Particle-Lung Interactions
LUNG BIOLOGY IN HEALTH AND DISEASE
Executive Editor Claude Lenfant Former Director, National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland
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The opinions expressed in these volumes do not necessarily represent the views of the National Institutes of Health.
Particle-Lung Interactions Second Edition
edited by
Peter Gehr University of Bern Bern, Switzerland
Christian Mühlfeld University of Giessen Giessen, Germany
Barbara Rothen-Rutishauser University of Bern Bern, Switzerland
Fabian Blank University of Bern Bern, Switzerland
Informa Healthcare USA, Inc. 52 Vanderbilt Avenue New York, NY 10017 # 2010 by Informa Healthcare USA, Inc. Informa Healthcare is an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 1-4200-7256-0 (Hardcover) International Standard Book Number-13: 978-1-4200-7256-3 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequence of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Particle-lung interactions / edited by Peter Gehr . . . [et al.]. — 2nd ed. p. ; cm. — (Lung biology in health and disease ; 241) Includes bibliographical references and index. ISBN-13: 978-1-4200-7256-3 (hardcover : alk. paper) ISBN-10: 1-4200-7256-0 (hardcover : alk. paper) 1. Pulmonary toxicology. 2. Particles. 3. Lungs—Dust diseases. 4. Pulmonary endothelium. 5. Pulmonary surfactant. I. Gehr, Peter. II. Series: Lung biology in health and disease ; v. 241. [DNLM: 1. Lung–metabolism. 2. Aerosols. 3. Air Pollutants. 4. Air Pollution—adverse effects. 5. Allergens. 6. Inhalation Exposure. 7. Lung—immunology. W1 LU62 v.241 2009 / WF 600 P273 2009] RC720.P37 2009 616.2’00471—dc22 2009023883 For Corporate Sales and Reprint Permissions call 212-520-2700 or write to: Sales Department, 52 Vanderbilt Avenue, 7th floor, New York, NY 10017. Visit the Informa Web site at www.informa.com and the Informa Healthcare Web site at www.informahealthcare.com
Introduction
The current and rightfully ever increasing concerns for air pollution associated with climate changes, and its health effects, make this second edition of Particle-Lung Interactions extremely timely. Years back, the interest in pollution, the associate particles, and irritant gases focused on indoor situation, but soon the interest was extended to the air that we breathe, wherever we are. In fact, it seems that the concerns about outside air particles soon became greater than those about indoor air particles, primarily because of the public health implications. Unquestionably, the interest in this matter has considerably increased during the last 10 years or so, especially since the publication of the first edition of Particle-Lung Interactions. Furthermore, as the editors of this new edition— Drs. Peter Gehr, Christian Mühlfeld, Barbara Rothen-Rutishauser, and Fabian Blank—point out in their Preface, two new “things” have appeared. First, the increased knowledge and interest in nanoparticles, which are now well-defined materials. The focus on their effect on health and conversely their use to improve drug delivery have become a very productive research area. Second, the interactions of these particles with organs, especially the respiratory tract and the lung, have shifted the focus of their impact to the cellular level. The scientific community is working hard to determine the mechanisms of these interactions and their impact at the subcellular and molecular levels. It is also recognized that although the lung is the port of entry of these nanoparticles, the particles will also impact on other organs, such as the cardiovascular system, if absorbed. The inclusion of this monograph in the series Lung Biology in Health and Disease will inform its readers about a dynamic field, but it will also stimulate basic and clinical research on an important, even critical, area. As the executive editor of this series, I express my thanks to the editors and authors of this volume for the opportunity to present it to our readership. Claude Lenfant, MD Vancouver, Washington, U.S.A.
vii
Preface
Leonardo da Vinci (1452–1519), prototype of the universal genius, was a painter, musician, architect, and an engineer. He was also a sculptor and an anatomist. In his Anatomical Atlas he added the short comment above the trachea, “dust is harmful.” Today we know that Leonardo da Vinci was right. During the past decades, a growing body of epidemiological and experimental literature has provided evidence that inhalation of “dust,” that is, airborne particles in the widest sense, substantially contributes to creating adverse health effects. The characteristics of the particles that may be inhaled with every breath depend on many factors, like season, climate, and environment. Similarly diverse are the health effects of particles, ranging from respiratory to cardiovascular diseases, from asthma over increased susceptibility to viral infections to myocardial infarction. In recent years, there has been evidence that the fraction of nano-sized particles, that is, particles smaller than 100 nm (0.1 mm) are of particular concern. When the first edition of this book entered the market, the editors anticipated that the field of particle-lung interactions was about to enter a new era. Cautiously, the editors mentioned the likely changes in the preface: Since this monograph first took shape, the field of particle-lung interactions has expanded considerably. The “bad” particles are currently ultrafine particles released into the environment from combustion processes. The “good” particles are those carrying insulin into the lungs. The treatment of diabetes via the inhalation route will most likely become the first approved aerosol treatment for a systemic disease. Nevertheless, we both hope that established knowledge and controversial issues are sufficiently reflected in the volume on particle-lung interactions.
In 2007, Joachim Heyder and Peter Gehr were asked by the publisher of Claude Lenfant’s series on Lung Biology in Health and Disease, Informa Healthcare, whether they would be interested in publishing a second edition of their volume, number 143, on Particle-Lung Interactions. Due to various circumstances Joachim Heyder was not able to work on the second edition. Therefore, Peter Gehr asked three young researchers around him to co-edit the next edition. This gave rise to the new editor team for this second edition on Particle-Lung Interactions. Although Joachim Heyder’s wisdom, editorial skills and fundamental and comprehensive knowledge cannot be overrated, the composition of the new editorial team provided different advantages.
ix
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Preface
Throughout all the work of editing, we experienced a very close and respectful collaboration. Two things have changed, since the appearance of the last book. Whereas nano-sized particles were briefly mentioned by only a few authors earlier, nanomaterials are now a major aspect in this second edition. A totally new field dealt with in many chapters, which is rapidly gaining significance, is manufactured nanoparticles. Publications in this field are still rather sparse but rapidly growing in number. A lot of experimental work is still needed, and this is particularly true for the good particles as they were called in the preface of the first edition. The hope expressed almost 10 years ago that the treatment of diabetes via the inhalation route will most likely become the first approved aerosol treatment for a systemic disease was abruptly destroyed when Pfizer stopped the production of insulin, apparently due to poor sales. Since then, Lilly and Novo Nordisk have also dropped their plans to produce inhalable insulin. The lung is a major target of ambient air pollution. The relationship between increased concentrations of air pollution and adverse health effects in children, subjects with asthma and COPD, and vulnerable adults is well documented. Major adverse health effects include not only increased respiratory symptoms, decreased lung function, and increased hospitalization but also altered mucociliary clearance, COPD, asthma, and increased mortality. Several chapters in this book deal with the various diseases associated with particle inhalation. A number of in vitro studies have been performed to investigate how fine and nano-sized particles enter tissues and cells of the lungs and to determine what effect they may have. In contrast to fine particles that are taken up by cells by phagocytosis, nanoparticles may enter cells by an endocytic or by another, yet to be defined mechanism. This circumstance could be a major cause for the enhanced adverse health effects of nano-sized particles. Many publications have shown that the main cellular effect of exposure to combustion-derived nanoparticles is the production of reactive oxygen species, which is a major factor in inflammation and toxicity. There is increasing evidence that these nanoparticles may pose a particular danger because of their high content of organic chemicals and the pro-oxidative potential. The key properties associated with these particles are their large surface area and the presence of metals and organics, all of which have the potential to produce oxidative stress. Moreover, the same particles may also have genotoxic effects. These aspects are discussed in this book. Studies investigating the interactions of the lungs with these minute particles have also been stimulated by advances in nanotechnology. This technological branch has been overwhelming us with newly designed materials that due to different physicochemical behaviors offer great advantages in many applications, ranging from antibacterial clothing to car lacquers and to medicinal particles. The latter have the potential not only of drug delivery via
Preface
xi
inhalation for systemic treatment but also of targeting drugs to specific cell types and even organelles. This might significantly reduce the side effects of current therapeutics or lead to new developments in diagnostics and therapy. However, due to their minute size, potential toxicity should be considered for manufactured nanoparticles as well. In most studies, investigators found that these artificially produced nanoparticles were associated with the generation of reactive oxygen species by cells exposed to the particles. A lot of work is still needed to determine whether the conclusions drawn for combustion-derived particles can be extrapolated to manufactured nanoparticles. Since both nanotechnology in its widest sense and nanotoxicology have been recognized to have a great future impact, governmental programs in the whole world are supporting both research fields financially. The concern about possible hazardous effects of manufactured nanoparticles crosses the borders of scientific curiosity quickly and enters broad public awareness. Some of the fears associated with nanoparticles are related to the lessons learned from the deleterious effects of inhaled asbestos particles. This time particle researchers are working hard to assess the risks associated with manufactured nanoparticles well in advance to avoid the health consequences and financial burdens that may follow after exposure. While the exposure of combustion-derived nanoparticles affects everyone, it is obvious that the exposure to manufactured nanoparticles may primarily affect people working in the production of nanoparticles, their processing, or application. These important concerns have caused the development of a whole new scientific field known as “nanotoxicology.” In January 2006, the first international congress on nanotoxicology took place in Miami and a second one was held in September 2008 in Zurich. Both journals and books entirely devoted to the subject have entered the market. Nevertheless, a great amount of work still needs to be done. In the light of the new developments, the second edition of this book has to face the dilemma that the established knowledge from the first edition needs to be combined with the vastly growing knowledge gained during the past few years, a process that will surely continue. This dilemma is aggravated by the publishers’ request to shorten the book compared to the first edition. Therefore, the second edition of this book has been rearranged completely. Fundamental aspects on pulmonary structure and function as well as different kinds of particles have been shifted into the first and second part of this book to avoid repetition of these aspects in different chapters. The third part deals with the inhalation of particles, their deposition and retention in the respiratory tract as well as their clearance and translocation. The two following parts are devoted to the interaction of particles with biological structures, starting at the systemic and organ level (part 4) and finishing with the cellular and molecular effects inhalation of particles may have (part 5). We provide the reader with a
xii
Preface
combination of both established knowledge and, hopefully, exciting new observations. We are well pleased with the result of the second edition and we warmly thank our colleagues for their extraordinary support. Without their special effort, it would have never been possible to finish this publication. We are grateful to the staff of Informa Healthcare for their invaluable and professional assistance in editing this book. The interactions with the authors as well as with the publisher were always stimulating. We do hope that the result will serve its purpose and that this book will take its place among the well-received monographs in the series in Lung Biology in Health and Disease. Peter Gehr Christian M€ uhlfeld Barbara Rothen-Rutishauser Fabian Blank
Contributors
Matthias Amrein
University of Bern, Bern, Switzerland
Fabian Blank Michael Bur
University of Calgary, Calgary, Alberta, Canada
Saarland University, Saarbrücken, Germany
Christopher Carosino
University of California, Davis, California, U.S.A.
Daniel L. Costa U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, U.S.A. Ken Donaldson
University of Edinburgh, Edinburgh, U.K.
Mark W. Frampton New York, U.S.A. Peter Gehr
University of Rochester Medical Center, Rochester,
University of Bern, Bern, Switzerland
Peter J. Gerber
University of Berne, Berne, Switzerland
Beatriz Gonzalez-Flecha Massachusetts, U.S.A.
Harvard School of Public Health, Boston,
Robert N. Grass Institute for Chemical and Bioengineering, ETH Zurich, Zurich, Switzerland Francis H. Y. Green Stephanie Hein Andreas Henning
University of Calgary, Calgary, Alberta, Canada
Saarland University, Saarbrücken, Germany Saarland University, Saarbrücken, Germany
Jens M. Hohlfeld Fraunhofer Institute of Toxicology and Experimental Medicine, and Hannover Medical School, Hannover, Germany
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Contributors
Katherine Horvath University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A. Ilona Jaspers University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A. Malcolm King
University of Alberta, Edmonton, Alberta, Canada
Wolfgang G. Kreyling Helmholtz Zentrum München—German Research Center for Environmental Health, Institute for Lung Biology and Disease (iLBD) and Focus Network Nanoparticles and Health, Neuherberg, Germany Claus-Michael Lehr Zoya Leonenko
Saarland University, Saarbrücken, Germany
University of Waterloo, Waterloo, Ontario, Canada
Ludwig K. Limbach Zurich, Switzerland
Institute for Chemical and Bioengineering, ETH Zurich,
Winfried Mo¨ller Helmholtz Zentrum München—German Research Center for Environmental Health, Institute for Lung Biology and Disease (iLBD) and Clinical Cooperation Group Inflammatory Lung Diseases, Gauting, Germany Amy K. Madl University of California, Davis, and ChemRisk, LLP, San Francisco, California, U.S.A. Christian Mu¨hlfeld* Matthias Ochs{
University of Bern, Bern, Switzerland
University of Bern, Bern, Switzerland
Kent E. Pinkerton
University of California, Davis, California, U.S.A.
Barbara Rothen-Rutishauser
University of Bern, Bern, Switzerland
Jonathan M. Samet Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, U.S.A. Samuel Schu¨rch
University of Calgary, Calgary, Alberta, Canada
Current affiliations: * University of Giessen, Giessen, Germany. { Hannover Medical School, Hannover, Germany.
Contributors
xv
Carsten Schleh Fraunhofer Institute of Toxicology and Experimental Medicine, and Hannover Medical School, Hannover, Germany Otmar Schmid Helmholtz Zentrum München—German Research Center for Environmental Health, Institute for Lung Biology and Disease (iLBD) and Focus Network Nanoparticles and Health, Neuherberg, Germany Saarland University, Saarbrücken, Germany
Marc Schneider
Holger Schulz Helmholtz Zentrum München—German Research Center for Environmental Health, Institute for Lung Biology and Disease (iLBD) and Focus Network Nanoparticles and Health, Neuherberg, Germany Joel Schwartz U.S.A.
Harvard School of Public Health, Boston, Massachusetts,
Manuela Semmler-Behnke Helmholtz Zentrum München—German Research Center for Environmental Health, Institute for Lung Biology and Disease (iLBD) and Focus Network Nanoparticles and Health, Neuherberg, Germany Paul A. Solomon Nevada, U.S.A.
U.S. Environmental Protection Agency, Las Vegas,
Wendelin J. Stark Institute for Chemical and Bioengineering, ETH Zurich, Zurich, Switzerland Tobias Stoeger Helmholtz Zentrum München—German Research Center for Environmental Health, Institute for Lung Biology and Disease (iLBD) and Focus Network Nanoparticles and Health, Neuherberg, Germany Vicki Stone Mark J. Utell U.S.A.
Edinburgh Napier University, Edinburgh, U.K. University of Rochester Medical Center, Rochester, New York,
Contents
Introduction Claude Lenfant . . . . . . . vii Preface . . . . . . . . . . . . . . . . . . . . . . . . ix Contributors . . . . . . . . . . . . . . . . . . . . xiii Part 1: Structure and Function of the Lung
1. Functional Aspects of Lung Structure as Related to Interaction with Particles . . . . . . . . . . . . . . . . . . . . . . . . . Christian M€ uhlfeld and Matthias Ochs
1
Part 2: Particle Characteristics
2. Ambient Tropospheric Particles . . . . . . . . . . . . . . . . . . . . . Paul A. Solomon and Daniel L. Costa
17
3. Manufactured Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . Robert N. Grass, Ludwig K. Limbach, and Wendelin J. Stark
38
4. Particulate Carriers for Pulmonary Drug Delivery . . . . . . . Stephanie Hein, Andreas Henning, Michael Bur, Marc Schneider, and Claus-Michael Lehr
60
Part 3: Inhalation of Particles
5. Deposition, Retention and Clearance, and Translocation of Inhaled Fine and Nano-Sized Particles in the Respiratory Tract . . . . . . . . . . . . . . . . . . . . . . . . . . Winfried M€ oller, Wolfgang G. Kreyling, Otmar Schmid, Manuela Semmler-Behnke, and Holger Schulz
79
Part 4: Health Effects of Particle Inhalation
6. The Epidemiology of Particle Health Effects . . . . . . . . . . . . Joel Schwartz xvii
108
xviii
Contents
7. Cardiovascular Consequences of Particles . . . . . . . . . . . . . . Mark W. Frampton, Mark J. Utell, and Jonathan M. Samet 8. Autonomic Mediation of the Cardiac Responses to Particle Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Beatriz Gonzalez-Flecha
120
137
9. Effects of Airborne Particles on Respiratory Viral Infection Ilona Jaspers and Katherine Horvath
151
10. Airborne Particles and Structural Remodeling of the Lungs Amy K. Madl, Christopher Carosino, and Kent E. Pinkerton
167
Part 5: Cellular and Molecular Mechanisms of Particle Health Effects
11. Effect of Particles on Mucus and Mucociliary Clearance . . . Malcolm King
193
12. The Role of Surfactant in Particle Exposure . . . . . . . . . . . . Francis H. Y. Green, Samuel Sch€ urch, Matthias Amrein, Peter J. Gerber, and Zoya Leonenko
204
13. Nanoparticle–Cell Membrane Interactions . . . . . . . . . . . . . Barbara Rothen-Rutishauser, Fabian Blank, Christian M€ uhlfeld, and Peter Gehr
226
14. Mechanisms and Processes Underlying Toxicological Responses to Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vicki Stone and Ken Donaldson
243
15. Aeroallergen-Lung Interactions . . . . . . . . . . . . . . . . . . . . . Carsten Schleh and Jens M. Hohlfeld
266
16. Dose-Response Relationships . . . . . . . . . . . . . . . . . . . . . . . Tobias Stoeger and Otmar Schmid
288
Index
....
305
1 Functional Aspects of Lung Structure as Related to Interaction with Particles ¨ HLFELD* and MATTHIAS OCHS{ CHRISTIAN MU University of Bern, Bern, Switzerland
I.
Organization of the Lung
The organization of the lung, that is, the arrangement of the composing structures, differs from that of other human organs as, at the end of a normal breath, the lung consists of about 80% air, 10% blood, and only 10% tissue. In general, the latter is composed of structures lining the airspaces and the blood vessels and, in between, interstitial structures providing mechanical and metabolic features of lung function. In addition, the lung contains a second circulation that provides blood to the tissue itself, a neuroendocrine system, an immune system, and a covering layer at the “outer” surface of the lung—the visceral pleura. Functionally, the pulmonary tissue needs not only to be strong enough to separate air and blood effectively but also to provide a large surface area and a thin tissue barrier for gas diffusion between air and blood. To meet these functional demands, the lung possesses an air-conducting zone (airways) and a gasexchange region (alveolar region), the two of which differ significantly with respect to their qualitative composition and quantitative contribution to lung structure. Therefore, despite its small volume, pulmonary tissue has a high degree of complexity with over 40 different cell types and highly specialized noncellular components. Obviously, a small chapter like this cannot cover all aspects of lung structure, and the reader may refer to more comprehensive articles for further reading (1). However, as part of a well-focused book, this chapter provides the possibility of highlighting certain aspects of lung structure in greater detail than it is usually done in more comprehensive articles. It aims at describing those structures that are most relevant to the focus of this book on nano-sized particles (NSP). In contrast to larger-sized particles, NSP are deposited effectively in the gasexchange region of the lung (see chap. 5), which is why we chose to describe the structures of the alveoli as a portal of entry in the beginning. This includes the interaction between particles and surfactant (chap. 12) and the alveolar epithelium (chaps. 13 and 14). NSP have been shown to be translocated to the circulation, which provides a possible explanation for cardiovascular particle effects (chap. 7); however, parts of these effects have also been related to the activation of the nervous system of Current affiliations: * University of Giessen, Giessen, Germany. { Hannover Medical School, Hannover, Germany.
1
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M€ uhlfeld and Ochs
the lung (chap. 8). Furthermore, there is a large body of evidence for interactions between particles and the pulmonary and systemic immune system (chaps. 9 and 15). We have therefore decided to focus the second part of this chapter on the structures known to be affected by particle exposure and constituting possible portals of exit or effect, namely, the capillary endothelium, the nervous system, and the immune system.
II.
General Design of the Air-Blood Barrier
At the level of alveoli, we find the barrier separating the airspaces from the vasculature—the air-blood barrier across which gas exchange takes place and along which NSP may enter the systemic circulation. The air-blood barrier is made of three tissue compartments: the alveolar epithelium (with the alveolar lining layer containing surfactant on top), the interstitium (including cellular elements as well as the connective tissue fiber scaffold in the extracellular matrix), and the capillary endothelium. The total surface area available for gas exchange in the human is about 140 m2, nearly the size of a singles tennis court. At the same time, the arithmetic mean thickness of the air-blood barrier is only about 2 mm (2). Over about half of this surface, we find the so-called thin parts of the air-blood barrier where the basement membranes of alveolar epithelium and capillary endothelium are fused, thus minimizing the interstitium. At the other half, termed the thick parts of the air-blood barrier, the two basement membranes are separated, thereby leaving room for interstitial cells (mainly fibroblasts, but also mobile cells like macrophages), elastic fibers, and bundles of collagen fibrils (Fig. 1). These different parts of the air-blood barrier are likely to behave differently with respect to the fate of inhaled particles.
Figure 1 Schematic drawing of the air-blood barrier. Surfactant forms a surface film (SF) at the
air-liquid interface. Under the watery hypophase, the alveolar epithelium consists of the large alveolar epithelial type I cells (AECI) with their thin processes and the cuboidal alveolar epithelial type II cells (AECII). Inside the septum, capillaries (C) are embedded in a network of collagen fibrils (co) and elastic fibers (el) and interstitial cells (IC).
Functional Aspects of Lung Structure as Related to Particles
III.
3
Surface Structures Lining the Portal of Entry for Particles
A. Surfactant
Once particles have entered alveoli, the first structure they come in contact with is the pulmonary surfactant layer at the air-liquid interface. The pulmonary surfactant system protects alveolar integrity in several ways. (i) It lowers surface tension at the air-liquid interface, thus keeping alveoli open. (ii) It prevents fluid fluxes into the alveolar lumen by lowering alveolar surface tension, thus keeping alveoli dry. (iii) It contributes several important components to the lung’s innate immune system, thus keeping alveoli clean. These biophysical (i and ii) and immunomodulatory (iii) functions are performed by a sophisticated system of remarkable biochemical and morphological complexity (for review, see Refs. 3–5). The morphological complexity (and beauty) of the surfactant system can only be revealed by electron microscopy of carefully prepared tissue samples. Type II alveolar epithelial cells (see below) synthesize, store, secrete, and to a large extent, recycle all components of surfactant. Therefore, an intracellular surfactant compartment within type II cells [in specialized lysosome-related organelles termed “lamellar bodies” (6)] and an intra-alveolar surfactant compartment can be distinguished. The alveolar lining layer that covers the alveolar epithelium as a thin and continuous film consists of the surface film and an aqueous hypophase underneath. Secreted surfactant components can be found in the surface film as well as within the hypophase. The intracellular and intraalveolar metabolism of surfactant is schematically summarized in Figure 2. Biochemically, surfactant is a mixture of lipids, mainly saturated phospholipids, and proteins, among them are the surfactant apoproteins SP-A, -B, -C, and -D. The surfactant proteins have important functions in surfactant subtype assembly, surfactant biophysics, surfactant homeostasis, and innate immunity. While the hydrophobic SP-B and SP-C are tightly associated with surfactant lipids, thereby fundamentally influencing the biophysical characteristics of surfactant (integral surfactant proteins), the hydrophilic SP-A and SP-D belong to the collectin protein family and are mainly involved in intraalveolar surfactant subtype assembly and innate immunity (associated surfactant proteins). In the alveolar lumen, SP-A is mainly found in the corners of tubular myelin figures whereas SP-D is mainly present as a free protein in the hypophase. Together with SP-A, SP-B is important for tubular myelin formation, thus stabilizing active surfactant forms (the “large aggregates” fraction of surfactant obtained by bronchoalveolar lavage), while the hydrophobic proteins SP-B and SP-C and, in conjunction, SP-A can enhance the adsorption of phospholipids into the surface film. SP-A might also inhibit surfactant secretion and stimulate surfactant reuptake of “spent” surfactant (the “small aggregates” fraction of surfactant obtained by bronchoalveolar lavage), which enters the type II cells as small unilamellar vesicles (Table 1). Interestingly, the lung collectins SP-D and, to a somewhat lesser degree, SP-A have also been localized outside the respiratory system, for example, in the gastrointestinal, urinary, and genital tract; mesothelium; synovial membrane; middle ear; lacrimal apparatus; and skin (7–9), indicating that they can exert their functions at a variety of mucosal surfaces. Accordingly, their expression at these extrapulmonary sites is increased under inflammatory conditions.
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M€ uhlfeld and Ochs
Figure 2 Schematic drawing of the current view of surfactant metabolism. The complex mixture
of surfactant lipids and proteins is synthesized in alveolar epithelial type II cells via nuclear transcription, translation at the rough endoplasmic reticulum (rER), posttranslational processing in the Golgi apparatus (Golgi), and further processing and transport in MVB. Some surfactant components might bypass the Golgi apparatus and MVB. Surfactant lipid and the hydrophobic proteins SP-B and -C are assembled in so-called CB showing structural characteristics of MVB and LB. Surfactant is stored in LB and secreted via this organelle by exocytosis. An alternative route by direct exocytosis of small vesicles and/or MVB is known for SP-A but may also be valid for other components, for example, SP-D. After exocytosis to the HY, LB are unpacked and, upon addition of SP-A, form a lattice of surfactant membrane, named tubular myelin (TM), from which the SF consisting of a monolayer and, in places, a surface-associated surfactant reservoir can be generated. HY and SF are termed the alveolar lining layer (ALL). Surfactant that is removed from the airliquid interface is either reused or degraded. “Spent” surfactant has the morphological appearance of UV and can be taken up and degraded by alveolar macrophages (AM), be cleared via the airways, or be taken up by alveolar epithelial type II cells. There, the surfactant components may be degraded, enter de novo synthesis, or be transported to CB or LB for resecretion. The surfaceactive “large aggregates” (LA) fraction of intra-alveolar surfactant obtained by bronchioalveolar lavage largely corresponds to TM whereas the inactive “small aggregates” (SA) fraction largely corresponds to UV. Thus, the functional status of the surfactant system is directly reflected in intraalveolar surfactant subtype composition. Abbreviations: MVB, multivesicular bodies; CB, composite bodies; HY, hypophase; LB, lamellar bodies; SF, surface film; UV, unilamellar vesicles.
B. Alveolar Epithelium
All along the lower airway tree, from the trachea to the about 480 million alveoli in the human (10), there is an uninterrupted layer of epithelial cells. The structural properties, however, differ considerably between the conducting zone and the gas-exchange zone. The pseudostratified epithelium of trachea and bronchi becomes a simple columnar epithelium in bronchioli. At the beginning of the gas-exchange region, there is an
Functional Aspects of Lung Structure as Related to Particles
5
Table 1 Characteristics and Functions of SP-A, -B, -C, and -D
Protein
Characteristics
Functions
SP-A
Hydrophilic; collectin; 26–38 kD (monomer); usually octadecamer (six triplet monomers)
SP-B
Hydrophobic; 8–9 kD
SP-C
Hydrophobic; 3.5–4.2 kD
SP-D
Hydrophilic; collectin; 39–46 kD (monomer); usually dodecamer (four triplet monomers)
Surfactant homeostasis (surfactant secretion inhibition, tubular myelin formation, large aggregate stability, surfactant recycling); immunomodulation (interaction with pathogens, interaction with immune cells, antioxidative functions, antimicrobial functions) Surfactant homeostasis (tubular myelin formation, surface film formation and stability) Surfactant homeostasis (surface film formation and stability) Surfactant homeostasis (small aggregate structure and reuptake); immunomodulation (interaction with pathogens, interaction with immune cells, antioxidative functions, antimicrobial functions)
Abbreviation: SP, surfactant-associated protein.
abrupt transition to a thin squamous epithelium. At all levels, the epithelium consists of more than one cell type. Usually, we find at least a mosaic of lining cells and secretory cells. At the level of alveoli, we find two cell types: the squamous type I cells that form the actual lining and the cuboidal type II cells that have secretory functions. Although type II cells are more numerous than type I cells (16% vs. 8% of all alveolar cells), type I cells cover about 95% of the alveolar surface (11,12). The alveolar epithelium is “sealed” by tight occluding junctions between epithelial cells (13). With their thin extensions, type I cells spread out broadly over the epithelial basement membrane. They have long been considered as the “silent” alveolar epithelial cell type, providing only passive barrier functions. However, recent studies strongly suggest that type I cells are actively involved in alveolar ion and fluid homeostasis (14,15). Type II cells serve two main functions: they produce and secrete surfactant (see above) and they act as progenitor cells for the regeneration of the alveolar epithelium under physiological and pathological (cuboidal metaplasia in acute lung injury) conditions, being able to differentiate into both type I and type II cells. They have thus been referred to as “defender of the alveolus” (16,17). Besides surfactant components with immunomodulatory functions, type II cells are also capable of secreting a variety of proinflammatory mediators, including a broad profile of eicosanoid metabolites, cytokines, and chemokines, thereby actively contributing to the modulation of the inflammatory status in the alveolus (17). C. Surface Structures and Particles
There is a growing body of literature on the interaction between particles and surfactant as well as the alveolar epithelium. Several chapters of this book deal with diverse functions of these structures as related to particles. Interactions between particles and
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M€ uhlfeld and Ochs
surfactant are addressed by Green et al. (chap. 12). In addition, the chapters on aeroallergen-lung interactions by Schleh and Hohlfeld (chap. 15) as well as Jaspers and Horvath (chap. 9) refer to the immunomodulatory functions of surfactant proteins A and D. Translocation across the alveolar epithelium is addressed by Mo¨ller et al. (chap. 5) and Rothen-Rutishauser et al. (chap. 13). Toxicological responses and the pharmaceutical significance of the alveolar epithelium are discussed by Stone and Donaldson (chap. 14) as well as Hein and colleagues (chap. 4), respectively.
IV.
Pulmonary Structures Related to the Portals of Exit of Inhaled Particles
A. Capillary Endothelium
Like the alveolar epithelium, the endothelium lining alveolar capillaries is made of a thin but uninterrupted squamous cell layer. The alveolar capillary endothelium is of the continuous type, that is, in contrast to, for example, the kidney or the liver, there are no pores in the endothelial cells. Compared to the alveolar epithelium, the occluding junctions between capillary endothelial cells are rather leaky, resulting in a higher permeability (13). Like type I cells of the alveolar epithelium, capillary endothelial cells contain numerous caveolae, plasma membrane invaginations that can be used for endo- and transcytosis. Thus, the lining cells of both the alveolar and the capillary side of the air-blood barrier are equipped for exerting caveolae-mediated transport functions. The Capillary Endothelium and Particles
Although the barrier function of the capillary endothelium is often in the focus, its metabolic function needs to be stressed both for local pulmonary reactions to particle exposure and as a source for the release of mediators to the systemic circulation. B. Nervous System
The innervation of the lungs consists of efferent and afferent neurons whose axons reach the extra- and intrathoracic airways mainly by the vagal nerve or by spinal nerve branches. The efferent innervation of the lungs can be divided into sympathetic nerves, cholinergic parasympathetic nerves (18), nonadrenergic noncholinergic parasympathetic nerves (19), and the so-called efferent function of sensory nerves (20). The afferent innervation of the lungs can be structured into slowly adapting stretch receptors (SAR), rapidly adapting stretch receptors (RAR), C fibers, and sensory receptors associated with neuroepithelial bodies (NEB). A unifying classification of the afferent neurons is difficult, if not impossible, due to the complexity and the plasticity of the pulmonary innervation. For example, useful ways of characterizing the afferent nerve phenotypes include conduction velocity and cell diameter, ganglion origin, modality, neurotransmitters, immunoreactivity for certain cellular markers, and morphology (for review, see Refs. 21 and 22). In addition, the phenotype of the neurons with respect to the expression of neurotransmitters may be altered by changes in the pulmonary environment (23,24). Stimulation of afferent nerves has a variety of possible effects including
Functional Aspects of Lung Structure as Related to Particles
7
Figure 3 Peribronchiolar nerve in the mouse lung. (A) Low-power electron micrograph of the bronchiolar epithelium with Clara cells and ciliated cells as well as the underlying interstitium. The incomplete rectangle indicates the region from which the high-power micrograph (B) is taken. A small nerve consisting of unmyelinated axon bundles (thin arrow), a myelinated axon (thick arrow), and a vas nervorum (asterisk) is shown. Due to incomplete fixation, the myelin sheath (arrow head) shows an artificial enlargement of the distance between the membranes, particularly on the left side of the myelin sheath.
aspiration reflex, apnea, changes in blood pressure, lower-airway mucosal secretion, bronchoconstriction, and cough (25). A peribronchiolar nerve is shown in Figure 3. Efferent Innervation
The preganglionic sympathetic nerves arise from the upper six thoracic segments of the spinal cord and project on the postganglionic neurons in the paravertebral sympathetic ganglia. The main function of the sympathetic innervation is the innervation of the blood vessels and the submucosal glands with only limited innervation of human airway smooth muscle (26). The preganglionic cholinergic parasympathetic nerves start in the vagal nuclei of the brainstem and reach intramural airway ganglia with the vagus nerves where they synapse with postganglionic neurons that innervate blood vessels, submucosal glands, and airway smooth muscle. The cholinergic parasympathetic innervation is abundant in proximal airways and decreases to the periphery (27). Its main effects are bronchoconstriction, mucus secretion, and bronchial vasodilation. While the “efferent” axon reflexes of sensory neurons also lead to bronchoconstriction, the nonadrenergic noncholinergic parasympathetic nerves have bronchodilatory effects. There is evidence that these effects are mediated by nitric oxide (19,28). Interestingly, autoradiographic studies of the human lung revealed that muscarinic receptors are abundant in submucosal glands and airway ganglia, but are also moderately present in intrapulmonary bronchi, airway smooth muscle of large and small airways, and also the alveolar wall (29). Similarly, b-adrenoreceptors were found abundantly in the airway epithelium, alveolar walls, and submucosal glands, and to a lesser extent, over airway and vascular smooth muscle (30).
8
M€ uhlfeld and Ochs Afferent Innervation
The respiratory tract has a trigeminal (nasal mucosa innervation), vagal (tracheobronchial innervation), and spinal (parts of bronchopulmonary innervation) afferent innervation. Accordingly, the cell bodies of the respective neurons lie within the trigeminal ganglia, the nodose (inferior), and jugular (superior) vagal ganglia or the dorsal root ganglia, respectively. While the nodose ganglia are derived from epibranchial placodes, the jugular ganglia are of neural crest origin (31,32). The different embryonic origin is also related to a different phenotypic modality of the associated fibers (21,33). According to conduction velocity and cell diameter, three main afferent fiber types innervating the lungs can be identified, namely, Ab and Ad as well as C fibers. Ab fibers are myelinated with relatively high conduction velocity. Ad fibers are also myelinated but thinner, and exhibit a slower conduction velocity. C fibers are unmyelinated and possess the slowest conduction velocity of the three subtypes (21). In the dorsal root ganglia, and presumably also in the vagal ganglia, the size of the cell bodies in the ganglia is positively correlated with conduction velocity (34). The mechanosensors SAR and RAR are myelinated fibers, mainly of the type Ad, in contrast to the C fibers, which are thought to be involved in the detection of thermal, inflammatory, and nociceptive stimuli. The differentiation of the low threshold mechanosensors SAR and RAR is based on their adaptation to prolonged deep inspiration. SAR typically discharge with changes in lung volume (tidal ventilation) and respond to sustained lung inflation in a nonadapting manner. In contrast, RAR also discharge during tidal breathing but react with a discharge burst upon maintained inflation and rapidly adapt to preinflation values. C fibers are relatively insensitive to mechanical stimuli but very sensitive to chemical stimuli, for example, to the experimentally applied capsaicin (35). According to the accessibility to chemicals injected into the bronchial or pulmonary arteries, they are classified into bronchial or pulmonary C fibers. Most of the afferent fibers in the airways are either glutamatergic or tachykinergic fibers (36,37). However, other transmitters and neuropeptides are expressed by subsets of afferent nerves, for example, calcitonin gene-related peptide (38), vasoactive intestinal peptide (39), or neuropeptide Y (40). The receptors of RAR, SAR, and C fibers are the free nerve endings, and the only receptor of higher order known in the airways so far are NEB. These highly specialized pulmonary endocrine cells are normal components of the intrapulmonary airways and become more frequent to the peripheral airways (22). The cells of NEB contain secretory granules that store ATP, serotonin, and several neuropeptides, including bombesin, calcitonin gene-related peptide, enkephalin, among others (41). Although the function of NEB has been speculative for many years, it has now become clear that their role in lung physiology changes during ontogeny. In the fetal mammal, they influence lung growth; in the postnatal lung, they have an oxygen-sensing function; and in adults, NEB have important functions in airway regeneration but are also related to the development of various types of lung cancer (42,43). The Nervous System and Particles
The methodological difficulties in studying the nervous system of the lung and, in particular, the neuronal plasticity explains why there is so little information on interactions between particles and the pulmonary nervous system. However, in a later
Functional Aspects of Lung Structure as Related to Particles
9
Figure 4 Flow chart of the immune system of the lung. This schematic drawing provides an
overview on the cellular and noncellular components of the immune system.
chapter of this book, Gonzalez-Flecha (chap. 8) deals with the neural origin of particleassociated reactive oxygen species. It will be interesting to understand which parts of the pulmonary nervous system are sensitive to particle exposure and what mechanisms drive the responses described in chapter 8. C. Immune System
The whole respiratory tract, from the nares or lips to the alveoli, contains a set of defense mechanisms that compensates for the entering of particles and microorganisms during respiration. The effectiveness of this defense system is illustrated by the fact that the large alveolar surface area of normal lungs is free of infectious agents. The main function of the respiratory tract defense system is to recognize and eliminate potentially harmful particles in an appropriate, non-overreacting way. The characteristics of the defense system change according to the anatomical location and consist of cellular and noncellular mechanisms, the latter including mechanical properties and soluble proteins. Most of the immune cells of the respiratory tract can be subdivided into groups according to the compartments in which they reside and the functional and phenotypic specializations associated with the anatomical location (Fig. 4). Noncellular Components
The noncellular components of the pulmonary defense system include sneezing and coughing, mucociliary clearance, and the barrier properties of the airway and alveolar epithelia. Sneezing and coughing bring about turbulence and shear forces strong enough to remove free or liquid-bound material such as debris, mucus, and other particles from
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M€ uhlfeld and Ochs
the nose and the airways, respectively (44). The airways are covered by a mucus blanket that is moved to the throat by the beat of the cilia of ciliated airway epithelial cells. Thus, particles that are deposited in the airways become trapped in the mucus layer and are transported with the mucus. This is termed the “mucociliary clearance” or “mucociliary escalator” (45). All air-conducting and gas-exchanging surfaces of the respiratory tract are covered by an epithelium, the characteristics of which depend on the localization. As mentioned above, the epithelium of the alveolar region is very thin and consists of two cell types, whereas the pseudostratified epithelium of the airways is composed of many different cell types. In addition to these mechanical properties, there are various liquid compartments in the airspaces, which contain protective proteins, such as surfactant proteins A and D (see above), components of the complement system, antimicrobial peptides, and immunoglobulins. Many of these proteins opsonize inhaled particles but some also have direct antimicrobial functions (46). Airway epithelial cells synthesize a large number of antimicrobial peptides including lysozyme, defensins, and lactoferrin (47). Cellular Components
According to the compartment, there are four types of macrophages in the lung: alveolar, airway, interstitial, and intravascular macrophages. Pulmonary macrophages are derived from blood monocytes and therefore originate from the bone marrow. This was originally proven for alveolar macrophages by Thomas and coworkers (48). In contrast to airway and alveolar macrophages, which can be isolated by bronchoalveolar lavage, intravascular and interstitial macrophages are not easy to obtain and therefore less well investigated. Intravascular macrophages are thought to act as sentinels of inflammatory proteins and antigens reaching the lung via the bloodstream. They can bind to and ingest microparticles, endotoxins, and particles circulating in the blood (49). Interstitial macrophages reside underneath the alveolar epithelium in regions where lymphatic vessels begin, that is, in connective tissue sleeves at the periphery or the center of acini. They are thought to migrate through the epithelium and replace alveolar macrophages, but may also take up antigens that have translocated over the alveolar epithelium. After antigen uptake, they may either migrate to the lymph vessels or become permanent residents of the interstitial space (50). Airway and alveolar macrophages are the first cellular defense line against inhaled particles. They are found within the mucus blanket of the airways or the watery hypophase of the alveoli and have a high capacity of phagocytosis (51). Airway macrophages usually exit the lungs through the mucociliary clearance, whereas alveolar macrophages may move from their alveolar localization either to the interstitial space or to the airways from where they take the same route as airway macrophages (52). From the interstitial space, they may move to the lymphatics (53) or become permanent residents of the interstitium. Alveolar macrophages possess the ability to move in an amoeboid manner along the alveolar epithelium by transient connections of their pseudopodia to the epithelial cells. Thus, they may move from one alveolus to another through pores of Kohn (Fig. 5). Uptake of antigens and pathogens by alveolar macrophages is enhanced by opsonization or inflammatory stimuli (e.g., interferon g) and is facilitated by scavenger receptors (e.g., MARCO) or mannose receptors and toll-like receptors (e.g., TLR-2), the importance of which is best
Functional Aspects of Lung Structure as Related to Particles
11
Figure 5 Alveolar macrophage in the mouse lung. This electron micrograph shows an alveolar macrophage, probably on his way from one alveolus to another through a pore of Kohn. Intracellularly, a large volume of phagolysosomes with lamellar body–like appearance is seen. The small nuclear profile on the right upper side of the macrophage indicates that this macrophage is not sectioned centrally.
documented by the detrimental effects of deficiency of these receptors (54–58). Above that, alveolar macrophages express a variety of other receptors, which enable their response to inflammatory stimuli and interaction with other cells of the defense system (58). While earlier data indicated that the life span of alveolar macrophages is in the range of a few months (59,60), recent studies in a mouse model have suggested that alveolar macrophages are quite long-lived cells with a life span not much shorter than the animal itself (61). Dendritic and Langerhans cells are antigen-presenting cells that are mainly found in the epithelium of the larger airways with their frequency becoming lower to the periphery, although other localizations, including alveolar septa, subpleural tissue, and alveoli, have been described as well (62–64). They have long, branched dendritic cell processes and an irregular, folded nucleus. In the tissues, they form a network the density of which may be increased by a variety of stimuli, including exposure to interferon g (65), cigarette smoking (66), and allergen inhalation (67). After antigen uptake, dendritic cells may either move to regional lymph nodes to activate lymphatic T cells (68) or they may stay in the lung and present antigen to local T lymphocytes (69). As shown in Figure 4, pulmonary lymphocytes can be classified into lymphocytes associated with the epithelial surface or lymphoid aggregates, interstitial/intraepithelial
12
M€ uhlfeld and Ochs
lymphocytes, and intravascular lymphocytes (70). Lymphocytes in lymphoid aggregates (bronchus-associated lymphatic tissue, BALT) are not prominent in human lungs, except for infection and chronic inflammation. The functions of pulmonary lymphocytes are cytotoxic actions against cells infected with viruses, antibody and cytokine production, and immune tolerance (71). Plasma cells, which produce antibodies, are relatively abundant not only around the acini of the seromucous glands of bronchi but also in the interstitium around bronchioles. In the airways, the main immunoglobulin is IgA, whereas in the alveoli, IgG predominates (72). Granulocytes (neutrophils, eosinophils, and basophils) are relatively rare in the normal human lung, but their frequency may increase due to certain stimuli or as a characteristic feature of a disease such as asthma. Mast cells contain metachromatic secretory granules that contain heparin and histamine and have a scroll-like appearance in the electron microscope. Mast cells are present in normal lungs but their number may increase in various diseases (44). The different cells of the pulmonary defense system are not functionally distinct players, but they communicate with each other by a great amount of cytokines and receptors and there is an increasing body of literature on this topic (73–77). The Immune System and Particles
As complex as the immune system of the lung are the interactions between inhaled particles and the pulmonary immune system. Alveolar macrophages, for example, have long been thought of as mere removers of the alveoli. However, their ability to interact with various cell types of the immune system as well as alveolar epithelial cells highlights the complex nature of the system as a whole. In this book, two different but clinically very important aspects of interactions between inhaled particles and the pulmonary immune system are addressed by Schleh and Hohlfeld (chap. 15) as well as Jaspers and Horvath (chap. 9).
