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Pulmonary Toxicology : Disposition, Metabolism and Enzyme Kinetics
Anthony J. Hickey, Ph.D., D.Sc. School of Pharmacy, UNC-Chapel Hill, NC
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Introduction Lung Deposition Clearance Mechanisms Lung Cells
Mucociliary Transport Cell Transport Absorption Lung Cells Enzyme Expression Metabolism Conclusion
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Nasal Passages T-B Airways Pulmonary Parenchyma Lymph Nodes B l o d G I T r a c t
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Mucus blanket Cilia Columnar epithelial cells
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FRACTION CLEARED PER DAY (X103)
MICE RATS PEOPLE DOGS AND GUINEA PIGS 100 10 1 0.1 FRACTION CLEARED PER DAY (X103) DAYS AFTER INHALATION
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1 µm polystyrene latex; 30 min; 60x
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Thompson, 1992
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Passive Diffusion Facilitated Diffusion Active Transport
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MOLECULAR WEIGHT (daltons)
RAT RABBIT DOG SHEEP FETAL LAMB MAN, AEROSOL DOG, AEROSOL 100 10-1 10-2 10-3 10-4 10-5 CLEARANCE (min-1) MOLECULAR WEIGHT (daltons) Effros and Mason, 1985
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Cross section of two stages
Aerosol Throat Cross section of two stages Airflow Snapwell™ containing epithelial cell monolayer Pre-separator 1 -The idea behind this was size selective delivery of particles based on what each cell type would see. 2 3 Petri dish 4 5 Vacuum 6 To vacuum pump
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Confluent monolayer of the small airways epithelial cells
Airflow Single Stage of the Cascade Impactor Showing Orifices Transwell® Dish Containing Epithelial Cell Monolayers Petri Dish Vacuum
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Relationship between clearance from the lungs and molecular weight of FITC-dextrans
Arrows indicate the positions of 4.4, 9.5, 21.2, 38.9 and 71.2 kD markers. Dotted lines represent the range of the data obtained from the different species.
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Comparison of relative permeability coefficients determined using in vitro model and relative in vivo clearance from the lungs for FITC-dextrans (4.4:9.5kD; 4.4:21.2kD; 4.4:38.9kD; and 4.4:71.2kD) FITC-Dextran Average MW (kD) 9.5 21.2 38.9 71.2 Relative Permeability: Papp(4.4kD)/Papp(x kD), (n=3) 3.0 2.9 4.9 12.9 Relative In-vivo Clearance: Cl(4.4kD)/Cl(x kD) 4.4 6.2 6.6 20
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Introduction Lung Deposition Clearance Mechanisms Mucociliary Transport Cell Transport Absorption Lung Cells Enzyme Action Expression Distribution Conclusion
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Cells of the Airway Epithelium
PUTATIVE FUNCTION Ciliated columnar Mucus movement Mucus (goblet) Mucus secretion Serous Periciliary fluid Clara (nonciliated epithelial) Surfactant production, xenobiotic metabolism Brush Transitional form of ciliated epithelial cell Basal Progenitor for ciliated epithelial cell and goblet cell Intermediate Transitional cell in differentiation of basal cell Neuroendocrine Chemoreceptor, paracrine function Alveolar Type I Alveolar gas exchange Alveolar Type II Surfactant secretion, differentiation to type I cell Alveolar Macrophages Pulmonary defense Mast Immunoregulation
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The rate of first-order kinetic reaction:
One-substrate mechanism: E + S ES E + P k1 k2 k3
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Dependence of initial rate of
reactant concentration for a simple first- or second-order chemical reaction. Dependence of initial rate of substrate concentration for a typical enzyme-catalyzed reaction.
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A Lineweaver-Burk plot (based on Michaelis-Menten Equation)
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Catalytic cycle of microsomal carboxylesterase (left) and microsomal epoxide hydrolase (right), two α/β-hydrolase fold enzymes.
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Drug and Xenobiotic Metabolism
Glucuronic Acid Carboxyamide PHASE I DRUG OH SH NH3+ CO2- PHASE II DRUG DRUG Functionalization Conjugation SO4- Glutathione Cytochrome P450s Monooxygenases Dehydrogenases Oxidases Esterases Glucuronosyltransferases Sulfotransferases Acetyltransferases Methyltransferases Glutathione S-Transferases MDR1 (P-Glycoprotein) EXCRETION Courtesy: Matt Redinbo
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Enzymatic Systems in the Respiratory Tract
Phase I CYP-450s Flavin containing mono-oxygenases (FMA) Monoamine oxidase (MAO) Aldehyde dehydrogenase NADPH cP450 reductase Esterases Epoxide hydrolase
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Enzymatic Systems in the Respiratory Tract
Phase II conjugating enzymes Glutathione S-transferase (GST) Sulfotransferase N-acetyltransferase methyltransferase
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Summary of P-450 Isozymes Reported in the Rat and Rabbit Nasal Cavities
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Some P-450 Isozymes Reported in Lungs of Various Species
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Some P-450 Isozymes Reported in Lungs of Various Species (Cont’d)
Isozyme Comments
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General pathways of xenobiotic biotransformation and their major subcellular location.
