Pulmonary Toxicology : Disposition, Metabolism and

Enzyme Kinetics

Anthony J. Hickey, Ph.D., D.Sc.

School of Pharmacy, UNC-Chapel Hill, NC

• Introduction

• Lung Deposition

• Clearance Mechanisms– Mucociliary Transport– Cell Transport– Absorption

• Lung Cells

• Enzyme Expression

• Metabolism

• Conclusion

NasalPassages

T-B Airways

PulmonaryParenchyma

LymphNodes

Blood

GI

Tract

MucusblanketCilia

Columnarepithelial cells

MICE

RATS

PEOPLE

DOGS AND GUINEA PIGS

100 200 300

100

10

1

0.1

DAYS AFTER INHALATION

FRAC

TIO

N CL

EARE

D PE

R DA

Y (X

103 )

1 µm polystyrene latex; 30 min; 60x

Thompson, 1992

Passive Diffusion

Facilitated Diffusion

Active Transport

MOLECULAR WEIGHT (daltons)

CLEA

RANC

E (m

in-1)

RAT

RABBIT

DOG

SHEEP

FETAL LAMB

MAN, AEROSOL

DOG, AEROSOL

100

10-1

10-2

10-3

10-4

10-5

101 102 103 104 105 106

Effros and Mason, 1985

Aerosol

To vacuum pump

Cross section of two stages

Throat

Pre-separator

1

2

3

4

5

6

Airflow

Vacuum

Snapwell™ containing epithelial cell monolayer

Petri dish

Confluent monolayer of the small airways epithelial cells

Vacuum

Airflow

Single Stage of the Cascade ImpactorShowing Orifices

Petri Dish

Transwell® Dish Containing Epithelial Cell Monolayers

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.

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

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)

• Introduction

• Lung Deposition

• Clearance Mechanisms

– Mucociliary Transport

– Cell Transport

– Absorption

• Lung Cells

• Enzyme

– Action

– Expression

– Distribution

• Conclusion

Cells of the Airway EpitheliumCELL 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

E + S ES E + Pk1

k2

k3

][][][

Skdt

Pd

dt

Sd

The rate of first-order kinetic reaction:

One-substrate mechanism:

A. Dependence of initial rate of reactant concentration for a simple first- or second-order chemical reaction.

B. Dependence of initial rate of substrate concentration for a typical enzyme-catalyzed reaction.

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

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

Drug and Xenobiotic Metabolism

DRUG DRUG

OH SH

NH3+ CO2

-

DRUGG

luc

uro

nic

A

cid

SO4- G

luta

thio

ne

Ca

rbo

xy

am

ide

PHASE I PHASE II

Functionalization Conjugation

EXCRETION

MDR1 (P-Glycoprotein)

Cytochrome P450s

Monooxygenases

Dehydrogenases

Oxidases

Esterases

Glucuronosyltransferases

Sulfotransferases

Acetyltransferases

Methyltransferases

Glutathione S-Transferases

Courtesy: Matt Redinbo

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

Enzymatic Systems in the Respiratory Tract

• Phase II conjugating enzymes– Glutathione S-transferase (GST)– Sulfotransferase– N-acetyltransferase– methyltransferase

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

Some P-450 Isozymes Reported in Lungs of Various Species

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

Isozyme Comments

General pathways of xenobiotic biotransformation and their major subcellular location.

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

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

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

• Introduction

• Lung Deposition

• Clearance Mechanisms

– Mucociliary Transport

– Cell Transport

– Absorption

• Lung Cells

• Enzyme

– Action

– Expression

– Distribution

• Conclusion

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)

Structures of Some Acute Pulmonary Toxins

J.J. Fenton, Toxicology: A Case-Oriented Approach, CRC Press, Boca Raton, FL 2002.

Diesel Exhaust Particles

Solid carbon core (primary particle size of10-80 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.

ROS FormationDEP

QuinonesRedoxCycling

ROS

PAHs

ROS

Hydroquinone

NQO-1

CYP1A1

Also from:-activated macrophages-recruited neutrophils

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.

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.

Normal AntioxidantDefense

Inflammation Toxicity

HighGSH/GSSG

Ratio

LowGSH/GSSG

Ratio

Level ofOxidative

Stress

Adapted from Xiao, et al. (2003). J Biol Chem. 278(50).

Hierarchical Oxidative Stress Response

Cell or Tissue Response

Scanning electron micrograph of an alveolar macrophage

Macrophages as a host cell for infectious microorganisms

Mycobacterium tuberculosis

Toxoplasma gondii

NONO2

-

NO3-

pHH2O2

OHO2

O2

SODO2

-

NADPLysosomalenzymes NH4

+

NADPHNH4+

GLSTLAM

ConclusionParticle deposition and distribution from the lungs is

mediated by a number of mechanismsConventional 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