V. Summary The present chapter has highlighted various aspects of lung structure as far as they are related to particle exposure. We are convinced that a broad knowledge of the normal structural and functional properties of the lung is essential for a scientific examination of particle-lung interactions. In a short chapter like this, we had to be very selective and focus on those aspects that we have nominated as the “portal of entry” and the “portals of exit” of inhaled particles. Both these terms should be reflected upon in a general way, as pointed out in the small passages about particles and the respective structural compartment. Within this book, we hope that this uncommon portrait of the lung helps the reader find her or his way through the subsequent chapters. Looked at from a broader perspective, this image may also stimulate the meeting of diverse areas in the field of particle-lung interactions and promote the investigation of the cross talk between the different structures and help in establishing structure-function relationships, thus providing a more integrated picture of the diverse interactions between particles and the lung.
Functional Aspects of Lung Structure as Related to Particles
13
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48. Thomas ED, Ramberg RE, Sale GE, et al. Direct evidence for a bone marrow origin of the alveolar macrophage in man. Science 1976; 192:1016–1018. 49. Matute-Bello G, Frevert CW, Martin TR. Animal models of acute lung injury. Am J Physiol Lung Cell Mol Physiol 2008; 295:L379–L399. 50. Geiser M. Morphological aspects of particle uptake by lung phagocytes. Microsc Res Tech 2002; 57:512–522. 51. Kiama SG, Cochand L, Karlsson L, et al. Evaluation of phagocytic activity in human monocyte-derived dendritic cells. J Aerosol Med 2001; 14:289–299. 52. Kilburn KH. A hypothesis for pulmonary clearance and its implications. Am Rev Respir Dis 1968; 98:449–463. 53. Thepen T, Claassen E, Hoeben K, et al. Migration of alveolar macrophages from alveolar space to paracortical T cell area of the draining lymph node. Adv Exp Med Biol 1993; 329:305–310. 54. Hogg N, Selvendran Y, Dougherty G, et al. Macrophage antigens and the effect of a macrophage-activating factor, interferon-gamma. Ciba Found Symp 1986; 118:68–80. 55. Arredouani M, Yang Z, Ning Y, et al. The scavenger receptor MARCO is required for lung defense against pneumococcal pneumonia and inhaled particles. J Exp Med 2004; 200: 267–272. 56. Arredouani MS, Palecanda A, Koziel H, et al. MARCO is the major binding receptor for unopsonized particles and bacteria on human alveolar macrophages. J Immunol 2005; 175:6058–6064. 57. Armstrong L, Medford AR, Uppington KM, et al. Expression of functional toll-like receptor2 and -4 on alveolar epithelial cells. Am J Respir Cell Mol Biol 2004; 31:241–245. 58. Palecanda A, Kobzik L. Receptors for unopsonized particles: the role of alveolar macrophage scavenger receptors. Curr Mol Med 2001; 1:589–595. 59. Godleski JJ, Brain JD. The origin of alveolar macrophages in mouse radiation chimeras. J Exp Med 1972; 136:630–643. 60. Matute-Bello G, Lee JS, Frevert CW, et al. Optimal timing to repopulation of resident alveolar macrophages with donor cells following total body irradiation and bone marrow transplantation in mice. J Immunol Methods 2004; 202:25–34. 61. Murphy J, Summer R, Wilson AA, et al. The prolonged life-span of alveolar macrophages. Am J Respir Cell Mol Biol 2008; 38:380–385. 62. Holt PG, Schon-Hegrad MA. Localization of T cells, macrophages and dendritic cells in rat respiratory tract tissue: implication for immune function studies. Immunology 1987; 62: 349–356. 63. Sertl K, Takemura T, Tschachler E, et al. Dendritic cells with antigen-presenting capability reside in airway epithelium, lung parenchyma, and visceral pleura. J Exp Med 1986; 163: 436–451. 64. von Garnier C, Filgueira L, Wikstrom M, et al. Anatomical location determines the distribution and function of dendritic cells and other APCs in the respiratory tract. J Immunol 2005; 175:1609–1618. 65. Gong JL, McCarthy KM, Telford J. Intraepithelial airway dendritic cells: a distinct subset of pulmonary dendritic cells obtained by microdissection. J Exp Med 1992; 175:797–807. 66. Soler P, Moreau A, Basset F, et al. Cigarette-smoking induced changes in the number and differentiated state of pulmonary dendritic cells/Langerhans cells. Am Rev Respir Dis 1989; 139:1112–1117. 67. McWilliam AS, Napoli S, Marsh AM, et al. Dendritic cells are recruited into the airway epithelium during the inflammatory response at mucosal surfaces. J Exp Med 1996; 184: 2429–2432. 68. Vermaelen KY, Carro-Muino I, Lambrecht BN, et al. Specific migratory dendritic cells rapidly transport antigen from the airways to the thoracic lymph nodes. J Exp Med 2001; 193:51–60.
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69. Julia V, Hessel EM, Malherbe L, et al. A restricted subset of dendritic cells captures airborne antigens and remains able to activate specific T cells long after antigen exposure. Immunity 2002; 16:271–283. 70. Pabst R. The immune system of the respiratory tract. In: Busse W, Holgate ST, eds. Asthma and Rhinitis. Cambridge, MA: Blackwell Scientific, 1994:415–425. 71. Little FF, Wilson KC, Berman JS, et al. Lymphocyte- and macrophage-mediated inflammation in the lung. In: Fishman AP, Elias JA, Fishman JA, et al. eds. Fishman’s Pulmonary Diseases and Disorders. 4th ed. New York: McGraw-Hill, 2008:291–305. 72. Burnett D. Immunoglobulins in the lung. Thorax 1986; 41:337–344. 73. Holt PG, Schon-Hegrad MA, Oliver J. MUC class II antigen-bearing dendritic cells in pulmonary tissues of the rat. Regulation of antigen presentation activity by endogenous macrophage populations. J Exp Med 1988; 167:262–274. 74. Holt PG, Oliver J, Bilyk N, et al. Down-regulation of the antigen-presenting cell function(s) of pulmonary dendritic cells in vivo by resident alveolar macrophages. J Exp Med 1993; 177:397–407. 75. Reynolds HY. Advances in understanding pulmonary host defense mechanisms: dendritic cell function and immunomodulation. Curr Opin Pulm Med 2000; 6:209–216. 76. Mantovani A, Muzio M, Garlanda C, et al. Macrophage control of inflammation: negative pathways of regulation of inflammatory cytokines. Novartis Found Symp 2001; 234: 120–131. 77. Kuipers H, Lambrecht BN. The interplay of dendritic cells, Th2 cells and regulatory T cells in asthma. Curr Opin Immunol 2004; 16:702–708.
2 Ambient Tropospheric Particles PAUL A. SOLOMON U.S. Environmental Protection Agency, Las Vegas, Nevada, U.S.A.
DANIEL L. COSTA U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, U.S.A.
I.
Introduction
Atmospheric particulate matter (PM) is a complex mixture of solid and liquid particles suspended in ambient air (also known as the atmospheric aerosol). Ambient PM arises from a wide range of sources and/or processes, and consists of particles of different shapes, sizes, and compositions, with an array of physicochemical properties (1,2). Because particle size best relates to particle aerodynamics, diameter is most often used when describing ambient PM, most notably with regard to its atmospheric transport, lung deposition, and sampling for scientific or regulatory purposes. Particle number concentration, surface area, and volume (where particle volume particle density ¼ mass) are typically used to describe the size distributions of particles comprising ambient PM (Fig. 1). When coupled with chemical composition and PM optical properties (and sometimes charge), one obtains a more complete appreciation of the complex physicochemical nature of ambient PM. PM of most interest to health scientists is that which is considered inhalable, that is, it enters human airways, including but especially beyond the nose and mouth (4–7). Such PM encompasses a size-range from a few nanometers to *10 mm aerodynamic diameter (AD). AD is a function of particle density and shape and is the prime descriptive parameter for PM that is collected or segregated by inertial measurement methods, including the respiratory tract. With standardization of individual particles as unit-density spheres with the same settling velocity as irregularly shaped particles, as described as an “aerodynamic diameter,” widespread application and comparisons of ambient particles can be achieved (for additional detail see Refs. 2 and 8). Further, it is common to find ambient PM described by its MMAD (mass median AD), which is the normalized particle AD with half the total mass of the particles in the air residing above that number and half the mass below (2). The geometric standard deviation (GSD), also referred to as sigma g (sg), describes the statistical distribution associated with MMAD and defines the heterogeneity of the particle sizes around that median value. MMAD (or its volume equivalent as is sometimes used) is important since it is widely utilized by both atmospheric and health scientists; in the latter case, to predict size-related deposition patterns in the respiratory tract.
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Figure 1 Typical number and volume distributions of atmospheric particles with the different
modes. Source: Adapted from Figure 3-2 in Ref. 3.
Historically, airborne particles have been categorized into three major size fractions or modes based on their mass (volume) or number concentration distributions (Fig. 1). These empirically defined size ranges are termed ultrafine (<0.1 mm, UF), fine (<2.5 mm, PM2.5, which includes UF PM), and coarse (PMc, 2.5 mm > PM <10 mm). The fine and coarse modes in this case are defined aerodynamically in terms of their MMAD with a minimum value between the two modes at around 2 to 2.5 mm AD. Ultrafine particles, due to their very small individual particle masses even when considered collectively, are best characterized by analytical methods that do not depend on mass, as they have no mass-dependent aerodynamic properties. Hence, number counts (methods are described below) are often used to define this mode. Table 1 presents a comparison of UF, fine, and coarse PM size fraction characteristics as to how they are formed, their sources, composition, and atmospheric lifetimes. By convention (and historically), 2.5 mm AD has become the operationally defined separation diameter between fine and coarse size fractions since there is a natural dip between these two modes in the size distribution (Fig. 1). Moreover, studies of PM deposition in the respiratory tract have shown that fine particles (including UF) penetrate into the deep lung, the region most prone to injury. Thus, PM2.5 has come to define the U.S. “fine particle” National Ambient Air Quality Standard (NAAQS) (9). PM10 (particles less than 10 mm AD) is also a size fraction of interest to the health and regulatory communities and includes all three modes below 10 mm AD. Within PM10,
Largely soluble, hygroscopic, and deliquescent
Probably less soluble than accumulation mode
Solubility
Composed of
Formed by
Combustion, high-temperature processes, and atmospheric reactions Condensation Nucleation Coagulation Condensation Reactions of gases in or on particles Coagulation Evaporation of fog and cloud droplets in which gases have dissolved and reacted Sulfate Sulfate, nitrate, ammonium, and Elemental carbon hydrogen ions Metal compounds Elemental carbon Organic compounds with very low Large variety of organic compounds saturation vapor pressure at ambient Metals: compounds of Pb, Cd, V, Ni, temperature Cu, Zn, Mn, Fe, etc. Particle-bound water
Accumulation (0.1 to 2.5 mm size range)
Formation processes
Ultrafine
Fine
Table 1 Comparison of Ambient Fine (
(Continued )
Suspended soil or street dust Fly ash from uncontrolled combustion of coal, oil, and wood Nitrates/chlorides/sulfates from HNO3/HCl/SO2 reactions with coarse particles Oxides of crustal elements (Si, Al, Ti, Fe) CaCO3, CaSO4, NaCl, sea salt Pollen, mold, fungal spores Plant and animal fragments Tire, brake pad, and road wear debris Largely insoluble and nonhygroscopic
Mechanical disruption (crushing, grinding, abrasion of surfaces) Evaporation of sprays Suspension of dusts Reactions of gases in or on particles
Breakup of large solids/droplets
Coarse
Ambient Tropospheric Particles 19
Minutes to hours Grows into accumulation mode Diffuses to raindrops <1 to 10s of km
Atmospheric half-life Removal processes
Source: Adapted from Table 2-2 in Ref. 5.
Travel distance
Combustion Atmospheric Transformation of SO2 and some organic compounds High temperature processes
Sources
Ultrafine
Accumulation (0.1 to 2.5 mm size range)
Days to weeks Forms cloud droplets and rains out Dry deposition 100s to 1000s of km
Combustion of coal, oil, gasoline, diesel fuel, wood Atmospheric transformation products of NOx, SO2, and organic compounds, including biogenic organic species (e.g., terpenes) High-temperature processes, smelters, steel mills, etc.
Fine
Resuspension of industrial dust and soil tracked onto roads and streets Suspension from disturbed soil (e.g., farming, mining, unpaved roads) Construction and demolition Uncontrolled coal and oil combustion Ocean spray Biological sources Minutes to hours Dry deposition by fallout Scavenging by falling rain drops <1 to 10s of km (small size tail, 100s to 1000s in dust storms)
Coarse
Table 1 Comparison of Ambient Fine (
20 Solomon and Costa
Ambient Tropospheric Particles
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PMc, as defined above, is also referred to as “thoracic” particles due to their predominant deposition in the larger lung airways. This chapter examines in more detail sampling methods, physicochemical characteristics, and sources of UF, fine, and coarse PM, since these size modes are presently of most interest to the health community. While each size mode has been linked to various health outcomes, there is evidence to support the notion that each may have unique health risks (10).
II.
General Issues
When compared with PM2.5, UF PM and PMc have relatively short lifetimes (hours or less) in ambient air—the former being lost to coagulation or condensation to larger sizes (typically between 0.1 mm and about 2 mm), peaking at around 0.7 mm to 1 mm, while the latter being removed by deposition due to gravitational settling (Table 1). PM2.5 on the other hand, can have long residence times in ambient air, several days to weeks (2,11), and hence this size range is most likely to interface with the inhabitants of a given urban airshed. Residence time also relates to transport time in air, whereby fine PM has been known to travel thousands of kilometers (e.g., Canadian Wildfires of 2004: Refs. 12–16). Thus, one airshed, be it urban or rural, can impact distant airsheds (5,12). Consequently, long residence times can complicate exposure studies since regional (100–1000 km transport distances) and even continental-scale (1000s of km) pollution can superimpose upon and mix with urban (10–100 km), neighborhood (1–10 km), and finer scale pollution. As a result of regional transport and the mixing of secondary and primary pollutants with urban and finer-scale pollution, the highest concentrations of PM2.5 generally occurs within or just downwind from urban areas (12,13,17). Therefore, this intermixing of pollutants on different spatial and temporal scales, along with atmospheric processing, makes it challenging to accurately attribute PM concentrations and composition observed at a receptor (e.g., human) back to specific PM sources. Currently, mass concentration (mg/m3) is the regulatory indicator of PM2.5 and PM10 identified by the U.S. Environmental Protection Agency (EPA). The use of a mass index stems from the associated health effects of PM derived from the ambient data reported in the mass-based monitoring network (see Refs. 5 and 6 and Figure 2.2 in Ref. 6). In 2006, EPA revised the PM2.5 daily NAAQS (35 mg/m3 from 65 mg/m3) keeping the annual average PM2.5 NAAQS unchanged (15.0 mg/m3) while removing the PM10 annual average standard but maintaining the daily PM10 standard (150 mg/m3) (9). PM10 values have been used as an interim estimate of PMc, a size-range previously thought to be of less concern but which has recently garnered attention regarding potential health outcomes (more specific PMc methods have not yet been widely deployed). Using mass (PM2.5 or PM10) as the exposure indicator for health outcomes has long been criticized (18) as being nonspecific without appreciation for basic toxicological principles. This criticism has encouraged considerable research attempting to link individual or multiple physicochemical properties of PM as more “appropriate” exposure indicators and subsequently to health outcomes. However, despite the existence of several plausible “causative” hypotheses linking health effects to physicochemical properties, no single or group of physicochemical properties of PM has been identified as the primary causative agent (e.g., metals, organics, number
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concentration) (19), which would thus merit a change in the mass-based regulations. Therefore, it is important for health researchers to appreciate the complexity of ambient PM if studies are to be appropriately targeted and relevant for risk assessment and component/source attribution. Unlike gas phase criteria pollutants (e.g., ozone or sulfur dioxide), measurements of PM are complicated since ambient PM is a dynamic mixture composed of many chemical species (100s–1000s) that vary in space and time. Size and composition can sometimes change rapidly (minutes to hours), due to variations in source emissions (anthropogenic and natural) and meteorology (2,3,12,13,20–25). Measurements are further complicated because gas phase emissions undergo chemical reactions in air, forming secondary particles [e.g., sulfate, nitrate, secondary organic aerosol (SOA)] that mix with directly emitted or primary particles. Chemical components in liquid (including fog and raindrops) as well as solid particles also undergo atmospheric processing changing the chemical composition of those particles. On the sampling side, some organic and inorganic (e.g., NH4NO3) species are semivolatile, having fractions in both the condensed and gas phases, simultaneously confounding sample collection with sampling artifacts. These sampling artifacts include adsorption of gas phase species by the filter or collected particles (a positive artifact) or loss of semivolatile species from the collected particles (a negative artifact). Comprehensive PM characterization is also restricted since no single measurement method can provide a measure of all PM modes by mass and their respective chemical components and physical properties (e.g., size distribution or number concentration). Until the past few years, national PM monitoring networks and many long-term research studies have primarily collected filter samples for PM mass and composition determination for 24-hour sampling periods and typically on a 1-in-3 day or 1-in-6 day sampling schedule. Considerable information can be lost (e.g., diurnal variation) with these relatively long sampling times and intermittent sampling periods (12,26,27). Ideally, implementation of real-time, continuous methods would best fill these information gaps, but while there is some progress in this area, few continuous methods are ready for routing or even long-term research networks (26). Nevertheless, several recent reviews describe relevant measurement methods that can assist the health community to better link ambient pollution to health effects (8,23,26,28–31). The above discussion illustrates that measuring the chemical and physical properties of PM can be challenging. It is important that health scientists are aware of the dynamics and diversity of PM mass and its chemical and physical properties if they are to make real headway into the assessment of causative agents and sources. It is also important to take care to avoid overinterpretation of correlation with restrictive data since a complete physicochemical analysis of PM is not yet possible. Moreover, when looking at human exposure/health effects linked to ambient PM or perhaps better to ambient sources of PM, not only do we need to measure and understand outdoor PM impacts but we also need to be able to separate exposure and effects from indoorgenerated PM. Studies have shown that, in general, indoor PM consists of a significant fraction (up to *70%, but highly variable) of particles that have penetrated from outdoors. These particles are usually in the PM2.5 mode, in contrast to UF PM and PMc, which do not penetrate indoors very well for physical reasons. This composite of outdoor-indoor PM exposure might suggest that fine PM should be the predominant PM of health interest. In fact, the bulk of the health literature on PM does relate to the fine PM fraction; however, there is evidence tying health outcomes to UF and coarse PM that is
Ambient Tropospheric Particles
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difficult to interpret in this outdoor-indoor context, and questions in this context remain. Hence, the better we understand ambient PM, specifically its physicochemical properties and sources, the more likely we are to gain insights into the PM properties and sources of greatest health risk. EPA’s PM Supersites Program and related studies (1,12), the NARSTO PM assessment (32), research published by Pandis (3), and EPA’s PM criteria documents (5,33) provide more detailed information on PM characterization, atmospheric processes, and source-receptor relationships.
III.
PM Measurement Methods
Historically, PM has been measured for regulatory and monitoring purposes by collecting PM on filters for 24-hour sampling periods on specified sampling days (6th day, 3rd day, and more recently, daily in specified locations). PM2.5 and PM10 are monitored widely in U.S. national regulatory networks for compliance with health-based NAAQS, mostly in populated areas, and for visibility deterioration in rural areas and U.S. national parks [Interagency Monitoring of Protected Visual Environments (IMPROVE), http://vista.cira.colostate.edu/improve/]. Both networks measure mass and chemical composition using filter-based methods. Mass of PM collected on a filter is typically determined by weighing the filter in the laboratory under controlled conditions before and after sampling, the difference between pre- and postweights being the collected mass. Size selective inlets allow for the inertial separation of PM10 or PM2.5 from larger particles (>10 mm) that occur in air, but are believed to be of little health consequence. In the case of PM2.5 as collected in EPA’s Federal Reference Monitoring (FRM) network, samplers are tightly controlled as to inlet flow rate and filter temperature, since variations in these parameters can result in inaccurate data. Some sampling stations for PM2.5 in the FRM network are designated for chemical analyses as part of EPA’s national Chemical Speciation Network (CSN) (34) (http://www.epa.gov/ttn/amtic/speciepg.html) as well as at all IMPROVE sites. UF PM and PMc data are much sparser than PM2.5 or PM10 since NAAQS are not in place for these fractions of PM. Generally, these size fractions are associated with special studies [e.g., epidemiology studies, programs like the Supersites Program (12) or collected by states conducting special studies (e.g., California)]. Various filter media (e.g., Teflon, quartz fiber, nylon) are used to collect ambient PM for chemical characterization. The choice of media depends on the PM component of interest and the chemical analysis method employed. As already noted, use of filters can bias the measurement values positively or negatively, relative to what is in the air. For example, semivolatile components [e.g., ammonium nitrate (NH4NO3)] or semivolatile organic species (SVOC) are known to volatilize from inert Teflon filters during sampling, transport, and storage (12 and references within, 23,35). On the other hand, organic gases adsorb (positive artifact) onto quartz fiber filters and collected particles during sampling, transport, and storage (12 and references within, 23,36,37). Use of denuders to remove gas phase interferences and reactive back-up filters to collect volatilized PM components helps to minimize both types of sampling artifacts (23,36). For example, MgO-coated honeycomb denuders followed by nylon filters are used in the PM2.5 CSN. Over the past 5 to 10 years, significant progress has occurred in the development of continuous and semicontinuous (herein referred to as continuous) methods for
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specific chemical components and physical properties of PM. These methods allow not only for automated daily averaged data to be obtained but also for the collection of data on the order of an hour or less. These shorter sampling periods afford better understanding of atmospheric processes, source attribution, and perhaps eventually, acute health effects from short duration PM exposures (e.g., exposure on freeways while driving to work). Continuous methods are less impacted by sampling, handling, and storage artifacts (26). Perhaps the most significant measurement advancement in the past decade has been methods that measure the chemical composition of single particles or composition integrated over a size range of particles from a few or tens of nanometers to about 2.5 mm (26,38–40); as a class referred to as particle mass spectrometers. These methods can help to provide refined source attribution and exposure assessments with reduced uncertainty that can help reveal linkages to health outcomes. While ultrafine particles are typically defined as particles less than 100 nm AD, an operationally defined size of about 150 nm was used in a continuous method developed within the Supersites Program to measure UF PM mass (41). Good agreement was obtained with estimates of UF PM mass by other methods. However, since UF PM only comprises a small fraction of the fine particle mass (3) (Fig. 1), making it difficult to measure accurately, most measures of UF PM are in terms of number concentration or size distribution. These methods include the condensation particle counter (CPC) using either water (WCPC) or butanol as the condensing agent to measure particle number concentration and the scanning mobility particle sizer (SMPS) or nano-SMPS to measure size distributions in the 3 to 600 nm size range. Chemical composition of UF PM can be determined in the laboratory on Micro-Orifice Uniform Deposit Impactors (MOUDI) stages that collect less than 100 nm particles on filters or in real time using particle mass spectrometers (e.g., RSMS-II) or the Aerosol Mass Spectrometer (AMS) (26 and references within). Effort to further develop continuous methods that measure the chemical composition of UF and fine particles, including particle mass spectrometers, is ongoing (26,31,40). Coarse particle concentrations are measured either by taking the difference between simultaneous collocated measurements of PM10 and PM2.5 (42) or by separating fine and coarse particles by inertia using either real or virtual impaction (43,44). Continuous indirect measurements of PMc mass can be estimated based on size distribution data using an Aerodynamic Particle Sizer (APS) (600 nm–10 mm diameter particles) (45,46) and applying shape and density factors to the measured size distribution data. Real impactors separate particle sizes using an impaction plate, real surface where particles of a given size range are collected. Virtual impactors use an air boundary (virtual plate) to separate fine and coarse PM, with each fraction being collected and analyzed on separate filters, although a small fraction of fine PM is collected with the PMc (43,44,47). Virtual impactors are preferred since particle bounce is eliminated. A continuous fine and coarse PM sampler based on virtual impaction is commercially available [Filter Dynamics Measurement System-Tapered Element Oscillating Microbalance (FDMS-TEOM)] and is based on initial work by Sioutas and Solomon (48) and Misra et al. (47). Continuous chemical speciation methods for PMc have not been developed to date. For most PM-related properties, total measurement uncertainty can only be obtained by comparison of a new method to existing or historical methods, where confidence has been built over time. This approach to estimate measurement uncertainty is needed since in-field analytical standards do not exist for most PM measurement
Ambient Tropospheric Particles
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methods (23,26). It is important to reiterate that the FRM for PM2.5 and PM10 mass are not analytical standards but regulatory standards, and mass concentrations obtained by these samplers may not always represent the aerosol present in the air due to negative sampling artifacts (12,23,26,31). Current science indicates that the continuous FDMSTEOM represents the most accurate measure of mass concentration in air, and mass values are often higher than the FRM (26,31) since negative artifacts are essentially eliminated. Results for PM2.5 mass and chemical composition from in-field comparison studies indicate the following measurement uncertainties for data collected with timeintegrated filter-based methods: 20% for mass, 30% to 35% for NO3 and NHþ4, 10% for SO2 4 , 20% to 50% for organic carbon (OC), and 20% to 200% for elemental carbon (EC), where the range for OC and EC depends on whether the same or different analytical methods are used (31,49). Uncertainty estimates for continuous methods for mass and chemical components are provided in Chow et al. (31) and Weber et al. (50), and are similar to or slightly better than those reported for integrated methods. Clearly, any characterization of ambient PM is going to have a number of contributing variances, including those owing to the nature of PM emissions and formation processes as well as their spatial and temporal distributions. Not to be underappreciated, of course, is the introduction of sampling artifacts and analytical uncertainties. These uncertainties only add to the difficulties in studying PM component-attributed health outcomes, but must be realized in any summary judgments regarding attributable-causation of PM properties.
IV.
Characteristics of Ambient PM
The spatial and temporal characteristics of PM mass and composition are strongly influenced by the strength and location of anthropogenic and natural emissions sources, topography, meteorology, and atmospheric processing. Particles can also be characterized as being “primary” or “secondary.” Primary particles are emitted directly in to the air and observed in all the three size modes. Secondary particles form from gas phase emissions (precursor species, such as, SO2, NOx, and >C8 VOC) that react in the atmosphere to form new particles by nucleation and/or where less volatile compounds condense onto existing particles [see Pandis (3) for an overview and Seinfeld and Pandis (2) for detail]. Secondary products are mostly observed in the fine mode, including UF PM. Reactions that form secondary particles can occur in the gas phase or condensed (particle) phase, such as in cloud or fog droplets. Oxidation of precursor gases such as SO2 and NOx results initially in acidic species (i.e., sulfuric and nitric acids) that are usually neutralized by ammonia (the only strong gas phase base in air) to (NH4)2SO4, NH4HSO4, and/or NH4NO3—depending upon the amount of ammonia available. Higher molecular weight gas phase organic compounds (typically >C8) can be oxidized to less volatile compounds, which can condense onto existing particles. Many of these reaction products are semivolatile (e.g., NH4NO3 in equilibrium with gas phase NH3 and HNO3), and the fraction in either phase depends on temperature, RH, and gas phase concentrations of the semivolatile component (2). The semivolatile nature of some PM components also adds complexity to their measurement and association with health outcomes. The smallest UF particle size range (<10 nm, nucleation mode) consists of fresh particles formed in situ from the gas phase by nucleation. These particles have a high coagulation rate growing into the larger UF mode, until they are diluted. Larger UF particles (10–100 nm) also have a significant primary particle component (e.g., EC), and
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grow by condensation or through heterogeneous reactions in droplets or on particles into the fine size range between 0.1 and about 2 mm AD (accumulation mode) (Fig. 1). Thus, a significant fraction of fine PM is secondary in nature (20). Coarse mode particles (Fig. 1) are typically produced by mechanical abrasive processes, and therefore consist mostly of primary particles with a significant fraction being related to crustal material (soil) and biological material (2,5,51,52). There can be overlap, however. The lower end (tail) of the coarse particle size distribution often extends into the “fine” particle size range, in part due to the collection efficiency of inertial samplers, so crustal material is often found in the PM2.5 size range as well. As indicated, a variety of processes define the multimodal particle size and mass distributions of ambient PM, but because various sources and atmospheric processes impact these size ranges differently, the chemical composition of the three modes are different. Temporal distributions can also vary among the three modes due to the impact of meteorology (e.g., temperature and RH), atmospheric residence time (a function of particle size), and atmospheric processing. A. Ultrafine Particles
UF PM composition and particle number vary considerably in space and time driven by sources of primary particles and the mechanism of formation for secondary particles. Extensive measurements of UF PM size distributions and chemical composition were conducted during the Supersites Program (12). UF particles in high concentrations (UF particle events) can be observed near combustion sources, which may occur any time of the day; in the afternoon, especially during the summer due to photochemistry; and in the morning over large regional areas (simultaneously over several 1000s km2, e.g., Pittsburgh, PA and Philadelphia, PA), referred to as regional nucleation bursts (53). OC is often a major fraction of UF PM (see Figure 3 in Ref. 54), except near certain industrial sources such as in Houston, Texas, where trace metals seem to dominate most wind directions (see Figure 18 in Ref. 55), or during regional nucleation bursts as observed in the greater Pittsburgh, Pennsylvania, area where sulfate dominates initially (53,56) (Fig. 2). These morning regional nucleation events were not expected, lasted for several hours, and occurred on clean days when competition for coagulation and condensation was low and particles were able to nucleate instead, with the new particles being initially acidic (i.e., H2SO4). Solomon et al. (12) pointed out possible health implications of this finding since UF PM and particle acidity may impact health as these nucleation bursts tended to occur when people might be outdoors exercising, to a greater extent on days when pollution levels are low. UF PM is also observed in high number concentrations on and near freeways with rapid decrease away from the freeway (58). Their decrease in number is faster during the summer than the winter due to stabilization of the volatile components under cooler wintertime conditions. Out of the tail pipe, initial particle formation is due to nucleation of sulfuric acid (H2SO4) particles with rapid growth (1–3 seconds) to above 10 nm by condensation of organic gases (59). As the particles move from the roadway to ambient background (within 90–300 m), they undergo evaporation and/or condensation; coagulation and deposition appear unimportant. This process changes the size distribution and composition of on- and near-roadway particles so that people living near the freeway (within a few hundred meters) may experience an UF PM size distribution and composition that others do not. Current routine ambient monitoring networks do not capture
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Figure 2 Diurnal behavior of ultrafine chemical composition (top) and number size distribution
(bottom) are illustrated on a day with intense new particle formation (September 12, 2002). Top panel reports composition in the 33 to 60 nm vacuum aerodynamic diameter size window. Bottom panel reports electrical mobility diameter. Except for the dark area at the top of the lower panel, the darker the gray, the higher are the particle number concentrations. Prior to 09:00, the ultrafine particles are mainly organic and have a mode at 20 to 30 nm, consistent with primary emissions from combustion sources. At around 09:00, new particle formation is evident at sizes below 10 nm. This is followed by growth of the new particles and a rapid increase in the fraction of sulfate (initially) and ammonium sulfate (later) in the ultrafine particles. Aerosol chemistry is measured by an aerosol mass spectrometer, for particles with vacuum aerodynamic sizes from 33 to 60 nm, or approximately 18 to 33 nm physical diameter. More information on the September 12, 2002 and other events are given by Zhang et al. (57). Source: Figure courtesy of Stanier C, University of Iowa, Iowa City (previously at Carnegie Mellon University, Pittsburgh, PA, in association with the Pittsburgh Supersites Project) and Zhang QI, University of Colorado, Boulder (from J Geophys Res 110, D07, journal cover figure and caption).
these small-scale variations in PM, in any size range, since the networks were designed to capture urban to regional-scale pollution. However, health effects may well occur as a result of the high pollution levels observed on these finer spatial scales, such as near roadways (60). In general, UF PM tends to have at least two peaks during the day (2,12,61–63). In the morning, UF PM peaks in most locations during rush-hour periods, with higher number concentrations observed during the winter than the summer. UF PM peaks during the mid-afternoon are due to photochemical nucleation. Hence, the composition of the two peaks may be different with unknown health or study-design implications. A third less pronounced afternoon peak occurs due to traffic in the winter or with seasonal activities like residential wood burning.
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PM2.5 mass and chemical composition vary in space and time driven by variations in source impacts, meteorology, atmospheric processing, and the timescale used for averaging data. Figure 3A illustrates the importance of averaging time showing the significant loss in short-term variability information within daily average values. The same may be said for spatial variability, which is also smoothed with longer timescale measurements (12,13). High frequency data, for example, can also reveal aging nuances that are missed with longer sampling periods. For example, as shown in Figure 3B, sulfate and SO2 can be decoupled over several hour periods, indicating the likely impact of aged aerosol (labeled b in Fig. 3B) versus fresher plumes high in unprocessed SO2 (labeled a in Fig. 3B) (49,65). Secondary and primary components of PM2.5 influence both the spatial and temporal variability of this size mode. Secondary components (sulfate, nitrate, and SOA) comprise a major fraction of PM2.5 in all parts of the United States, during all seasons, and during all times of the day. These secondary components can become dominant
Figure 3 Temporal distribution of dry PM2.5 mass, PM2.5 sulfate, and SO2. Top plot shows relationship of PM2.5 mass (continuous thin solid line) to sulfate (dotted line) relative to the 24-hour average values (bars) of each. Note large deviations of the 1-hour data from the 24-hour average values and the good correlation between sulfate and PM2.5 mass. Lower plot shows the relationship between sulfate (thin solid line) and SO2 (dotted line). Note the two are correlated at times, perhaps from the same distance source or sources and not at other times, suggesting different sources. Note periods when SO2 was high and sulfate low (labeled “a”) and more frequent periods when sulfate was high and SO2 was low (labeled “b”) showing likely impact of plumes (a) from power plants outside the city and regional impact of more aged aerosol (labeled b). Weber (mass and sulfate) and Edgerton (SO2) collected these data. Source: Adapted from Figure 7 in Ref. 64.
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throughout geographical regions (e.g., sulfate in the eastern United States, nitrate in parts of the western United States). Differences in relative amounts of secondary PM are also responsible for differences on urban versus regional scales as well as temporal variations (diurnal, seasonal, and annual average) in PM2.5 (20). Urban, neighborhood, and smaller scales also can be strongly influenced by primary emissions (e.g., primary OC and EC from motor vehicles such as diesel engines and from residential wood combustion) (12,21,66). The predominance of light duty diesel in Europe distinguishes its near road environments somewhat from those in the United States, notably in the levels of EC. In the United States, annual average PM2.5 mass is highest in the eastern third of the country and California where the NAAQS is most often violated (24). In the eastern United States, major urban areas, large SO2 sources and source areas (e.g., the Ohio River Valley), significant amounts of biogenic hydrocarbons, often sufficient ammonia, and favorable meteorology drives the composition and resulting variations in PM2.5 mass. Long-range transport of fine PM and PM precursors (the latter form fine PM during transport) also contribute significantly to PM2.5 levels in the eastern United States, particularly during the summer (13), resulting in relatively homogeneous PM levels over large spatial scales (e.g., mid-Atlantic and northeastern United States), although, urban and downwind urban areas are slightly higher as noted above. The major chemical components of PM2.5, as illustrated in (Fig. 4), include sulfate, nitrate, ammonium, organic carbon, elemental carbon, and crustal material [the latter often estimated from the sum of Al, Fe, Si, and Ca after converting to their common soil-related oxides (68)] (1,22,24,25,69). A large fraction of PM2.5 is composed of secondary species (20), especially during the summer in the eastern United States, with sulfate and OC, including SOA as major components, whereas in the winter nitrate increases significantly in cooler locations, and sulfate decreases with both components becoming about equal, and OC often being the highest component. In California, especially in the winter, nitrate and primary OC are the major components with important contributions from EC due to residential wood combustion. In the summer, nitrate and OC are still the major components, but sulfate and crustal material increase from their winter values. Similar compositions are observed elsewhere as shown in Figure 4 for Leads, United Kingdom. In general, OC (primary plus secondary) appears to be a fairly consistent and major fraction of PM2.5 year round at all locations accounting for 20% to 50% of the PM2.5 mass on average, with SOA being a higher fraction in the summer. C. Coarse Particles
Direct measurements of PMc mass and composition are much more limited than those of fine PM. Recent health effects studies have prompted more attention to PMc and its monitoring (4,5,10,70–72). The fraction of PMc in PM10 varies due to source impacts and meteorology since PMc comprises mostly primary components. The western United States, for example, has higher concentrations of PMc (more windblown dust) than in the eastern United States, with PMc to PM10 ratios often greater than 0.5, whereas in the east, the ratio is usually less than 0.5 (73). Variations in the PMc to PM10 ratio ranging from 90% to 10% are observed elsewhere in the world (4).
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Figure 4 Comparison of typical major component composition of airborne particulate matter
from the eastern and western United States and Leads, United Kingdom. Source: Adapted from Figures 1 and 2 in Ref. 4, and Ref. 67.
As mentioned above, PMc generally has a shorter residence time in air than fine PM, except under special circumstances (e.g., transport of Asian or Saharan dust; 5,74). For that reason, its spatial distribution is more a function of local source impacts leading to a more heterogeneous spatial distribution (5,75,76). The chemical composition of PMc is dominated by trace elements, mostly those associated with crustal material (estimated as defined above) usually associated with windblown dust. Industrial processes (e.g., smelters, kilns), tire wears, and construction activities also contribute to trace metals in PMc. Other major components include carbon derived from tire wear and pieces of pollen, spores, and other biological material, and ions, such as nitrate, sulfate, and chloride from sea salt spray (4,5,52,62,76,77). Nitrate in PMc often results from the interaction of sea salt and nitric acid (NaCl þ HNO3(g) ¼ NaNO3 þ HCl(g)) emanating
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from sea spray, as does sulfate and chloride in PMc. However, little is known about the composition of the organic fraction of PMc. D. Related Properties of PM
Significant advances have resulted during the past five to eight years with regard to determining particle density (28) and aerosol water content (or particle bound water; PBW) (78) in airborne PM. With regard to density, data obtained in multiple locations indicate that PM below 2.5 mm has a density of about 1.5 to 1.7 gm/cm3, which falls within the expected range for particles composed mostly of sulfate, nitrate, and organic carbon, and about 10% trace elements (79). On the other hand, aerosol water content, especially when acidic, may influence the fraction of soluble components (e.g., trace metals) readily available to dissolve in lung fluids. For example, higher water content was observed during the summer when the fine PM fraction was acidic (78–80), which by inference not only makes available water-soluble species to lung fluids, but as an acidic milieu, may solubilize some components, such as transition metals that might otherwise not solubilize in neutral lung fluids.
V. Estimating Source Contributions at Receptor Locations Source- and receptor-oriented approaches are typically used to quantitatively estimate (i.e., including uncertainty estimates) source contributions at the receptor (presumably the exposed individual). Source-oriented approaches start at the source and use fundamental knowledge of the complex atmospheric system to model the emissions and the fate of those emissions to a receptor (81,82). Receptor-oriented models start with the measured concentrations at the receptor location and estimate the contribution of the source impact using statistical or similar methods (e.g., factor analysis) (66,83). Both models have their strengths and weaknesses. While source-oriented models can be used to estimate future PM concentrations based on predicted changes in emissions, receptor-oriented approaches cannot, since they derive from measurements at the receptor. Source-oriented models are more efficient at modeling secondary components of PM whereas receptor-oriented approaches are more efficient at modeling primary components. Thus, source- and receptor-oriented approaches are best applied simultaneously or as hybrids (82) to obtain the most accurate representation of the impact of source emissions at receptor locations. Advances allowing inclusion of high-time resolution chemistry and meteorological data continue to improve these models and their performance (12,27,66). With increasing emphasis being placed on attributing health effects to sources (if not PM components) to streamline and make more efficient control strategies, the importance of scientifically defensible attribution models is a growing need in the regulatory community.
