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 Lung Cells
Mucociliary Transport
Cell Transport
Absorption
Lung Cells
Enzyme Expression
Metabolism
Conclusion

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

4. Mucus
blanket
Cilia
Columnar
epithelial
cells

5. 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

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

7. Thompson, 1992

8. Passive Diffusion
Facilitated Diffusion
Active Transport

9. 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

10. 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

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)
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

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

15. 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

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

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. 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

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

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

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 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):

34. 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

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
-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).

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
NH4+
NADP 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