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Distribution of Enzymes
Upper respiratory tract Olfactory epithelium: CYP450 & NADPH CYP450 levels < liver, but activities >> than liver Epoxide hydrolase, carboxylesterase, aldehyde dehydrogenase activity > respiratory Phase II enzymes: GST, glucoronyl transferases, sulfotransferases
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Distribution of Enzymes
Lower respiratory tract Tracheobronchial region CYP450 throughout FMO absent in larynx and trachea Bronchiolar region Clara cells: CYP450 isozymes NADPH cP450 reductase FMO, GST, UDP-GT, and epoxide hydrolase Type II pneumocytes
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Distribution of Enzymes
Alveolar Macrophages: No CYP450 Type I cells No metabolic activity Susceptible to toxicity e.g. butylated hydroxytoluene is severely toxic to Type I cells
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Introduction Lung Deposition Clearance Mechanisms Mucociliary Transport Cell Transport Absorption Lung Cells Enzyme Action Expression Distribution Conclusion
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Pulmonary Enzyme Systems
CYP450 mono-oxygenase Metabolism of endogenous FA’s, steroids, and lipid soluble xenobiotics Note: some metabolism leads to bioactivity or carcinogens (e.g. benzo[a]pyrene) NADPH Cytochrome P450 reductase Identical to hepatic enzyme Activates toxicity of paraquat and nitrofurantion (reduction of nitro grp free radical regenerates parent drug and superoxide anion lipid peroxidation and depletion of cellular NADPH)
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Structures of Some Acute Pulmonary Toxins
J.J. Fenton, Toxicology: A Case-Oriented Approach, CRC Press, Boca Raton, FL 2002.
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Diesel Exhaust Particles
Solid carbon core (primary particle size of 10-80 nm, agglomerates of nm). Adsorbed hydrocarbons. Liquid condensed hydrocarbon particles. Sulfates, nitrates, metals, or trace elements. -This a cartoon representation of a typical accumulation mode DEP. -The particles are an aggregation of primary particles. -Primary particles are made of elemental carbon with a layer of adsorbed and condensed hydrocarbons. -Also there are small levels of sulfates, nitrates, metals, and other trace elements. -This adsorbed layer comprises % of particle mass. -This layer is thought to be very important to the health effect caused by DEP because of the reactivity of many of the compounds. -The hundreds of compounds adsorbed to the surface of DEP include at least 15 polyaromatic hydrocarbons (PAHs) or nitro-polyaromatic hydrocarbons (N-PAHs) that are considered to be potential or probable carcinogens by the US National Toxicology Program. -PAHs and nitro PAHs up to 1% of particle mass. Adapted from Marano, et al. (2002). Cell Biol Toxicol. 18(5):
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ROS Formation DEP Redox Cycling Quinones PAHs CYP1A1 ROS ROS NQO-1
-ROS are formed in response to DEP exposure by several pathways. -ROS are formed during metabolism of PAHs to quinones by phase I metabolizing enzymes. -ROS can then be formed continuously by quinoid redox cycling until the quinoids are metabolized to hydroquinones by phase II metaboling enzymes. CYP1A1 ROS ROS NQO-1 Also from: -activated macrophages -recruited neutrophils Hydroquinone
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Role of epoxide hydrolase in the inactivation of benzo[a]pyrene 4,5-oxide and in the conversion of benzo[a]pyrene to its tumorigenic diolepoxide.
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Two-Electron Reduction of Menadione to a Hydroquinone, and Production of Reactive Oxygen Species During its One-Electron Reduction to a Emiquinone Radical Casarett and Doull’s Toxicology: The Basic Science of Poisons, C.D. Klaassen Ed., 6th Ed. McGraw-Hill, New York, NY 2001.
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Hierarchical Oxidative Stress Response
High GSH/GSSG Ratio Low GSH/GSSG Ratio Level of Oxidative Stress -This is a graphical representation of that HOSR with caused by the production of ROS. -Level of OS on the y axis and cellular response on the x. -At low stress levels antioxidant defense proteins are induced, followed by a proinflammatory response and ultimately resulting in apoptosis/necrosis if the level of stress is high enough. -There has been a number of connections made between cellular effect following exposure and disease. Normal Antioxidant Defense Inflammation Toxicity Cell or Tissue Response Adapted from Xiao, et al. (2003). J Biol Chem. 278(50).
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Scanning electron micrograph of an alveolar macrophage
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Macrophages as a host cell for infectious microorganisms
Mycobacterium tuberculosis Toxoplasma gondii pH NO NO2- NO3- H2O2 OH O2 O2 NH4+ NADPH SOD O2- Lysosomal enzymes NH4+ NADP GL ST LAM
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Conclusion Particle deposition and distribution from the lungs is mediated by a number of mechanisms Conventional enzyme kinetic analysis may be used to characterize activity in lung tissue (fluids or cells). There are a number of cell types throughout the respiratory tract exhibiting differential enzyme expression and activity. Local metabolism of xenobiotics may result in toxicity (metabolism of drugs may result in efficacy or inactivation). Pathogens act, in part, by suppressing metabolism
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