VI.
Summary and Conclusions
Since 2000, significant progress has been made in the development and evaluation of continuous methods to measure UF, fine, and coarse PM and their major physicochemical properties. The most significant advancement during this time has occurred
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with the development of particle mass spectrometers able to measure either the chemical composition of single particles (*3 nm to as large as 2.5 mm) (single particle mass spectrometers) or the size distribution (tens of nm to 2.5 mm) of individual components (AMS). Continuous methods improve our understanding of the spatial and temporal distribution of the chemical and physical properties of PM that should provide new detail and insights to aid health research, including a better understanding of acute effects on hourly or shorter duration. Time-integrated filterbased methods are robust and provide reliable measurements (if care is taken to account for sampling artifacts) over varying lengths of time to relate to temporally associated health outcomes on timescales of a day to years. Caution is always needed when using filter-based systems since sampling artifacts can impact potential measurement results. The FDMS-TEOM currently allows for the most accurate measure of PM mass relative to what is in the air, since it simultaneously provides estimates of the non- and semivolatile components of PM. When several methodologies are combined, a more complete and realistic perspective of the PM exposure can be obtained. PM mass, composition, and physical properties can vary spatially across all size modes, particularly over short timescales (hours to days). Spatial distributions, especially for PM2.5, become more homogeneous over longer timescales (especially seasonal and annual). Ideally, PM measurements and health outcomes should be temporally paired to minimize misclassification errors. Interestingly, there is considerable consistency worldwide in terms of the major components of PM within size modes, although relative proportions of individual components may vary among locations. Specific local source impacts can lead to significantly different local levels than observed on regional scales. Secondary components are important contributors in larger UF and PM2.5, whereas primary particles are more important in PMc as well as the smaller UF PM, although the processes of formation and composition differ substantially within these size modes. The major PM components, although varying in proportions by size range, location, and season are sulfate, nitrate, ammonium, organic carbon, elemental carbon, and crustal material. Regional nucleation bursts, occurring over thousands of square kilometers, create large numbers of initially acidic (sulfate dominated) UF particles under relatively “clean” conditions. These events may have unexpected health implications not appreciated with standard PM monitoring and misperceptions of low exposure potential. Much more needs to be learned about the spatial and temporal variation of PMc and the sources and composition of organics in the coarse mode. Recent studies have indicated adverse health effects from PMc, suggesting the need for better characterization, especially on shorter timescales since PMc health effects primarily appear to be acute, and since PMc varies more over space and time than does fine PM. Health outcomes are increasingly being associated, sometimes with surprising specificity, with the various PM size modes. It follows that their respective compositions likely play a significant role in resulting health effects. However, gains in the temporal aspects of sampling and analysis to approximate the dynamics of ambient PM will have to be made to achieve the level of understanding likely needed to refine or improve regulatory indicators.
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3 Manufactured Nanoparticles ROBERT N. GRASS, LUDWIG K. LIMBACH, and WENDELIN J. STARK Institute for Chemical and Bioengineering, ETH Zurich, Zurich, Switzerland
I.
Introduction
Nanotechnology is shaping the future. The current broad, multidisciplinary technology development clearly shows how nanoparticles have become a key ingredient in commodity products affecting our daily life. Next to an exponentially growing market and economic excitement over the promises of nanoparticles, we often forget that nanoparticles have already been used industrially for nearly a century. Both widespread production and the use of these manufactured nanoparticles have already led to human and environmental exposure. While such arguments may offer partial relief from fears assigned to presumed toxic nanomaterials, the sheer volume of novel nanomaterials becoming presently accessible makes the evaluation of nanoparticle-related risks a very relevant and current topic. In the first part of this chapter, we will describe the commercial production and specific applications of nanoparticles with a focus on the dominant class of inorganic materials. The second part of this chapter introduces three important concepts for an accurate physical description and treatment of nanoparticles as a basis for a scientific risk evaluation. Starting from purely physical basics, we will work our way to the elaboration of exposure scenarios and a preliminary model for the evaluation of nanoparticle-related risks to humans and the environment. To keep this chapter within a reasonable length, we will focus on concepts that are common to most manufactured nanoparticles.
II.
Advantages and Applications of Nanoparticles
Following the specification of the British Standards Institution (1), a nanoparticle is defined as a particle with one or more dimensions at the nanoscale (<100 nm). This therefore includes spherical particles of diameters below 100 nm (Fig. 1A), nonspherical particles with at least one dimension less than 100 nm (Fig. 1B) as well as aggregates of such particles (Fig. 1C). Common to all of these structures are material properties, which differ from the properties of the same material at the bulk state (i.e., >1 mm). These properties include, among others, optical, surface, and electromagnetic effects. The source of these effects has been explained mainly by the confinement of energetic states within the nanoparticle or the very large surface-to-volume ratio of sufficiently small
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Figure 1 Schemes (top) and TEM (bottom) of characteristic nanoparticles. Materials on the TEM
images are flame-made titania (A) (82), barium fluoride (B) (64), and bismuth (C) (6). Abbreviation: TEM, transmission electron microscope.
structures. Or, in other words, since a nanoparticle is small, a significant part of its constituents are exposed on the particle surface. Applications of manufactured nanoparticles today try to take advantage of these effects and largely lie within the areas of polymer reinforcement (e.g. carbon black in tires), heterogeneous catalysis, chemical mechanical polishing (CMP) for computer chip manufacturing, magnetic recording media, cosmetics, and sensors (2). The research field of nanotechnology, however, is rapidly advancing and novel applications are being proposed in academic literature on a daily basis and now include optoelectronics (e.g., quantum dots) (3–5), thermoelectrics (6), battery materials (7,8), biomaterials (9–11) (Fig. 2), high wear alloys (12–15), and biotechnology (16,17). To give a better idea of industrial nanotechnology and the applied materials, some applications are briefly described below. A. Heterogeneous Catalysis
In heterogeneous catalysis, the extremely large surface area (often 50–200 m2/g) of nanoparticles is utilized to take advantage of surface-molecule interactions at specifically designed surface sites [e.g., vanadia/titania (18), silica/titania (19)]. Structurally, homogeneous nanoparticles are preferably applied for the dispersion of catalytically active noble metals (20–25) and include base materials consisting of carbon black (21,26) or thermally and chemically stable oxides (27) (e.g., Al2O3, CeZrO4). For industrial application, the particles are usually packed to catalyst beds, through which
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Figure 2 The use of nanocomposites in biomedical applications opens new possibilities:
poly(lactic-co-glycolic acid), PLGA, fibers (A) were loaded with 40 wt% of tricalciumphosphate nanoparticles resulting in a biodegradable cotton wool like–implant material (B). The structure of the material allows an easy application during operation (C) and a high bioactivity, which is evident from the deposition of hydroxyapatite on the surface of the fibers (D) (10).
the chemicals pass during reaction. Since 90% of all chemical transformations involve catalytic processes, nanoparticle applications are significantly affecting global energy needs, green processing, and sustainable manufacturing. B. Chemical Mechanical Polishing
The fabrication of computer chips, waveguides, and hard drives involves planarization of surfaces as a major processing step (28). This can be achieved by CMP, which flattens the corresponding chip or hard-drive surfaces and integrated circuit structures through the combined action of chemical and mechanical forces. More specifically, a rotating patterned wafer is pressed against a rotating polishing pad while feeding an abrasive polishing slurry containing nanoparticles and chemical agents. Currently used ultrapure nanoparticles are mostly selected from silica, alumina, and ceria. The total market
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size for nanoparticle-based CMP slurries has been estimated to US$440 million in 2003 (29), utilizing about 25,000 tons of nanoparticles (assuming a price of $20/gallon and a 30 wt% particle loading). C. Nanobiomaterials
Hard tissue repair requires formation of tight bonds between existing and new bone or dental materials. Calcium phosphates and certain bioglasses (SiO2-CaO-P2O5-Na2O) have therefore found widespread application in a variety of bone grafts for repair and regeneration of bone defects arising from trauma, tumor, and osteoporosis (30–32). Most recent investigations have shown that the use of nanomaterials instead of standard, granular materials is highly beneficial, resulting in a higher reactivity (11), faster dissolution rate (9), and easier processability with correspondingly shorter operation time (33). More advanced properties targeting specific tissues and surgery can be obtained from incorporation of bioactive ceramic nanomaterials into degradable polymers (Fig. 2) within so-called nanocomposites (34). These new hybrid materials can now be formed to blocks, layers, or fibers and combine the high bioactivity of specific nanoparticulate compositions with the mechanical properties of polymers (10,35). D. Magnetic Nanoparticles
Magnetic nanoparticles find a range of applications in electronics [e.g., information storage (36)] and medicine. Such particles have been used as contrast agents for magnetic resonance imaging (MRI) (37,38) and have been suggested for hyperthermia treatment of tumors and magnetically directed drug delivery (39–42). Other applications requiring large amounts of magnetic nanoparticles are magnetically aided procedures used for the separation and sorting of cells (43,44), proteins (45,46), or even organic chemicals (47). Also, the application of classical iron oxide particles, carbon-coated metals (iron and cobalt) with optional surface functionalization has most recently become available commercially (48). The use of surface functionalized magnetic nanoparticles is significantly increasing the speed and specificity of medical diagnostics (49,50). E.
Commodity Applications
The market for nanotechnology in food has been estimated to reach US$7 billion in 2007 (51), but the full potential of this market has certainly not been realized yet. Most large food companies are running specific research programs to take advantage of the nano food market. Possible food applications start with the use of nanoparticles in functional packaging. Here, nanoparticles can be used to modify the permeation behavior of foils, to design barrier properties (thermal, mechanical), or to develop active antimicrobial and antifungal surfaces (52). Ideas currently pursued further include nanotechnology-based sensors for the surveillance of food quality inside packaging (53,54). The addition of manufactured nanoparticles to foodstuff may be used for flavor encapsulation (55), flavor enhancement, or as pigments for coloration. A further application and demand for nanoparticles emerges from the cosmetic industry. The small particles are used as pigments or to create specific coloring effects. Undoubtedly, the best-known application in cosmetics is the use of nanoparticulate titania and zinc oxide in sunscreens (56), an
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over $400 million business. In a study of 1200 different sunscreens authorized for supply in Australia (December 2005), about a third contained either titania or zinc oxide nanoparticles. The current rapid growth for nanoparticles requires large-scale, industrial manufacturing plants. On the basis of the sheer number of new applications within the nanotechnology market sector [estimated to grow to US$1 trillion annually by 2015 (57)], there will be a vigorous growth in this industry, not only in production volume but also in diversity.
III.
Industrial Nanoparticle Manufacture
Estimates on the total amounts of nanoparticles produced worldwide often show great divergence, ranging up to 10 megatons (58) a year. The main materials produced are carbon black as well as single oxide materials such as silica, titania, alumina, iron oxide, zinc oxide, ceria, and zirconia. The production scale of newer, more complex, oxidic materials such as indium tin oxide (ITO) (59,60), barium titanate (61,62), and battery materials (7,8) are strongly increasing. Manufacturing strategies for the large-scale production of nanoparticulate salts including phosphates (63), halides (64), carbonates (65), and sulfates (66) have recently been presented in literature, but the relevant industry is still in its infancy. The same can be said for metallic nanoparticles, where low cost synthesis methods for nickel (67–69), cobalt (70,71), iron (72,73), bismuth (13), copper (74), and many other metal nanoparticles are available, but the high reactivity and flammability of transition metal nanoparticles is hindering a faster commercial growth of this market. The synthetic methods leading to the corresponding nanoparticles are highly diverse, depending strongly on the production scale, material properties, and the final application. Generally, the processes can be divided into bottom-up and top-down processes. While top-down approaches for the production of particles are generally straightforward and simple, they are of very low energy efficiency (75) and mostly result in products with inhomogeneous particle sizes. Bottom-up or “self-assembly” approaches result in well-controlled nanoparticles, which have been built from smaller molecular building blocks. Beside “top-down” methods such as ball milling, processes for the large-scale manufacture of nanoparticles can generally be divided into gas-phase and liquid-phase methods. A. Liquid-Phase Methods
Here, an inorganic or metal-organic precursor is converted to nanoparticles in water or an organic solvent by the action of temperature, pressure, or a chemical reagent (76,77). The often complex and expensive postprocessing of the reaction mixtures resulting in large amounts of liquid waste limits the profitability of these processes (75). B. Gas-Phase Methods
Nanoparticles required at very large scale such as titania, carbon black, and silica are therefore preferably synthesized by gas-phase methods. Very similar to the liquid-phase methods, gas-phase synthesis employs an inorganic or metal-organic precursor compound.
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This precursor is then converted to the final nanoparticles at very high temperatures (>10008C) in flame, plasma, or laser reactors. The advantages of this process are short reaction times (often <100 ms), large production rates [>10 tons per hour for flames producing titania (78)], and a continuous operation. Since the processes operate in the gas-phase without particle-wall contact and no solvents or surfactants are employed, the purity of the final product is generally much higher than in liquid-phase processes (79). This helps to avoid costly postprocess operations. Whereas liquid-phase synthesis methods can be used to produce nanoparticles of nearly arbitrary shapes (e.g. cubes, spheres, etc.) and with extremely narrow size distributions (80), high-temperature gasphase processes are limited to self-similar, log-normal distributions (81). Furthermore, the control of particle morphology is very limited, often resulting in highly aggregated nanoparticles (Fig. 1) (82). On the basis of these limitations and the high energy demand of nanoparticle formation, a very large research community is working on improving existing synthesis procedures and developing new “green” nanoparticle production schemes (83,84).
IV.
Physical Behavior of Nanoparticles
To evaluate the risks emerging from nanoparticles, their properties and behavior have to be well understood. In this chapter, we want to focus mainly on the description of the physical behavior of nanoparticles in fluids. Because of the extremely small size of nanoparticles and the impossibility of seeing these structures with the naked eye or even conventional light microscopy, the behavior cannot be simply understood by intuition. The general idea that such particles behave just as their larger and visible counterparts is wrong and often leads to significant errors in scientific investigations. The following introduction of some straightforward physical concepts offers a basis for the rational and correct design of any experiment in nanotoxicology and helps to understand many, often classical, problems that have been encountered when handling nanoparticles in toxicological investigations. The concepts introduced here are unique to all materials sufficiently small and are mostly based on the fact that nanoparticles are very strongly affected by Brownian motion. This means that processes such as diffusion and aggregation always have to be accounted for when experimenting with nanoparticles. For example, if an air stream loaded with nanoparticles is fed through a tube, most particles will never leave the tube but will diffuse toward the tube wall and will collect there, or they make small groups of particles, so-called aggregates (Fig. 1). Very similarly, it has been shown that particle transport to cells may be limited by diffusion processes (85). The following section may not appear inviting to readers of medical and biological fields at first, but on a closer look, the required mathematics are very elemental and fortunately accessible to all researchers. A. Diffusion
Diffusion randomly moves small particles, atoms, or molecules of any material and is responsible for the propagation of odors in air and color dyes in water. The diffusion coefficient (the mobility) of nanoparticles (D) can be determined from the Einstein– Stokes equation with Cunningham correction (86). Simplified for air (at ambient
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conditions, Dair) and water (Dwater), this gives the diffusion coefficient as a function of the nanoparticle radius (R, in meters). 1:2 1017 1:1 107 2 1þ Dair ðm = secÞ ¼ RðmÞ RðmÞ Dwater ðm2 =secÞ ¼
2:2 1019 RðmÞ
To decide if diffusion will play a role in the processes or experimental system investigated, a characteristic length (Lc) can be calculated as a function (87) of the experiment time (t): pffiffiffiffiffiffiffiffi Lc ¼ 4Dt Figure 3 shows the length scale for diffusion processes (Lc) for standard conditions in air and water and shall serve as an aid to decide if diffusion processes have to be accounted for within an experiment. It must be added that diffusion is a statistical process and Lc is the distance an average particle travels along a concentration gradient. Some particles will have diffused further, some not at all. Furthermore, it may not be forgotten that nanoparticles are also transported with the fluid stream, by convection, turbulence, and mixing. For a hands-on example, if a nanoparticle stream is fed through a tube during intratracheal instillation, particle aggregation will take place in the air stream and the critical length is the length of the tube (for aggregation) or the diameter (for particle loss to the wall of the tube). Or, when exposing cell cultures to nanoparticle-containing liquids, the characteristic length is the mean path that the particles will travel in the cell culture wells before encountering a cell surface (typically a few millimeters). The effects of particle shape (e.g., nanotubes) are outside the scope of this work, but can partially be physically addressed by using sphere-equivalent (hydrodynamic) diameters. B. Check for Sedimentation and Diffusion Effects
A problem, often encountered when handling nanoparticles, is to evaluate whether their transport is governed by diffusion or sedimentation processes. This, of course, depends strongly on the size and density of the particles as well as the medium’s viscosity. The sedimentation velocity of nanoparticles is shown in Figure 3 for spheres in water and air for various particle densities. Comparing this data with the length scale of diffusion allows a discrimination of the major transport process. This approach has been experimentally validated by Limbach et al. (85), where human lung fibroblast cells were incubated in vitro with medium containing ceria (cerium oxide) nanoparticles (Fig. 4) and the transport of ceria into the cells was quantitatively measured over time. The presented data show that the cellular uptake of particles follows the diffusion behavior for small particles (*40 nm) and switches to the sedimentation behavior for larger particles (*300 nm). This work is an example of a cell-particle interaction scenario, where the particle transport and resulting particle contamination of the cells is limited by purely physical interactions.
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Figure 3 The average length a nanoparticle travels by diffusion in air (A) and water (B) can be
compared with the sedimentation velocity of the particle (C, D) and helps in identifying the most prominent transport process.
A further behavior of nanoparticles in fluids, which is often neglected and has led to considerable experimental error and misinterpretation, is particle aggregation. In nanotechnology, aggregation is the process where individual nanoparticles collide to form larger particles—aggregates. Two properties of these aggregates are important: first, they are extremely stable and, second, they form very rapidly, even in water. The reason for the high stability of nanoparticle aggregates is that the Van der Waals interactions between the individual particles are often much larger than any other force acting on the aggregate. Therefore, the aggregates cannot be disrupted or destroyed by simple shear forces, which act during mixing in biological systems or turbulence, although the individual nanoparticles are held together by solely physical forces. The stability of nanoparticle aggregates under various mixing conditions has been studied in detail for liquid- (88–90) and gas-phase (91,92) systems.
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Figure 4 The uptake of ceria nanoparticles into living human lung fibroblast cells is shown on the
transmission electron micrograph (A). After exposure of the cells with a nanoparticle-containing feed solution, the particles (black dots) can be found in vesicles (black arrows) inside the cell. The evolution of the particle mass taken up by the cells is shown on the right for 25 nm particles (B) and 320 nm particles (C). The experimental data was compared with calculated uptake rates based on particle diffusion (solid lines) and particle sedimentation (broken lines). While the uptake of 25 nm particles is limited by the diffusional transport of the particles, larger particles are transported toward the cell by sedimentation (85).
The second point is the velocity of aggregation—which, at these particle sizes, can be nearly instantaneous. The fact that nanoparticles aggregate in gaseous environments, such as the air we breathe is well established (93). That the same is true for more viscous environments such as water or blood is often overlooked. The underlying process is again Brownian motion leading to the statistical event of particle collision. Generally, the rate at which the aggregation process evolves in water depends on the particle radius [r (m)], the particle density [rp (kg/m3)], the particle concentration [C (kg/m3)], and the stability of the particle dispersion [W (-)]. To give a rough estimate of the aggregation velocity, the characteristic time of doublet formation [two particles sitting together, tagg (sec)] can be calculated (94): tagg ¼
W r3 p ; T kB C
17 twater agg ðsecÞ ¼ 7:6 10
W r3 p C
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This is the minimal mean time it takes for two individual particles to collide, resulting in a doublet. We can use this collision time to evaluate whether our experiment is influenced by aggregation or not. C. Stability of Nanoparticle Dispersions and Colloids
While the particle size and concentration are easily addressed through measurements, the dispersion stability (W) can only be described with some difficulty. A possible description of stability can be based on the zeta potential, which is a measure of the charge stabilization of the particles (95). This is, however, not a full description of the dispersion stability, since the particles can also be stabilized by other effects such as steric hindrance. Further, the stability or aggregation rate of particles in dispersion cannot be quantified from the zeta potential data. The situation in biological fluids is even more complex, where particles are usually coated with proteins (96,97), resulting in a totally changed surface chemistry and charge distribution. Furthermore, salt concentrations are high, leading to a destabilization of the dispersion through disintegration of the charge stabilization. To make a first practical approach under such biological conditions, it can be assumed that there is no dispersion stabilization (i.e., W ¼ 1), and aggregation is driven by pure Brownian motion (diffusion limited). Figure 5 shows the time of doublet formation for various particle concentrations and radii under these conditions. The figure further shows the evolution of the aggregate size under typical conditions. An important result of these theoretical considerations is that the behavior of nanoparticles strongly depends on their concentration. In biological exposure experiments, where the toxicological potential of a nanoparticle is tested at various concentrations, the effects of aggregation and sedimentation have to be accounted for.
Figure 5 The characteristic aggregation time is the average time it takes for the formation of doublet particles (A) and is a measure for the importance of aggregation-related effects. The evolution of the particle size due to diffusion, limited aggregation is shown on the right (B) for 10 and 25 nm particles at various concentrations. The data shown here was calculated for a particle density of 5000 kg/m3 and for water as dispersing medium.
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This will be illustrated by the following thought experiment: if a nanoparticle exposure experiment is run at extremely low concentrations, aggregation can be neglected and single nanoparticles may enter the exposed cells, leading to a toxic response. If the same experiment is run at higher nanoparticle concentrations, the uptake of single nanoparticles may be prevented by the formation of larger aggregates. This may lead to a counterintuitive reduction of the toxic effect. While these thoughts are speculative, they will illustrate that the small size and mobility of nanoparticles in aqueous media imply well-designed exposure systems accounting for the above described physical effects.
V. Risks of Nanoparticle Exposure The risks arising from nanomaterials and nanoparticle exposure have stimulated numerous public discussions, scientific disputes at conferences, during panel discussions with the manufacturing industry and are now even heard in bar room politics. Recently, a large array of reviews on the toxicology of nanoparticles has been presented (98–115) and ironically tends to double the number of original investigations on nanotoxicology. The general conclusion from all reviews is that the field of nanotoxicology is still young and more work has to be done to fully understand the risks of nanoparticles. From an industrial and sociological viewpoint this may be too late, as numerous products have already entered pilot stages or are under commercialization. We therefore want to show some very simple concepts to understand and proactively evaluate individual risks of specific nanoparticles. For this, we employ the concepts shown in the previous paragraph together with a simple chemical understanding of nanoparticles. A first simplification of the problem can be made by dividing risk into hazard potential and exposure. In mathematic terms, this accounts to: risk ¼ hazard potential exposure. To evaluate the exposure of a nanoparticulate material or product, the full life cycle of the nanoparticle in the product has to be investigated. This means that all processes and scenarios where human beings or the environment could come into contact with nanoparticluates have to be accounted for—from the production of the particles to their elimination. This leads to very difficult questions: How do we address exposure scenarios? How are manufactured nanoparticles eliminated from their life cycle? When and how do the particles stop being nano? When looking at exposure scenarios, the physical description and understanding of nanoparticles gain great importance. This can be better understood by looking at some illustrative examples [further examples can be found in Franco et al. (116)]. A. Gas-Phase Processing
If the nanoparticle production plant is not fully sealed from the worker’s activity area or the surrounding environment, particles will escape from the production plant and travel by convection and diffusion until aggregates large enough have formed and sedimentated (“fall to the floor”). Just as with gaseous chemicals, the distribution of the particles in the environment can be estimated with multimedia box models (117) accounting for diffusion, sedimentation, and aggregation.
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B. Commodity Products Containing Free Nanoparticles
By the use of such products, the consumer gets in direct contact with nanomaterials. Such products may be cosmetics; sunscreens, food additives, or cleaning products. The form of use or application of these products (e.g., spray vs. cream) will strongly affect the entry point into the human body (e.g., lung vs. skin) and, therefore, directly affect the risk potential of the product. Here, the direct exposure of the applicant and his environment can be evaluated by investigating the principles of use and deduct the emission rate. This process can be compared with the exposure assessment of cigarette smoking (118). C. Products with Bound Nanoparticles
Products containing nanoparticles fixed in a matrix, such as nanoparticles dispersed in a polymer as found in, for example, baseball bats, bicycle frames, or batteries. Although the exposure risk to such nanoparticles is clearly smaller, the exposure analysis is far more difficult, since in normal use the applicant does not come in contact with the particles. Here, emission could come from abrasion, wear, and inadequate use of the product. D. End of Life
To be able to solve the problem of nanoparticle exposure end-of-life scenarios of nanoparticles have to be addressed with great care. Processes resulting in an elimination of nanoparticles are chemical dissolution, sintering at elevated temperatures, and irreversible binding to or into solid media. Currently operated waste-processing measures such as incineration, land filling, and waste-water treatment have to be investigated as to their ability to eliminate nanomaterials. What happens with a carbon nanotube reinforced bicycle frame after use? It may be remodeled, polished, changed—a mechanic working on the frame will breathe in dust, creating scenarios we are too familiar with from earlier asbestos cases. There again, arguments come up calming critical voices that the dangerous fibers were inaccessible when used in paints or cements. Today, building renovation and existing asbestos cases exceed costs of over US$145 billion (119). All these examples should serve as thought-provoking impulses, as it is highly important that industry, regulatory bodies, and scientists are aware of the complexity of nanoparticle exposure, which cannot be pigeonholed to “nano is dangerous” or “nano is safe.” E.
Hazard Potential
The hazard potential, on the other hand, involves particle uptake into the human body as well as other living systems and the resulting toxicological potential. Little is known of how nanoparticles and living systems interact—even if first in vitro cell cultures and first in vivo studies are investigated (120–130). However, if the present data is carefully analyzed and combined with a chemical understanding of nanoparticles, a preliminary classification of the hazard potential can be drawn. The presented approach was included into the governmental report “Synthetische Nanomaterialien. Risikobeurteilung und Risikomanagement. Grundlagenbericht zum Aktionsplan” by the Swiss Federal Office for the Environment and the Federal Office of Public Health (131). Similar considerations are also the basis for nanomaterial legislation, which is under consideration in
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many countries [e.g., see the National Nanotechnology Initiative (132) for the United States and The Royal Society report (133) for the United Kingdom].
VI.
Preliminary Nanoparticle Hazard Potential Classification
The present chapter intends to combine the above physical understanding with a chemical understanding of nanoparticles and to use this knowledge to classify nanoparticles. The goal of this classification is to shed light on the sheer endless list of potentially hazardous particles. Nanoparticles made of materials that are intrinsically toxic or hazardous, such as lead or cadmium, are not discussed here as such materials are already under heavy reglementation. We want to understand the hazardous effects of nanoparticles, which would be regarded as “safe” in their bulk phase (e.g., ceria, titania, iron oxide). Specific hazard classes offer a tool to design the necessary toxicological and environmental studies that can assess the unique hazards associated with the use of particular nanoparticles. On the basis of our present understanding, two fundamental properties of nanoparticles can be used as guiding principles: degradation or persistence (134–136) and the chemical and catalytic activity of a material (100,137–139). A. Degradation
The persistence of a material determines its mean dwell time in an organism or ecosystem. This inherently defines whether a material interacts shortly (acute) or for a longer time with its environment or matrix. The uptake of nanoparticles in humans can in principle be considered as an (deliberate or accidental) introduction of the material into the organism. Existing guidelines for medical implants make clear material distinctions based on the dwell time of an alien substance (e.g., an implant). This is directly related to the definitions of pharmaceuticals versus medical implants in regulatory issues (e.g., ISO10993). In many aspects, rapidly degrading nanoparticles can, therefore, be regarded as similar to chemicals, while persistent materials become (nondeliberately) similar to medical implants. A first distinction is made, on the basis of a material’s degradation properties (similar to presently used ISO 10993), resulting in a classification as metabolizable/ degradable, semi-persistent, and persistent materials, and offers an estimate on the dwell time of the material. This may be illustrated by considering a specific material as degradable (first category) if it fully dissolves in its ionic or molecular constituents within six hours when immersed in a simulated body fluid. The second category of semi-persistent materials comprises nanoparticles that remain after six hours in simulated body fluid but degrade within, for example, two weeks. Materials that do not dissolve or degrade within this time frame are put into the third category of inert or persistent materials. B. Activity
The second criterion for classification concerns catalytic and chemical activity. The chemical activity of specific nanoparticles can have a mass-proportional effect (e.g., the effects of arsenic uptake are mass related) or a nanoparticle can have catalytic effects [interactions with the energy metabolism; oxidizing species (100,137–139); signaling
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Figure 6 To describe their hazard potential, nanoparticles are classified along their degradation
rate (A, B, C) and chemical activity resulting in nine risk classes (A1–C3). Persistent particles as well as highly active semi-persistent particles have been identified as high-risk materials (140).
pathways; etc.]. These catalytic effects can strongly increase the damaging action of a material, since the effects remain over a longer time, and the nanoparticles are not consumed during their interaction with the organism or the environment. This type of interaction is not only mass depending but may also significantly deviate from a composition-based risk assessment. It has to be remembered that, based on the very large surface areas of nanoparticles, these materials are often used intentionally for catalytical applications. The classification scheme considers catalytically nonactive, moderately active, and active nanoparticles (Fig. 6). C. Hazard Grid
On the basis of this concept and the descriptions above, three major groups of materials may be distinguished for toxicological assessment for humans and the environment (140): High risk: combinations of persistent and active materials Intermediate risk: all catalytically active nanoparticles Lowest risk: combinations of degradable and nonactive material properties This very simple classification allows the adoption of the toxicological screening program for the individual groups (Table 1). The screening approach for every group must be adapted to the groups’ specifics, which are described below. D. Persistent Nanoparticles
Persistent nanoparticles remain within an ecosystem or an organism for a prolonged time. Most of the fundamental properties and interactions are still poorly known. This
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Table 1 Prioritization of Nanoparticles for Toxicological Investigations
1—Not active 2—Low activity 3—Catalytically active
A—degradable
B—semi-persistent
C—persistent
Class A1 Class A2 Class A3
Class B1 Class B2 Class B3
Class C1 Class C2 Class C3
High-risk classes are highlighted in bold.
group therefore requires the highest degree of attention, security measures, and additional investigations. Particles of group C3 must be considered as hazardous and should be handled with special care. The more degradable groups A and B should be investigated for their metabolic pathways including potential excretion pathways (110,141,142). It must be demonstrated whether the materials leave the organism or the corresponding ecosystem (103,143– 145). If this is not the case, a long-term investigation must demonstrate full bio- and ecocompatibility before a potential use in consumer goods can be considered. On the basis of the current state of research, such materials should still be handled with great care. It must be noted that numerous persistent nanoparticles have been used in current product development and are handled without specific safety measures in many research laboratories. With the absence of acute toxic effects, few actions have been taken to shield such persons from particle uptake, while it must be expected that they carry these materials within their body for decades. In addition to all toxicology tests (established procedures from pharmaceutical industries), accumulation of particles in specific compartments, the generation of noxious degradation products, particle transformations, or masking (e.g., by protein or lipid adsorption) must be investigated (100,102,146). For all tests, application of the material must take into account a relevant procedure to efficiently simulate an incident. E.
Semi-persistent Nanoparticles
Toxicological tests during the degradation time and the clearance of the degradation products are required. Potential side reactions must be taken into account as locally, particles may accumulate for short times. However, accumulation of particles in the food chain is not expected. Group B3 should still be considered dangerous—here, a massive toxicological effect may arise on the time scale of weeks. It must be emphasized that a mere classification based on composition is not sensible as catalytic effects may be in orders of magnitude larger than any direct mass-related stoichiometric effects (137). An example based on iron/silica shall illustrate this consideration. On the basis of a mere compositional classification, a specific iron/silica nanoparticle may be considered rather unproblematic as silica is already in use for half a century and iron is a biogenic element. However, certain iron/silica samples have been designed as excellent catalysts for the oxidation of organic compounds on an industrial level. If such catalytically active particles are exposed to an organism, the oxidative damage is much larger than expected from the mere presence of minute amounts of iron and some silica (137).
Manufactured Nanoparticles F.
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Degradable Nanoparticles
This class of materials only briefly interacts with an organism or the environment. The materials are typically dissolved after uptake and can be evaluated with presently used methods. Specific tests for degradation could be adapted from existing protocols (ISO10993) The degradation products must be followed and investigated carefully and local accumulation of specific substances must be considered. The mobility of nanoparticles may help otherwise nontoxic materials to enter normally isolated areas. For degradable materials, the risk evaluation can be oriented largely along the presently used materials-based methods, as long as pharmacokinetics are taken into account (147).
VII.
Future Industrial Product Development
To enable a sustainable industrial development of nanotechnology, the advantages arising from a nanoproduct have to be weighed against its potential risks. These risks are not only limited to the direct use of the product but should also be considered during production, disposure, and presence of the material in the environment. It may therefore be insufficient to rely on the sense of responsibility of entrepreneurs. This makes governmental and regulatory actions necessary, which have to closely accompany the scientific and business developments of nanotechnology. This of course is hindered by the fact that the detailed mechanism of nanoparticle toxicity is far from being fully understood. However, simple concepts as described in this chapter can be utilized to classify the hazard potential and exposure risk of well-defined nanoparticle-based consumer products. The question which has to be answered is not whether nano is “good” or “bad,” but how can the rapidly evolving nanodevelopment be integrated into our society. The tools used for this integration have to be adapted for the physics and chemistry of nanoparticles and have to also account for diffusion, aggregation, and catalytic activity.
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4 Particulate Carriers for Pulmonary Drug Delivery STEPHANIE HEIN, ANDREAS HENNING, MICHAEL BUR, MARC SCHNEIDER, and CLAUS-MICHAEL LEHR Saarland University, Saarbru¨cken, Germany
I.
Introduction
Drug administration by inhalation is well established, but it has mainly been used for locally treating diseases in the lungs, such as asthma or chronic obstructive pulmonary disease (COPD). During the past two decades, though, increasing attention has been paid to using the healthy lung as a convenient route to treat diseases such as diabetes by aerosol delivery of insulin. Pulmonary delivery offers many advantages as a noninvasive method for both local and systemic drug delivery due to the characteristics of the lung. The lung has a large surface area, offers rapid absorption through the thin alveolar epithelium, there is low enzymatic activity, and it affords direct access to the circulation. To be effectively deposited in the lung, it is well-known that an aerosol must fulfill some requirements. The particle size of the administered drugs needs to have a median mass aerodynamic diameter between 1 and 5 mm, because smaller particles are exhaled while bigger particles will not reach the alveoli and are confined to the upper airways instead. Ultrafine particles of 5 to 10 nm are efficiently deposited and may remain in the deep lungs as well, but this size range is not used for aerosol medicines at the moment. This is probably due to a lack of appropriate formulation technologies that can generate ultrafine drug particles, along with intrinsic limitations on the dose of an active pharmaceutical ingredient that ultrafine particles can deliver within a reasonable aerosol volume or time of inhalation. Regardless of their size, particulate drug carriers for inhalation aerosol must be nontoxic and well tolerated. However, only few materials are approved by the United States Food and Drug Administration (FDA) for inhalation. Current aerosol drug products essentially nebulize the “naked” drug via different systems such as nebulizers, pressurized metered dose inhalers, or dry powder inhalers (1,2). Apart from a few propellants and solvents, excipients other than lactose or NaCl are virtually uncommon in pharmaceutical aerosols due to their unproven safety and a lack of regulatory approval for use in inhalation aerosols. Biodegradable pharmaceutical polymers such as poly(lactic-co-glycolic-acid) (PLGA) and chitosan are common in controlled release formulations for oral or even parenteral administration, but they have yet to be approved for use in humans by inhalation.
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Limited by a very short list of approved excipients, the development of inhalation medicines has so far focused on aerosol production to optimize deposition in preferred lung areas. Controlled release of the active ingredient from the carrier, which is a common strategy for many oral or parenteral drug formulations, has yet to be achieved for inhaled drugs. A controlled-release system for an inhaled drug is rather challenging because once it is deposited in the lungs, it will be prone to a variety of very efficient clearance mechanisms, such as mucociliary clearance in the upper airways (3) and macrophage clearance in the lower respiratory tract (4,5). These mechanisms are physiologically important to protect the body from inhaled particles a priori, regardless of whether they are toxic or not. Therefore, advanced particulate drug carriers must overcome these clearance mechanisms to achieve long-term sustained release or enhanced absorption into the bloodstream for systemically acting drugs. At the same time, however, such carriers must be absolutely biocompatible and well tolerated by the patient. To avoid any long-term accumulation in the lung or other compartments in the body, they must be biodegradable within an appropriate timeframe. Advanced carriers also offer an opportunity to target drugs to a specific site of action, for example, by triggering cell-specific uptake mechanisms, reducing side effects while decreasing the dose that is required for treatment. While there are already several excellent reviews and textbooks on pulmonary drug delivery in general (6,7), this chapter will focus on emerging technologies that are beyond the well-known devices and formulations for metered dose inhalers, dry powder inhalers, or nebulizers.
II.
Systemic Drug Delivery via the Lungs
The aim of aerosol delivery systems employed in systemic formulations is to provide good systemic bioavailability by allowing convenient, pain-free inhalation and rapid uptake into the bloodstream. This approach may be of interest whenever oral delivery of an active pharmaceutical ingredient is not feasible and intravenous injection is not desired. These formulations need to cross the diffusional barrier at the alveolar epithelial barrier and avoid the clearance mechanisms of the lung that will act to diminish or inhibit uptake. Another consideration for the inhaled formulation is whether the drug needs to act with a rapid onset of action or over prolonged period of release. A. Large Molecules
It is difficult to deliver large molecules like insulin or heparin to the systemic circulation with acceptable bioavailability by any route other than parenteral administration. Nebulizing and administering them via the lungs has been shown to be feasible, but the bioavailability is usually rather low. This may, however, be further improved if advanced particulate drug carriers and novel excipients are employed in conjunction with improved aerosol technology. There has been strong interest in developing an inhalable insulin formulation for many years due to an increase in diabetes mellitus patients and because many patients suffer from their daily regime of injections. The first FDA-approved product was Exubera1, which came on market in 2006. It was a microparticle powder produced by a
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spray-drying technique containing recombinant human insulin, mannitol, glycine, and sodium citrate. Only 10% of rapid acting insulin was absorbed into the systemic circulation compared to subcutaneous administered insulin (8). Although certainly an important pioneer, one must acknowledge that Exubera was in principle still a conventional formulation where no attempt was made to enhance absorption or modify release from the carrier. Exubera failed to gain wide acceptance, and in October 2007, Pfizer decided to phase out Exubera for mainly economical reasons. Other firms were also working on inhalable insulin formulations, but after the withdrawal of Exubera, Novo Nordisk (AERx1 insulin) and Eli Lilly (AIR1 insulin) decided to stop their clinical phase III trials because of economical reasons, too. One example of an enhanced insulin formulation is Technosphere1 insulin from MannKind Corporation (California, U.S.). It consists of pH-sensitive carrier particles and monomeric insulin, which is the bioactive form of insulin. The insulin is loaded onto pH-sensitive organic molecules that self-assemble into small particles under the neutral pH conditions in the lung (9). The monomers diffuse into the bloodstream and show rapid uptake with 30% to 50% bioavailability compared to subcutaneous administration (10,11). The FDA accepted submission for Technosphere1 insulin which is now called AFRESA1 in May 2009. Other formulations have been tested in vitro and in animals. Grenha et al. (12) developed insulin-loaded lipid/chitosan nanoparticle complexes that were spray-dried with mannitol into microspheres. Testing in vitro showed that the lipids provide controlled release of the insulin. Other groups have used large porous particles for delivering insulin into the systemic circulation (13,14). These particles were first introduced by Edwards et al. (14) and are characterized by large sizes (>5 mm) but small mass densities. These particles can be deposited into the deep lung, and since they are too big to be phagocytosed, they are an attractive delivery system for systemic drug application. To avoid accumulation in the lungs caused by the reduced phagocytosis, these particles need to be biodegradable. Another advantage is that the large porous particles aggregate less than other nonporous particles because smaller particles have stronger cohesive forces (15). Huang and Wang (16) encapsulated insulin into liposomes and administered them to mice and produced a decrease in plasma glucose levels compared to mice administered empty liposomes. Cagnani et al. (17) produced an inhalable insulin powder with spray-drying technique using clear mild acidic solutions of insulin. In vitro studies showed that these particles had respirable aerodynamic diameters and a “raisin-like” morphology that showed no agglomeration tendency. While these studies have all paid attention to insulin delivery, others have focused on improving the stability of the formulations. Amidi et al. (18) produced insulin-loaded microparticles with N-trimethyl chitosan and dextran as carriers using a supercritical fluid-drying technique and showed that the particle characteristics and the insulin structure were maintained for one year. All these carriers mentioned here could be employed in the lungs offering enhanced systemic delivery, perhaps even sustained release properties, and therefore allow diabetes patients to avoid daily injections. Another important macromolecule for systemic delivery via the lungs is heparin. To prevent deep vein thrombosis, a low molecular weight heparin (LMWH) was connected to a positively charged dendrimer to enhance absorption by reducing the negative surface charge density of the LMWH. The drug-dendrimer complex was administered to rats, and it was as efficacious as subcutaneously administered LMWH and had no toxic effects on the lungs (19). Yang et al. (20) tested LMWH formulations with
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tetradecyl-b-maltoside or dimethyl-b-cyclodextrin in vitro and in vivo and showed that both formulations enhance the pulmonary absorption of LMWH. They also showed that tetradecyl-b-maltoside formulations were more potent than dimethyl-b-cyclodextrin formulations. B. Small Molecules
Small molecules can also be systemically delivered via the lungs by inhalation. This is an attractive option when the drug molecules are not stable or water soluble enough to be delivered via the gastrointestinal tract, or when an extremely rapid onset of action is desired, such as analgesia. Some nebulized drugs are in clinical trials that are administered to the alveoli to achieve a rapid onset of action. MAP0004 is a dihydroergotamine mesylate (21) used to treat migraine; however, intravenous administration of this drug causes some serious side effects. When MAP0004 was applied to the lungs by a pressurized metered dose inhaler, there was a decreased rate of side effects in healthy volunteers. Another strategy for inhaled migraine therapy is described by Rabinowitz et al. (22,23). It is a single dose thermal aerosol device with a thin (about 5 mm) film of pure drug (rizatriptan). Breath activation of the device by patients causes rapid heating of this film and a vapor is formed in less than one second, followed by condensation of the vapor phase drug into aerosol particles during inhalation. The emerging solid particles are spherical with an amorphous form and a mass median aerodynamic diameter of 1 to 3 mm, which is optimal for alveolar deposition. Drugs delivered in this way will act rapidly, because the particles dissolve in the alveolar liquid lining upon deposition and rise immediately into the systemic circulation. Unfortunately, this delivery method is only suitable for drugs with specific properties: they have to be able to sublimate and need to be thermostabile because the drug is heated to 4008C in the device. About 175 different drugs, such as rizatriptan, fentanyl and zolpidem have been used with this technology without thermal decomposition. In pain control, there is not only a need for a rapid onset of action but drug plasma levels also need to be maintained above a minimum level for a prolonged period of time while avoiding an initial peak that exceeds the maximum tolerance level and undesired drug effects. From a pharmacokinetic point of view, a controlled-release delivery system addresses all these issues. There is a strong incentive to develop inhalable controlled-release formulations to treat pain in cancer patients. There have been some attempts to use nebulized fentanyl for pain relief (24) and the early stages of development are summarized by Farr and Otulana (25). One product is a composition of free and liposome-encapsulated fentanyl (AeroLEFTM), and it passed the phase II trials in 2007. The free fentanyl provides a rapid onset of analgesia while an extended period of analgesia is achieved with the liposomeentrapped fentanyl (26). Liposomes show many advantages for pulmonary delivery because they are made of different phospholipids, such as dipalmitoylphosphatidylcholine (DPPC) and dilauroylphosphatidylcholine (DLPC), which naturally occur in the lung and therefore are compatible with lung surfactant. Hydrophilic as well as lipophilic substances can be incorporated into liposomes, and they enable sustained drug release. Liposomes are not as stable as microparticles, though some attempts have been made to prolong their stability by producing a liposomal dry powder by lyophilization (27,28) or spray drying (29).
