Environmental Toxicology 26 Benzo[a]pyrene OR R = sulphate or glucuronic acid • Polycyclic aromatic hydrocarbon. • Environmental carcinogen. • Cell cultures from rodents, fish and humans Principles of Environmental Toxicology 27 Heavy Metal Toxicity - Pb • Absorbed via Ca channels as divalent ion. • Capable of reacting with a variety of binding sites. – Protein precipitation. • Specific toxic effect depends on rxns with ligands that are essential for the living system. • Metal ligands are formed with sulfhydryl groups, as well as amino, phosphate, imidazole, and hydroxyl groups of enzymes and essential proteins. Principles of Environmental Toxicology 28 Heavy Metal Toxicity - Pb, 2 • Sensitivity of a system and degree of interference determines clinical effects. – Digestion/respiration → absorption. –Liver → detoxication. –Kidney → excretion. • Antidotes are competing ligands. N NO OH O OH O HO O HO EDTA Principles of Environmental Toxicology 29 Heavy Metal Toxicity - Pb, 3 • Metallic lead absorbed most efficiently by the respiratory tract. • 10% of ingested lead is absorbed. – Small intestine. – Lead salts are soluble in gastric juices; absorbed. • Plasma to blood cells – erythrocytes. • After oral ingestion: – 60% bone (also hair, teeth). – 25% liver (hepatocytes). – 4% kidney (renal tubules). – 3% intestinal wall. Principles of Environmental Toxicology 30 Heavy Metal Toxicity - Pb, 4 • Some endpoints. – Sulfhydral enzyme inhibition. – K transport in RBC inhibited • Anemia. – Porphyrinuria. • Excreted chiefly in feces and urine. • Chelating agents: –Ca -EDTA. – Penicillamine. – Dimercaptrol (BAL). C H2 CH C H2 HO SH SH 2,3-Dimercapto-propan-1-ol 6 Principles of Environmental Toxicology 31 Case Study: Elevated PbB Associated with Illicitly Distilled Alcohol, Alabama 1991 • The use of automobile radiators containing lead-soldered parts in the illicit distillation of alcohol (i.e., "moonshine") is an important source of lead poisoning among persons in some rural Alabama counties. • In 1991, eight persons were diagnosed with elevated blood lead levels (BLLs) at a local hospital. • 9 patients had been evaluated for alcohol- related medical conditions at the hospital. Manifestations included generalized tonic-clonic seizures (six), microcytic anemia (five) (hematocrit mean: 32.1%), encephalopathy (two), upper extremity weakness (one), and abdominal colic (one). BLLs ranged from 16 ug/dL to 259 ug/dL (median: 67 ug/dL). MMWR (1992) 41(17);294-295 Principles of Environmental Toxicology 32 Case Study: “Moonshine” Lead Toxicity • Seven patients required hospitalization for 48 hours or longer (range: 2-18 days). Three of these received chelation therapy; initial BLLs were 67, 228, and 259 ug/dL. One patient, whose BLL was 67 ug/dL, died during hospitalization from alcohol- withdrawal syndrome complicated by aspiration pneumonia. • Patients reported moonshine ingestion ranging from 0.2 L per day to 1.5 L per day. • The lead contents of specimens of moonshine confiscated from two radiator- containing stills in the county in 1991 were 7400 ug/L and 9700 ug/L, compared with nondetectable amounts (less than 1.0 ug/L) in municipal water from the county. • Consumption of 0.5 L per day of moonshine containing 9700 ug/L lead would result in a steady state BLL of approximately 190 ug/dL. Principles of Environmental Toxicology 33 Elimination of Toxicants • Urinary. • Fecal. • Respiratory. • Other: –Saliva. –Sweat. – Milk (transfer to child). – Nails, Hair, Skin. – Cerebrospinal fluid. Hughes Principles of Environmental Toxicology 34 Kidney Principles of Environmental Toxicology 35 Renal Macrostructure Renal cortex Renal medula Ureter Bovine Principles of Environmental Toxicology 36 Renal Filtration Microstructure 7 Principles of Environmental Toxicology 37 Renal Histology Tubules Glomerulus Microscopic Principles of Environmental Toxicology 38 Urinary Excretion • Glomerular filtration • Tubular secretion • Tubular reabsorption Principles of Environmental Toxicology 39 Fecal Excretion • Excretion in bile to intestine. – Active transport of toxicant parent and metabolites. – Highly soluble Phase II metabolites (large, ionized) • Excretion into the lumen of the GI tract. – Direct diffusion from capillaries. NLM Principles of Environmental Toxicology 40 Exhaled Air • Gas phase xenobiotics. • Passive diffusion from blood to alveolus via concentration gradient. – The total alveolar epithelial surface area within an average adult human lung has been estimated to be as large as 100-140 m 2 . Gray's Anatomy 1918 . 1 Biotransformation and Elimination of Toxicants Principles of Environmental Toxicology Instructor: Gregory Möller, Ph.D. University of Idaho Principles of Environmental Toxicology 2 Learning. Objectives • Explain the role of biotransformation in toxicokinetics. • Describe how biotransformation facilitates elimination of toxicants. • Distinguish between Phase I and Phase II reactions. •. Consumption of 0.5 L per day of moonshine containing 9700 ug/L lead would result in a steady state BLL of approximately 190 ug/dL. Principles of Environmental Toxicology 33 Elimination of Toxicants •