Pulmonary Toxicology : Disposition, Metabolism and Enzyme Kinetics

1. Pulmonary Toxicology : Disposition, Metabolism and Enzyme Kinetics Anthony J. Hickey, Ph.D., D.Sc. School of Pharmacy, UNC-Chapel Hill, NC

2. Introduction • Lung Deposition • Clearance Mechanisms • Mucociliary Transport • Cell Transport • Absorption • Lung Cells • Enzyme Expression • Metabolism • Conclusion

3. Nasal Passages G I T r a c t B l o o d T-B Airways Pulmonary Parenchyma Lymph Nodes

4. Mucus blanket Cilia Columnar epithelial cells

5. 100 10 1 0.1 MICE RATS PEOPLE DOGS AND GUINEA PIGS 100 200 300 FRACTION CLEARED PER DAY (X103) DAYS AFTER INHALATION

6. 1 µm polystyrene latex; 30 min; 60x

7. Thompson, 1992

8. Passive Diffusion Facilitated Diffusion Active Transport

9. 100 10-1 10-2 10-3 10-4 10-5 RAT RABBIT DOG SHEEP FETAL LAMB MAN, AEROSOL DOG, AEROSOL 101 102 103 104 105 106 CLEARANCE (min-1) MOLECULAR WEIGHT (daltons) Effros and Mason, 1985

10. Aerosol Throat Cross section of two stages Airflow Snapwell™ containing epithelial cell monolayer Pre-separator 1 2 3 Petri dish 4 5 Vacuum 6 To vacuum pump

11. 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

12. 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.

13. 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)

14. Introduction • Lung Deposition • Clearance Mechanisms • Mucociliary Transport • Cell Transport • Absorption • Lung Cells • Enzyme • Action • Expression • Distribution • Conclusion

15. Cells of the Airway Epithelium

16. k1 k3 E + S ES E + P k2 The rate of first-order kinetic reaction: One-substrate mechanism:

17. 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.

18. A Lineweaver-Burk plot(based on Michaelis-Menten Equation)

19. Catalytic cycle of microsomal carboxylesterase (left) and microsomal epoxide hydrolase (right), two α/β-hydrolase fold enzymes.

20. OH SH DRUG DRUG DRUG NH3+ CO2- Drug and Xenobiotic Metabolism Glucuronic Acid Carboxyamide PHASE I PHASE II Functionalization Conjugation SO4- Glutathione Cytochrome P450s Monooxygenases Dehydrogenases Oxidases Esterases Glucuronosyltransferases Sulfotransferases Acetyltransferases Methyltransferases Glutathione S-Transferases MDR1 (P-Glycoprotein) EXCRETION Courtesy: Matt Redinbo

21. 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

22. Enzymatic Systems in the Respiratory Tract • Phase II conjugating enzymes • Glutathione S-transferase (GST) • Sulfotransferase • N-acetyltransferase • methyltransferase

23. Summary of P-450 Isozymes Reported in the Rat and Rabbit Nasal Cavities

24. Some P-450 Isozymes Reported in Lungs of Various Species

25. Isozyme Comments Some P-450 Isozymes Reported in Lungs of Various Species (Cont’d)

26. General pathways of xenobiotic biotransformation and their major subcellular location.

27. 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

28. 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 • CYP450 isozymes • NADPH cP450 reductase

29. 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

30. Introduction • Lung Deposition • Clearance Mechanisms • Mucociliary Transport • Cell Transport • Absorption • Lung Cells • Enzyme • Action • Expression • Distribution • Conclusion

31. 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)

32. Structures of Some Acute Pulmonary Toxins J.J. Fenton, Toxicology: A Case-Oriented Approach, CRC Press, Boca Raton, FL 2002.

33. Diesel Exhaust Particles Solid carbon core (primary particle size of 10-80 nm, agglomerates of 50-1000 nm). Adsorbed hydrocarbons. Liquid condensed hydrocarbon particles. Sulfates, nitrates, metals, or trace elements. Adapted from Marano, et al. (2002). Cell Biol Toxicol. 18(5): 315-320.

34. ROS Formation DEP Redox Cycling PAHs Quinones CYP1A1 ROS ROS NQO-1 Also from: -activated macrophages -recruited neutrophils Hydroquinone

35. 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.

36. 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.

37. Hierarchical Oxidative Stress Response High GSH/GSSG Ratio Low GSH/GSSG Ratio Level of Oxidative Stress Normal Antioxidant Defense Inflammation Toxicity Cell or Tissue Response Adapted from Xiao, et al. (2003). J Biol Chem. 278(50).

38. Scanning electron micrograph of an alveolar macrophage

39. 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 NADP NH4+ GL ST LAM

40. 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