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III.
Controlled Loco-Regional Delivery to the Lungs
The field of inhalation therapy was established many years ago to develop drug delivery systems to treat loco-regional diseases like asthma and COPD. These early drug formulations often showed no controlled or sustained release because the particles weren’t modified to circumvent the clearance mechanisms of the lungs. Therefore, new particulate systems were developed to prolong residence times of the drug particles that are used to treat several lung diseases. A. Asthma/COPD
There is a wide range of aerosol drug products on the market for the treatment of asthma and COPD. Most of these formulations are made of drug particles mixed with lactose as a carrier material and are administered by a dry powder inhaler or by nebulizing the drug dissolved/dispersed in a propellant with a pressurized metered dose inhaler. Although these formulations achieve efficient pulmonary deposition, they are not designed to provide sustained or controlled release. If they are long acting (e.g., b-agonists), it is due to the pharmacological half-life of the drug and not the delivery system. Indeed, the particle technology of these conventional formulations aims to improve the aerodynamic properties, and thus the deposition rate of the aerosol particles. Some of the new approaches in this area try to find formulations that show sustained release properties so as to reduce the dose frequency for patients and improve bioavailability in the lung. Arya et al. (30) coated budesonide particles with a very thin film of polylactic acid using the pulse laser ablation technique. They administered coated and uncoated budesonide intratracheally to neonatal rats and observed higher AUC levels in the lung with coated budesonide, while the systemic exposure of budesonide was reduced compared to uncoated budesonide. In another study, the poly-(ethylene oxide)-block-distearoyl phosphatidyl-ethanolamine polymer (mPEG-DSPE) was used to prepare beclomethasone loaded micelles (31). The lyophilized beclomethasone loaded polymeric micelles showed high entrapment efficiency, and in vitro drug release studies showed a sustained release over six days. In another study, salbutamol acetonide was incorporated into solid lipid microparticles (SLMs) after increasing its lipophilicity (32). SLMs show physicochemical stability and compatibility and no acute toxicity in vivo in rats (33), and in vitro release studies demonstrated that salbutamol acetonide SLMs had a slower release of the drug than pure salbutamol acetonide. Thus, SLMs promise to provide sustained pulmonary drug delivery, which in turn will reduce the number of doses required by patients. Liposomes have also been considered as another carrier for lung delivery of antiinflammatory drugs. Saari et al. (34) investigated the distribution of 99mTc-labeled beclomethasone dipropionate DLPC and DPPC liposomes in healthy volunteers. They found that the clearance of DPPC liposomes was slower than DLPC liposomes, perhaps because of the different phase transition temperatures, but in both formulations, about 80% of the deposited radioactivity remained in the lungs 24 hours after inhalation. Learoyd et al. (35) produced chitosan-based terbutaline sulfate particles where chitosan acted to modify drug release. Different molecular weights of chitosan were used and high molecular weights of chitosan increased the duration of terbutaline release.
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B. Pulmonary Arterial Hypertension
Pulmonary arterial hypertension is a severe lung disease with increased pulmonary arterial pressure resulting in right ventricular failure (36). The vasodilator iloprost is FDA-approved for pulmonary administration, but because of its short-acting properties, multiple inhaled doses (6–9/day) are necessary (37). Therefore, Kleemann et al. (38) developed iloprost-containing liposomes for sustained release. Liposomes containing DPPC and cholesterol showed good stability and iloprost loading efficiencies, so further investigations are under way in vivo to develop a suitable carrier system for prolonged iloprost release in the lungs. C. Immunosuppressives
Pulmonary drug delivery is being evaluated for loco-regional application of immunosuppressive drugs to lung transplant patients. Intravenous or oral formulations of Tacrolimus are available for therapy of lung transplantation but they are poorly tolerated. Sinswat et al. (39) created nanostructured aggregates containing amorphous (with lactose) or crystalline tacrolimus nanoparticles by an ultrarapid freezing technique. These aggregates could be delivered by nebulization and showed high drug absorption in the lungs of mice. Another immunosuppressive drug, cyclosporine A, is very hydrophobic so aerosol formulations were based on ethanol and propylene glycol dissolutions (40,41), but these excipients were sometimes poorly tolerated in animals as well as humans. Thus, cyclosporine A liposomes were produced, and they were efficiently absorbed into lung tissue, and the formulations were well tolerated (42,43). In another approach, Chiou et al. (44) produced cyclosporine A powders with confined liquid impinging jets (CLIJ) technique and subsequent spray drying. They optimized this technique to obtain suitable particles for pulmonary delivery of proteins. All these advanced formulations of cyclosporine A promise to reduce systemic plasma levels and thus toxicities to other organs like kidneys. From the above discussion, it is clear that majority of approaches improve bioavailability, control the release properties, or reduce the dose frequency for the drug with the aim of improving patient compliance and the quality of therapy. However, one problem remains unsolved. There is no existing technology able to inhibit or circumvent the clearance mechanisms of the respiratory tract. As a consequence, the potential of such carriers to act as a platform for sustained drug delivery for longer periods cannot be entirely exploited, even when it is possible to design aerosol drug carriers that show sustained release profiles for 24 hours or longer. New carrier systems are needed that avoid clearance and achieve a powerful drug depot in the respiratory tract.
IV.
Drug Targeting within the Respiratory Tract
Targeted delivery of drugs is particularly important when the therapy causes severe side effects, such as in the treatment of lung cancer. These drugs are often administered to the systemic circulation, but to achieve an acceptable drug level at the site of action, high plasma levels may be required and can produce side effects in other tissues. Therefore, targeting drugs to the lungs by inhalation therapy promises to protect other tissues and reduce side effects.
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Delivering vaccines by the pulmonary route is easy, fast, and noninvasive, and therefore a powerful strategy in the fight against infectious diseases, particularly in the developing world. Furthermore, this immunization route allows mass vaccination campaigns to be carried out without the need for medical personnel. Many pulmonary vaccines are in development for several infectious diseases such as influenza (45,46), measles (47–49), diphtheria (50), and hepatitis. Pulmonary vaccine delivery can induce local immune responses in the lungs as well as systemically (51). Below the pulmonary epithelium, there is an array of immune cells, such as antigen-presenting cells, that continuously sample inhaled antigens and subsequently present these antigens to T cells, and the bronchoalveolar lymphoid tissue (BALT) that is induced by local infection (52). Local activation of the pulmonary immune response has the advantage of targeting pathogens directly at the port of entry and is suitable for diseases like influenza. Several formulations for intranasal administration of influenza vaccine have already been tested and shown to elicit a modest systemic immune response (45,53). Smith et al. (54) encapsulated inactivated or subunit split influenza virus vaccines into spray-dried microparticles containing DPPC as well as DSPC and administered them intratracheally to mice and rats. This formulation showed improved local bioavailability to the BALT, and increased antigen loading of antigenpresenting cells, IgG antibodies, and T cell responses locally as well as systemically. In another study, an influenza subunit vaccine powder stabilized by inulin was prepared by spray freeze-drying and delivered to the lungs of mice (46). This formulation produced enhanced IgG and IgA levels compared to the conventional intramuscular administered influenza vaccine, proving that the modified vaccine can enhance local and systemic antibody production. Another infectious disease that is transmitted by the airborne route is measles. Several research groups administered wet mist aerosols of live attenuated measles vaccine to people and the immune response was greater compared to injected vaccine (55,56). However, the stability of the vaccine was a big problem because of cold-chain maintenance caused by the thermolability of the vaccine. Therefore, different research groups have developed powder vaccines with increased stability. De Swart et al. (47) administered two different dry powder measles vaccines to macaques, but the vaccination was less efficient than intramuscular vaccination or nebulized vaccination. Thus, more work is required to improve the composition of the dry powders to obtain a formulation that can boost serum antibody levels with acceptable properties for administration by dry powder inhalers. B. Anti-infectives
One third of the world population is infected with tuberculosis (57). The treatment of tuberculosis is a great challenge, because Mycobacterium tuberculosis invades and replicates within macrophages. Drugs against tuberculosis are given orally and over a long period, though side effects and a high dose frequency result in many interruptions to therapy. Targeting macrophages could decrease systemic exposure, reduce the dose that is needed, and decrease side effects, though a special targeting strategy is needed to both channel the drugs into infected macrophages and provide prolonged drug
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release once it is delivered. Therefore, different formulations for pulmonary administration are being developed. Pandey et al. (58) produced biodegradable PLG nanoparticles with three anti-tubercular drugs (ATDs) (rifampicin, isoniazid, and pyrazinamide) and administered the aerosolized nanoparticles to infected guinea pigs. They found that the bioavailability of all three drugs was increased compared to intravenous administration and that the drugs remained above a therapeutic concentration for 11 days after inhalation. Sharma et al. (59) tried to improve the bioavailability of ATDs by producing bioadhesive wheat germ agglutinin-coated PLG nanoparticles with ATDs. Wheat germ agglutinin was used because it is known to bind to the alveolar epithelium (60), and the results showed that the concentrations of the nebulized ATDs were in a therapeutic range for about 15 days. Other investigations have employed alginate nanoparticles (61). As already discussed above, liposomes are well suited for administration to the lungs because their similarity to surfactant prevents them from acting as irritants once deposited in the lungs. Zaru et al. (62) designed different rifampicin-loaded liposomes and showed that rifampicin-liposomes were less toxic to alveolar epithelial cells (A549) compared to the free drug. Stealth liposomes are sterically stabilized liposomes that avoid elimination through the reticuloendothelial system (63) and are used for intravenous cancer therapy [e.g., Caelyx1/Doxil1 (stealth liposomal doxorubicin)]. Deol et al. developed stealth liposomes for pulmonary delivery by modifying the surface with O-stearylamylopectin to increase the affinity for the lung tissue of mice. The encapsulated drugs isoniazid and rifampicin showed reduced toxicity for peritoneal macrophages in infected mice compared to free drugs (64,65). Another targeting strategy exploits the mannose receptors that are expressed on alveolar macrophages through mannosylation of liposomes. Wijagkanalan et al. (66) reported efficient targeting of mannosylated liposomes to alveolar macrophages after intratracheal instillation to rats, as did Chono et al. (67) when they administered ciprofloxacin-loaded mannosylated liposomes for pulmonary intracellular parasitic infections. In another study, ciprofloxacin nanoparticles were encapsulated in large porous particles and showed controlled release over two to four weeks (68). Other groups have developed microspheres with different polymers to act as carrier systems for anti-infective drugs. Takenaga et al. (69) demonstrated that lipid microspheres loaded with rifampicin could be delivered to alveolar macrophages in vitro as well as in vivo with reduced side effects in the liver. Hirota et al. (70) examined the phagocytic activities of alveolar macrophages to rifampicin-containing PLGA microspheres of different sizes. They found that 3 mm particles were the most efficient for drug delivery to alveolar macrophages. Inhalable PLGA microspheres have been investigated for the treatment of tuberculosis by several other research groups (71–74). Capreomycin is used in the treatment of multidrug-resistant tuberculosis, but it shows severe side effects when it is administered intravenously. Garcia-Contreras et al. (75) developed large porous capreomycin sulfate particles and administered them to the respiratory tract of guinea pigs, reporting a decrease in both inflammation and bacterial burden in the lung tissue. New approaches for delivering anti-infectives to the respiratory tract are not limited to tuberculosis therapy though. Tobramycin is an anti-infective that is used to treat Pseudomonas aeruginosa, which often exists in cystic fibrosis (CF) patients. Pilcer et al. (76) formulated lipid-coated tobramycin particles and showed that they were in a
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respirable range, and that the lipid coating reduced agglomeration, further improving drug deposition. In another approach, moxifloxacin was loaded onto chitosan microspheres that were cross-linked with glutaraldehyde and tested in an in vitro model with Calu-3 cells. In these experiments, the microspheres retarded the absorption of moxifloxacin compared to free moxifloxacin (77). C. Pulmonary Gene Therapy
CF disease is caused by mutations in the gene that is encoding the CF transmembrane conductance regulator. CF is characterized by abnormal mucus production, inflammation in the respiratory tract, and chronic bacterial infection (78). Treatment of CF by gene therapy is an interesting field because by replacing the defective gene with a gene transfer vector, mucus production can be normalized and infection suppressed. Therefore, CF transmembrane conductance regulator gene transfer was one of the first targets of gene therapy. There are other genetic disorders like a-1 antitrypsin deficiency (79) or hemophilia (80) where gene therapy could beneficially affect the aetiopathology, but one of the biggest gene therapy fields is concerned with the treatment of different types of cancer (81). Nebulization of naked plasmid DNA leads to low transfection rates and poor stability of the DNA (82). Therefore, DNA must be delivered to the mucosal surface of the lung by carrier systems that protect the DNA from enzymatic degradation, improve long-term expression of the antigen, and enhance transfection efficiency. There are two different types of DNA carriers, viral and nonviral vectors, and both have their own advantages and disadvantages. The viral vectors that are used for DNA delivery have a high efficiency in gene transfer even though they have been modified to eliminate their pathogenicity. Since DNA delivery by viral vectors is not based on particle technologies, they will not be discussed in this review, except to note that they are immunogenic (83), which is a major disadvantage compared to nonviral vectors, because multiple dosing therapies are not possible. Nonviral vectors can be administered in multiple doses, but gene transfer is less efficient than viral vectors. Most of the carriers are positively charged so that they can interact with the DNA, which is negatively charged, by complexation or adsorption. Nonviral vectors need to be biocompatible, nontoxic, and able to carry DNA across various cellular barriers to the nucleus. For these reasons, liposomes and polymers are perfectly suited carriers for DNA delivery, and they are also easy to generate. Chitosan is a very popular polymer for gene delivery that has been utilized by many research groups (84–88) because of its mucoadhesive properties (89). Li and Birchall (88) developed lipid/polycation condensed plasmid DNA chitosan particles and showed that the in vitro deposition of chitosan-modified powders was higher than unmodified powders, and that the level of reporter gene expression was enhanced. Another polymer that is used for pulmonary gene delivery is polyethylenimine (PEI) (90– 92). Kleemann et al. (93) developed TAT-PEG-PEI conjugates to deliver plasmid DNA and reported enhanced DNA protection and higher transfection efficiencies in vivo compared to unmodified PEI. Cationic lipids, such as lipofectin, have also been used as carriers for gene delivery. Bhattarai et al. (94) administered lipofectin polymer [poly (p-dioxanone-co-L-lactide)-block-poly(ethylene glycol)] micelles with the tumor suppressor gene PTEN to C57BL/6 mice with a melanoma and observed significantly improved gene expression of PTEN in the lungs without any toxicity and longer survival times.
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It is also worth noting that many of these delivery particles have also been employed to deliver antisense DNA/RNA or siRNA to the lungs to treat several diseases by gene therapy. Similar to plasmid DNA, these smaller nucleotide sequences also need to be formulated with a carrier system that can protect them and enhance transfection rates. D. Lung Cancer Therapy
The therapy of cancer, especially lung cancer, is still very toxic for the patient because most cytostatic drugs are not sufficiently specific in their action. Drugs against lung cancer are administered systemically and they act systemically, causing serious side effects in healthy organs such as the liver, heart, or kidneys. Thus, pulmonary administration offers the opportunity to achieve higher local effects and even sustained release in the lung while reducing systemic exposure to cancer drugs. Several approaches have been adopted to target different cancer drugs to the lungs. Hitzman et al. (95) administered aerosolized lipid-coated nanoparticles loaded with 5-fluorouracil to hamsters with squamous cell carcinoma of the lung. They chose lipid-coated nanoparticles because earlier studies had shown that these particles have sustained release properties (96,97). 5-Fluorouracil levels were much lower in the serum than in the lungs in these experiments, indicating effective local exposure, and sustained release can be achieved with this approach. Paclitaxel-loaded albumin nanoparticles were approved for injection by the FDA in 2005 to treat breast cancer (98), and while there are no published studies on their effectiveness in the lungs, the potential of this technology for inhalation therapy deserves to be investigated. Liposomes have been used in rats by Zhang et al. (99) to achieve sustained release of 9-nitrocamptothecin in the lungs while reducing accumulation in other tissues. In another approach, the toxic effects of cisplatin were reduced by sustained release lipid inhalation targeting (SLIT) (100). SLIT-cisplatin is a dispersion of cisplatin encapsulated in lipid vesicles that releases 50% of the dose immediately while the other 50% remains in liposomes for sustained release (101). This phase I study showed that the administration of SLIT-cisplatin is feasible and safe; unfortunately the deposition efficiency (10–15%) was very low. Some cell-specific targeting systems have the potential to further improve cancer therapy in the respiratory tract. For example, lectin-functionalized liposomes bind specifically to the tumor-derived cell line A549 (60,102) and therefore may act as an effective targeting system. Abu-Dahab et al. (102) in our laboratories investigated the effect of nebulization on the stability of lectin-functionalized liposomes and their binding to A549 cells with promising results. A more specific target may be the transferrin receptor, which is overexpressed in many human tumor cells. Anabousi et al. (103,104) examined the uptake levels and cytotoxicity of transferrin-conjugated liposomes and showed enhanced uptake with increased cytotoxicity. Additive PEGylation of these liposomes increased their stability for aerosolization. Finally, an interesting emerging target for cancer therapy is telomerase because it is present in most human cancers (105) [see Shay and Wright (106) for a recent review]. Inhibiting telomerase may represent a novel therapy for lung cancer, except for specific telomerase inhibitors like the antisense oligonucleotide 20 -O-methyl-RNA (2-OMR) which need a special carrier system to exert a biological effect in targeted cells. Beisner et al. (107)
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Figure 1 AFM image of a nanosphere DNA complex. Bar represents 150 nm.
administered this telomerase inhibitor in different liposomal formulations containing DOTAP (N-[1-(2,3-dioleoyloxy)]-N,N,N-trimethylammonium propane methyl-sulfate), which is a cationic lipid, or a mixture of DOTAP and cholesterol to A549 cells. These reagents enhanced transfection of A549 cells and efficiently inhibited the telomerase. Nafee et al. (108) used chitosan-coated PLGA nanoparticles, which were developed by Kumar et al. for plasmid DNA delivery (Fig. 1) (109), as a carrier for the antisense oligonucleotide 2-OMR. Because of a cationic surface modification by chitosan, PLGA is able to form nanoplexes with nucleotide-based drugs, protecting these molecules from premature degradation and facilitating their cellular uptake. Taetz et al. (110) used cationic chitosan/PLGA nanoparticles to deliver 2-OMR to A549 cells and observed enhanced uptake of 2-OMR nanoplexes into A549 cells, efficient telomerase inhibition, and significant shortening of telomeres compared to 2-OMR alone. Obviously, these kinds of nanotechnology-based carrier systems represent an interesting new platform for the safe and efficient delivery of telomerase inhibitors in the context of lung cancer.
V. Future Prospects In this chapter, we have outlined several strategies and technologies for inhalable drug delivery particles (Table 1). There are many aerosol formulations on the market for the treatment of “classical” lung diseases, like asthma and COPD, and until now, significant improvements have been achieved mainly by improving the aerosol properties of the formulations. The technology already exists to produce a variety of particles, such as nanoparticles, liposomes, and large porous particles that can be efficiently deposited by aerosol inhalation in the lungs—even under pathophysiological conditions. More work is needed though to control what happens to these particles after they are deposited in the respiratory tract. Indeed, establishing a depot for prolonged release from these particles still appears to be rather difficult. To make further progress, the particles must be able to
Ø 10–1000 nm
Ø 20 nm–100 mm
Ø 50 mm (carrier) Ø 1–3 mm (drug crystals) Sufficient solubility in either aqueous or lipidic phase (fentanyl, cyclosporine A, iloprost, beclomethasone)
Stable against production procedure (antituberculosis drugs)
Nanoparticles: (PLGA, chitosan) Solid NP (matrixcontrolled)
Solid, crystalline drugs (conventional asthma drugs)
Requirements on drugs (examples)
Liposomes
Microparticles: large lactose carrier particles with adherent pure drug crystals
Particle type
High stability High carrier capacity
Similar to lung surfactant Biocompatible Biodegradable Incorporation of hydrophilic or lipophilic drugs Controlled release
111,112
References
Materials not approved for pulmonary delivery
(Continued )
12,58,59,95,108, 110,113,114
16,26,34,38,42, Stability problems 43,62,66,67, Short shelf-life 99,102–104 High production cost Low encapsulation efficiency Large scale production difficult Effective aerosolization not trivial
No controlled release
Good stability Free of critical excipients
Disadvantages
Advantages
Table 1 Overview of Different Particle Types for Pulmonary Drug Delivery
Particulate Carriers for Pulmonary Drug Delivery 71
Amorphous solid particles
Large porous particles
Note: the symbol Ø means diameter (d).
Ø 1–3 mm
Ø > 5 mm
Ø 10–1000 nm
Requirements on drugs (examples)
Thermostabile, sublimable drugs (rizatriptan, fentanyl, zolpidem)
Advantageous for drugs with rapid clearance and/or poor bioavailability (insulin, heparin)
Core-shell NP Needs protection (membrane-controlled) from enzymatic breakdown (insulin, tacrolimus)
Particle type
Disadvantages
Less aggregation Reduced phagocytosis by macrophages
Only a few drugs are suitable for this procedure
Sophisticated production
Complex behavior Incorporation of hydrophilic or in physiological lipophilic drugs environment (risk of aggregation) Protection of sensible Effective drugs aerosolization not Controlled release trivial Large surface Biodegradable High number of particles leading to a high number of sites of deposition (macrophage targeting in therapy of tuberculosis)
Rapid onset No additives Particle generation in situ
Advantages
Table 1 Overview of Different Particle Types for Pulmonary Drug Delivery (Continued )
22,23
13,14
References
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evade the clearance mechanisms of the lungs such as macrophages and mucociliary clearance without affecting them; otherwise the integrity of the respiratory tract could be compromised. More work is also required to find an effective and safe way for enhancing the absorption of high-MW drugs across the cellular barriers of the pulmonary epithelia. We have learned from the research on so-called absorption enhancers that changing the fluidity of cellular membranes—by modulating the tightness of intercellular junctions in order to affect a general increase—is unlikely to be an effective strategy Diseases affecting the lung, such as CF and cancer can alter the properties of the tissue in many ways such as affecting mucus characteristics, generating chronic inflammation, or producing unventilated lung areas. This can make it difficult to target drugs to the desired site of action. Therefore, alternative drug delivery strategies are also needed to effectively treat these diseases. In this regard, some progress is being made in at least one of these areas, namely, the penetration of mucus by particulate drug carriers (115). Due to the (understandably) strict emphasis on safety, the complex regulatory hurdles of the drug registration process, and other peculiarities of the pharmaceutical market, the conversion of innovative delivery technologies into marketed drug products is a rather slow process. Setbacks may occur sometimes for economic rather than scientific reasons, such as that seen recently with inhaled insulin. However, the progress that has been made in pulmonary drug delivery over the past few years is nevertheless impressive and we are sure new therapies will continue to be developed, even if the road ahead is long and winding.
References 1. Atkins PJ. Dry powder inhalers: an overview. Respir Care 2005; 50(10):1304–1312. 2. Geller DE. Comparing clinical features of the nebulizer, metered-dose inhaler, and dry powder inhaler. Respir Care 2005; 50(10):1313–1321. 3. Lansley AB. Mucociliary clearance and drug delivery via the respiratory tract. Adv Drug Deliv Rev 1993; 11(3):299–327. 4. Moeller W, Haeubinger K, Kreyling WG, et al. Particle clearance from the human respiratory tract. Atemwegs- und Lungenkrankheiten 2005; 31(7):342–351. 5. Sibille Y, Reynolds HY. Macrophages and polymorphonuclear neutrophils in lung defense and injury. Am Rev Respir Dis 1990; 141(2 I):471–501. 6. Patton JS, Byron PR. Inhaling medicines: delivering drugs to the body through the lungs. Nat Rev Drug Discov 2007; 6(1):67–74. 7. Bechtold-Peters K, Luessen H, eds. Pulmonary Drug Delivery—Basics, Applications and Opportunities for Small Molecules and Biopharmaceutics, Apv—Pharma Reflexion. Vol 2. Aulendorf, Germany: Editio Cantor Verlag, 2007. 8. Owens DR, Zinman B, Bolli G. Alternative routes of insulin delivery. Diabet Med 2003; 20 (11):886–898. 9. MannKind Corporation. Technosphere1 insulin system: how does the Technosphere1 insulin system work? Available at: http://www.mannkindcorp.com/technosphere_insulin. aspx. Accessed: June 2008. 10. Richardson PC, Boss AH. Technosphere1 insulin technology. Diabetes Technol Ther 2007; 9(suppl 1):65–72.
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5 Deposition, Retention and Clearance, and Translocation of Inhaled Fine and Nano-Sized Particles in the Respiratory Tract ¨ LLER WINFRIED MO Helmholtz Zentrum Mu¨nchen—German Research Center for Environmental Health, Institute for Lung Biology and Disease (iLBD) and Clinical Cooperation Group Inflammatory Lung Diseases, Gauting, Germany
WOLFGANG G. KREYLING, OTMAR SCHMID, MANUELA SEMMLER-BEHNKE, and HOLGER SCHULZ Helmholtz Zentrum Mu¨nchen—German Research Center for Environmental Health, Institute for Lung Biology and Disease (iLBD) and Focus Network Nanoparticles and Health, Neuherberg, Germany
I.
Introduction
The concept of dose is fundamental to the discipline of toxicology. For inhaled particles, dose considerations require information on particle deposition, retention, and clearance, and on particle translocation into circulation and secondary target organs. In this chapter, we summarize the state of knowledge on these processes with a particular focus on the increasing amount of ultrafine and nano-sized particles (NP), and we discuss the main governing parameters including environmental, particle, anatomic, and physiological factors. Although workplace exposure to airborne particles may play a significant role for specific professions (miners, welders, etc.), exposure to ambient particles is unavoidable for the whole population. Particulate matter (PM) in ambient air is a complex mixture of multiple chemical components ranging from a few nanometers in size to tens of micrometers (1). Primary particles are directly emitted as liquids or solids from sources, such as biomass burning; incomplete combustion; volcanic eruptions; resuspension of road, soil, and mineral dust; sea salt; and biological materials. Secondary particles, on the other hand, are formed by gas-to-particle conversion in the atmosphere. In addition, airborne particles undergo various physical and chemical interactions and transformations (atmospheric aging), including changes of particle size, structure, and composition. Ambient particle number and mass concentrations typically vary in the range of about 102 to 105/cm3 and 1 to 100 mg/m3, respectively (2). The significance of all these different particle characteristics on particle dosimetry is illustrated in section II. In general, the predominant chemical components of ambient PM are sulfate, nitrate, ammonium, sea salt, mineral dust, organic compounds, and black or elemental carbon. Although some of the chemical components present in ambient PM are toxic, none of them reach concentrations of
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toxicological relevance such as found for occupational exposures (3). Hence, adverse health effects are unlikely to be related to a single PM component, but more likely to a complex, possibly synergistic interaction of multiple components in varying physical structures with the respiratory tract and other target organs. The deposited mass of inhaled particles is crucial for dose assessment. The physical mechanisms of particle deposition and human deposition data are illustrated in section III. In the past, the most common metric for particle abundance has been “particle mass concentration,” and daily averages range nowadays from 20 to 50 mg/m3 in European and North American cities (PM10, particle mass concentration in the size range 10 mm aerodynamic diameter). Since for ambient PM, the mass concentration is dominated by the coarse and accumulation mode fraction, the presence of ultrafine and NP (diameter 100 nm) is not reflected by PM mass. However, there is evidence that adverse health effects in urban areas are also associated with particle number concentration (4), which is dominated by ultrafine particles (UFPs). The importance of NP for human health is also expected to rise in the near future due to many new developments in nanotechnology, taking advantage of the unique properties of engineered NP. Besides deposition, retention and clearance determine dose assessment and toxicity, as discussed in section IV. On the walls (epithelium) of the respiratory tract, particles first come into contact with the mucous or serous lining fluid. Water-soluble particle compounds will be dissolved, bound to proteins, often metabolized in the lining fluid, and eventually transferred to the blood, undergoing further metabolization and possible excretion. In this way, they have the potential to reach any organ (5–7) and to induce toxic effects far from their site of entry into the lungs. The nonsoluble residue particles will remain in the lungs for longer times, until they are removed by one of the regional clearance processes. There is evidence that NP behave differently compared with fine particles. As illustrated in section V, NP may penetrate into circulation and thereby translocate to other organs. NP specific toxicity can thereby reach other organs in the body. Of central importance in toxicology is the exposure-dose-response paradigm (see sect. V.D). Hence, the dose retained in the lungs (or organism) for a certain period of time will vary depending on the properties of the particles. Because of retarded clearance of NP from the body, NP may accumulate not only in the lung but also in other organs of the organism.
II.
Relevant Particle Parameters Regarding Particle Dosimetry
A. Particle Size, Shape, and Density
Figure 1 depicts the various size fractions of a typical ambient aerosol (8). Depending on the sources, the relative significance of these size fractions varies, but generally particles between a few nanometers up to about 10 mm are found under ambient conditions. As mentioned above, ambient (combustion derived) particles less than 0.1 mm are referred to as UFPs in the toxicological literature, and particles with at least one dimension below 0.1 mm are commonly termed “nanoparticles” (NP, manufactured) in nanotechnology (9). In this review, we will not further discriminate between combustion-derived and manufactured particles and will therefore use the term NP both for UFP and NP.
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Figure 1 Particle size distribution of ambient aerosol by three characteristics: mass, surface area, and number. In addition, size ranges of different particle modes are given. Source: From Ref. 8.
Evaluation of the size fraction alone as a modulating factor in PM toxicity is difficult since it is not independent of chemical composition, that is, certain size fractions tend to contain certain chemical components, for example, metals in the fine and crustal materials in the coarse size fraction. Furthermore, there are clear differences between particles in different size fractions in terms of total and regional dosimetry within the respiratory tract, as further discussed in sections III to V. Particle shape varies from perfect spheres such as droplets to fibers, and more recently, nanotubes of extremely small diameters of few nanometers and of lengths in the micrometer and millimeter range. Nonspherical particles are described by the aspect ratio of diameter and length. Particle shape is known to affect the aerodynamic (gravitational and inertial) as well as the diffusional behavior of the particles. Due to the alignment of fibers along the streamlines of the inhaled gas, particles of several tens of mm length can penetrate deep into the lung. Just recently it was shown that particle shape also affects phagocytosis by macrophages (10). Alveolar macrophages (AM) with diameters in the size range of 10 to 15 mm are not able to completely engulf and clear such fiber particles from the alveolar epithelium. Therefore, long fibers can stay for extended periods of time in the lung and can be the origin of cancerous transformations (11). Particle density, usually expressed in g/cm3, refers to the mass/unit volume of the particle itself. Liquid particles and ground or crushed particles have a density equal to that of their parent material. Generally, the mean particle density is the sum of the densities of all gaseous, liquid, and solid compounds weighted by their volume fractions within the particle. However, nonspherical and/or hollow particles (e.g., smoke and fume particles) may have effective densities significantly smaller than those predicted
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from their chemical composition and smaller than 1 g/cm3 due to their irregular shape and the presence of internal voids (12). Particle density is taken into account by the definition of the aerodynamic diameter of aerosol particles, which is the diameter of a unit density sphere having an equivalent settling velocity in air to that of the particle. Measurements of the mean apparent density of all particles of an ambient aerosol are integrating over all particle densities and have been based on simultaneous measurements of various aerosols parameters (12,13). Particle transport in the air and deposition onto surfaces is determined by the diffusion, sedimentation, and impaction properties of the particles. The former is described by the thermodynamic diameter (frequently also referred to as mobility diameter) and the latter two are characterized by the aerodynamic diameter (14), where the diffusion-dominated size regime comprises particles up to about 0.1 mm and aerodynamic effects are dominating above about 1.0 mm. Between 0.1 and 1.0 mm both diffusion and sedimentation contribute to particle transport (15). The mobility diameter and aerodynamic diameter depend on particle material (density), shape, and geometric size. B. Particle Composition and Hygroscopicity
The complex heterogeneity of aerosol composition does not only vary between particles but also within individual particles. Compounds can be distinguished by their volatility or solubility in aqueous or lipid solvents versus compounds, which cannot escape the particle structure by evaporation or dissolution/desorption processes. In this case, it is helpful to distinguish the extractable shell from the stable core of these particles. Note that shells may not only exist as an outer layer around the particle core but can intrude into a porous core. The core of a particle has to be distinguished from its surface. Particle matrices of solid particles may be made up of mono- and/or polycrystalline lattice structures versus amorphous molecular assemblies (1,16). Liquid particles may consist of different solvents (aqueous versus organic) containing numerous solutes in each of their phases. The surface may comprise of molecules of the crystalline structure extending to the particle surface or may contain a molecular layer coating the particle core with a ligand, and so on. This coating may consist of different molecular constituents than the core. Since the surface provides the interface to the outside, the surface structures and composition may undergo chemical and physical changes, which will not necessarily affect the particle core (17). Many natural and anthropogenic airborne particles are unstable, tending to change size as a result of condensation of gaseous species or evaporation of volatile components, such as water (18). The near saturation conditions in the respiratory tract (378C, 99.5% relative humidity) promote vapor transport between air in the airway lumen and hygroscopic and nonsoluble micron-sized and nanoparticles (nonsoluble matter can serve as condensation nuclei in supersaturation conditions) (19). Many pharmaceutical formulations used in aerosol therapy contain hygroscopic substances. In contrast to vapor absorption and condensation on nano- and micron-sized particles, aqueous UFPs (droplets) are expected to experience evaporation and shrink because of the high vapor pressure required to stabilize them. Any of these mechanisms alters the aerodynamic and diffusive properties of inhaled particles (via changes in size and density) and modifies their deposition characteristics (20) (see section “Particle Deposition of Pharmaceutical and of Hygroscopic Aerosols”).
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Table 1 Number and Surface Area of Different Sized Spherical Particles of Unit
Density for a Given Mass of 100 ng Particle diameter (nm) 2 20 200 2000
Particle number
Surface area (mm2)
2.4Eþ13 2.4Eþ10 2.4Eþ07 2.4Eþ04
300 30 3 0.3
C. Particle Surface Parameters
Particle surface area has been identified as one of the most promising particle metrics (compared to particle mass) in toxicological studies. Particle surface area is described by various parameters, resulting in considerable confusion. The simplest surface area is calculated as the spherical surface of an equivalent diameter, which had been determined by any kind of size-determining methodology. Figure 1 illustrates the strong increase of particle number and surface area of a given particle mass with decreasing particle diameter. The “active surface area” is used in aerosol science and takes only the outer surface area into account, which is able to interact with other aerosol particles, for instance, during coagulation (21,22). In contrast, the BET surface area (BrunauerEmmett-Teller) determined by N2 or other gas molecule absorption describes the entire porous surface including sub-nanometer pores (23). The rise in particle surface of a given mass with decreasing diameter is illustrated in Table 1. For a given mass, particle surface increases linearly with decreasing particle diameter. From Table 1, one can also conclude that particles consisting of aggregates of much smaller units may contain a 10- to 100-fold higher internal surface areas compared with compact particles of the same size. Specific interaction with the biological environment will generally be based on the properties of the particle surface area and less likely on the core of the particle, as will be further discussed. If particles are suspended in liquids (such as the lining fluid of the lung), the structure of polar molecular assemblies at the surface of the particle may lead to a surface charge, such that the particle appears to have a net charge in an electrical field. Usually, this phenomenon is measured as the zeta potential, which determines the particle mobility in an external electrical field (24). Since surface molecules are not tightly bound, the surface charge undergoes changes depending on the fluid media properties, such as ionic strength, proton concentration, and of other constituents.
III.
Total and Regional Particle Deposition
A. Particle Transformation During Inhalation
While airborne, the aerodynamic properties of inhaled particles, the flow dynamics during breathing, and the anatomy of the respiratory tract will determine their deposition probability in the respiratory tract. When entering the human respiratory tract, the environmental conditions change, including a rise in temperature to near 378C and a near humidity saturated atmosphere. Hygroscopic growth due to water uptake will affect
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the size, density, and shape of the particles, affecting the deposition probability in the various regions of the respiratory tract (see sect. III.C). In addition, chain-like aggregated particles may undergo structural transformations under the surface tension of an aqueous film taken up in the high humidity of the respiratory tract, which may lead to compaction, and hence reduction of their mobility diameter (25–28). B. Mechanisms of Particle Deposition and Modeling
Particle deposition in the respiratory tract is determined predominantly by three different mechanisms, which move particles out of the streams of inhaled and exhaled air toward the airway walls. These mechanisms are relevant for different size ranges: (i) sedimentation by gravitational forces acting on particles more than 0.5 mm aerodynamic diameter; (ii) impaction caused by their inertia in branching airways acting on particles more than 1.5 mm aerodynamic diameter; and (iii) diffusional motion of particles less than 0.5 mm (thermodynamic diameter) by thermal (Brownian) motion of the air molecules. Each mechanism becomes relevant for the given size range and can coexist with other mechanisms, as illustrated in Figure 2. These physical mechanisms of particle motion affecting particle deposition work through three important components (aerosol properties and physiology) during breathing: (i) particle properties (size, density, and
Figure 2 Bronchial (including bronchiolar, BB & bb, dotted lines) and alveolar (AI, full lines)
particle deposition of unit density particles in the human respiratory tract according to ICRP Publication 66 (29). Data are shown for two different breathing scenarios: exposure during sleeping (nose breathing, thin lines) and exposure during heavy exercise (mouth breathing, bold lines). The arrows indicate the particle diameter regime of the different particle deposition mechanisms in the respiratory tract.
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shape) and their possible dynamic change during breathing; (ii) geometry of the branching airways and the alveolar structures; and (iii) breathing pattern determining the airflow velocity and the residence time in the respiratory tract, including breathing through the nose in comparison to oral breathing (30). The respiratory tract can be considered as a series of three filtering units: the extrathoracic region (ET, including the nose, mouth, pharynx, and larynx), the bronchial (BB) and bronchiolar (bb) conducting airways (including trachea), and the alveolar interstitial (AI) region (29,31,32). Larger particles are primarily deposited in the nose and in larger airways due to impaction, because they cannot follow the streamlines of the airflow at bifurcations. Inhalation flow rate is a major factor controlling deposition by impaction. Therefore, larger particles are filtered out of the air stream and cannot penetrate down to the deep lung. Smaller particles can pass through the large airways and are deposited in the lung periphery due to sedimentation. Longer residence times in smaller structures enhance deposition by sedimentation. Very small particles (UFP and NP, smaller than 100 nm) behave like diffusing gas molecules and stochastically disperse due to Brownian motion. The smaller the particles, the more effective the Brownian motion. In addition, longer residence times enhance the deposition due to Brownian motion. In the particles size range of 0.3 to 0.5 mm, all deposition mechanisms are rather ineffective (15), resulting in a minimum in the total particle deposition curve of the human respiratory tract, as shown in Figure 2 according to the empirical International Commission on Radiological Protection (ICRP) model (29), which is a summary of all experimental data being available until its release in 1994 (15,33–36). The simulations shown in Figure 2 represent two typical breathing scenarios, exposure during sleeping and heavy exercise. Monodisperse particles of unit density were used in the calculations. Exposure during sleeping assumes nose breathing and has a mean ventilation rate of 0.45 m3/hr. Exposure during heavy exercise represents mouth breathing and has a mean ventilation rate of 3.0 m3/hr. Total deposition is not very different between the two scenarios (data not shown), but regional deposition differs significantly. Compared to heavy exercise, the extrathoracic deposition of both, coarse (>2 mm) and ultrafine (<0.1 mm) particles is larger during sleeping, mainly due to nose breathing, since the nose serves as an effective filter for both size regimes. This results in lower bronchial (BB & bb) deposition of larger particles during sleeping. Heavy exercise has maximal BB deposition at about 5-mm aerodynamic diameter. For the fine and coarse particle size regime, alveolar deposition is maximal at about 3-mm aerodynamic diameter, with little difference between the two breathing scenarios. In the UFP size regime, the two breathing scenarios have significant impact on both BB and alveolar deposition. In the alveolar region, both breathing scenarios have maximum deposition between 10 and 100 nm, where heavy exercise has higher alveolar deposition for particles smaller than 60 nm. This is primarily caused by the lower filtering of the upstream BB and extrathoracic compartments during heavy exercise due to shorter residence times. BB and extrathoracic deposition of particles below 10 nm is more and more dominant, and heavy exercise exceeds BB deposition during sleeping below 5-nm particle size. The decline of BB and alveolar deposition for very small particles is due to the rising extrathoracic deposition. For toxicological dose assessments, the ventilation rate also has to be taken into account, which differs between the two breathing scenarios by a factor of 6.7.
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Figure 3 Deposition probability in the human airways (bronchial and bronchiolar, BB & bb, generations 1–16) and the alveolar region (AI, generations 16–23) based on the MPPD computer code (32,39). For 250 and 20 nm particles, deposited particle mass fractions are either plotted for each generation (left panels) or relative to the surface area of each generation (right panels). Model parameters were: tidal volume = 625 mL, breathing frequency = 12/min, oral breathing, 5-lobe asymmetric model by Yeh and Schum (40).
Even though the fraction of fine PM is small in thoracic conducting airways, the toxicologically more relevant PM deposition per unit airway surface area may often exceed that of the gas exchange region, because of its 100-fold larger surface area [adult lungs *140 m2 (37)] when compared to that of airways (38). This is demonstrated in Figure 3 for 20 nm and 250 nm particles; where the deposition probabilities are calculated in the airways (lung generations 1–16, BB according to ICRP model) and in the alveolar region (generations 16–23, AI according to ICRP model) using the Multiple Path Particle Deposition (MPPD) computer code (32,39). For both particle sizes, deposited mass fractions are either plotted per generation or relative to the surface area of each generation. While the largest amount of both particle sizes is found in the alveolar region, the highest particle mass per epithelial surface area is found in the upper airways.
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C. Particle Deposition of Pharmaceutical and of Hygroscopic Aerosols
Particle deposition in the respiratory tract has a high variability between subjects (41,42), which result from varying lung anatomy and breathing pattern, and therefore, deposition measurements have been performed under spontaneous and standardized breathing maneuvers. Since aerosol inhalation is also considered as topical or systemic therapy, this varying deposition implies great uncertainty in the administered dose. To reduce this variability, standardized breathing maneuvers are recommended (43), such as implemented in the AKITA inhalation device (Activaero GmbH, Gemu¨nden, Germany) (44). Effective aerosol drug delivery to the lung has to consider the effects of particle size and the dispersity of the aerosol. Most pharmaceutical applications use metered dose inhalers or jet nebulizers, which generate polydisperse (broad size range) particles between 4 and 6 mm aerodynamic diameter (45,46). A large fraction of these particles deposits in the mouth cavity, at the larynx, or at the first bifurcations in the lung, and only a minor fraction enters the small airways or the lung periphery (47). This effect of extrathoracic deposition is enhanced in patients with airway obstructions due to smaller airway calibers and increased breathing frequencies. One approach to overcome this limitation is to use porous particles as drug carriers (48,49), which are up to 10 mm in geometric diameter, but which behave aerodynamically like 3 mm particles due to their low density. There are new therapeutic options using nanocarriers (50,51), but since they are mostly delivered within solutions, using standard medical nebulizers, their deposition characteristic is determined by these devices, as discussed above. When inhaling environmental aerosols or aerosol drugs, hygroscopic properties have to be considered. Aerosol particles containing salt ingredients will increase their particle diameter in the near humidity-saturated atmosphere of the lung due to an uptake of water vapor, forming droplets of larger size. This causes a shift in the particle size distribution, including changes in the characteristic deposition pattern. In the fine particle size regime, this general increase in particle diameter is associated with an increase in total particle deposition (19,52–55). In the UFP size regime, this effect is contrary. Due to the decrease of deposition probability with increasing diameter, there is a decrease of deposition after hygroscopic growth, which was confirmed experimentally, as illustrated in Figure 4 (56). In contrast to vapor absorption and condensation on nanoand micron-sized hygroscopic particles, aqueous UFPs are expected to experience evaporation and shrink because of the high vapor pressure required to stabilize them. D. Particle Deposition in Susceptible Individuals and Patients with Lung Diseases
There are only few studies on particle deposition in the respiratory tract of children and none in infants. Since the geometry of the conducting airways and the alveoli in children with developing lungs differs from the adult lungs particle deposition, probability will also change. Alveolarization of the lungs of one-year-old human infants is complete, but the entire lung volume is much smaller than the adult lungs and, hence, the developing lungs of infants and children have smaller calibers (airways and alveoli) compared with adults (57,58). Therefore, the physical factors mentioned above cause increased particle deposition, as confirmed in experimental studies (59–62). In addition, NP inhalation may bear an increased risk for the developing lungs of children compared to adults, since
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Figure 4 Total deposition fraction for Sebacate oil (nonhygroscopic) and for salt particles
(hygroscopic) at rest and during exercise. Source: From Ref. 56.
toxicological effects of NP may impair the development and growth of certain lung structures and may enhance the prevalence for allergic diseases (63–65). Lung diseases often change the geometry of the lung, followed by changes in breathing pattern and particle deposition (30). Airway obstructions in asthma are caused by smooth muscle contraction due to allergic responses and local inflammations, but the obstructions are mostly reversible. Increased fine particle deposition in small airways could be confirmed in women with nonspecific asymptomatic BB hyperresponsiveness, which is associated with early inflammation of small airways, and which can therefore be considered as a precursor of asthma (66). Increased deposition of UFPs was demonstrated in chronic asthma (67). Obstructions seen in COPD patients are due to local chronic inflammation and mucus hypersecretion, and these are mostly irreversible. Narrower airways imply higher particle deposition due to impaction of micron-sized particles at bifurcations (67–70). On the other side, emphysema seen in many COPD patients can imply lower particle deposition in the lung periphery due to increased sizes of small airways and alveoli (71). Increased UFP deposition in COPD patients was also found in previous studies (72,73); note in these studies, a full breath inhalation maneuver during spontaneous breathing was used. Therefore, these studies cannot discriminate deposition between airways and lung periphery. Increased UFP deposition in small airways was confirmed in smokers and COPD patients using the aerosol bolus inhalation technique (74).
IV.
Particle Retention and Clearance
A. Particle Clearance from the Airways
The airways are covered by a mucus layer, which is transported by beating cilia toward the larynx from where it is swallowed into the gastrointestinal (GI) tract. Mucus transport velocities between 10 and 20 mm/min in the trachea and subsequently
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decreasing velocities in airways of decreasing calipers confirm a fast removal of particles being deposited in the airways and on the mucus layer (75,76). Extrapolating this to the whole system of airways suggests that particles being deposited there should clear within about 24 hours. Particles deposited in the lung periphery are subject to macrophage-mediated clearance mechanism, which follows a completely different time scale. Particles deposited in the entire lung will therefore be eliminated from the lung by a two-phase process. Radiopneumography was introduced by Albert and Arnett (77) to study regional particle deposition and clearance kinetics. Although this interpretation of the two-phase elimination/retention kinetics was accepted over a period of 40 years, it had to be revised recently because of two observations. l
l
UFPs can be eliminated in short periods of time from the lung periphery via the circulation without involvement of macrophages (78,79). A considerable fraction of particles deposited in nonalveolated airways after shallow bolus inhalation is long-term retained in these airways (80–82).
Both long- and short-term particle elimination from airways and lung periphery have to be considered. In principal, particles can be eliminated from airway surfaces by mucociliary transport (rapid) and by penetration into BB epithelium (slow) (83). They can be eliminated from the lung periphery by phagocytosis and subsequent transport within macrophages (slow) and via alveolar epithelium and translocation to circulation (rapid) (78,79). Biomedical aerosol research has therefore to cope with four rather than two mechanisms by which particles are eliminated from the respiratory tract. This has to be taken into account when the fate of therapeutic or toxic particles deposited in the lung is considered. Except for translocation of NP into circulation, these mechanisms were included in the ICRP Human Respiratory Tract Model of particle clearance (29), and the principal clearance mechanisms are illustrated in Figure 5. Insoluble particles are mechanically transported out of the various lung compartments toward the GI tract with various rates, following fecal excretion. In addition, there is translocation within the lung into interstitium and lymph nodes. Soluble particle compounds will move to circulation and be excreted via kidney and urine, if they are not otherwise metabolized. Translocation of insoluble NP from the blood pool to the GI tract and the kidney, and subsequent excretion via feces or urine has to be considered but seems to be small (79). A future revision of the ICRP model must include mechanical transport rates of NP from the different lung compartments to blood circulation, as illustrated by the dotted gray arrows in Figure 5. Clearance studies after shallow aerosol bolus inhalation, targeting particles primarily to the conducting airways, showed that there is a fraction of long-term retained particles, which depends on particle size (84,85). Different mechanisms can be considered for slow particle clearance in the airways, resulting in a loss of particles from mucociliary transport, such as (i) penetration of particles through the mucus into periciliary spaces (86,87) or (ii) deposition of particles in areas being transiently free of mucus at the time point of deposition (88). Smaller particles may have a higher probability of penetrating between cilia where they can be engulfed by airway phagocytic cells and/or dendritic cells (89) or where they can translocate into the epithelium. The fraction of long-term retained particles in the airways decreases with increasing particle
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Figure 5 Schematic view of the ICRP-66 particle clearance model from the different compart-
ments of the human lung [after (29)], including mechanical particle transport to the gastrointestinal tract (followed by fecal excretion, indicated by transport rates gET, gBB, gbb, gAI) and to the lymph nodes (indicated by transport rates lET, lBB, lbb, lAI). In addition, absorption of soluble fractions to the blood circulation is indicated by absorption rates sET, sBB, sbb, sAI, followed by urine excretion via the kidney. A future required revision describes NP translocation to the circulation (blood) and is illustrated by the gray arrows (indicated by translocation rates tET, tBB, tbb, tAI).
size. In a recent study it could be shown that only 25% of 100 nm carbon particles targeted to the airways by shallow aerosol bolus inhalation were cleared within 24 hours by mucociliary action (74). The remaining particle fraction was retained more than 48 hours and may follow other clearance mechanisms, such as uptake by phagocytic or epithelial cells (83). The retarded clearance of particles from the conducting airways has implications for not only dose assessment of inhaled toxicants and radiolabeled particles (81) but also infection transmission after inhalation of bacteria or viruses. However, there may be advantageous effects related to retarded particle clearance in the airways. Inhaled drugs undergoing retarded particle clearance in the airways would allow for their prolonged effect.
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B. Particle Clearance from the Lung Periphery
The absence of mucociliary action in the lung periphery results in much slower clearance kinetics. The chemical stability of the inhaled particles plays a crucial role, because the dominant fraction of micron-sized particles can be considered to be within AM. Acidic conditions within phagolysosomes of AM, including chelating agents, interact with the particles to dissolve or digest them and to inactivate bacteria or viruses (90,91). But insoluble particles can resist this dissolution and stay in the lung for longer time periods. Therefore, AM will determine the fate of these particles in the deep lung. This is included in the dose evaluation of the ICRP Human Respiratory Tract Model for inhaled radiolabeled aerosols by distinguishing between insoluble, moderately soluble, and soluble materials. However, macrophage-mediated particle removal may be impaired especially in the elderly, smokers, and subjects with lung diseases (92,93). Besides particle dissolution in AM, there is a minor mechanical particle transport from the peripheral lungs to the distal end of the ciliated airway epithelium and the mucociliary transport to the larynx and the GI tract. Note that there are significant species differences in this particle transport (94,95), which primarily result from different lung anatomy between rodents and man. The transport (clearance) rates of insoluble particles deposited in the different regions of the lung of man are shown in Figure 5. However, NP are less effectively taken up by macrophages but interact to a greater extent with epithelial lining cells than with larger particles (96). In contrast to fine particles deposited on the epithelial cells, NP will enter these cells rapidly, therefore are no longer accessible for phagocytosis by AM (97). In addition, clearance of NP may differ from that of fine and coarse particles. Recent studies in healthy subjects suggest that insoluble carbon particles (which is the primary fraction of combustion particles) do not clear from the lung within days, and are therefore long-term retained (73,74,98). These data support the hypothesis that inhaled NP may translocate into the epithelium and interstitial spaces and subsequently enter circulation, where they are transported to other organs of the body (83,99). However, the above-mentioned human studies on the clearance of NP were short-term studies over a few days. No human long-term studies are currently available, and animal studies on NP clearance from the lung are limited. Surprisingly, when lung clearance of inhaled iridium NP or micron-sized polystyrene particles was followed over six months in rats, there was no difference in the lung retention kinetics between the two particle types (100,101), suggesting that the long-term retention kinetics known for micron-sized particles can be used as a first approximation. C. Particle Clearance in Patients with Lung Diseases
Acute and chronic diseases can affect the clearance capacity of the respiratory tract and the residence time of foreign materials and organisms in the different regions of the respiratory tract. Acute and chronic infections can inhibit ciliary function, cause mucus hypersecretion, and alter the viscous properties of mucus, leading to retardation or complete inhibition of mucus transport (102,103). In addition, genetic diseases can cause ciliary dysfunctions, as in primary ciliary dyskinesia, or mucus dehydration and stiffening, as in cystic fibrosis. Mucociliary clearance dysfunctions are followed by bacterial colonization of the airways and the formation of bronchiectasis (104–106). In all these
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diseases, coughing plays a significant role in mucus transport and clearance, particularly in the upper generations of the tracheobronchial tree (107). Alveolar clearance is impaired by chronic cigarette smoke exposure and interstitial lung diseases such as sarcoidosis or interstitial pulmonary fibrosis (92,93). AM are suppressed in their function of phagocytosis, signal transduction, and intracellular digestion (108,109). The impairment of clearance has not only an influence on the dose of retained toxic or radioactive particles but also in the defense against inhaled bacteria or viruses. Impaired phagocytosis causes an impaired bacterial killing in the lung, and thereby supports the progression of bacterial colonilization and induction of bacterial or viral infection. Impaired clearance of NP in the diseased lung has been reported in various studies, such as in smokers and COPD patients (73,74). This impaired clearance of NP causes NP accumulation in the lung, and since these particles are attributed to a specific toxicity and reactivity due to their high surface area, this may influence formation of chronic lung diseases and the onset of exacerbations. This has been confirmed in COPD, asthma, and cystic fibrosis patients (65,110,111). D. Particle Transformation in the Epithelial Lung Lining Fluid and Biochemical Reactions
Once deposited, other than aerodynamic and thermodynamic particle, parameters become important. Even if the morphology of chain-aggregated particles persists until deposition on the epithelial surface, they may either compact there or disintegrate, depending on their surface properties and epithelial lining fluid properties (112). Because of the liquid layer on top of the epithelium (epithelial lining fluid, ELF; with water-insoluble surfactant film at the air-liquid interface), particle solubility and bioavailability will determine the subsequent metabolism and biokinetics of the various particle components. Water-soluble or lipid-soluble fractions will disperse from the particle and rapidly spread and dilute in ELF and will undergo completely different biochemical interactions with the liquid epithelial layer, when compared to the remainder core of the particle. Insoluble particles or residual particle cores deposited and retained in ELF may react with ions, chelating molecules, and proteins, which may result in functional changes of the latter as well as coating or complexing the particles. Thereby the particles may specifically bind to selected proteins and follow protein-mediated transport processes (113–115), carrying them from the epithelial surface into the circulation. Pathways may be paracellular across tight junctions between adjacent epithelial cells or transcellular by transcytosis as shown by Heckel and colleagues (116), and are sizedependent as discussed by Patton (6). A schematic in Figure 6 illustrates these pathways. E.
Nanoparticle Relocation within the Epithelium
Most of present textbooks state that all inhaled particles that deposited on the epithelial lung surface stay there until AM as the front line of the defense system phagocytize them for further disintegration and removal (117). In contrast, NP show a different behavior: (i) only a small fraction of deposited NP are phagocytosed by AM (118) and dendritic cells (89,97), and (ii) most NP disappear from the epithelial surface within one to two
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Figure 6 Suggested absorption of large [(A) transcytosis] and small [(B) tight junctions] mac-
romolecules across alveolar epithelium/endothelium into blood capillaries (basement membrane between epithelium and endothelium is not shown). Source: From Ref. 6.
days and stay within the lung tissue, that is, epithelium, interstitial spaces, and vascular endothelium (101). This was confirmed by sequential lung retention, clearance, organ/ tissue and bronchoalveolar lavage (BAL) measurements in healthy adult WKY rats over a six-month period after a single one-hour inhalation of 192Ir radiolabeled iridium NP. From three weeks to six months after inhalation, only about 20% of the lung retained Ir-NP were accessible by BAL (Fig. 7). In contrast, more than 80% of micron-sized particles retained in the lungs were accessible by BAL throughout the six-months period, and they all were retained in AM on the epithelium (117). On the basis of these observations, the authors conclude that more than 80% of the lung retained Ir-NP were translocated into lung tissues, and thereby not accessible by BAL. Hence, there is a strong size-dependent difference in particle relocation into and through the epithelium: most NP have immediate access to the lung tissue (97) while micron-sized particles do
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Figure 7 Fractions of retained 2 mm fine polystyrene (PSL) particles and 20 nm Ir-NP accessible by bronchoalveolar lavage. Source: From Ref. 101.
not. These observations were confirmed independently, showing that macrophages play a secondary role in uptake and clearance of inhaled titanium dioxide NP (96). Since only a minor fraction of these particles enters secondary target organs (101), they must reside in the epithelium for longer times.
V. Systemic Translocation and Uptake in Secondary Target Organs A. Why Is There So Much Interest in Translocation of Nanoparticles?
Both clearance and translocation refer to the migration of particles from their primary organ of intake (organ of deposition; here the lungs) into other (secondary) organs. However, in contrast to clearance, the process of translocation implies that the NP penetrate at least one body membrane. Among other parameters, the translocation rate depends on particle size, surface composition, and surface charge. Since the translocation rate becomes negligible for particles larger than about 200 nm, direct experimental evidence for translocation exists only for NP. After deposition on the lung epithelium, NP have to cross the air-blood barrier after they enter the circulation. NP in the circulation are rapidly filtered from the blood stream in the liver, and to a lesser extent, in the spleen. But there is more and more evidence that, based on specific surface properties, NP can circulate in the blood for longer times (119–121), and that significant fractions may be taken up in secondary organs, such as the brain, the kidneys, or the heart (122). In addition, transport via neuronal connections offers an alternative pathway
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of translocation as evidenced by the observed NP translocation from the nose into the brain through the olfactory bulb (123–125). A variety of NP were shown to initiate significantly stronger adverse reactions in biological systems than their larger-sized counterparts (126). This was associated with (i) the large number of accumulated NP, (ii) their drastically increased surface area/NP mass, and (iii) the nature of their surface eventually containing adsorbed toxic substances or reactive nanostructures. Compared to the site of entry, such as the lung, which has effective defense mechanisms, the secondary sites may be more susceptible, because similar mechanisms may not be present. In addition, since NP may not clear efficiently from the body, these irritants may accumulate and eventually trigger and/or mediate the onset of chronic diseases or carcinogenic processes (127). For example, the induction of neural disorders in the brain, such as Alzheimer’s disease (128), is discussed as a possible consequence of chronic NP exposure (129). The invention of new types of NP (such as carbon nanotubes and others) with unique properties may offer perspectives for novel diagnostic and therapeutic applications, which are currently investigated by the newly emerging discipline of nanomedicine (130) (see also chap. 4 on medical applications of NP). One of the active fields of research in nanomedicine focuses on deliberate modification of the surface properties of NP, with the objective of designing, testing, and optimizing characteristic biokinetic behaviors (passage of membrane boundaries and translocation to secondary organs) of medicinal NP to reach high target organ specificity (131). For example, drug delivery to the central nervous system via blood-borne NP requires surface modifications to facilitate translocation across the tight blood-brain barrier (BBB) via specific receptors (132– 134). In addition, NP may offer new therapeutic options in nonviral gene therapy, such as in cystic fibrosis by inhalation of NP gene vectors (135–137). B. Nanoparticle Translocation into Circulation and Secondary Target Organs
There is agreement that under normal conditions, excluding high dose exposure or exposure to toxic particles, micron-sized particles are not likely to be translocated toward the systemic circulation, neither through the air-blood tissue barrier (into capillaries) nor through the “lymph-blood barrier” in the tracheobronchial lymph nodes. Humans’ classical pathology has reported such particle loads in secondary target organs only under heavy exposure conditions, such as in coal mine and asbestos workers (138,139). Evidence of translocated NP, such as gold, silver, titanium dioxide, polystyrene, and carbon, and sizes ranging from 5 to 100 nm diameter originates from animal data, providing either evidence of particles in circulation (83,140,141), or secondary target organs (78,142–144), or thrombogenic effects of inhaled NP (145,146). There remains uncertainty about translocated fractions of NP beyond 5% of the delivered lung dose (147,148). However, completely balanced studies of the biokinetics of inhaled iridium NP indicate translocated fractions of about 1% of the deposited NP dose to the lungs of rats (100,149). Just recently, a similar estimated fraction of 1% to 2% of translocated 50 and 200 nm polystyrene particles was reported (150). The former studies have the disadvantage of using iridium NP being extremely rare in the environment and not yet used in nanotechnology. However, the physicochemical properties of the iridium particles labeled with 192Ir g-emitting isotope, for
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example, extremely low solubility, allowed quantitatively balanced biokinetic analyses. The iridium NP did not only translocate to the liver at about 0.1% to 0.5% of the lung dose but also to the spleen, the kidneys, the brain, and the heart at similar fractions. Uptake of the 15 to 20 nm particles in secondary target organs was about a factor of 2 to 3 higher than for 80 nm particles (100,149). Principally, two pathways of NP translocation from the surface of the alveoli and the airways toward the brain are possible: (i) along neuronal axons (123,151,152) or (ii) via pulmonary and systemic circulation (83,100,149). It needs to be emphasized that the iridium NP uptake in the brain was not via a neuronal pathway from the olfactory mucosa in the nose, since extrathoracic airways of the rats were bypassed when ventilated through an endotracheal tube. While translocation into the brain via the blood circulation intuitively seems likely because of the analogy to the uptake in other secondary organs, the extremely tight BBB represents a significant obstacle to easy translocation into the brain (132). However, it has been shown that surface modifications, such as coating of the NP with, for example, polysorbate 80 or apolipoprotein-E could overcome the BBB (153). Nevertheless, NP translocation into the brain via the olfactory neuronal pathway seems to be more effective, since it circumvents the tight BBB. Translocation of NP along nerve cells from the olfactory epithelium to the olfactory bulb was first reported by Howe and Bodian (154), for 30 nm polio viruses in monkeys, and later, for nasally deposited colloidal 50 nm silver-coated gold particles in squirrel monkeys (152). Recently, carbon and MnO-NP and even fine Fe2O3 particles were reported to translocate along the same pathway to the central nervous system, based on their presence in the olfactory bulb of rats after inhalation or nasal instillation (123–125,155). Finally, fluorescent 40 nm polystyrene NP were reported to translocate from the nerve endings in the tracheal epithelium along their neurons to their cell body in the ganglion nodosum and jugular ganglia in the neck of guinea pigs along neurons innervating the trachea (156). The magnitude of the translocated fraction, the transport mechanisms, and the ratedetermining parameters are currently under debate. Conflicting results on systemic translocation of NP to the systemic circulation after inhalation in human studies have been reported: while Nemmar et al. (157) have demonstrated a rapid uptake of radiolabeled carbon NP into the bloodstream within minutes, and subsequent uptake in the liver, other authors could not find NP amounts above the limit of detection (<1% of inhaled NP) outside the lungs (73,74,158,159). In all studies 99mTc-radiolabeled carbon NP were used together with gamma camera imaging. The observation that the translocated fraction is less than 1% is in agreement with animal studies by Kreyling et al. and Chen et al. (79,150). The high fraction of systemic translocation of radioactivity in the Nemmar study is likely to be attributable to leaching of the 99mTc radiolabel off the particle matrix. Although the exact translocated fraction is currently unknown in human studies due to insufficient detection efficiencies, even low translocated fractions can accumulate during chronic exposure and potentially result in high doses of NP numbers and surface area in secondary target organs (74), as illustrated in the following paragraphs. However, indirect evidence of particle translocation in humans comes from recent exposure studies on healthy subjects inhaling diluted diesel exhaust (160,161). Inhalation of dilute diesel exhaust impaired two important and complementary aspects of vascular function in humans: the regulation of vascular tone and endogenous fibrinolysis. Furthermore, dilute diesel exhaust exposure led to changes in EEG in human volunteers (162).
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C. Nanoparticle Accumulation in Secondary Target Organs During Chronic Exposure
Although accumulation of various types of NP in secondary target organs has been observed in several animal studies (78,79,83,140–143), there is very little information on exact organ-specific accumulation fractions and potential clearance mechanisms from secondary organs. One of the few long-term retention studies reports that the amount of 15 nm iridium NP in each of the secondary target organs did not increase with increasing retention time after a single one-hour exposure but peaked after one week at about 0.5%, as shown in Table 2 (100). Thereafter, fractions decreased again, but remained detectable below 0.1% of the initial lung deposit throughout the six-months period of observation. However, even though the mass fractions of iridium NP were rather low in secondary target organs, the number of NP is impressively high. Data at one week and six months after the single inhalation are shown for lungs and all secondary target organs— liver, spleen, heart, brain, and kidneys—in Table 2. More than 1 billion NP were found in each of the secondary target organs one week after a single one-hour exposure; and still more than 100 million NP were determined six months after the inhalation. In addition, the surface area of the retained iridium NP was calculated based on the BET specific surface area of 123 m2/g or 1500 m2/cm3 per mass or per volume of NP, respectively (163) (BET surface area was determined by nitrogen absorption measurements). One week after inhalation, the total surface area of the retained iridium NP is close to one square centimeter (in each secondary target organ), and after six months this value has dropped to 10% to 20%. In addition, extrapolating the single exposure to chronic (daily) exposure will result in high doses of NP numbers and surface area in the case of low-solubility NP materials, as further illustrated in section VI. From the limited number of studies, one can conclude that transport of lungdeposited particles to and subsequent accumulation in secondary target organs does occur at small but detectable amounts. There is evidence for the existence of clearance mechanisms from all secondary organs with about the same efficiency as the long-term
Table 2 Retained Mass Fractions (In Relation to Lung Retention Immediately After Exposure),
Corresponding Numbers, and Retained BET Surface Area of Insoluble 15 nm Iridium NP in the Lungs and in Secondary Target Organs of WKY Rats One Week and Six Months After a Single One-Hour Inhalation Retained mass fraction
Retained particles number
Particle surface area (cm2)
Organ
1 wk
6 mo
1 wk
6 mo
1 wk
6 mo
Lungs Liver Spleen Heart Brain Kidney
0,6 0,006 0,004 0,004 0,003 0,006
0,06 0,0005 0,0003 0,0005 0,0005 0,0001
7,00Eþ11 7,00Eþ09 4,66Eþ09 4,66Eþ09 3,50Eþ09 7,00Eþ09
7,00Eþ10 5,83Eþ08 3,50Eþ08 5,83Eþ08 5,83Eþ08 1,17Eþ08
1,26Eþ02 1,26Eþ00 8,42E01 8,42E01 6,32E01 1,26Eþ00
1,26Eþ01 1,05E01 6,32E02 1,05E01 1,05E01 2,11E02
The according surface area of the retained Ir-NP are calculated based on the BET surface area of 123 m2/g or 1500 m2/cm3 (per mass or per volume of particles, respectively). Source: From Refs. 79 and 100.
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clearance from the lungs. However, substantial uncertainties remain regarding the dependence of these findings on particle properties and whether similar effects are to be expected in humans. One of the puzzling issues is the fact that in spite of the expected uptake of smoke particles in secondary target organs of smokers, no such findings are reported in the literature. D. Exposure-Dose-Response Relationships
Of central importance in toxicology is the exposure-dose-response paradigm. Since all the different aspects of dose-response relationships in relation to in vitro and in vivo NP exposure are discussed in more detail in chapter 16 of this book, it will be illustrated here very shortly, since it is of central importance in dose assessment. With respect to inhaled particulate compounds, inhaled, deposited, and retained doses need to be differentiated. The initially deposited mass in a given region of the respiratory tract is subject to different clearance processes, depending on the site of deposition and physicochemical characteristics of the particle (see above). As a result, the dose retained at a certain time after deposition will change. The retained dose is determined by the biopersistence of an NP (164,165). Although doses are usually expressed per particle mass, other dose metrics, such as particle volume, particle surface area, or particle number may be more appropriate, depending on the elicited response and underlying mechanisms. For example, it has been hypothesized that the retardation of alveolar macrophage-mediated particle clearance occurring under overload condition in the lung is due to the phagocytized particle volume in macrophages rather than particle mass (166). While this hypothesis explained well the overload-induced prolonged alveolar clearance of poorly soluble and lowtoxicity particles (e.g., coal mine dust) (167,168), it did not correlate well with the lung burden of NP (coal mine dust, TiO2), causing clearance retardation (169,170). An early key study demonstrated that ultrafine TiO2 with an average particle size of 20 nm caused more inflammation in rat lungs than exposure to the same airborne mass concentration of fine TiO2 with an average particle size of about 250 nm (164,171). So far, TiO2 had been considered as a low-toxicity dust, and indeed had served as a negative control dust in many studies on particles toxicology. Interestingly, when the deposited TiO2 dose was expressed as particle surface area rather than particle mass, there was a common dose-response relationship of the inflammatory responses of these different sized TiO2 particles (172). Similarly, a unique dose-response function between inflammatory neutrophilic cell influx and the surface area of administered carbon NP obtained from different combustion processes with different sizes was observed in a mouse model (173). In addition, Sto¨ger and coworkers found a threshold of instilled carbon particle surface area in the lungs of 20 cm2, below which they did not observe any inflammatory response in the lungs. The importance of particle surface area for eliciting inflammatory responses in the lung has also been confirmed by several in vitro studies (174–177). The concept of particle surface as dose metric has now become generally accepted to be critical for explaining the biological, toxicological, and chemical activity of NP. However, other particle characteristics need to be considered as well, such as the number of retained NP. In an epidemiological study, it could be demonstrated that the number of particles correlated similarly as the particle mass with respiratory effects (4).
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VI.
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Conclusions: Application of Animal Dose-Response Relationships to Humans?
The probability of any biological effect of inhaled aerosol particles occurring in humans or animals depends on exposure, deposition, retention and clearance, translocation, and metabolic fate of particles in the lung and other organs of the body. UFP and NP not only show completely new physical and biochemical properties but their fate in the living organism also differs considerably in relation to inhaled fine and coarse particles, requiring the development of new dose metrics. Extrapolation of airway and lung doses between species must consider species-specific differences in evaluating dose-response relationships, as was discussed recently (95,178,179). Thus, extrapolation of deposition patterns from most healthy animal models can be performed at least to some extent, since the differences in anatomy and breathing conditions are widely known. However, subsequent particle retention, redistribution within the lungs, and clearance pathways toward other organs and out of the body differ consistently between rodents and man (93,94). As a result, extrapolation will only be possible under limited specific conditions, should be used with great caution, and may only be considered to represent a first rough estimate. Taking the long-term iridium NP kinetics after a single exposure in rats into account, we can estimate long-term accumulation in secondary target organs during a yearlong chronic exposure in man. Let us consider a daily unit dose to the lung; from this dose only 0.0005 is long-term retained in secondary target organs, as found in the liver, heart, and brain after six months (Table 2). The daily dose of insoluble NP to the human lung can be estimated from the following assumptions: l
l l l l
103 cm3 insoluble NP in inhaled air, that is, 10% of an average NP concentration of 104 cm3 104 L daily inhaled gas volume by an adult human 30% deposited fraction of the inhaled NP in the peripheral lungs ) 3 109 daily dose of deposited insoluble NP in the human lung ) 3 1011 long-term retained NP in the lung during one year exposure (assuming about 70% clearance of these insoluble NP from the lungs)
On the basis of these assumptions, 6 108 NP would have accumulated in each secondary target organ during one year of continuous exposure. Although the accumulated doses in secondary target organs are three orders of magnitude below the lung dose, it is indeed not negligible. Note that extrapolation from rodent data to man requires great caution as indicated by other interspecies differences observed between rodents and man. These first estimations show that the slow clearance of NP from the human lung and low translocation into secondary organs of the body can imply high numbers of particles being retained in these organs after chronic exposure, including high retained surface area, which may be the relevant parameter for triggering inflammatory responses in these organ tissues. These numbers may be higher in patients and susceptible persons (infants and children). In addition, these numbers can reach and exceed the number of cells of an organ, and thereby have to be considered as a potential hazard for the organ. This estimate also highlights the importance of controlling NP emissions and exposure of the general population.
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157. Nemmar A, Hoet PH, Vanquickenborne B, et al. Passage of inhaled particles into the blood circulation in humans. Circulation 2002; 105(4):411–414. 158. Wiebert P, Sanchez-Crespo A, Falk R, et al. No significant translocation of inhaled 35 nm carbon particles to the circulation in humans. Inhal Toxicol 2006; 18:741–747. 159. Mills NL, Amin N, Robinson SD, et al. Do inhaled carbon nanoparticles translocate directly into the circulation in humans? Am J Respir Crit Care Med 2006; 173(4):426–431. 160. Mills NL, Tornqvist H, Robinson SD, et al. Diesel exhaust inhalation causes vascular dysfunction and impaired endogenous fibrinolysis. Circulation 2005; 112(25):3930–3936. 161. Mills NL, To¨rnqvist H, Gonzalez MC, et al. Ischemic and thrombotic effects of dilute diesel-exhaust inhalation in men with coronary heart disease. N Engl J Med 2007; 357(11):1075–1082. 162. Cruts B, van Etten L, Tornqvist H, et al. Exposure to diesel exhaust induces changes in EEG in human volunteers. Part Fibre Toxicol 2008; 5(1):4. 163. Roth C, Ferron GA, Karg E, et al. Generation of ultrafine particles by spark discharging. Aerosol Sci Technol 2004; 38(3):228–235. 164. Oberdo¨rster G, Ferin J, Lehnert BE. Correlation between particle size, in vivo particle persistence, and lung injury. Environ Health Perspect 1994; 102(suppl 5):173–179. 165. Oyabu T, Ogami A, Morimoto Y, et al. Biopersistence of inhaled nickel oxide nanoparticles in rat lung. Inhal Toxicol 2007; 19(1 supp 1):55–58. 166. Morrow PE. Possible mechanisms to explain dust overloading of the lungs. Fundam Appl Toxicol 1988; 10(3):369–384. 167. Kuempel ED, Tran C-L, O’Flaherty EJ, et al. Evaluation of particle clearance and retention kinetics in the lungs of U.S. coal miners. Inhal Toxicol 2000; 12(suppl 3):397–402. 168. Kuempel ED, O’Flaherty EJ, Stayner LT, et al. A biomathematical model of particle clearance and retention in the lungs of coal miners. Regul Toxicol Pharmacol 2001; 34(1):69–87. 169. Cullen RT, Tran CL, Buchanan D, et al. Inhalation of poorly soluble particles. I. Differences in inflammatory response and clearance during exposure. Inhal Toxicol 2000; 12(12):1089–1111. 170. Tran CL, Buchanan D, Cullen RT, et al. Inhalation of poorly soluble particles. II. Influence of particle surface area on inflammation and clearance. Inhal Toxicol 2000; 12(12):1113–1126. 171. Ferin J, Oberdo¨rster G, Penney DP. Pulmonary retention of ultrafine and fine particles in rats. Am J Respir Cell Mol Biol 1992; 6(5):535–542. 172. Oberdo¨rster G. Toxicology of ultrafine particles: in vivo studies. Philos Trans R Soc London A 2000; 358(1775):2719–2739. 173. Stoeger T, Reinhard C, Takenaka S, et al. Instillation of six different ultrafine carbon particles indicates a surface area threshold dose for acute lung inflammation in mice. Environ Health Perspect 2006; 114(3):328–333. 174. Li XY, Gilmour PS, Donaldson K, et al. Free radical activity and pro-inflammatory effects of particulate air pollution (PM10) in vivo and in vitro. Thorax 1996; 51(12):1216–1222. 175. Faux SP, Tran CL, Miller BG, et al. In vitro determinants of particulate toxicity: the dosemetric for poorly soluble dusts. Reseach Report 154. Crown, Norwich, UK: Health and Safety Executive, 2003. Report No.:154. 176. Beck-Speier I, Dayal N, Karg E, et al. Oxidative stress and lipid mediators induced in alveolar macrophages by ultrafine particles. Free Radic Biol Med 2005; 38(8):1080–1092. 177. Monteiller C, Tran L, MacNee W, et al. The pro-inflammatory effects of low-toxicity lowsolubility particles, nanoparticles and fine particles, on epithelial cells in vitro: the role of surface area. Occup Environ Med 2007; 64(9):609–615. 178. Lippmann M, Schlesinger RB. Interspecies comparisons of particle deposition and mucociliary clearance in tracheobronchial airways. J Toxicol Environ Health 1984; 13(2–3):441–469. 179. Warheit DB. Interspecies comparisons of lung responses to inhaled particles and gases. Crit Rev Toxicol 1989; 20(1):1–29.
6 The Epidemiology of Particle Health Effects JOEL SCHWARTZ Harvard School of Public Health, Boston, Massachusetts, U.S.A.
I.
Introduction
Trying to describe the epidemiologic associations between inhaled particles and adverse health is a daunting task, which I will mostly duck, by referring to such comprehensive reviews as the U.S. EPA Air Quality Criteria Document for Particles. Necessarily, in a short chapter, I will need to be selective. In keeping with the general tone of the book, I will select pulmonary effects as one of my foci, but given the extensive literature on cardiovascular effects of particles (1,2), which presumably occur because either particles or signals leave the lung and enter systemic circulation (3–5), I will also touch on some topics related to those outcomes. Lastly, since the major concern about airborne particles derives from their association with mortality, I will discuss recent findings about that as well. Even this subset far exceeds the limitations of this chapter—hence I will focus on findings that I find intriguing and particularly informative. I apologize in advance for slighting some other important factors and acknowledge that this is a subjective choice. Some data does inform this choice, and I am particularly drawn to epidemiologic findings that seem consistent with toxicologic findings. In particular, there are recent findings indicating that metals are important in the health effects of particles (6,7), and that metal processing genes matter as well (8). Oxidative stress has also been well documented in toxicologic studies of particles, including a role in the cardiovascular effects. Finally, there is toxicologic data indicating that particles are associated with atherosclerosis (9), thrombosis (10), blood pressure changes, and ischemic responses (11). All these have parallel epidemiology, and these convergent findings may help explain the reported associations between particles and early death.
II.
Pulmonary Effects
Lung function is one of the strongest predictors of cardiorespiratory health and longevity (12,13), although the causal pathway is poorly understood (14). In the 1950s, a study examined the lung function of British postal workers and found that workers in large urban areas with greater pollution consistently had lower lung function. The advantage of this study was that the postal workers generally stayed in the same counties all of their careers and were of the same social status. Since then, many other workers have reported
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cross-sectional associations between lung function and particles. A recent example is the paper of Lubinski and coworkers (15), who reported decreases in the ratio of forced expiratory volume in one second (FEV1) to forced vital capacity (FVC), and FEV1 as a percent of predicted associated with increased exposure to particles in nonsmoking young Polish men. A study of the rate of lung function growth in children, using data from within city variations in Mexico City, reported reduced rates of growth associated with living in higher particle parts of town (16). What have been missing, until recently, were investigations of the effects of changes in particle exposure. Change in exposure will be an important theme of this chapter for several reasons. First, if particle-induced changes in health are permanent, and we have to wait for a new generation before seeing public health improvements follow the exposure reductions, that has important public health implications. It certainly dramatically affects any cost-benefit analyses. Second, showing that a change in exposure produces a change in response, more directly addresses the causality of the association. Finally, cross-sectional comparisons between lung function, mortality rates, or any other response and cross-sectional variations in exposure have the potential to be confounded by any unmeasured predictors of outcome that vary geographically. In contrast, examinations of change, while subject to potential confounding like any observational epidemiologic study, are subject to a completely different set of confounders. Things, such as socioeconomic status or ethnicity, that vary geographically, potentially confound a comparison of geographic variation in exposure and outcome. In contrast, examinations of year-to-year changes in exposure within location do not suffer that potential confounding. Other variables, which do vary from year-to-year, can confound, but are unlikely to be correlated with the potential confounders of the cross-sectional associations. Hence, if associations are seen using this very different study design as well, it provides greater confidence that the associations are causal. Whereas the beneficial effects of smoking cessation on lung function are well established (17), until recently, there had been few studies of the effects of reductions in air pollution on lung function. A dramatic effect of intervention was seen by Avol and coworkers (18). They identified 110 children from the USC Children’s Health Study who moved from the study area, and followed them up in their new home with pulmonary function testing identical to that in the main cohort. Subjects who moved to locations with higher PM10 concentrations had lower rates of annual growth in lung function, while children who moved to locations with lower PM10 levels than they had left showed higher rates of growth in lung function. This effect was increased in subjects who lived in the new location for at least three years. More recently, a similar finding has been reported in adults. The Swiss Cohort Study on Air Pollution and Lung Diseases in Adults (SAPALDIA) is a prospective study of a random sample of adults who were given pulmonary function tests in 1991 and again in 2002. This study exemplifies another key development in recent years in studies of particles. Using fixed monitoring sites to characterize community exposure, as was done in the original cross-sectional analysis of this study (19), is being superseded by individual estimates of exposure. These estimates are derived either from interpolation techniques (20,21), dispersion models (22), or hybrid models that combine dispersion models with land use regression techniques, and are increasingly common. Individual estimates permit characterization of exposure differences across and within communities,
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improving the statistical power to detect pollution-related effects, and reducing potential confounding by reducing correlation with town level covariates. Such a hybrid model was used in SAPALDIA. In analyses adjusted for town of origin, covariates, and baseline exposure, a decrease of 10 mg/m3 in the annual average subject-specific PM10 concentration between examinations was associated with a 9% decrease in the annual rate of decline in FEV1 [i.e., by 3.1 mL/yr; 95% confidence interval (CI), 0.03–6.2], and a 16% decrease in the annual rate of decline in forced expiratory flow between 25% and 75% of volume out (FEF25–75) (i.e., by 11.3 mL per second/yr; 95% CI: 4.3–18.2) (23). Hence, lung function appeared to recover if particle exposure was reduced, even in adults.
III.
Cohort Mortality Studies
In 1970, Lave and Seskin published a paper regressing age standardized mortality rates in U.S. cities against average particle concentrations in those cities (24). The advantage of that study was that the mortality experience of the entire population of each city was compared to the average of the population-oriented monitors in the city. While individual exposures differed from the mean exposure, it seemed reasonable to assume that the exposure error was Berkson (a statistical term for measurement error where a group of people vary randomly around a mean exposure), and hence produced no downward bias in the estimated effect, since the average of all persons’ experiences was being compared to the average exposure. The difficulty was that no individual level covariates (i.e., other factors that may explain the differences between cities in mortality rates) were controlled, raising questions about confounding (e.g., by socioeconomic status, smoking, or occupational exposures) and ecological bias. More recent studies have alleviated that problem by recruiting cohorts of individuals in various areas and measuring individual covariates. The problem with most of those cohort studies is that they are convenience samples, and unlike the study of Lave and Seskin, do not capture the population mortality experience or the population average difference from the monitored exposure. If the convenience sample differed in health and exposure from the population mean similarly in all locations, this would be less of a problem. However, there is no reason to believe this is true. Specifically, the American Cancer Society (ACS) cohort was recruited from friends of the volunteers of the society (25). The ACS volunteers in city A may represent a healthier, and less exposed subset of city A than they do in city B. This, clearly, can introduce bias into the estimates. This is problematic for two reasons. First, there is potential confounding if, for example, the cities with higher exposures had systematically less healthy subjects recruited. I know of no reason to assume that this bias will always be in the same direction in each study. However, it does introduce a greater uncertainty (above the statistical uncertainty derived from the standard error of the estimate) into the estimate from such a study. Moreover, studies with greater possibilities of the relation between sample health and population health varying from city to city have a greater risk of this additional uncertainty and potential bias. Second, there is no longer any reason to assume that the exposure error is predominantly Berkson. This, fairly unambiguously introduces a downward bias.
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These concerns apply to all the cohort studies, with the obvious exception of the Six City Study (26). The Six City Study chose a neighborhood within each city, recruited a random sample of that neighborhood, and put a population-oriented monitor in the middle of each neighborhood. Most subjects lived within a few kilometers of that central monitor, and the assumption of Berkson error seems valid. Further, bias due to differential sampling in different locations was eliminated by the random sampling. This means that the extra source of uncertainty, and extra downward bias, present in the other studies is not present in the Six City analysis (27), suggesting this study should be given greater consideration. In contrast, many recent risk assessments have treated it as generating the high estimate. Further support for the hypothesis of a downward bias in the ACS study comes from a recent reanalysis of the ACS study that only used monitors in the same county of residence of each subject to assign exposure. That study found a higher coefficient for the effects of sulfate particles on mortality than the original study (28). Even more intriguingly, a recent study examined only the 22,905 participants of the ACS study living in southern California using a geographic information system-based exposure model, and reported even larger effect size estimates for PM2.5 (21). Similarly, the Women’s Health Initiative study found a larger effect on mortality when used within city exposure estimates (29). Hence, the use of more localized measures of exposure, with resultant lower exposure error, generally has resulted in larger effect estimates. Again, a key finding for cohort studies of mortality has come from studies examining changes in exposure and changes in mortality rates. Most of the cohort studies, including the original Six City Study, have contrasted a surrogate for long-term exposure with long-term survival. They tell us that people live less long in more polluted cities. They do not, directly, tell us what mortality reduction accompanies a reduction in exposure. A cross-sectional analysis of mortality rates and air pollution cannot tell us that, no matter how sophisticated the Cox proportionate hazard modeling. In a follow-up of the Harvard Six City Study, Laden and coworkers provide precisely that estimate (30). They examined a further 10 years of mortality in the six cities. In some cities, there was a substantial drop in pollution between the first and second follow-up periods, in some cities there was a moderate drop, and in some cities there was little or no change. The mortality rate ratios followed the same pattern: where there was a substantial drop in pollution, there was a substantial improvement in life expectancy; where there was little change in pollution concentrations, there was little change in life expectancy. The slope for change in exposure was similar to, but slightly higher, than the cross-sectional slope. Again, if the mortality rates change within town as the air pollution changes, and those changes fit on the same dose-response curve as the original cross-sectional association, this provides substantial assurance that the association is not confounded, because the factors that are likely to confound an association of temporal change are usually different from those that might confound a cross-sectional study, and there is no reason for the confounding of two different estimates by different confounders to produce similar estimated effects for particles. Another follow-up analysis of the Six City Study looked at year-to-year changes in particle concentrations to examine two questions—does the dose-response continue below 15 mg/m3; and what is the lag between change in exposure and change in mortality rate (31). Schwartz and coworkers, using a penalized spline with up to 18 degrees of freedom, showed that the association was essentially linear down to 8 mg/m3, where
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Figure 1 Shows the weighted average of 32 alternative dose-response curves between PM2.5 and the risk of dying in each year of follow-up relative to the risk at the lowest exposure level. The weights are proportional to how well each model fits the data, using a Bayesian model averaging approach. The hatched lines show the 95% confidence interval of the estimated risk at each exposure level, based on a Jacknife estimate.
the data become sparse, and that the effects of reduced particle exposure on mortality appear to be mostly seen within two years. Because the uncertainties around the dose-response curve from fitting a particular model do not reflect the uncertainty in model choice, they also used model averaging, where multiple models are fit explicitly, and averaged, weighted by their probability of being correct given the data. These models explicitly included the possibility of thresholds at multiple different particle concentrations. Figure 1 shows the dose response curve averaging across 32 different models, including ones with a range of thresholds, illustrating this result. The finding of a rapid change in mortality risk associated with change in particle exposure in the Six City Study fits nicely with the similar report for lung function from the SAPALDIA study. Finally, the study of Zanobetti and Schwartz examined over 190,000 subjects discharged alive from hospitals following myocardial infarctions (32). They looked at year-to-year changes in exposure within city and year-to-year changes in survival probability, adjusted for long-term time trend, in separate survival analyses within each of 21 cities. They then combined the results across cities. This approach does not allow any differences in exposure across city to contribute to the association (which is only examined within city), and again focuses on temporal changes in particles and mortality. Hence, as in the Six City analysis above, the set of potential confounders is quite
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different from those in a traditional cohort study. They reported a significant association with PM10, with, in this susceptible subgroup, larger coefficients than were seen in the Six City Study.
IV.
Traffic Exposure
Another major recent theme in particle research is the potential for greater toxicity from traffic particles, which are enriched in ultrafine particles. While a number of studies have reported that acute changes in intermediary biomarkers of risk are associated with traffic particle exposure, the results of studies of chronic exposure are worthy of particular attention, since the outcomes are more serious. For example, the Netherlands Cohort Study reported larger effects on mortality from exposure to local, traffic-related particles (33). In a study of a susceptible subgroup, Medina-Ramon and coworkers followed up 1389 patients hospitalized with acute heart failure in Worcester, Massachusetts, for survival over five years (34). An interquartile range increase in daily traffic within 100m of home was associated with a mortality hazard ratio (HR) of 1.15 (95% CI: 1.05, 1.25), while mortality risk decreased with increasing distance to bus routes (HR: 0.88; 95% CI: 0.81, 0.96). The bus route finding suggests diesel engines in particular may be a source of high-risk particles. In a related study, Tonne et al. examined 5000 cases of myocardial infarction in Worcester and 10,000 controls, and reported a 4% increase (95% CI: 2,7) in risk of myocardial infarction per interquartile range change in traffic density within 100m of home, and a 5% increase (95% CI: 3–6) per kilometer closer to major roadway (35). While the chronic exposure studies have most commonly studied serious adverse events, the most dramatic finding with traffic is the study of Peters and colleagues, who reported an odds ratio of 2.92 (95% CI: 2.22–3.83) for having a myocardial infarction while being in traffic in the past few hours (36). Other evidence for adverse cardiovascular effects of traffic particles comes from acute studies of intermediary markers of cardiovascular health. For example, O’Neill et al. (2005, 2007) reported that black carbon, a surrogate for traffic particles, was associated with a 13% decline in flow-mediated dilation of the brachial artery, which measures the ability of the peripheral arteries to dilate, and a 27% increase in serum markers of arterial inflammation (37,38). Similarly, black carbon showed a stronger association than PM2.5 with ST segment depression and heart rate variability (HRV) (39,40). A study putting participants on a bus, which enhances their exposure to traffic particles, also reported evidence that such exposures influenced HRV (41). One of the most intriguing recent findings about traffic particles is the report of Franco-Suglia et al. (42). They used a land use regression model of black carbon concentrations in Boston and estimated chronic exposure to traffic particles for school-aged children. Black carbon was associated with decrements in cognitive function of the order of 2 IQ points. This is a major potential health hazard that requires confirmation. However, it does have some toxicological support (3). Specifically, ultrafine particles, whose major source of ambient exposure is from traffic, have been shown in experimental protocols to translocate from the nose up the olfactory nerve into the brain, with exposures shown not only in the olfactory bulb but
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also in the striatum frontal cortex and cerebellum (43,44). This in turn is associated with increased inflammatory responses in the brain (45,46) as well as changes in neurotransmitter levels (47). More recently, exposure to diesel exhaust has been shown to produce changes in EEG patterns in human volunteers, indicative of cortical stress (48). Calderon-Garciduenas and coworkers have produced a series of studies of small populations of children and dogs in Mexico City and a control, much less polluted Mexican city. They have reported, in dogs, greater rates of prefrontal lesions, neuroinflammation, gliosis, and ultrafine particle deposition in Mexico City. MRI evaluation of the children’s brain also revealed greater prefrontal lesions, and autopsy studies of accident victims showed upregulation of cyclooxygenase-2, ILb, and CD14 in victims living in more polluted locations (49–51). The association between airborne particles and mortality implies very large public health impact. For example, the Laden paper suggests that an average 5 mg/m3 decrease in PM2.5 concentrations in the United States would be associated with a 5% to 10% decrease in all cause mortality, which is 100,000 to 200,000 fewer deaths/yr. While the large number of such studies, and the recent use of individual exposure estimates, changes in exposure, and so on adds considerable confidence that these associations are real, understanding what mechanisms might lead to the particle-mortality association is clearly important to assessing the causality of these findings. There have been many such studies in recent years, and again, I focus here on those that have confirmatory human studies. Some of those potential pathways are discussed below.
V. Intermediary Biomarkers A. Oxidative Stress
Animal experiments indicate that reactive oxygen species, which have established relevance in the pathogenesis of cardiovascular disease (52), are potential mediators for particle effects on HRV and other cardiovascular endpoints (2,53–55). Diesel particles have also been shown to increase oxidative stress in endothelial tissue, inducing the production of heme oxygenase-1 (56). The viability of cell cultures of microvascular endothelial cells was impaired by diesel particles with an accompanying large increase in induction of heme oxygenase-1 (57). A series of studies have examined the role of genes related to oxidative stress on cardiovascular effects of particles, particularly HRV. Schwartz (2005) and Chahine (2007) have reported that subjects that were GSTM1 null or with the long variant of HMOX-1 had enhanced HRV response to airborne particles, including a significant three-way interaction (58,59). Other studies have focused on oxidative stress per se. For example, Rossner and colleagues examined bus drivers in Prague and reported increased levels of indicators of oxidative stress such as F-2 isoprostane and 8-OHdG in drivers compared to controls (60). B. Metals and Genes Related to Metal Metabolism
A number of toxicologic studies have implicated metals on particles with adverse health effects (7,61,62). Most interestingly, a recent study using knockout rats for divalent metal transport protein 1 (which moves metals from extracellular to intracellular space)
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showed increased toxicity following exposure to metal-rich particles (8). This suggests that genetic variations in metal processing genes may be important. Recently, Park and colleagues showed that polymorphisms in the HFE gene modified the PM2.5 association with HRV in a cohort of elderly men (63). This suggests that such polymorphisms matter in humans at more modest doses. C. Thrombosis
Animal studies and controlled human exposure studies have demonstrated that ambient particles can increase prothrombotic activity and even induce thrombosis in acute exposures, as well as contribute to atherosclerosis (10,64,65). Again, there is recent epidemiology that supports this finding. Baccarelli and coworkers reported an association of airborne particles with decreased clotting time, as well as between longer-term exposure, and the risk of deep vein thrombosis (66,67). This is consistent with the results of a controlled exposure study to diesel particles, which reported increased ST depression and alterations in fibrinolytic capacity (65). In addition, Kunzli et al. have reported (again using a GIS-based exposure model) an association with chronic atherosclerosis (68). D. Blood Pressure Changes
Controlled human exposure studies have recently indicated that airborne particles are associated with acute changes in blood pressure. One study reported an association between arterial diameter and particle exposure (69). A follow-up study reported a direct association with blood pressure (70). This is supported by observational epidemiology studies in panels of subjects in Germany and the United States (71,72). However, the most exciting development in this field is one of the few uses of a randomized control trial in environmental epidemiology. McCracken and coworkers randomized houses in rural Guatemala with unvented open fires for cooking to either receive a chimney stove or not. Women over age 45 who cooked were examined in both groups, and a significantly lower blood pressure was found in the intervention group (73). At the end of the trial, the control group was given the same stove, and the investigators returned to measure blood pressure in the controls, using a pre-post design. Again, stove intervention was associated with a reduction in blood pressure. Overall, a rapidly developing body of literature is providing considerable support for the findings of association between airborne particles and mortality, indicating that this is a major public health problem.
References 1. Brook RD. Is air pollution a cause of cardiovascular disease? Updated review and controversies. Rev Environ Health 2007; 22(2):115–137. 2. Brook RD, Franklin B, Cascio W, et al. Air pollution and cardiovascular disease: a statement for healthcare professionals from the Expert Panel on Population and Prevention Science of the American Heart Association. Circulation 2004; 109(21):2655–2671. 3. Peters A, Veronesi B, Calderon-Garciduenas L, et al. Translocation and potential neurological effects of fine and ultrafine particles a critical update. Part Fibre Toxicol 2006; 3:13.
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4. van Eeden SF, Tan WC, Suwa T, et al. Cytokines involved in the systemic inflammatory response induced by exposure to particulate matter air pollutants [PM(10)]. Am J Respir Crit Care Med 2001; 164(5):826–830. 5. Salvi S, Blomberg A, Rudell B, et al. Acute inflammatory responses in the airways and peripheral blood after short-term exposure to diesel exhaust in healthy human volunteers. Am J Respir Crit Care Med 1999; 159(3):702–709. 6. Ghio AJ, Cohen MD. Disruption of iron homeostasis as a mechanism of biologic effect by ambient air pollution particles. Inhal Toxicol 2005; 17(13):709–716. 7. Molinelli AR, Madden MC, McGee JK, et al. Effect of metal removal on the toxicity of airborne particulate matter from the Utah Valley. Inhal Toxicol 2002; 14(10):1069–1086. 8. Ghio AJ, Piantadosi CA, Wang X, et al. Divalent metal transporter-1 decreases metal-related injury in the lung. Am J Physiol Lung Cell Mol Physiol 2005; 289(3):L460–L467. 9. Suwa T, Hogg JC, Quinlan KB, et al. Particulate air pollution induces progression of atherosclerosis. J Am Coll Cardiol 2002; 39(6):935–942. 10. Nemmar A, Hoet PH, Dinsdale D, et al. Diesel exhaust particles in lung acutely enhance experimental peripheral thrombosis. Circulation 2003; 107(8):1202–1208. 11. Wellenius GA, Coull BA, Godleski JJ, et al. Inhalation of concentrated ambient air particles exacerbates myocardial ischemia in conscious dogs. Environ Health Perspect 2003; 111(4): 402–408. 12. Tockman MS, Pearson JD, Fleg JL, et al. Rapid Decline in FEV1. A new risk factor for coronary heart disease mortality. Am J Respir Crit Care Med 1995; 151:390–398. 13. Hole DJ, Watt GC, Davey-Smith G, et al. Impaired lung function and mortality risk in men and women: findings from the Renfrew and Paisley prospective population study. BMJ 1996; 313:711–715. 14. Donaldson K, Stone V, Seaton A, et al. Ambient particle inhalation and the cardiovascular system: potential mechanisms. Environ Health Perspect 2001; 109(suppl 4):523–527. 15. Lubinski W, Toczyska I, Chcialowski A, et al. Influence of air pollution on pulmonary function in healthy young men from different regions of Poland. Ann Agric Environ Med 2005; 12:1–4. 16. Rojas-Martinez R, Perez-Padilla R, Olaiz-Fernandez G, et al. Lung function growth in children with long-term exposure to air pollutants in Mexico City. Am J Respir Crit Care Med 2007; 176(4):377–384. 17. Scanlon PD, Connett JE, Waller LA, et al. Smoking cessation and lung function in mild-tomoderate chronic obstructive pulmonary disease. The Lung Health Study. Am J Respir Crit Care Med 2000; 161:381–390. 18. Avol EL, Gauderman WJ, Tan SM, et al. Respiratory effects of relocating to areas of differing air pollution levels. Am J Respir Crit Care Med 2001; 164(11):2067–2072. 19. Ackermann-Liebrich U, Leuenberger P, Schwartz J, et al. Lung function and long term exposure to air pollutants in Switzerland. Study on Air Pollution and Lung Diseases in Adults (SAPALDIA) Team. Am J Respir Crit Care Med 1997; 155(1):122–129. 20. Schwartz J. Air pollution and blood markers of cardiovascular risk. Environ Health Perspect 2001; 109(suppl 3):405–409. 21. Jerrett M, Burnett RT, Ma R, et al. Spatial analysis of air pollution and mortality in Los Angeles. Epidemiology 2005; 16(6):727–736. 22. Cyrys J, Hochadel M, Gehring U, et al. GIS-based estimation of exposure to particulate matter and NO2 in an urban area: stochastic versus dispersion modeling. Environ Health Perspect 2005; 113(8):987–992. 23. Downs SH, Schindler C, Liu LJ, et al. Reduced exposure to PM10 and attenuated age-related decline in lung function. N Engl J Med 2007; 357(23):2338–2347. 24. Lave LB, Seskin EP. Air pollution and human health. Science 1970; 169(947):723–733. 25. Pope CA III, Burnett RT, Thun MJ, et al. Lung cancer, cardiopulmonary mortality, and long-term exposure to fine particulate air pollution. JAMA 2002; 287(9):1132–1141.
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26. Ferris BG Jr., Dockery DW, Ware JH, et al. The Six-City Study: examples of problems in analysis of the data. Environ Health Perspect 1983; 52:115–123. 27. Dockery DW, Pope CA III, Xu X, et al. An association between air pollution and mortality in six U.S. cities. N Engl J Med 1993; 329(24):1753–1759. 28. Willis A, Jerrett M, Burnett RT, et al. The association between sulfate air pollution and mortality at the county scale: an exploration of the impact of scale on a long-term exposure study. J Toxicol Environ Health A 2003; 66(16–19):1605–1624. 29. Miller KA, Siscovick DS, Sheppard L, et al. Long-term exposure to air pollution and incidence of cardiovascular events in women. N Engl J Med 2007; 356(5):447–458. 30. Laden F, Schwartz J, Speizer FE, et al. Reduction in fine particulate air pollution and mortality: extended follow-up of the Harvard Six Cities Study. Am J Respir Crit Care Med 2006; 173(6):667–672. 31. Schwartz J, Coull B, Laden F, et al. The effect of dose and timing of dose on the association between airborne particles and survival. Environ Health Perspect 2008; 116:64–69. 32. Zanobetti A, Schwartz J. Particulate air pollution, progression, and survival after myocardial infarction. Environ Health Perspect 2007; 115(5):769–775. 33. Hoek G, Brunekreef B, Goldbohm S, et al. Association between mortality and indicators of traffic-related air pollution in the Netherlands: a cohort study. Lancet 2002; 360(9341): 1203–1209. 34. Medina-Ramon M, Goldberg R, Melly S, et al. Residential exposure to traffic-related air pollution and survival after heart failure. Environ Health Perspect 2008; 116(4):481–485. 35. Tonne C, Melly S, Mittleman M, et al. A case-control analysis of exposure to traffic and acute myocardial infarction. Environ Health Perspect 2007; 115(1):53–57. 36. Peters A, von Klot S, Heier M, et al. Exposure to traffic and the onset of myocardial infarction. N Engl J Med 2004; 351(17):1721–1730. 37. O’Neill MS, Veves A, Sarnat JA, et al. Air pollution and inflammation in type 2 diabetes: a mechanism for susceptibility. Occup Environ Med 2007; 64(6):373–379. 38. O’Neill MS, Veves A, Zanobetti A, et al. Diabetes enhances vulnerability to particulate air pollution-associated impairment in vascular reactivity and endothelial function. Circulation 2005; 111(22):2913–2920. 39. Gold DR, Litonjua AA, Zanobetti A, et al. Air pollution and ST-segment depression in elderly subjects. Environ Health Perspect 2005; 113(7):883–887. 40. Rich DQ, Schwartz J, Mittleman MA, et al. Association of short-term ambient air pollution concentrations and ventricular arrhythmias. Am J Epidemiol 2005; 161(12):1123–1132. 41. Adar SD, Gold DR, Coull BA, et al. Focused exposures to airborne traffic particles and heart rate variability in the elderly. Epidemiology 2007; 18(1):95–103. 42. Suglia SF, Gryparis A, Wright RO, et al. Association of black carbon with cognition among children in a prospective birth cohort study. Am J Epidemiol 2008; 167(3):280–286. 43. Elder A, Gelein R, Silva V, et al. Translocation of inhaled ultrafine manganese oxide particles to the central nervous system. Environ Health Perspect 2006; 114(8):1172–1178. 44. Wang B, Feng WY, Wang M, et al. Transport of intranasally instilled fine Fe2O3 particles into the brain: micro-distribution, chemical states, and histopathological observation. Biol Trace Elem Res 2007; 118(3):233–243. 45. Kleinman MT, Araujo JA, Nel A, et al. Inhaled ultrafine particulate matter affects CNS inflammatory processes and may act via MAP kinase signaling pathways. Toxicol Lett 2008; 178(2):127–130. 46. Campbell A, Oldham M, Becaria A, et al. Particulate matter in polluted air may increase biomarkers of inflammation in mouse brain. Neurotoxicology 2005; 26(1):133–140. 47. Tin Tin Win S, Mitsushima D, Yamamoto S, et al. Changes in neurotransmitter levels and proinflammatory cytokin mRNA expressions in the mice olfactory bulb following nanoparticle exposure. Toxicol Appl Pharmacol 2008; 226(2):192–198.
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67. Baccarelli A, Zanobetti A, Martinelli I, et al. Effects of exposure to air pollution on blood coagulation. J Thromb Haemost 2007; 5(2):252–260. 68. Kunzli N, Jerrett M, Mack WJ, et al. Ambient air pollution and atherosclerosis in Los Angeles. Environ Health Perspect 2005; 113(2):201–206. 69. Brook RD, Brook JR, Urch B, et al. Inhalation of fine particulate air pollution and ozone causes acute arterial vasoconstriction in healthy adults. Circulation 2002; 105(13): 1534–1536. 70. Urch B, Silverman F, Corey P, et al. Acute blood pressure responses in healthy adults during controlled air pollution exposures. Environ Health Perspect 2005; 113(8):1052–1055. 71. Ibald-Mulli A, Timonen KL, Peters A, et al. Effects of particulate air pollution on blood pressure and heart rate in subjects with cardiovascular disease: a multicenter approach. Environ Health Perspect 2004; 112(3):369–377. 72. Zanobetti A, Canner MJ, Stone PH, et al. Ambient pollution and blood pressure in cardiac rehabilitation patients. Circulation 2004; 110(15):2184–2189. 73. McCracken JP, Smith KR, Diaz A, et al. Chimney stove intervention to reduce long-term wood smoke exposure lowers blood pressure among Guatemalan women. Environ Health Perspect 2007; 115(7):996–1001.
7 Cardiovascular Consequences of Particles MARK W. FRAMPTON and MARK J. UTELL University of Rochester Medical Center, Rochester, New York, U.S.A.
JONATHAN M. SAMET Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, U.S.A.
I.
Introduction
Increases in the concentration of fine particles in the air are associated with increases in mortality. The excess deaths are attributable to both cardiovascular and respiratory causes. The association has been observed in many cities worldwide; and perhaps most remarkably, remains strong at low particle concentrations previously thought to be without adverse health effects. Although the mechanisms by which particulate pollution induces health effects at such low mass concentrations remain unclear, considerable evidence has accrued focusing on the role of oxidative stress. Determining the biological mechanisms involved has been identified as a high-priority research need in the United States (1) and other countries. As suggested by the word cardiovascular, this chapter will explore hypotheses and evidence linking particle exposure with both cardiac and vascular dysfunction and disease. We will begin by considering the epidemiological database and its implications for mechanisms of health effects. The following sections explore the cardiovascular diseases that may confer increased susceptibility to the effects of particle exposure, and examine the experimental approaches being taken to move from speculation to understanding of the mechanisms involved.
II.
Implications of Epidemiological Data
A. Overview
Cardiovascular diseases are a leading cause of death worldwide and a substantial proportion of adults have preclinical cardiovascular disease. Consequently, any adverse effect of airborne particles on cardiovascular morbidity and mortality has substantial public health implications. Additionally, persons with cardiovascular disease are widely recognized as a population at increased risk from exposure to airborne particles and other air pollutants. A rapidly increasing body of epidemiological evidence links airborne particles to a wide range of cardiovascular effects, ranging from elevation of levels of biomarkers of injury to increased cardiovascular mortality (Table 1). These studies have been conducted throughout the world, in developed and developing
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Table 1 Health End Points Considered in Recent Studies of Particulate Matter
Health end points Long-term exposures
Short-term exposures
Cardiovascular mortality Blood markers of cardiovascular risk (fibrogen, platelets, white blood cells) Histopathologic markers of subclinical chronic inflammatory lung injury Cardiovascular mortality Cardiovascular hospital admissions Stroke mortality and hospital admissions Myocardial infarction Hypoxemia Heart rate Heart rate variability Inflammatory and related markers Cardiac arrhythmia/cardiac arrest/sudden out-of-hospital coronary deaths ST-segment depression Cardiac repolarization changes Blood pressure/arterial vasoconstriction/vascular reactivity and endothelial function
Source: Adapted from Ref. 3.
countries. This chapter provides a brief overview of this now substantial body of evidence, much of it reported in the last decade. The scope of the findings and its consistency in linking particulate matter to adverse health effects would not have been anticipated in the early 1990s, when concern began to mount about the public health consequences of airborne particles. More in-depth coverage can be found in the Environmental Protection Agency’s Criteria Document for Particulate Matter (1), in the 2004 statement of the expert panel convened by the American Heart Association (2), and in the 2006 comprehensive review written by Pope and Dockery (3). The epidemiological evidence is complementary to the diverse experimental studies on the cardiovascular toxicity of particulate matter. A variety of observational study designs have been used to investigate the cardiovascular consequences of airborne particulate matter (Table 2). These designs focus on effects at the individual and population levels and characterize effects on shorter and longer-term time frames. Many well-known studies are exemplary of these designs. B. Summary of Findings
The Harvard Six Cities Study, for example, is a cohort study on air pollution exposure and mortality that now spans several decades; exposure was characterized with measurements made across follow-up and all-cause and cause-specific mortality risks among participants were analyzed in relation to average concentration of particles in each of the cities (4). For acute effects, both time-series analysis and the case-crossover design have been used. In the time-series approach, the association between daily counts of events,
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Table 2 Epidemiological Study Designs Used to Investigate Cardiovascular Effects of PM
Individual level Design Cross-sectional survey Panel study Cohort study Case-crossover Population level Cross-sectional, ecological Time-series designs
Features Observations made at one point in time; comparisons made along exposure gradient Individuals, often with disease, followed for a short period with repeated exposure and outcome assessments People are followed for a long period with exposure estimated at the start, and possibly repeated Exposures compared during critical time window when event occurred versus windows without events Rates compared by levels of PM Association between daily event rates and PM levels on same and recent days assessed
Abbreviation: PM, particulate matter.
for example, cardiovascular hospital admissions, is examined in relation to pollutant levels on the same or recent days, controlling for other potentially relevant, time-varying factors, such as temperature. The National Morbidity, Mortality, and Air Pollution Study (NMMAPS) is a multicity, time-series study of particulate air pollution and daily mortality; one of the outcomes considered was the daily count of cardiovascular deaths (5,6). In the case-crossover design, the effects of time-varying exposures, such as particulate air pollution, are assessed by comparing exposure during a “case” period when an event occurs with exposure during a “control” period when the person was at risk but an event did not take place (7). For example, Peters and colleagues (8) carried out a case-crossover study of air pollution exposure, including particles, and acute myocardial infarction; exposures were compared for a window of risk corresponding to the hour before the event. They found that exposure to traffic was associated with increased risk, although exposure to airborne particles was not. The panel study is another design that assesses changes in outcome measures in relation to air pollution exposure. Typically, a relatively small group of participants, often a group considered to be susceptible, is followed for weeks or months with repeated observation of outcomes and tracking of exposures. For example, Delfino and colleagues (9) followed 19 children with asthma over a two-week period, measuring their lung function and using a nephelometer to assess exposures on a continuous basis. Inverse associations with lung function level were found with both estimated personal exposure and central-site concentration of particulate matter. The evidence from the epidemiological studies, interpreted in the context of other relevant research findings, has been found sufficient to conclude that airborne particulate matter is a cause of excess morbidity and mortality from cardiovascular disease (1–3). The evidence comes from hundreds of time-series studies of mortality and admissions to hospitals, including several multicity studies such as NMMAPS, cohort studies of longer-term mortality, panel studies, and studies of biomarkers. Much of the renewed concern about the health effects of airborne particles was motivated by the findings of time-series studies in the early 1990s. Figure 1, taken from
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Figure 1 Excess risk estimates for total nonaccidental, cardiovascular, and respiratory mortality in single-pollutant models for U.S. and Canadian studies, including aggregate results from two multicity studies (denoted in bold print). PM increments: 50 mg/m3 for PM10 and 25 mg/m3 for PM2.5 and PM10–2.5. Results presented from time-series studies that did not use GAM or were reanalyzed using GLM. Abbreviations: PM, particulate matter; GAM, Generalized Additive Model; GLM, Generalized Linear Model. Source: From Ref. 1.
the 2004 Criteria Document (1), gives estimates for the increased daily cardiovascular mortality associated with three different indicators of airborne particulate matter. The estimates are consistently increased and higher than those for all-cause mortality, presumably reflecting the greater susceptibility of persons with cardiovascular disease to particulate matter. One consideration in interpreting the time-series findings is the extent to which the observed associations reflect only a brief life shortening. The results of longer-term cohort studies are informative in this regard; they also show increased risk (Fig. 2) with the implication that airborne particulate matter poses a significant public health problem and does not merely advance the time of death of frail people, sufficiently ill to be poised to die, a phenomenon often referred to as mortality displacement (3). The major studies carried out within the United States provide consistent evidence of increased risk for cardiovascular mortality associated with exposure to PM2.5. The excess risk from the cohort studies is an order of magnitude larger than that from the time-series studies, and if such an excess holds across the full lifespan, airborne particulate matter would be anticipated to lead to substantial life shortening. The epidemiological evidence also links airborne particulate matter to cardiovascular morbidity. Figure 3, also from the Criteria Document (1), summarizes the timeseries studies on admission to hospital and emergency room visits for cardiovascular and
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Figure 2 Adjusted relative risk ratios for cardiovascular-related mortality associated with a
10-mg/m3 contrast in PM2.5 for selected long-term exposure studies. Source: From Ref. 3.
respiratory diseases. Numerous studies, again including multicity studies, link airborne particulate matter to this set of outcomes. Other research has explored various preclinical markers. Many have addressed heart rate and heart rate variability (HRV) using time-series approaches. Pope and Dockery (3) summarize 19 studies. In general, there is an indication that HRV is reduced in association with higher levels of exposure to particulate matter, suggesting that inhaled particles have an effect on the autonomic nervous system. In addition to presumed action via the autonomic nervous system, inhaled particles have been hypothesized as having adverse effects by causing inflammation. Pope and Dockery (3) also summarized studies that considered various biomarkers of inflammation and oxidative stress (Table 3). Associations were observed for a wide array of nonspecific biomarkers, including the level of C-reactive protein (CRP), blood cell counts, and exhaled nitric oxide. Reports with similar findings have been published since the review was published (27–29). There is concern that ultrafine particles (UFP, diameter < 0.1 mm) may be important contributors to health effects. Epidemiological studies of PM health effects generally utilize available measurements of ambient PM mass, such as PM10 or PM2.5. However, UFP contribute little to PM mass and yet dominate ambient particle number.
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Figure 3 Excess risk estimates for hospital admissions and emergency department visits for cardiovascular and respiratory diseases in single-pollutant models from U.S. and Canadian studies, including aggregate results from one multicity study (as denoted in bold print). PM increments: 50 mg/m3 for PM10 and 25 mg/m3 for PM2.5 and PM10–2.5. Results presented from time-series studies that did not use GAM or were reanalyzed using GLM. PM effect size estimates ( 95% confidence intervals) are depicted from the studies listed. Abbreviations: PM, particulate matter; GAM, Generalized Addictive Model; GLM, Generalized Linear Model. Source: From Ref. 1.
Ambient monitoring networks generally do not measure particle number, so there are limited UFP exposure data for use in epidemiological studies. Studies in Erfurt, Germany, have demonstrated a relationship between UFP exposure and increased cardiorespiratory mortality (30). Forastiere et al. (31) found a relationship between UFP counts and fatal out-of-hospital cardiac events. In Europe, in a large study of patients with stable coronary artery disease who underwent biweekly exercise tests, particle exposure was associated with increased ST depression on the electrocardiogram, indicating cardiac ischemia (32). Both UFP and larger particle exposures have been associated with changes in blood markers of vascular and systemic inflammation (33). In summary, a wide range of epidemiological evidence shows adverse effects of airborne particulate matter on cardiovascular health. The evidence has been judged sufficient to conclude that particulate air pollution causes both cardiovascular mortality and morbidity. Uncertainty as to the underlying mechanisms and limitations of the epidemiological evidence are barriers to quantitatively estimating risks at the low concentrations that now prevail in the United States and several other more developed countries.
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Table 3 Summary of Human Studies of Particulate Air Pollution Effect on Various Pulmonary or
Systemic Inflammation and Related Markers of Cardiovascular Risk Primary sources Peters et al. 1997 (10), Peters et al. 2001 (11) Seaton et al. 1999 (12)
Tan et al. 2000 (13)
Salvi et al. 1999 (14), Salvi et al. 2000 (15)
Pekkanen et al. 2000 (16)
Ghio et al. 2000 (17), Harder et al. 2001 (18), Gong et al. 2003 (19), Ghio et al. 2003 (20), Huang et al. 2003 (21), Ghio and Huang 2004 (22) Sorensen et al. 2003 (23)
Adamkiewicz et al. 2003 (24) Pope et al. 2004 (25) Ruckerl et al. 2006 (26)
Exposure type, place, subjects 1985 pollution episode, Augsburg, Germany, adults Estimated personal exposure to PM10, Belfast and Edinburgh, U.K., elderly adults Elevated PM10 levels during forest fire episodes, Singapore, 19- to 24-yrold healthy men Diesel exhaust, exposure chambers, health nonsmoking young adults
Ambient air pollution including PM10, London, male and female office workers Exposure to CAPs in exposure chambers, volunteer adults
Personal monitoring of PM2.5 and carbon black, Copenhagen, young adults Ambient PM2.5 Steubenville, OH, elderly adults Ambient PM2.5, Utah, elderly adults Ambient PM, Erfurt, Germany, 57 males with coronary heart disease
PM associations Increased blood plasma viscosity and CRP Increased CRP, reduced red blood cells
Elevated polymorphonuclear leukocyte band cells
Elevated neutrophils, lymphocytes, mast cells, endothelial adhesion molecules, IL-8, GRO-cx in airway lavage, bronchial tissue and/or bronchial epithelium; also increased neutrophils and platelets in peripheral blood Higher plasma fibrinogen concentrations
Somewhat mixed results, but small increases in neutrophils and fibrinogen consistent with mild inflammatory responses to PM Small increases in markers of oxidative stress Increase in airway inflammation as measured by exhaled nitric oxide Elevated CRP Elevated CRP
Abbreviations: PM, particulate matter; CRP, C-reactive protein. Source: Adapted from Ref. 3.
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C. Populations at Risk
In considering susceptibility, the likelihood of an adverse response to an inhaled pollutant depends on the degree of exposure to the pollutant and individual characteristics that determine the susceptibility of the exposed person. The relation between exposure and response may have different forms, depending on the mechanism by which the pollutant causes disease. If some minimum degree of exposure is required to produce disease, then a threshold is present. For most inhaled pollutants, the evidence indicates increasing risk with increasing exposure. Epidemiological studies suggest that the observed increases in mortality occur among individuals with underlying cardiovascular disease, particularly, ischemic heart disease. Recent studies also suggest that people with type II diabetes are at substantially increased risk for cardiovascular events related to increased particle levels (34). Additionally, because heart disease is the leading cause of death in the United States, even relatively small effects from particulate air pollution on cardiovascular mortality would have major public health consequences. The principal diagnostic labels assigned to this potentially susceptible population include coronary artery disease or ischemic heart disease, inclusive of persons with a history of angina, myocardial infarction, or coronary artery bypass surgery. Persons with coronary artery disease have atherosclerotic narrowing of the coronary arteries, which deliver blood to the heart. Additional cardiovascular consequences of atherosclerosis include congestive heart failure, atrial and ventricular arrhythmias, and reduced systemic blood flow. Short-term elevations of PM have also been shown to increase ischemic stroke mortality (35). The increasing numbers of U.S. adults with metabolic syndrome (MS) is a growing public health concern. The various definitions of MS include abdominal obesity, hyperlipidemia, hypertension, and abnormal glucose metabolism, all of which are important risk factors for atherosclerotic cardiovascular disease. Recent epidemiologic studies have demonstrated that the presence of certain component abnormalities of MS, such as obesity, hypertension, and diabetes, may further increase susceptibility to PM-associated cardiac effects. Recently, Chen and Schwartz (36) conducted a secondary analysis of white blood cell counts and MS data from the NHANES-III and PM10 data from the EPA Aerometric Information Retrieval System. In 2978 adults with no existing cardiovascular disease, they observed a statistically significant association between long-term PM exposure and white blood cell counts with the lower counts in the least polluted areas. The findings linking an increased inflammatory response across subpopulations with more MS component abnormalities support the increased susceptibility to PM-associated cardiovascular responses. Particle exposures may affect cardiovascular health by indirect mechanisms. For example, the obstructive sleep apnea syndrome is a very common condition, with wellknown adverse cardiovascular effects. It occurs in individuals whose body habitus is characterized by a narrow posterior pharyngeal airway: the relaxation of upper airway musculature during the rapid eye movement phase of sleep leads to upper airway obstruction, apnea, hypoxemia, and subsequent arousal. The hypoxemic episodes are often associated with changes in cardiac rate and rhythm, and sufferers are at increased risk for adverse cardiac events and for the development of pulmonary hypertension and cor pulmonale. Exposure to particulate air pollution could either worsen upper airway
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obstruction through nasal or pharyngeal irritation or enhance the severity of hypoxemic episodes by increasing ventilation-perfusion mismatching (37). The increases in mortality associated with particulate air pollution are greatest among the elderly. The prevalence of atherosclerotic vascular disease increases with age. Many physiological changes associated with aging may increase susceptibility to particle effects. Many components of the cardiovascular system are affected by aging, including myocardial contractility, electrical conduction, and valvular function. The finding of enhanced susceptibility in the elderly is supported by experimental data from animal models (38) and human exposure chamber studies (39). In a recent cohort study of 63,940 postmenopausal women in the Women’s Health Initiative Observational Study (40), the reported increase in mortality due to cardiovascular disease and stroke associated with PM2.5 (increased by 76–83% for each 10 mg/m3 increment of PM2.5) was much higher than what (9–18%) had been estimated previously in the general population (4).
III.
Cardiovascular Diseases of Concern and Their Pathogenesis
Exposure to increased levels of particulate matter has been associated with increased cardiovascular events including hospitalizations for myocardial infarction, congestive heart failure, arrhythmias, angina, and stroke. Changes in blood viscosity, decreased HRV, ST-segment depression, increased discharges of implanted defibrillators, increased blood pressure, and increased markers of thrombosis and inflammation have been associated with increases in particulate air pollution in panel and cohort studies. The development of atherosclerosis is a unifying feature in cardiovascular disease. The clinical manifestations of atherosclerosis are well known and include coronary heart disease, stroke, and peripheral vascular disease. An initial lesion in atherosclerosis involves the intima of the artery, which may begin in childhood with the development of fatty streaks. Early accumulation of cholesterol in the artery causes a reduction in arterial distensibility. More advanced lesions of atherosclerosis occur with smooth muscle cell migration into the intima and macrophage infiltration evolving into fibrous plaques; these events occur with increased frequency with aging (41). Advanced lesions, which do not become revascularized, lead to narrowing of the vessel lumen and ischemia with clinical consequences depending on the vessel site: angina with obstruction of the coronaries, stroke with obstruction of the carotids, and claudication with obstruction of the peripheral vessels. Multiple factors contribute to the pathogenesis of atherosclerosis, including endothelial dysfunction, dyslipidemia, inflammation, smoking, and air pollution. As atherosclerotic plaques develop and expand, they acquire their own microvascular network extending from the adventitia through the media and into the thickened intima. These thin-walled vessels are prone to disruption, leading to hemorrhage within the substance of the plaque (42). Atherosclerosis is generally asymptomatic until the plaque stenosis exceeds 70% or 80%, which can produce a critical reduction in flow, for example, with coronary blood flow to myocardium. These large lesions can produce typical symptoms of angina pectoris. However, acute coronary and cerebrovascular syndromes (unstable angina, myocardial infarction, sudden death, and stroke) are
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typically due to rupture of plaques with less than 50% stenosis. Tissue factor is the primary initiator of coagulation and, in advanced atherosclerosis, is found in the plaque. Tissue factor, as well as other factors such as enhanced platelet activity, may contribute to the development of thrombosis following plaque rupture. Tissue factor also plays a role in the progression of atherosclerosis via coagulation-dependent and coagulationindependent mechanisms.
IV.
Mechanisms
It is now clear that exposure to ambient PM is associated with mortality and morbidity from cardiovascular disease. There appear to be both acute and long-term cardiovascular consequences of PM exposure. It is less clear how inhaling particulate matter into the lungs can cause effects on the heart and blood vessels. A. Evidence for Direct Effects of Particles on the Cardiovascular System
One key consideration is whether cardiovascular effects are direct or indirect; that is, do particles gain access to the circulation and directly perturb function of blood vessels and the heart? Cell culture studies (43,44) show that UFP (<100 nm diameter) differ from larger particles in the way they enter cells: they appear to be able to diffuse through the lipid cell membrane, entering free into the cytoplasm, not enclosed in a membrane vesicle. From there they can enter the nucleus or mitochondria, and interfere with cellular energy processes. They can cross the epithelium, enter vascular endothelial cells, and even red blood cells, and be thereby transported to other organs. UFP activate blood platelets (45,46) and could enhance vascular thrombosis. However, the degree to which this happens in humans inhaling ambient air particles, and whether transported particles cause health effects, is in question. The data on translocation of UFP in humans are conflicting (47,48). In a recent study (49), nonsmokers, smokers, and COPD patients inhaled 100 nm carbon particles labeled with technetium 99m (Tc) and radioactivity was traced over 24 hours. Most of the deposited radioactivity was retained in the lung; there was no radioactivity detected in the liver. A cumulative 1.1% of the radioactivity was detected in the urine, which could be accounted for by the measured amount of leaching of Tc from the particles. Thus, translocation of 100 nm particles, if it occurred, was below the limits of detection in this study. It remains possible that, even if intact particles do not enter the bloodstream in quantities sufficient to elicit direct effects, particle chemical components, reactive oxygen species, or secondary reaction products may enter the circulation and exert effects beyond the lung. B. The Role of Pulmonary Inflammation
Another of the key unresolved questions in the cardiovascular effects of PM exposure is the following: Are the cardiovascular effects a consequence of PM-induced pulmonary inflammation (33)? Exhaled NO (eNO) is considered a marker of airway inflammation. Some epidemiological studies have shown associations between increases in ambient
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PM or exposure to traffic and eNO concentrations in children with asthma (50,51). Exposure to relatively high concentrations of diesel exhaust, a mixture of PM and gases, induced airway inflammation (52,53). However, inhalation of elemental carbon UFP did not affect pulmonary function, inflammatory cells in induced sputum, or eNO in healthy and asthmatic adults (54,55). Exposure to concentrated ambient fine particles induced mild inflammatory responses as assessed by bronchoalveolar lavage (17,56). However, clinical studies have generally not found increases in markers of systemic inflammation, such as CRP, following PM exposure. Overall, the inflammatory effects of PM, at concentrations comparable to ambient appear to be relatively mild and may not explain the epidemiological findings of increased cardiovascular mortality and morbidity associated with PM exposure. It is likely that other, more direct mechanisms also play a role. Figure 4 provides a schematic summary of some of the pathways that may be involved in PM cardiovascular effects. Two of the primary pathways involve (i)
Figure 4 Hypothesized mechanistic pathways for effects of exposure to PM. Abbreviations: NO, nitric oxide; TF, tissue factor; ET, endothelins; HRV, heart rate variability; MI, myocardial infarction; PM, particulate matter.
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perturbation of vascular function or endothelial dysfunction, and (ii) alterations in the autonomic nervous system, possibly induced by PM effects on the airways. C. Endothelial Function
Endothelial dysfunction is the underlying and earliest abnormality in atherosclerotic vascular disease (57). A healthy endothelium maintains vascular homeostasis, including control of blood pressure, regulation of leukocyte traffic, and control of thrombosis (58) primarily through production of NO via action of NO synthases (NOS) on L-arginine. Local NO release by endothelial cells diffuses to vascular smooth muscle to maintain vasodilatation, and also diffuses into the blood, where it reduces platelet adhesion and aggregation, reduces leukocyte adhesiveness and expression of adhesion molecules, downregulates leukocyte NADPH oxidase function, scavenges superoxide anion (59), and prevents coagulation by inhibiting tissue factor expression in endothelial cells. NO also reacts quickly with hemoglobin and plasma proteins. These complex interactions may allow NO to regulate vascular tone in response to hypoxia, via release of nitrite (60). Many of these effects are opposed by endothelin-1, a potent vasoconstrictor (61). Endothelial NO is reduced and endothelial function is impaired in cigarette smoking, diabetes, hyperlipidemia, and atherosclerosis (57). Endothelial dysfunction precedes the development of atherosclerotic plaques. Superoxide and other reactive species are released in the inflamed vascular wall by leukocytes and may be generated by reactive chemical species delivered on the surface of inhaled particles (62). Depletion of NO occurs in part because of NO reactions with O2–, producing peroxynitrite, a strong oxidant. Both O2– and peroxynitrite participate in the oxidation of low-density lipoprotein, with further damage to the endothelium (63). PM effects on endothelial function could explain both acute and long-term cardiovascular effects of PM. The human circulatory system is equipped with two vascular beds with markedly differing physiology. The pulmonary system operates under a low pressure, has thinwalled conduit vessels, and a network, all of capillaries closely apposed to the alveolar air spaces for the purposes of gas exchange. Blood leukocytes, including polymorphonuclear leukocytes and monocytes, squeeze slowly through this capillary bed, so that more than 60% of blood leukocytes are in the pulmonary circulation under resting conditions. The systemic circulation, which includes the coronary arteries, is more extensive, operates under a much higher pressure, with thick-walled vessels. Shear stress plays an important role in systemic vascular function. In both vascular beds, NO plays a key role in maintaining vascular homeostasis and patency, and in controlling inflammation. Systemic hypertension and pulmonary hypertension are both associated with reduced bioavailability of NO. D. PM Effects on Vascular Function
There is growing evidence that exposure to combustion-related PM alters endothelial function in both the systemic and pulmonary vascular beds. In human clinical studies, one-hour exposures to freshly generated diesel exhaust at a PM concentration of 300 mg/ m3 impaired vasodilatory responses in the forearm in healthy subjects (64). In patients with previous myocardial infarction, two-hour exposures to *300 mg/m3 diesel exhaust
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increased cardiac ischemia during exercise and impaired acute endothelial release of plasminogen activator, a response that favors coagulation (65). Animal models of coronary artery disease indicate exposure to PM can worsen ischemia (66). Pulmonary vascular function may also be affected by pollutant exposure. Inhalation of 50 mg/m3 carbon UFP for two hours, with intermittent exercise, altered peripheral blood leukocyte phenotype (67) and reduced the pulmonary diffusing capacity for carbon monoxide (54), findings which are consistent with effects on pulmonary vascular function. Inhalation of UFP also reduced forearm reactive hyperemia, a measure of systemic vascular responsiveness, and reduced plasma nitrate levels, findings consistent with reduced NO availability and subtle systemic vascular effects of UFP exposure (68). In contrast, exposure of healthy subjects and patients with stable coronary artery disease to concentrated ambient particles that were low in combustion products did not elicit alterations in systemic endothelial function (69). PM vascular effects thus appear to differ with particle source and composition. E.
PM Effects on the Autonomic Nervous System
The second major pathway, effects on the autonomic nervous system, may contribute to, or serve as a marker of, adverse cardiac effects such as arrhythmias or myocardial dysfunction. Animal studies using whole-organ chemiluminescence suggest PM effects on the autonomic nervous system may be mediated via oxidative stress. Rats exposed to PM via instillation or inhalation showed increased oxidative stress in the heart, which was prevented with the antioxidant N-acetylcysteine, and by blockade of the autonomic nervous system with either atenolol or glycopyrrolate. Reduced HRV has been found to be a risk factor for cardiovascular events (66). A number of panel studies of ambient air pollution exposure have shown relationships between HRV and PM exposure. For example, Adar et al. (70) found HRV parameters to be reduced with increased exposure to traffic-related pollution. Forty-four nonsmoking seniors, aged more than 60 years, some with cardiac risk factors and coronary heart disease, participated in four two-hour trips to downtown St. Louis between March and June of 2002, aboard a diesel-powered bus. The investigators found that exposure to PM2.5 and black carbon was associated with reduced HRV. However, not all studies have found PM effects on HRV, and the specific HRV effects have not been entirely consistent. For example, Riediker et al. (71) monitored nine young (average age 27 years) healthy nonsmoking male North Carolina Highway Patrol troopers on four consecutive days while working a 3 PM to midnight shift and riding in gasoline-powered patrol cars. In-vehicle PM2.5 (average of 24 mg/m3) was associated with increased HRV associated with in-vehicle PM2.5 exposure, a finding that differs from the negative association observed by Adar et al. The differing findings regarding HRV may be due to differences in age, cardiovascular fitness, or prevalence of heart diseases among the subjects in the two studies. PM effects on HRV in human clinical studies have also been inconsistent. In summary, evidence exists for multiple mechanistic pathways by which PM exposure may adversely influence cardiovascular health. Key pathways include changes in autonomic nervous system function, effects on endothelial function, and effects on blood coagulation. These mechanisms are likely involved in both acute and long-term cardiovascular consequences of PM exposure. More clearly defining the mechanisms
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involved in PM cardiovascular effects, and the particle characteristics most important in these effects, should help in the development of preventive strategies.
V. Summary Exposure to particles in the air is associated with increased mortality in the elderly with chronic heart or lung disease. The mortality data are coherent with morbidity data and data on hospitalizations for cardiovascular disease, and suggest a sizeable pool of chronically ill individuals are susceptible to the acute and chronic exposure to particles in the air. Worldwide, outdoor PM pollution is estimated to cause 50,000 excess deaths annually. The experimental database has dramatically improved our understanding of the mechanisms by which exposures to very low levels of particles exacerbate both cardiovascular and respiratory diseases. In addition, efforts to identify and link specific components of the PM mix with various PM-associated health effects are intensifying. Key PM characteristics hypothesized to promote responses are size and surface area, metal content, organic content, and ability to generate reactive oxygen species. As we better understand these relationships, the tool of source apportionment takes on increasing importance for controlling exposures and reducing the health impacts.
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8 Autonomic Mediation of the Cardiac Responses to Particle Exposure BEATRIZ GONZALEZ-FLECHA Harvard School of Public Health, Boston, Massachusetts, U.S.A.
I.
Introduction
Three hypotheses have been put forth in the past few years to explain the mechanisms of cardiotoxicity by ambient particulate matter (PM). Namely, that the cardiac effects of PM are mediated by: (i) increased autonomic influence on the heart, (ii) proinflammatory cytokines or chemokines produced by lung cells and released into the blood stream, and/or (iii) direct effect of ultrafine particles or soluble particle components that could leach out of the particles and reach the heart. The ability of fine PM to affect the autonomic input on the heart and to trigger pulmonary and systemic inflammatory responses, as well as that of ultrafine particles or some soluble components to reproduce some of these effects in animal models is well documented (1,2). Yet, the question on whether these occurrences are mechanistically related to the cardiotoxicity of PM remains unanswered. This chapter reviews some of the work in our laboratory implicating the changes in vagosympathetic balance triggered by PM and the accompanying increases in oxygen free radicals (ROS), as critical steps in the mechanism leading to PM induced cardiotoxicity. Inflammation is a well-known response of the lungs to the inhalation of gaseous or particulate pollutants. Pulmonary inflammation has been reported in response to inhalation and instillation exposures to PM (2,3) and is discussed in detail in previous chapters of this book. In the model discussed here, one of inhalation exposure to concentrated ambient particles (CAPs) in rats, inflammatory responses are detectable 24 hours after the end of the exposure (4). Cardiac responses, on the other hand, are detectable within the first few minutes of exposure and therefore are unlikely to be attributable to inflammatory mediators. The direct effects of soluble components or ultrafine particles cannot be ruled out, but they seem unlikely since their concentrations are estimated to be below the trace amounts found in biological samples. For example, the maximal amount of iron (the more abundant metal in Boston CAPs samples) potentially delivered by average CAPs aerosols is estimated to be *4 mg or 70 nmol (5). Assuming an intrapulmonary deposition fraction similar to that calculated for Boston CAPs in healthy humans [*20%, (6)], the maximal yield would be 14 nmol of Fe (5). Copper complexes (7,8), vanadium, manganese (9), and other metals (10) are required at millimolar or submillimolar concentrations to trigger responses in the lung. Similarly, a quick calculation of the level of
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ultrafine particles in the PM2.5 aerosols used in our last study (5) yields an approximately 0.4% of the CAPs mass (i.e., *5 mg/m3). This amount is about 30-fold lower than that used by Elder et al. to simulate episodes of high increases of ultrafine particles in urban air (11,12). Nonetheless, although it is clear that CAPs aerosols cannot deliver concentrations of ultrafine or metals in the toxic ranges, we cannot rule out the possibility that a combination of several redox-active components or ultrafine particles could approach the thresholds for activation of cellular responses at the hot spots for deposition in the lung, where the concentrations are estimated to be thousands of times higher (13). This could account for the triggering of pulmonary responses as well as responses in other tissues, provided that they are initiated by activation of pulmonary receptors at the deposition hot spots. Our experimental approach is based on the use of two technologies currently available in the laboratory, the Harvard Ambient Particle Concentrator, and the measurements of organ chemiluminescence (CL) for the in vivo determination of oxidative stress. The Harvard Ambient Particle Concentrator [complete description in (14)] utilizes the principle of virtual impactors to concentrate ambient particles with aerodynamic diameter less than 2.5 mm (size cut 0.1–2.5 mm) for subsequent aerosol exposure of animals, without altering particle composition or size distribution (15,16). Each exposure is characterized by measurements of integrated CAPs mass concentration (gravimetric) and trace metal concentrations (X-ray fluorescence), and by continuous monitoring of black carbon, a surrogate for elemental carbon, and particle number concentration (NC). These measurements accurately define the exposure scenario, a critical point since variations in PM composition are an important source of variability in the biological effects among different studies. Boston ambient PM2.5 is typical of northeastern urban fine particle pollution, comprised of both local and regional sources including oil combustion; traffic-derived PM; secondary production of sulfates, nitrates, and organics via photochemical reactions; soil/crustal material; and marine aerosol (17). The studies discussed here were carried out between 2001 and 2007. The average CAPs composition in these studies was very similar, with average maximal variations of 0.5- to 2-folds in total mass, Al, S, Cl, V, Mn, Zn, and Br concentrations with respect to the mean values for all studies. The CAPs mass concentrations ranged from 100 to 950 mg/m3, with extreme values of *2.5 mg/m3 for a few days in 2002 and 2003. Detailed compositions data is given in each publication (4,5,18,19). In average, this sample is representative of human “real world” exposures in some transport environments (20) and highly populated cities (21). A. In Vivo Measurements of Oxidative Stress
The determination of in situ CL was developed as an assay to measure the steady-state concentrations of oxygen free radicals in situ in intact organs, in real-time, and in a noninvasive way (22–25). Organ CL is a low-intensity emission in the visible range mainly due to the decay of excited states of molecular oxygen (singlet oxygen) and excited carbonyls (22,24), which are formed during the termination steps of the chain reaction of lipid peroxidation (26). Organ CL follows the square of the intracellular
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concentration of H2O2 (27,28), a unique experimental advantage of the technique since small variations in [H2O2] are exponentially reflected in the values of CL. Organ CL has been successfully used previously to show increased oxidative stress in the lung of rats exposed to Paraquat (29) or hyperoxia (30), the perfused lung ex vivo (31), the liver and brain of rats exposed to hyperbaric hyperoxia (32), and the heart of mice treated with Doxorubicin (33). Here we review the use of this technique to assess oxidative stress in a model of heart toxicity by PM and in that way, to study the role of ROS on the cardiac effects of PM. B. Cardiac Oxidant Stress After Particle Inhalation
Adult Sprague-Dawley rats exposed to CAPs, but not control inert particles, for periods of one to five hours show significant increases in the steady-state concentrations of oxidants in the lung and heart but not in liver (18). The CAPs-dependent two-fold increases in oxidants in the lung and heart are associated with oxidant-dependent lung and heart edema, and with significant increases in the serum levels of lactate dehydrogenase (LDH) and of creatine kinase (CK), indicating an injurious effect of CAPs at environmentally relevant concentrations. While lung oxidants increase, in average, immediately upon exposure to CAPs, significant oxidative stress in the heart is observed only after a one-hour lag phase (18). Also, while the increases in lung CL are associated to the CAPs concentrations of redox-active metals (Fe, Cu, Mn, and Zn), heart CL shows strong associations with Al, Si, Fe, and Ti, metals considered as reporters of crustal components of CAPs (18). These differences clearly point to different mechanisms accounting for CAPs-induced oxidative stress in the lung and heart. C. Organ CL as a Predictor of Oxidative Damage
CL is an early and sensitive marker of oxidative stress, and therefore it can predict the development of cellular, subcellular, or tissue damage when and if they are caused by ROS. This is illustrated most clearly in our early work using an in vitro version of the CL assay to estimate mitochondrial damage in heart biopsies from patients undergoing revascularization surgery (34). Open-heart surgery requires that the heart is subjected to ischemia/reperfusion, and the amount of damage associated to this procedure can be accurately estimated by measuring the increases in heart CL in biopsies taken before and after the surgery. In this model, since ischemia/reperfusion in the heart causes mainly mitochondrial damage, the increases in the values of CL correlated with increases in the levels of mitochondrial damage measured by electron microscopy [r ¼ 0.88, p < 0.001 (34)]. Electron micrographs of human heart biopsies were examined morphometrically under a grid and mitochondrial damage was scored using the criteria of Kloner. Mitochondrial damage was graded 0 through 4 based on severity of swelling and membrane integrity (34). In the model of exposure to CAPs aerosols, increases in heart CL precede and are associated to mild tissue damage (18), are not observable in rats exposed to filtered air, and are abrogated by pretreatment with antioxidants like N-acetyl cysteine (4) or MnTBAP [manganese (III) tetrakis (4-benzoic acid)porphyrin] (35). Furthermore, rats exposed to particle-free (filtered) ambient air for periods of one to five days show a progressive decrease in the levels of lung, heart, and liver CL over time (Fig. 1) (30)
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Figure 1 Spontaneous CL from the lung, liver, and heart of animals exposed to filtered air for
one, three, and five days. At the end of each period, animals were anesthetized and the in situ CL of the lung (filled circles), liver (empty circles), and heart (empty squares). Each point represents the mean of 4 to 6 determinations SEM. Abbreviation: CL, chemiluminescence. Source: Data from Ref. 30.
showing that CL measurements correlate with the levels of ROS, specifically superoxide anion, produced by PM. The fact that increases in CL precede tissue damage indicates that oxidative stress is directly involved in the mechanism of damage, and this is confirmed by the abrogation of PM-induced lung inflammation (4) and changes in cardiac rhythm (19) by antioxidants. Thus, it follows that CL can be used as a marker of oxidative stress and a predictor of oxidative damage by PM.
II.
Experimental Evidence Supporting the Neural Hypothesis
Ambient particles may elicit cardiovascular effects, in part, through the autonomic nervous system. Support for this hypothesis has come from a number of studies showing that short-term exposure to particles is associated with changes in autonomic function as assessed by heart rate variability (HRV: the beat-to-beat alterations in heart rate) (14,36– 41). Measures of HRV derived from electrocardiographic recordings provide a quantitative, noninvasive marker of cardiac autonomic nervous system control (Task Force of the European Society of Cardiology and the North American Society of Pacing and
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Electrophysiology 1996). These studies have generally found that increased levels of PM are associated with changes in heart rate variability that are consistent with perturbations of both sympathetic and parasympathetic activity leading to relative sympathoexcitation in some cases, and to relative vagal predominance in others [reviewed in (1)]. These results and some mechanistic studies in our laboratory led to the formulation of the working hypothesis illustrated in Figure 2. Some of the steps in this pathway have been experimentally tested and will be discussed below.
Figure 2 Schematic representation of the hypothetical mechanism of production of ROS, acti-
vation of autonomic responses, and cardiac effects by ambient air particles. Abbreviation: ROS, reactive oxygen species.
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If autonomic signals are important for the lung-to-heart communication after PM deposition, it follows that dissecting the neural connections innervating the heart should abrogate PM effects on cardiac ROS and function. Animal models of syngeneic heart transplantation utilize mice that receive a syngeneic heart transplanted into the abdomen via the descending aorta and inferior vena cava (Fig. 3) (42,43). This beating heart model is used primarily in studies of cardiac transplant rejection, especially with tissue mismatches. For our purposes, we used mice with genetically identical tissues (isografts), so rejection does not occur (44–47). The syngeneic heart transplantation model allowed us to investigate the effect of CAPs on the intact native heart and the transplanted heart where no direct autonomic nervous connection is present. If CAPs effects on heart CL require intact autonomic innervation, the native heart will show effects while the transplanted heart should show no effect. On the other hand, if the cardiac effects were due to inflammatory mediators of particle components that would reach the heart via the bloodstream, both hearts would show analogous responses. When mice with syngeneic transplanted hearts were exposed to CAPs aerosols (CAPs mass concentration: 350 mg/m3), the control hearts (with normal innervation and perfusion) responded to the inhalation as the hearts of control animals, with a two-fold increase in their levels of oxidants (Fig. 3). In contrast, the hearts transplanted into the abdomen (with perfusion but no innervation) failed to respond to the same stimuli. These results indicate that CAPs effects on heart CL require an intact cardiac innervation.
Figure 3 PM effect on heart CL in a model of heart denervation. The cartoon on the left shows the anatomical localization of the transplanted heart (Isograft). Mice were exposed to CAPs aerosols (CAPs mass concentration: 350 mg/m3) or filtered air (Sham) for five hours. Bars represent the mean of four determinations SEM. * p < 0.01. Abbreviations: CL, chemiluminescence; CAPs, concentrated ambient particles.
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B. Neural Signals from the Lung
A reasonable conclusion from the previous data would be that the autonomic connections between the lung and heart must be intact for the cardiac effects of PM to manifest. Hence, we would expect that denervating the lung would abrogate PM effects on cardiac oxidants and function, in a similar way that cardiac denervation did. We used a model of transection and reanastomosis of the left lung in rats (48,49). This model is used primarily in studies of mucociliary transport and lung transplantation. For our purposes, this model allowed us to investigate the effect of PM on the intact native lung (right lung) and the denervated lung (left lung) where no direct autonomic nervous connection is present. If PM effects on heart CL require intact autonomic innervation, instillation of PM on the native lung should show cardiac effects while the instillation in the denervated lung should show no effects. Urban air particles (UAP: Standard Reference Material 1649: Urban Dust, NIST, Washington, United States) were instilled into the left or right lung of denervated animals five days after surgery, when the baseline of CL in the denervated lung had returned to control values (n ¼ 6, p > 0.05). The results for animals instilled on the denervated (left) lung are shown on Figure 4. Consistent with the previous results, denervation of the lung abrogates the cardiac response to PM. The responses to UAP in the control (right) lung were equivalent to those of control animals (50) (manuscript in preparation). UAP have similar proportions of trace metals than the CAPs aerosols used in these studies but are richer in Fe, Br, and Pb (19). Additionally, UAP includes a range of
Figure 4 PM effect on heart CL in a model of lung denervation. UAP were instilled into the
denervated lung, and the cardiac responses were measured by means of heart CL. Bars represent the mean of four determinations SEM. * p < 0.01. Abbreviations: PM, particulate matter; CL, chemiluminescence; UAP, Urban air particles; Den, denervated.
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particles sizes, including not only fine respirable particles but also some coarse nonrespirable ones. C. PM-Induced Sympathovagal Imbalances and Cardiac ROS
Rats exposed to either intratracheal instillation of UAP or to inhalation of CAPs (mass concentration: 740 300 mg/m3) show changes in cardiac oxidants and function as a result of the exposure. Heart CL and heart rate variability (SDNN, the standard deviation of beat-to-beat intervals) were significantly increased in rats exposed to PM (19). The electrocardiogram (ECG) of healthy individuals exhibits periodic variation in beat-tobeat intervals, a fluctuation predominantly mediated by respiratory gating of parasympathetic efferent activity to the heart. Reduced HRV is then used as a marker of reduced vagal activity and of higher risk of sudden cardiac death. On the other side, increases in HRV would be indicative of increased vagal activity, but these changes have not shown any clear association with risk of sudden cardiac death. Consistent with the results presented below, the increases in HRV found in this model are therefore indicative of increased vagal tone on the heart after PM exposure, but not necessarily of a positive effect of PM on the heart. Using heart CL as a marker of oxidant stress and a predictor of changes in heart rate variability, we tested the implication of autonomic stimulation in the responses to CAPs by using sympathetic and parasympathetic antagonists. Both atenolol (a b-1 receptor antagonist) and glycopyrrolate (a muscarinic receptor antagonist) abrogated the effects of PM on heart CL. Consistently, analysis of the high and low frequency components of HRV indicated that instillation exposure to PM leads to increases in both sympathetic (50%) and parasympathetic (110%) stimulation with a predominance of parasympathetic stimulation (19). This study clearly shows a mechanistic sequence connecting PM exposure, autonomic activation, increases in cardiac ROS, and changes in cardiac function. Increased production of ROS and autonomic activation appear to occur simultaneously and form a self-enhancing loop, since inhibition of either one of these steps inhibits the other. PM deposition in the lung is thus immediately sensed, resulting in a complex imbalance in cardiac autonomic activity with activation of both sympathetic and parasympathetic neurons. The predominance of one over the other in a given model may be determined by other physiological imbalances (i.e., preexisting inflammation, endothelial damage, etc.) or to specific components present only in some PM. D. ROS as Central Mediators of Cardiac Dysfunction by PM
Changes in heart rate variability due to PM exposure can be prevented by preadministration of N-acetyl cysteine, a generic antioxidant, clearly connecting increases in ROS with changes in cardiac rhythms (19). To date, we can only speculate about the source of PM-induced ROS in the heart. However, a novel finding of our studies is that autonomic stimulation (i.e., intravenous injection of either sympathetic or parasympathetic agonists) increases heart CL (19). Furthermore, as described above, both sympathetic and parasympathetic antagonists prevent the development of cardiac oxidant stress by PM. These observations point to autonomic excitation of cardiac receptors as a critical point for the increase in production
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of ROS. Both sympathetic activation and acetylcholine can, after receptor activation, cause oxidant stress in cardiomyocytes (51,52). Furthermore, blockade of both sympathetic and parasympathetic receptors lead to decreases in ROS (53). In this way, receptor-mediated effects on intracellular sources of ROS by both sympathetic and parasympathetic transmitters could cause the observed increases in cardiac oxidants after PM exposure. Although this would contradict the basic physiological rule that the sympathetic and parasympathetic systems exert opposite effects on the heart (i.e., sympathetic stimulation increases heart rate whereas parasympathetic stimulation decreases it), the chemical/cellular processes leading to increased ROS production are expected to be more ubiquitous and dependent on nonspecific electron transfer processes (54) rather than to highly specific and controlled transmission through either sympathetic or parasympathetic neurons. E.
Pulmonary Reflexes: The Lung-Heart Link?
The responses discussed in the previous sections show that increases in PM levels in ambient air are sensed immediately. One possible mechanism for such a sensing would be the activation of pulmonary reflexes. The lung is equipped with a variety of receptors to sense the presence of irritants, oxidants, or other noxia. Cardiopulmonary reflexes include bradycardia, apnea, and hypotension (55,56) and are aimed to reduce the amount of inspired pollutant transported into the blood stream (57). Pulmonary chemoreflexes initiated by nocireceptors such as the transient receptor potential (TRP) vanilloid receptor family affect cardiovascular function by altering the vagosympathetic tone on the heart (58). Several reports show that activation of vanilloid (capsaicin) receptors by PM induces some of the known responses to PM in the lung, including inflammatory cytokine release (59) and apoptosis (60), and influences inflammatory sensitivity (61). Our question was whether activation of these or similar receptors, and the concomitant triggering of autonomic responses, link the pulmonary and cardiac responses to PM. Capsazepine (CPZ), a selective antagonist of TRPV1 receptors, given either intraperitoneally or as an aerosol, prevented increases in heart CL by CAPs (mean concentration: 218 23 mg/m3) (5). Continuous monitoring of cardiac function in the same model showed that inhalation exposure to CAPs aerosols leads to decreases in heart rate and in the length of specific ECG intervals. The observed increases in P-wave duration and QT interval and decreases in QRS and Tpe (the time interval between the peak of the T-wave and the end of the T-wave) indicate cardiac current abnormalities leading to significant changes in conduction velocity and ventricular repolarization (5). These changes were detectable immediately upon exposure, maintained throughout the five hours of CAPs inhalation, and prevented by CPZ administration showing a central role for pulmonary reflexes initiated by TRPV receptors in the cardiac effects of PM. The connection between increases in oxidants and cardiac dysfunction after PM exposure, shown in the previous section, is confirmed in this study. Both metal components of PM and PM-induced increases or ROS could account for the activation of TRP receptors. Aerosolized H2O2 triggers airway reflexes via TRPV1/TRPM2-dependent stimulation of vagal lung afferents in rats (62). Similarly, copper complexes and strong oxidizing agents (diamide and chloramines) activate TRPV1 receptors via irreversible oxidation of cysteine residues on the extracellular domain (7,8). As discussed above, although these effects require millimolar concentrations
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of oxidants or metals, one could envision that a combination of redox-active metals and ROS could approach these levels at the hot spots of PM deposition in the lung and activate TRP receptors in this way. Although these studies were carried out with samples enriched in PM2.5, the contribution of ultrafine PM (UFP) to these outcomes cannot be ruled out, as shown by the association between changes in QT segment and Tpe duration with particle NC (5). The potential adverse cardiovascular effects of inhaled UFP are currently under intensive investigation. Toxicological studies have elegantly shown high pulmonary deposition of UFP (63,64), translocation (65,66), and effects outside the lung (67), including evidence showing effects mediated by alterations in the autonomic nervous system (68). Epidemiological studies also suggest a role for UFP and related components in the development of adverse cardiovascular effects of PM. However, few studies address directly the role of UFP, mostly due to the lack of accurate exposure data. Increases in particle NC have been shown to be associated with increased risk for ST depression in humans during submaximal exercise tests (69), small decreases in systolic and diastolic blood pressure in patients with coronary heart disease (70), decreases in both time- and frequency-domain of HRV in young healthy subjects (71), decreases in low-to-high frequency ratio of HRV (72), and decreases in T-wave amplitude in males with ischemic heart disease (73). Interestingly, in some cases the reported effects of UFP were independent of ambient levels of PM2.5 (69) and even opposite, depending on the source (72). On the contrary, other outcomes such as blood pressure and autonomic control of heart rhythm showed associations with both UFP and PM2.5 (5,70,73), suggesting that some mechanistic pathways are sensitive to particle size whereas others, including the effects on the autonomic nervous system, are activated by specific particle components.
III.
Summary
Although pulmonary inflammation is a well-known response to PM, some critical health effects of particle exposure manifest before inflammation sets in and thus seem to be mediated by other mechanisms. The “neural hypothesis” is based on the experimental observation that exposure to PM affects the autonomic nervous system in many ways, leading to complex imbalances in sympathovagal tone in the heart. Our results indicate that these imbalances are triggered by activation of irritant receptors expressed in C-fibers and other neurons in the pulmonary tract, and lead to significant alterations of cardiac electrophysiology and rhythms. ROS are essential mechanistic components in this pathway, as they are required for the activation of irritant receptors in the lung, and for the electrophysiological changes in the heart. The production of ROS in the lung seems to be associated, at least in part, to the PM levels of redox-active metals (18) and may be due to Fenton-type reactions catalyzed by them or to redox-cycling of organic components present in PM as semiquinones (74,75). On the other hand, ROS production in the heart is associated with crustal components of PM (18) and could be attributed to receptor-mediated effects on intracellular sources of ROS (19), a consequence of the increased autonomic tone on the heart after PM exposure. Intriguing points for further investigation include the potential involvement of other TRP channels expressed in C-fibers and other neurons in the respiratory tract, the
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possible implication of ultrafine particles or soluble components in this or other cardiac responses, and the specific PM components responsible for these activations. Most of the postulates presented here seem to apply to human exposures as well; however, further validation of these mechanisms in relevant human exposure models is warranted. Examples of such validations could include testing for prevention of PM-induced changes in ECG in individuals treated with antioxidants in controlled exposure experiments, or for associations of polymorphisms in TRP receptors and sensitivity to PM in epidemiological studies. Being PM, a complex mixture of organic and inorganic components that varies in proportion daily, it seems reasonable to expect different biological pathways to be activated by different particle components, each of which may be highly abundant on a given day and negligible on the following. This variability underscores the importance of studies that evaluate statistically the association of individual components with different mechanisms and health outcomes.
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60. Agopyan N, Bhatti T, Simon SA. Vanilloid receptor activation by 2- and 10-microm particles induces responses leading to apoptosis in human airway epithelial cells. Toxicol Appl Pharmacol 2003; 192:21–35. 61. Veronesi B, Oortigiensen M, Roy J, et al. Vanilloid (capsaicin) receptors influence inflammatory sensitivity in response to particulate matter. Toxicol Appl Pharmacol 2000; 169:66–76. 62. Ruan T, Ho CY, Kou YR. Afferent vagal pathways mediating respiratory reflexes evoked by ROS in the lungs of anesthetized rats. J Appl Physiol 2003; 94(5):1987–1998. 63. Chalupa D, Morrow P, Oberdo¨rster G, et al. Ultrafine particle deposition in subjects with asthma. Environ Health Perspect 2004; 112:879–882. 64. Daigle C, Chalupa D, Gibb F, et al. Ultrafine particle deposition in humans during rest and exercise. Inhal Toxicol 2003; 15:539–552. 65. Nemmar A, Hoet P, Vanquickenborne B, et al. Passage of inhaled particles into the blood circulation in humans. Circulation 2002; 105:411–414. 66. Oberdo¨rster G, Sharp Z, Atudorei V, et al. Extrapulmonary translocation of ultrafine carbon particles following whole-body inhalation exposure of rats. J Toxicol Environ Health A 2002; 65:1531–1543. 67. Elder A, Oberdo¨rster G. Translocation and effects of ultrafine particles outside of the lung. Clin Occup Environ Med 2006; 5:785–796. 68. Harder V, Gilmour P, Lentner B, et al. Cardiovascular responses in unrestrained WKY rats to inhaled ultrafine carbon particles. Inhal Toxicol 2005; 17:29–42. 69. Pekkanen J, Peters A, Hoek G, et al. Particulate air pollution and risk of ST-segment depression during repeated submaximal exercise tests among subjects with coronary heart disease: the Exposure and Risk Assessment for Fine and Ultrafine Particles in Ambient Air (ULTRA) study. Circulation 2002; 106:933–938. 70. Ibald-Mulli A, Timonen K, Peters A, et al. Effects of particulate air pollution on blood pressure and heart rate in subjects with cardiovascular disease: a multicenter approach. Environ Health Perspect 2004; 112:369–377. 71. Chan C, Chuang K, Shiao G, et al. Personal exposure to submicrometer particles and heart rate variability in human subjects. Environ Health Perspect 2004; 112:1063–1067. 72. Timonen K, Vanninen E, de Hartog J, et al. Effects of ultrafine and fine particulate and gaseous air pollution on cardiac autonomic control in subjects with coronary artery disease: the ULTRA study. J Expo Sci Environ Epidemiol 2007; 16:332–341. 73. Henneberger A, Zareba W, Ibald-Mulli A, et al. Repolarization changes induced by air pollution in ischemic heart disease patients. Environ Health Perspect 2005; 113:440–446. 74. Valavanidis A, Fiotakis K, Bakeas E. Electron paramagnetic resonance study of the generation of reactive oxygen species catalysed by transition metals and quinoid redox cycling by inhalable ambient particulate matter. Redox Rep 2005; 10:37–51. 75. Squadrito GL, Cueto R, Dellinger B, et al. Quinoid redox cycling as a mechanism for sustained free radical generation by inhaled airborne particulate matter. Free Radic Biol Med 2001; 31(9):1132–1138.
9 Effects of Airborne Particles on Respiratory Viral Infection ILONA JASPERS and KATHERINE HORVATH University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A.
I.
Introduction
Particulate Matter (PM) is one of the six “criteria” pollutants for which the United States Environmental Protection Agency (U.S. EPA) is required by the Clean Air Act to set National Ambient Air Quality Standards (NAAQS). Similarly, the European Commission has set standards for PM levels in European countries, and the World Health Organization (WHO) has set worldwide guidelines for ambient PM levels. Exposure to other airborne particles, such as cigarette smoke particles, is less well regulated, despite recent worldwide efforts to minimize exposure to secondhand tobacco smoke (SHS) by prohibiting smoking in public places and indoor workplaces. While there is no shortage of reviews that focus on the health effects of airborne particles, specific information concerning the effects of exposure to such particles on respiratory infections is lacking. Therefore, this review will focus on the current evidence involving potential interactions between airborne particles and respiratory viral infections. A. Respiratory Virus Infections
Most upper respiratory infections (URIs), which include the common cold, flu, pharyngitis, as well as epiglottitis and laryngotracheitis (croup), are caused by viruses (1). Although over 200 different viruses can be the cause of URIs, the most common viruses that are usually associated with these infections include rhinovirus, respiratory syncytial virus (RSV), influenza, parainfluenza, coronavirus, and adenoviruses (1). Common symptoms of URIs include runny and stuffy nose, sneezing, cough, sore throat, headache, and fever. Lower respiratory infections (LRIs) include bronchitis, bronchiolitis, and pneumonia, of which, bronchitis and bronchiolitis are usually caused by viruses. Among some of the viruses commonly associated with LRIs are RSV, parainfluenza, adenovirus, and influenza (1). The usual symptoms of LRIs are cough, fever, and chest pain. In addition to causing morbidity and even mortality in otherwise healthy individuals, respiratory virus infections are also a major cause for exacerbations of chronic lung diseases, such as asthma and chronic obstructive pulmonary disease (COPD) (2–6). Especially in pediatric populations, respiratory virus infections are considered to be the major cause for exacerbation of allergic airways disease and asthma-related hospitalization (2,7,8). Thus, in addition to causing symptoms associated with URIs and LRIs,
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respiratory virus infections can also cause symptoms such as increased allergic inflammation or airflow obstruction (9). Considering that respiratory allergies and infections are the most common form of illness in the United States and Europe and account for more missed days from work or school than any other disease, it is important to understand potential associations between exposure to airborne particles and increased susceptibility to or exacerbation of respiratory virus infections.
II.
Airborne PM and Virus Infections
A. Epidemiologic Evidence
A large number of epidemiological studies have noted positive associations between levels of PM and the incidence of morbidity and mortality, including those involving cardiovascular and respiratory conditions (10–20). However, information on ambient PM characteristics, sources, and health effects are the focus of other chapters within this book. Therefore, we will focus on summarizing current information on the effects of ambient PM on respiratory virus infections. A number of studies have examined potential links between ambient PM and respiratory infection. Dockery et al. performed a cross-sectional study in which they examined the association of air pollution with respiratory health in children (21). In this study, the authors found that rates of chronic cough, bronchitis, and chest illness were positively associated with levels of ambient PM. In addition, a study performed in Finland compared the incidence of URIs in children living in a polluted city to those living in reference cities. The authors of this study found that children living in the polluted city that had higher levels of PM showed an increased incidence of URIs (22). Since NO2 was also elevated in the polluted city, the authors were unable to show that the effects were solely due to the elevated levels of PM. The association between respiratory infections and ambient PM was also examined in a well-known study performed in Utah Valley. Data were collected over a three-year period during which a local steel mill was on strike for a year. This presented a unique opportunity to correlate the effects of ambient PM in a more controlled environment since the levels of PM while the mill was open were nearly double the levels observed when the mill was closed. In this study, an association was found between the increased levels of ambient PM and hospital admissions for pneumonia among other respiratory illnesses in both children and adults (23,24), suggesting a link between enhanced levels of ambient PM and increased incidence of respiratory infections. More compelling evidence that increased ambient PM levels enhance respiratory virus infections comes from a study conducted by Pope et al., which examined the association of long-term exposure to PM and mortality (23). This study demonstrated that an increased risk of mortality from pneumonia and influenza was associated with a 10 mg/m3 increase in the levels of PM over the course of a few years (23). More recently, potential association between ambient air pollution, including PM, and increased fatality of individuals affected by the outbreak of the severe acute respiratory syndrome (SARS) has been examined (25). Using an air pollution index (API), which was derived from ambient concentrations of PM, SO2, NO2, CO, and O3, this study found an association between API and increased fatality due to SARS. More specifically, their analysis suggested that SARS patients living in regions with a high
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API were twice as likely to die from SARS as compared to patients living in regions with low API (25). Although the API used in this study does not specifically identify particular ambient air pollutants associated with the increased SARS mortality, a subsequent study indicated that a 10 mg/m3 increase in the levels of PM, SO2, or NO2 increased the relative risk of daily SARS mortality (26). Thus, mortality because of highly pathogenic viruses such as SARS could be potentially enhanced by exposure to ambient PM. There are several studies examining the effects of short-term fluctuations in ambient PM levels on hospital admissions for respiratory infections including pneumonia. For example, hospital admissions for pneumonia and asthma were significantly associated with PM levels of the previous three days (27). In addition, a study conducted in Germany examined the effects of short-term exposure to ambient air pollution on respiratory illness in children (28). The authors of this study found that a 60 mg/m3 increase in PM was associated with a 27% increase in croup cases (28). Similarly, a recent study looking at the effects of exposure to ambient PM and potential association with respiratory infections in children living in Toronto demonstrated that short-term increases (1–7 days) in ambient PM levels were associated with an increase in hospital admissions for respiratory infections in children younger than 15 (29). Furthermore, a recent study showed that a 10 mg/m3 increase in same-day levels of ambient PM was associated with increased hospital admissions for respiratory infections (10). Taken together, these studies provide epidemiologic evidence that exposure to elevated ambient PM levels and even short-term variations in PM levels can have significant consequences on infection-related respiratory morbidity and even mortality. B. Experimental Studies
A number of in vitro and in vivo laboratory studies have also investigated the effects of exposure to PM and respiratory viral infection. Exposure to ultrafine carbon black (CB) was used to examine the effects of airborne particles on RSV infection in mice (30). Specifically, mice were instilled with 40 mg of CB or saline vehicle followed by instillation with RSV or media the following day. Instillation of ultrafine CB particles prior to infection resulted in a suppressed early response to RSV infection followed by a later increase in inflammation and exacerbation of the overall infection (30). In a companion study, the same group first infected mice with RSV followed by instillation of ultrafine CB particles three days post infection, which also increased the morbidity associated with RSV infections (31). Thus, exposure to ultrafine CB either before or after infection with RSV modified immune responses and increased morbidity associated with the infection. Other studies have examined the effects of PM exposure on the ability of macrophages to respond to RSV infection. In the context of a viral infection, the role of macrophages in the antiviral defense response is to phagocytize the virus and release immune mediators. However, exposure to PM reduced the ability of macrophages to phagocytize RSV and decreased the production of RSV-induced immune and inflammatory mediators (32,33), suggesting a blunted immune response. Thus, exposure to either concentrated ambient PM or ultrafine CB particles can significantly affect respiratory virus-induced inflammation and morbidity, which may at least partially be caused by the decreased ability of macrophages to phagocytize the virus and mount an effective antiviral immune response against the invading pathogen.
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Epidemiological data and experimental studies suggest that diesel exhaust (DE) is a major contributor to adverse health effects associated with exposure to particulate air pollutants (34,35). There are a number of studies that have specifically examined the effects of DE exposure on the susceptibility and response to respiratory viral infection, which is why it is being discussed separately here. A study conducted by Hahon et al. examined the effects of chronic DE exposure on the susceptibility to respiratory viral infection (36). In this study, mice were exposed to 2 mg/m3 of DE for one, three, or six months followed by infection with influenza virus. The authors observed higher virus growth levels and decreased interferon (IFN) levels in mice exposed to DE for three and six months compared to air-exposed mice. Additionally, antibodies against influenza virus hemagglutinin were lower in mice exposed to DE for three or six months. Although the exposure concentrations used in this study were rather high, these studies provided evidence for potential interactions between exposure to DEP and respiratory virus infections in mice. A more recent study looked at the effects of short-term DE exposure on the susceptibility and response to RSV infection in mice (37). In this study, mice were exposed to 300 mg/m3 or 1000 mg/m3 of DE for 6 hr/day for seven consecutive days followed by infection with RSV. The authors found that DE-exposed mice were more susceptible to RSV infection, as shown by an increase in RSV gene expression in the lungs of mice exposed to either dose of DE. In addition, exposure to DE also caused an enhancement of the inflammatory response to RSV infection, as marked by increased levels of bronchoalveolar lavage fluid (BALF) inflammatory cells and lung mucus cell metaplasia (37). Studies in our laboratory have shown that exposure to DE enhances the susceptibility to influenza infections in epithelial cells in vitro and mice in vivo (38). Specifically, our data indicate that exposure of mice to 0.5 mg/m3 DE for 4 hr/day for five days prior to infection with influenza resulted in enhancement of the influenza-induced inflammatory response and pulmonary injury (39). Although the concentrations of DE used are rather high and would be more equivalent to occupational exposures rather than ambient exposures, these studies demonstrate that exposure to DE can enhance the susceptibility and response to respiratory viral infections, and may result in an enhancement of the resulting morbidity of infection. As indicated above, respiratory virus infections are the major cause for asthmarelated hospital admissions and exacerbation of allergic inflammation. Since exposure to airborne particles, such as DE particles, can increase the susceptibility to respiratory virus infections, it seems plausible that there could be potential interactions between exposure to airborne particles and virus-induced exacerbation of allergic inflammation. Using a mouse model of ovalbumin-induced allergic airway inflammation, we examined whether exposure to DE particles prior to infection with influenza affects markers of allergic inflammation. Briefly, as outlined in Figure 1A, mice were sensitized and challenged with ovalbumin followed by instillation with DE particles 24 hours prior to infection with influenza A. Mice were sacrificed 24 hours post infection and examined for markers of allergic inflammation. Figure 1B shows that exposure to DE particles prior to infection with influenza significantly enhances the number of eosinophils in the BALF. Thus, in the context of allergic airways disease, exposure to airborne particles could increase the susceptibility to respiratory virus infections and consequently enhance virus-induced exacerbation of allergic inflammation.
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Figure 1 Exposure to DEP prior to infection with influenza increases allergic inflammation.
(A) Outline of experimental protocol. C57BL/6 mice were sensitized intraperitoneal (i.p.) and challenged with ovalbumin (Ova) prior to instillation with 25 mg DEP or saline vehicle, followed by intranasal instillation of 500 PFU influenza A/PR/8 24 hours afterward. Mice were sacrificed 24 hours postinfection and examined for markers of allergic inflammation. (B) Differential cell count of BALF. *, Significantly different from all other groups; p < 0.05.
D. Tobacco Smoke and Virus Infections
Tobacco smoke is a major contributor of indoor airborne particles. Chronic exposure to cigarette smoke (either passive or mainstream) is associated with increased severity of and susceptibility to respiratory virus infections (40). The notion that passive exposure to environmental tobacco smoke (ETS) is a significant risk factor for respiratory virus
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infections in children was proposed more than 30 years ago (41), and since then, epidemiological studies have confirmed that exposure to ETS is associated with greater rates of URIs and LRIs in children (42–44). There is evidence indicating that exposure to ETS is associated with increased prevalence and severity of respiratory infections in children, as well as the development of bronchiolitis in infants (45–48). In addition, exposure to side-stream (SS) cigarette smoke has been shown to significantly alter the immune response to RSV infection in mice (49). This study demonstrated that exposure of neonatal mice to SS followed by infection with RSV resulted in decreased levels of Th1 cytokines, such as interleukin (IL)-12 and IFNg, increased the number of BALF eosinophils and increased the expression of viral proteins as compared with mice infected with RSV following exposure to air (49). Thus, passive exposure to cigarette smoke either prior or concurrent to respiratory virus infections modifies immune defense responses and enhances the severity of the infection. Similar to passive exposure to cigarette smoke, there is ample evidence that smokers are more susceptible to respiratory virus infections. For example, epidemiological studies suggest that smokers are more susceptible to influenza virus infections than nonsmokers (40,50–53), yet the mechanisms mediating these effects are not known. Recently published observations indicate that in mice, chronic exposure to mainstream cigarette smoke can either increase or decrease influenza virus-induced primary antiviral immune and inflammatory responses, depending on the initial infectious dose (53). In this study, adaptive immune responses, as marked by influenzaspecific antibody production, remained unaffected by the cigarette smoke exposure (53). Similarly, analysis of an influenza outbreak in the Israeli army demonstrated that while smokers were more susceptible to influenza virus infections, influenza-specific antibody levels were not decreased in smokers (51,54). Chronic exposure of mice to cigarette smoke and subsequent infection with a replication-deficient adenovirus reduced the number of dendritic cells (DCs) in the lung and decreased the ability to mount an adaptive immune response, as marked by decreased virus-specific antibodies (55). Thus, chronic exposure to mainstream cigarette smoke may either affect innate, adaptive, or both arms of the antiviral immune response, and these effects may be virus specific. E.
Indoor Air Pollution and Virus Infections
There is also epidemiological evidence linking other sources of indoor airborne particles, such as burning of biomass, to respiratory infections. Biomass fuels consist of wood, dung, charcoal, and agricultural residues and are mainly used in less-developed countries. Associations have been noted between exposure to cooking fires and development of acute respiratory infections in both adults and children (56,57). A study conducted in Yemen showed that in children less than three months of age, exposure to either cigarette smoke or the use of cooking fuels other than gas, such as biomass, was associated with an increased risk for severe RSV hypoxia (58). There is also epidemiological evidence showing an association between exposure to biomass combustion and deaths caused by respiratory infections in children (59–61). Additionally, associations between exposure to wood smoke and LRIs in children have been observed in several studies (62–65). Taken together, these studies indicate that exposure to indoor airborne particles derived from the burning of biomass in cooking stoves or wood smoke
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is associated with increased susceptibility to and severity of respiratory virus infections, especially in children.
III.
Potential Mechanisms
From the wide range of epidemiological evidence and experimental studies, it is clear that exposure to airborne particles can exacerbate adverse health effects associated with respiratory viral infections. This raises questions about potential mechanisms whereby exposure to these agents can impact host susceptibility and response to viral infection. Given the complexity of potential interactions between host responses to both invading pathogens and environmental agents, the mechanisms behind these responses are likely to be quite convoluted and may differ greatly between pathogens, pollutants, as well as individuals. Therefore, this summary will provide an overview of potential mechanisms that could be involved in the alteration of host immunity caused by exposure to airborne particles. A schematic of potential mechanisms mediating interactions between airborne particles and respiratory viruses is shown in Figure 2. A. Oxidative Stress
One possible driving force for many of the adverse health effects elicited by exposure to airborne particles is oxidative stress. It can have a wide range of effects on cellular function, including immune responses. All of the airborne particles mentioned herein have been shown to induce oxidative stress, and exposure to these agents may result in the production of free radicals, which can have damaging effects on the lung (38,66,67). Since epithelial cells lining the respiratory tract are the primary targets for invading pathogens as well as inhaled airborne particles, there may be a link between the homeostatic balance of oxidants and antioxidants in these cells and their susceptibility and response to viral infection. This notion is supported by a study that observed decreased susceptibility to influenza infections in mice supplemented with reduced
Figure 2 Schematic of potential interactions between airborne particles and respiratory viruses.
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glutathione (GSH) (68). Studies conducted in our laboratory support these observations and have shown that pretreatment of respiratory epithelial cells with GSH reverses the DE-induced enhancement of influenza infections (38). Furthermore, oxidative stress induced by exposure to airborne particles may enhance the morbidity of the resulting infection through an increased inflammatory response. Generally, antioxidants have been suggested to be protective against respiratory virus-induced inflammation and injury. Specifically, studies by Beck and Matthews showed that the pathogenesis of a viral infection was exacerbated in mice that were in a state of oxidative stress through nutritional deficiency of the trace element selenium (69). These studies were confirmed in subsequent in vitro studies that demonstrated that selenium deficiency modified the ability of bronchial epithelial cells to produce cytokines in response to influenza virus infections (70). In addition, treatment with the antioxidant butylated hydroxyanisole decreased RSV-induced lung injury (71). Yet another study showed that transgenic mice overexpressing extracellular superoxide dismutase showed less lung injury and inflammation after infection with influenza compared to wild-type mice (72). Similarly, overexpression of hemoxygenase 1 (HO-1) decreased influenza-induced lung injury in mice (73). Together, these studies provide evidence that homeostatic balance between cellular oxidants and antioxidant is an important modifier of virus-induced responses and thus provides a potential mechanism contributing to the enhanced susceptibility and response to viral infections seen after exposure to airborne particles. B. Macrophages
Another aspect in which exposure to airborne particles may exert their effects on host immunity is through modulation of macrophages, which play an important role during initial innate immune defense responses. The two main roles of these cells are phagocytosis of debris and pathogens and antigen presentation. Their role as phagocytes is a crucial part of the innate defense response. Phagocytosis of viruses plays an important role during viral clearance and, consequently, limits the propagation of the viral infection. Studies have shown that exposure to airborne particles can reduce the ability of macrophages to phagocytize or inactivate viruses (32,33). This suppressed phagocytic internalization may be caused by cytoskeletal dysfunction in macrophages exposed to PM (74). In addition, exposure of macrophages to PM increases apoptotic cell death (75) and causes mitochondrial dysfunction (76). This may occur through interaction of airborne particles or particle components with scavenger receptors located on the cell surface of macrophages (77). Thus, suppression of macrophage function or increased apoptotic cell death could affect both the host’s susceptibility and response to a viral infection. C. Epithelial Cells
As indicated earlier, epithelial cells represent the major site for viral entry and viral replication. Consequently, particle-induced modifications at the level of the epithelium are likely to significantly affect the ability to respond to and protect against viral infections. Specifically, work from our laboratory has shown that exposure to DE (38) and cigarette smoke condensate (CSC) (Jaspers et al., unpublished observations) enhances the ability of influenza virus to attach and/or enter epithelial cells. Infection of
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Figure 3 Exposure to CSC increases protease activity. Apical washes were collected from differentiated human nasal epithelial cells exposed to either vehicle or 1 mg/mL CSC for 24 hours prior to infection with influenza. Trypsin activity was measured as colorimetric cleavage of the peptide Boc-Gln-Ala-Arg-MCA and normalized against a standard curve of trypsin. *, Mean significantly different from vehicle control; p < 0.05. Abbreviation: CSC, cigarette smoke condensate.
epithelial cells with influenza virus requires proteolytic cleavage of the hemagglutinin present on the surface of the virus particles prior to entry into the cell (78). The expression of these trypsin-like serine proteases (tryptases) is localized to cells lining the respiratory tract, which has been hypothesized to be responsible for the pneumotropic character of the influenza virus (79). The level of proteolytic activity derived from this tryptase can be regulated by the amount of tryptase synthesized and released by lung epithelial cells as well as by several antiproteases, such as secretory leukocyte protease inhibitor (SLPI) and elafin, which control the activity of the proteases. Therefore, we examined whether exposure of airborne particles, such as cigarette smoke particles, could modify the proteolytic activity present on the surface of epithelial cells. Briefly, differentiated human nasal epithelial cells were exposed to 1 mg/mL CSC 24 hours prior to infection with influenza virus and examined for changes in proteolytic activity of apical washes. Figure 3 shows that exposure to CSC prior to infection with influenza increases proteolytic cleavage of a peptide containing the same amino acid sequence as the influenza virus hemagluttinin. Thus, modification of epithelial-derived protease activity, which is required for activation and infectivity of certain viruses, could provide another mechanism by which exposure to airborne pollutants enhances susceptibility to viral infections. Similarly, exposure to DE particles increases the expression of ICAM-1 and lowdensity lipoproteins (80), both of which are receptors for human rhinovirus (81), suggesting that exposure to DE could increase the susceptibility to rhinovirus infections. The receptor for coxsackievirus and adenovirus (CAR) mediates attachment and infection of a number of adenoviruses and is located at or near epithelial tight junctional complexes (82). Disruption of epithelial tight junctions enhances access to the receptor and thus increases the susceptibility to infection with adenoviruses. Exposure to airborne pollutants such as cigarette smoke has been shown to cause disruption of tight junctional
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complexes in epithelial cells (83), suggesting a potential mechanism of enhancing susceptibility to infection with adenoviruses. Another important part of the host’s innate immune defense against respiratory viruses is played by surfactant proteins (SP). The hydrophilic SPs, SP-A and SP-D, are involved in the pulmonary defense against invading pathogens and are secreted by epithelial cells including alveolar type II cells and Clara cells and in the lung (84). SP-A and -D bind pathogens, facilitate phagocytosis, and regulate inflammation. Reduced expression or altered function of SP-A or -D can have a wide range of detrimental effects on the innate response to a respiratory viral infection, such as enhanced susceptibility to infection, increased inflammation, or decreased phagocytosis, as demonstrated by a variety of studies using SP-A and -D knockout mice (85–87). Studies conducted in our laboratory as well as others’ have shown that exposure to airborne particles can decrease the expression and alter the function of SP-A and -D (39,88,89), which is associated with enhanced susceptibility to respiratory virus infections. Taken together, these studies demonstrate that modification of epithelial cell integrity, epithelial cell surface receptor expression, and release of SPs are among potential mechanisms mediating enhanced susceptibility to virus infection following exposure to airborne particles. D. Dendritic Cells
In addition to epithelial cells by themselves, the interplay between the airway epithelium and resident antigen presenting cells could play an important role in modifying the response to invading pathogens and airborne particles. Resident DCs fulfill the pivotal task of mobilizing both innate and adaptive immune cells by secreting chemokines to attract inflammatory cells to the site of infection and by migrating to peripheral lymph nodes to initiate an adaptive T cell response (90). DCs are predominantly located on the basolateral side of the epithelium and “sample” their microenvironment for antigens and PM through extensions of “dendritic-like” processes into the airway lumen (91–93). Ambient particle-induced effects on DCs result from a combination of direct DC activation via sampling of the airway lumen and from chemokines and maturation factors secreted from the epithelium itself in response to pollutants. Interactions between airway epithelium and DCs have been studied to understand how exposure to airborne particles can cause a Th2 allergic response through the use of co-culture systems of DCs and airway epithelial cells (91,93). Exposure of the epithelium to pollutants causes the epithelium to release factors promoting DC development. In addition, exposure of bronchial epithelial cells to concentrated ambient PM caused release of MIP3a by these cells, a chemokine that causes DC migration (94). Furthermore, exposure to DE particles (DEP) causes human bronchial epithelial cells to induce DC maturation by increasing CD83 expression and increasing the ability of DCs to stimulate T cell proliferation in a granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent manner (95). Thymic stromal lymphopoietin (TSLP) has been presented as a possible player in this mechanism because TSLP released from DEP-treated human bronchial epithelial cells induces DC polarization in a Th2-dependent manner (96). In response to viral infections, DCs produce a number of cytokines/chemokines, including IFN-regulated chemokines MIG (CXCL9), IP-10 (CXCL10), and I-TAC
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(CXCL11) to activate Th1-type immune responses as part of the normal immune response (90). However, the predominantly Th1-type immune response aimed at clearing the infection can be altered by exposure of DCs directly to airborne particles, such as those contained in DE and cigarette smoke. For example, exposure to airborne particles has been shown to modify characteristic DC responses including modification of expression of costimulatory molecules such as CD40, CD80, CD83, and CD86 (97–99), decreased release of IL-12 (100), expression of the endocytosis receptor CD206 (99), and suppressed ability to activate DCs (101). In a study of BALF in smokers versus nonsmokers, DCs demonstrated increased expression of CD80, CD86, and CD1a with a decreased expression of the lymph node homing receptor CCR7 in smokers (102). In addition, COPD patients who smoke have lower numbers of bronchial mucosal DCs (103), while mice chronically exposed (24 weeks) to CS showed an increase in DCs in the airway and lung parenchyma with increased expression of CD40 and CD86 that correlates with alveolar wall destruction (104). Although whether or not absolute numbers of DCs in the lung are augmented by exposure to CS or other airborne particles is not completely understood, it does potentially create a DC phenotype with a modified expression of costimulatory molecules, which is likely to alter their ability to orchestrate an antiviral immune response. In addition, exposure to airborne particles may affect the ability of DCs to orchestrate T cell responses, which is supported by studies in which DEP-treated DCs cocultured with allogenic CD4þ T cells showed increased Th2 cytokines IL-13 and IL-18 with a suppressed Th1 IFNg response (97,100). Similarly, Vassallo et al. showed that CS inhibits DC-mediated T cell proliferation whereas nicotine alone does not (105). Here, CS-exposed DCs drive a Th2 response by increasing IL-4 secretions and decreasing IFNg in a mixed lymphocyte reaction. Furthermore, when DCs are challenged with viruses, exposure to CS has been shown to interfere with the characteristic T cell responses. Chronic CS exposure for two to four months decreased DCs in pulmonary tissue in mice and decreased both the total number of T cells and the ratio of CD4:CD8 T cells upon infection with adenovirus compared to air controls (55). Thus, exposure of DCs to airborne particles modifies the ability of DCs to interact with T lymphocytes and therefore interferes with an effective T cell response against the invading virus.
IV.
Concluding Remarks
The studies mentioned within this review demonstrate how exposure to airborne particles can alter host immunity to respiratory viral infections. Given the number of individuals that contract some kind of respiratory virus each year, potential interactions between exposure to airborne particle and respiratory virus infections has significant public health implications for people throughout the world. As more studies are conducted, we will continue to improve our understanding of potential mechanisms upon which exposure to airborne particles increases the susceptibility to and severity of respiratory virus infections, and identify potential subpopulations that are predominantly affected by such interactions. In addition, with the apparent rise in production, emission, and exposure to nano-sized particles, either ambient or industrial, there is a need to examine potential interactions between nanoparticles and respiratory virus infections.
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An important challenge for the future will be to further our understanding of the cellular and molecular mechanisms involved in pollutant-induced effects on the susceptibility to viral infections, which will yield potential targets for therapeutic strategies to overcome these effects.
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10 Airborne Particles and Structural Remodeling of the Lungs AMY K. MADL University of California, Davis, and ChemRisk, LLP, San Francisco, California, U.S.A.
CHRISTOPHER CAROSINO and KENT E. PINKERTON University of California, Davis, California, U.S.A.
I.
Introduction
The pathological effects of airborne particles have been documented in historical writings dating back for millennia. In the first century, for example, the Greeks described a lung disease that appeared to cause a wasting away of the body (1,2). By the 16th and 17th centuries, lung diseases among miners, smelters, and stone cutters were described by a number of physicians, and occasionally, autopsies were performed on workers, revealing the presence of dust in the lungs (1). Early occupational studies generally did not distinguish among lung diseases caused by different types of dust (e.g., coal, silica, metal, asbestos), chemicals, or bacterial agents. Originally, nearly all respiratory diseases were termed “phthisis” or “consumption.” It was not until the early to mid 1900s with the development of new medical diagnostic methods, such as Mycobacterium tuberculosis detection in sputum samples (a causative agent of tuberculosis and frequent confounder for diagnosing pulmonary disease caused by mineral dusts) and chest X rays, that pulmonary disease began to be defined according to specific agents (e.g., silicosis, asbestosis, coal miners’ pneumoconiosis). Chronic obstructive pulmonary disease, emphysema, and other forms of structural remodeling of the lungs (granulomas of berylliosis, interstitial fibrosis of hard metal disease, macules of siderosis) are additional pulmonary diseases that were historically identified through occupational exposure to airborne particles. In more recent times (specifically, the second half of the 20th century), other nonoccupational sources of airborne particulates (i.e., ambient air pollution, tobacco smoke, diesel exhaust) have been reportedly associated with lung remodeling, ultimately leading to lung structural changes and functional deficits, and, in some cases, mortality among healthy and susceptible populations. This chapter reviews how different types of airborne particles and fibers can lead to very different lung remodeling, depending on their physicochemical characteristics, biopersistence, and internal dose. This chapter will also touch upon the theoretical or known mechanisms by which some airborne particles cause site-specific or broad-based structural changes in the lungs.
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II.
General Concepts Leading to Site-Specific Particle Effects in Lung Remodeling
The site of action through which airborne particles elicit biological changes in the lungs is often dictated by particle respirability, biopersistence (durability), and retention (dose), as well as physicochemistry. Depending on their source, airborne particles can be comprised of a complex mixture of carbon, metals, and polyaromatics, such as in the case of combustion particles, or they may exist in a relatively pure form, but may represent a range of physical characteristics (e.g., asbestos or man-made vitreous fibers). Physicochemical characteristics not only influence a particle’s ability to deposit in certain regions of the respiratory tract but also its propensity to be retained within or translocated to specific regions or cellular compartments in the lungs. While the respirability and probability of particle deposition in the respiratory tract is based on many factors (e.g., size, solubility, shape, density), particle size best determines by what mechanism and to what extent airborne particles will enter and deposit in the nasopharyngeal, tracheobronchial, and alveolar regions of the lung. These factors also affect the extent and location in which particles induce inflammatory, fibrogenic, and biochemical processes that can ultimately lead to structural remodeling of airways and gas exchange regions of the lungs. Larger diameter particles (i.e., >2–3 mm diameter) act primarily by inertial mechanisms and will preferentially deposit in the upper respiratory tract, whereas smaller particles (i.e., <100 nm diameter) act by diffusion and will deposit both in the nasopharyngeal and tracheobronchial regions, and, to a greater extent, in the alveolar or gas exchange region of the lungs. Particle deposition models suggest that up to 90% or more of inhaled particles of 100 nm in diameter or less will deposit in the respiratory tract, with approximately 50% depositing in the alveoli (3). Agglomeration of individual particles can change the size and aerodynamic properties of inhaled particles; therefore, while subunits of airborne particles may be nanoscale in size, agglomeration or aggregation may cause such airborne particles to act similarly to larger-sized particles. The propensity of fibrous particles to deposit in the respiratory tract is largely determined by fiber diameter, and, to a lesser extent fiber length. Fiber length has little impact on respirability up to a length of about 20 mm, but the deposition of longer fibers is inversely related to the length of longer fibers. While fibers up to 3.5 mm in diameter have been detected in the lungs of asbestos workers, fibers of this dimension may represent the very upper bound limit of respirability (4–7). A number of studies have shown that nearly all fibers deposited in the pulmonary region of the lung are thinner than 0.7 mm (8–12). Once particles are deposited within the respiratory tract, the mechanism by which they are cleared from the lungs depends not only on the site of deposition but also on the size of the particle itself (Fig. 1). Solid particles are cleared from the lungs through a variety of mechanisms (i) sneezing, coughing, and removing mucus from the nasopharyngeal region, (ii) direct or macrophage-mediated transport along the mucociliary escalator and subsequent elimination by the gastrointestinal tract, (iii) direct or macrophage-mediated transport across the bronchiolar or alveolar epithelium and subsequent clearance by the systemic circulation or interstitial lymphatics, and (iv) physicochemical processes, including dissolution, leaching, and physical breakdown of particles. Because the particle’s aerodynamic or structural properties may not allow it to maneuver the complex branching patterns of the bronchioles, airway bifurcations and the central acinus of the bronchoalveolar duct junction are often regions in the lungs that
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Figure 1 Transport pathways of insoluble particles in different regions of the respiratory tract.
not only receive the highest deposited particle dose but are also frequently the site of pathological changes and structural remodeling (Fig. 2). Chrysotile asbestos inhalation studies show that these fibers can preferentially deposit on airway bifurcations, induce cellular proliferation at the bronchoalveolar duct junction, and lead to focal “hot spots” of fibrosis following acute exposure (Fig. 3) (13–19). The pattern of disease and structural remodeling of the lungs, however, can often be specific to the agent and the size of the particle. Macules (nonpalpable nonfibrotic pigmented lesion), for example, are often observed in respiratory bronchioles adjacent to focal emphysema in coal workers’ pneumoconiosis (CWP), distinct fibrotic nodules within the parenchyma are seen in chronic silicosis, and noncaseating granulomas can follow lymphatic routes along the airway in chronic beryllium disease (CBD) (20). The mechanisms by which different particles can cause unique patterns of remodeling of the airways or parenchyma, in many cases, is not well understood; however, a complex interplay between particle kinetics, inflammation, and cell signaling pathways that influence cellular injury and repair is usually involved. One concept that seems to be consistent across several types of particles, however, is that increased particle retention provides an opportunity for reactive particle surfaces to interact with the bronchiolar and alveolar epithelium at initial sites of deposition, as well as at more distant locations where particles reach through translocation. This issue of particle translocation and effects occurring distant from the deposition site has received considerable attention for nanosized particles, primarily because nano-sized particles have a potentially high efficiency for deposition, target both the upper and lower regions of the respiratory tract, are retained in the lungs for a longer period of time, induce more oxidative stress, and cause greater
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Figure 2 Scanning electron micrograph of the bronchiole-alveolar duct junction (BADJ) in the
rat lung. This transition zone in the lung from conducting airway to alveolar gas exchange is a primary site for particle deposition. Source: From Ref. 155.
Figure 3 Higher magnification of the first alveolar duct bifurcation seen in Figure 2. Fibers of
chrysotile asbestos preferentially litter the alveolar surfaces within this region of the BADJ. Source: From Ref. 155.
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inflammatory effects than their fine-sized equivalents (21–27). Well studied agents such as coal, silica, asbestos, and beryllium, as well as more recently evaluated particles, such as engineered nanomaterials and particles associated with ambient air pollution, will be reviewed to illustrate how different particles can cause remodeling in the lungs.
III.
Structural Remodeling in the Lungs by Particles in Occupational Settings
A. Coal and Coal Workers’ Pneumoconiosis
Utilized as a fossil fuel, coal is derived from organic material from sedimentary strata and is comprised of a complex mixture of minerals, trace elements, and organic compounds. Depending on the source of the deposit, coal can also contain varying amounts of crystalline silica. Although coal mining has been undertaken since the 1700s, documentation of CWP in the form of black fibrotic lung lesions did not appear until approximately 1830, and CWP was not differentiated as a distinct disease from silicosis until about 1930 (20,28,29). Even so, public awareness of the constant threat and often sudden reality of disaster, disease, and death endured by coal miners and their families did not finally come about until after the 1968 Farmington mine disaster in West Virginia. This tragic event involved an explosion in Consolidation Coal Co.’s No. 9 mine near Farmington, West Virginia that sealed the mine and effectively created a tomb for 78 miners. The deaths of 222 miners in 1967, 311 in 1968, and then the 78 in Farmington spurred President Nixon, in March 1969, to submit a proposal for a strong new Federal Coal Mine Health and Safety Act (30). Once this Act was passed, the National Institute for Occupational Safety and Health (NIOSH) developed several surveillance programs for monitoring underground coal miners’ health. As a result of NIOSH’s programs, the disease rate of CWP decreased from 11% to 4 % over the past 30 years (30). Despite this decrease in disease rate, however, there were still over 15,000 deaths from CWP reported in the decade prior to the 2002 NIOSH health surveillance report titled “The Work-Related Lung Disease Surveillance Report” (31). The pathology of CWP has a number of features that make it a distinct disease irrespective of the heterogeneity of coal mine dust composition and the mining region of origin. CWP is generally classified as “simple” or “complicated” depending on the extent of fibrotic involvement of the lungs. Simple CWP is classified according to the number, size, and shape of small opacities on a chest X ray, using a system of standard reference films established by the International Labour Organization (ILO) (20). Persons with milder forms of simple CWP may not exhibit any clinical symptoms whereas complicated CWP (progressive massive fibrosis) may display signs of chronic airflow obstruction, restrictive defects, uneven ventilation/perfusions, reduced diffusing capacity, and low oxygen saturation that can often lead to pulmonary hypertension and cor pulmonale (20). The mechanisms that are believed to underlie the pathogenesis of CWP are similar to those for crystalline silica and silicosis, and include (i) direct cytotoxicity, (ii) activation of oxidant production by lung phagocytic cells, (iii) release of proinflammatory cytokines from lung cells, and (iv) secretion of growth factors (20,32–34). Other features that are often observed with complicated forms of CWP include coal dust macules (soft lesions comprised of dust-laden macrophages and often associated with focal emphysema), coal dust nodules (firm lesions comprised of dust-laden macrophages in a fibrotic stroma),
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rheumatoid nodules (eosinophilic necrotic tissue surrounded by layers of fibroblasts and macrophages), diffuse interstitial fibrosis, emphysema, and chronic bronchitis. It should be noted that coal dust nodules that are composed of collagen and reticulin arranged concentrically are generally indicative of exposure to crystalline silica (20). Emphysema in coal workers is commonly centriacinar in location but is also observed in panacinar, irregular, and paraseptal form (20,35,36). The mechanism underlying the pathogenesis of emphysema in coal workers appears to be similar to that of cigarette smoke, involving excessive release of proteases, reactive oxygen species (ROS), and inhibition of defense mechanisms (20,33,37–40). It should be noted, however, that although these other pathological features appear to be consistent in severe forms of CWP, in many epidemiologic studies it is often difficult to tease apart the effects of coal dust and cigarette smoke. B. Silica and Silicosis
Silicosis was recognized as an occupational lung disease very early in the history of occupational medicine and industrial hygiene (41). Around the late 1800s, the increasingly widespread use of pneumatic tools and automatic machinery, particularly in the mining and stone-cutting industries, created very dusty work environments. Because few dust-control measures were used and respiratory protection was not worn by workers, disease and mortality rates during these years were significantly higher among workers in “dusty trades” compared with other occupations (42). Crystalline silica, the causative agent of silicosis, is an abundant rock-forming mineral and is present in different forms or polymorphs in the environment. While each form is composed of units of silicon dioxide (SiO2), the forms differ in their atomic spacing, lattice structure, and angular relation (43). Quartz is most prevalent in the environment and, consequently, in the workplace as well; it is used in abrasives, cleaners, ceramics, electronics, fillers, optics, polishes, and refractory materials. Cristobalite and tridymite, other polymorphs of crystalline silica less commonly found in rocks or soils, may occur in industrial operations involving the heating of quartz or amorphous silica, such as during the calcining of diatomaceous earth or brick manufacturing (43). Despite its early recognition as an occupational hazard, silica continues to pose a major health hazard, with estimates of approximately 1.7 million U.S. workers exposed to respirable crystalline silica in industries and occupations (i.e., construction, sandblasting, and mining); it has been attributed to the over 15,000 silicosis deaths occurring over the past three decades (44,45). There are three forms of silicosis: chronic, accelerated, and acute (43). Chronic silicosis is what many consider the “classic” form of silicosis and can develop after ten years or more of sufficiently high silica exposure. Chronic silicosis may be described as simple silicosis, where individual fibrotic nodules are solitary and less than 1 cm in diameter, or as conglomerate silicosis (also referred to as “complicated silicosis” or “progressive massive fibrosis”), wherein the nodules become confluent and eventually replace the lung parenchyma (46). Patients with simple silicosis may not display any symptoms of pulmonary dysfunction. Accelerated silicosis, which usually manifests within 5 to 10 years of high silica exposure, is similar to chronic silicosis, with the onset of fibrosis being more rapid, irregular, and diffuse. Acute silicosis, also referred to as “alveolar lipoproteinosis,” can occur within weeks to five years after initial exposure to very high airborne concentrations of silica (46). With this form, the nodular pattern of fibrosis is completely absent.
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Exposure to crystalline silica leads to tissue fibrosis through a cascade of cellular events. Silica is not readily degraded by macrophage lysosomal enzymes and is directly cytotoxic to macrophages and polymorphonuclear leukocytes. Macrophages that engulf silica particles become necrotic and release lysosomal contents and ROS, leading to local recruitment of polymorphonuclear leukocytes and ultimate damage to the surrounding tissue (47–51). A cyclical process of macrophage recruitment, ingestion, and cell death can cause a focal point of immune activity and fibrosis in the lung, resulting in chronic inflammation (47–52). A number of studies have shown that silica-induced toxicity appears to be most closely correlated with factors that can influence molecular or cellular interactions with the particle surfaces. Multiple lines of evidence suggest that the formation of ROS, reactive nitrogen species (RNS), or other highly reactive molecules may play key roles in the development of the cell injury, proliferation, apoptosis, and fibrogenesis associated with silica exposure (47–51,53). These reactive species can be generated directly via chemical reactions with the silica particle surfaces or indirectly through interactions of silica with various cell types. In vitro studies have shown that freshly fractured silica particles generate more ROS and are more toxic to alveolar macrophages than aged silica particles (54). The role of particle surface properties in the pathogenesis of disease is also supported by evidence that surface modifications or coatings have been shown to decrease the fibrogenicity of silica particles in vivo (55). In addition, in vitro studies have demonstrated that hydroxyl or superoxide radicals are formed in the presence of freshly fractured silica and that crystalline silica is a potent stimulant of the increase in ROS production in alveolar macrophages (54,56,57). The sustained presence of ROS resulting from repeated attempts by alveolar macrophages to phagocytize silica particles can cause chronic cell injury or death and may promote fibrogenesis (47–51). With a clear understanding of the histopathological and clinical aspects of silicosis, recent research efforts have focused on characterizing the mechanisms by which it occurs and its apparent role in the development of lung cancer. Many of these studies have evaluated how particle surface chemistry, intercellular signaling pathways, and oxidant stress may induce inflammation and stimulate the immune system, ultimately leading to cell proliferation and tissue fibrosis following exposure to crystalline silica (47–51). Overall, the intercellular interactions and cell signaling pathways are complex, numerous cross-talk and feedback mechanisms exist, and species responses and forms of silica differ. Nevertheless, these studies have revealed some of the underlying mechanisms involved in the development of silica-induced lung injury. The mechanisms underlying silica-induced carcinogenesis in animals, and perhaps in humans, are not well understood. The International Agency for Research on Cancer (IARC) described experimental evidence for a direct genotoxic action of crystalline silica as “weak” (58). However, many of the pathways that are believed to be involved in the inflammatory and fibrogenic processes may also be important in the carcinogenic process. For example, the development of lung tumors in the rat appears to be dependent on coexistent chronic inflammation and cell proliferation (59). ROS produced as a result of silica exposure can interact with DNA in vitro to form potentially promutagenic lesions (55,60–63), and increased cell proliferation can lead to the development of cancer by increasing the likelihood that such lesions will become fixed before they can be repaired by the cell. It has been suggested that the formation of lung tumors in rats exposed to silica appears to be consistent with a nonspecific response to persistent
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inflammation and increased cell proliferation, and that it may therefore be appropriate to apply a threshold model of carcinogenesis to inhaled silica if silicosis is required for the development of silica-related lung cancer (64). C. Asbestos and Asbestosis
Once thought to be the “miracle” mineral, asbestos gained widespread use beginning in the early 1900s and has been reportedly incorporated in some 3000 different products because of its low cost and desirable qualities, such as heat and fire resistance; wear and friction characteristics; tensile strength; heat, electrical, and sound insulation; adsorption capacity; and resistance to chemical and biological attack (65). Peak use of asbestos in the United States reached nearly 1500 million pounds in the 1950s and has declined precipitously since the mid-1970s because of regulatory and societal concerns about the health effects caused by asbestos exposure (66). Asbestos is a general term for a variety of naturally occurring fibrous silicate minerals that fall into two groups: serpentine (chrysotile) and amphibole (amosite, crocidolite, tremolite, actinolite, anthophyllite). While amosite insulation was frequently used on board U.S. maritime vessels, between 95% and 99% of all asbestos used elsewhere in the United States was chrysotile (65,67). It has been estimated that between 1940 and 1979, some 27,000,000 persons in the United States had occupational contact with asbestos, with about 7% of this number engaged in primary mining, milling, or manufacturing asbestos and the largest exposed groups resulting from the construction and shipyard industries. It has also been estimated that some 4,000,000 persons were exposed to asbestos in shipyards during the Second World War (20,68). Asbestos exposure has been related to five different pulmonary lesions: benign asbestos-induced pleural disease, asbestos-induced small airway disease, asbestosis, lung cancer, and mesothelioma. Manifestations of benign asbestos-induced pleural disease include parietal pleural plaques, diffuse pleural fibrosis, rounded atelectasis, and benign asbestos effusion. Although the pathogenesis of these disorders is poorly understood, it is believed that transport of asbestos fibers to the pleura, either directly through the lung parenchyma or through the lymphatics, is involved (69). Pleural plaques consist of dense fibrous tissue usually free of any inflammatory reaction that can become calcified. A great majority of individuals with pleural plaques do not exhibit any clinical symptoms. Diffuse pleural fibrosis is generally nonspecific, consisting of dense collagenous tissue and various chronic inflammatory cells, including lymphocytes, macrophages, and plasma cells. Rounded atelectasis usually occurs as a result of contraction and buckling of fibrotic visceral pleura, leading to collapse and folding of the immediately adjacent lung parenchyma. Benign asbestos effusion can occur with or without an inflammatory infiltrate but is characteristically an exudate, with glucose and protein levels similar to those of plasma (69). Asbestos-induced small airway disease is exhibited by fibrosis of the respiratory bronchioles and alveolar ducts and is morphologically distinct, with the presence of pigment and asbestos bodies along with fibrosis and distortion of airway walls (20). Asbestosis is diagnosed when bilateral diffuse interstitial fibrosis of the lung is observed with the presence of asbestos bodies. Asbestosis is usually found in persons with sufficiently high exposures over many years, and has typically been documented in miners and millers, asbestos textile workers, and workers handling raw asbestos insulation. This disease is generally observed at doses in the range of 25 to 100 fibers/cubic
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centimeter-year (f/cc-yr) and greater, with amosite and crocidolite causing asbestosis at the low end of this range and chrysotile-induced asbestosis requiring doses in excess of 100 f/cc-yr (20,70–74). The pathogenesis of asbestosis involves a complex cascade of inflammatory, cytokine, and ROS responses, with the polymorphonuclear leukocyte and macrophage being primary effector cells in the process. Macrophages, as well as asbestos fibers themselves, produce ROS catalyzed by iron on the fiber surface, which in turn upregulates cell signaling pathways leading to production of inflammatory mediators, neutrophil chemoattractants, cell damage, and proliferation, and, ultimately, collagen formation. The central role of ROS in asbestosis has been confirmed through the prevention of inflammation and fibrosis by administering oxygen species scavengers such as catalase and iron chelators (75–81). Despite the role that ROS and proinflammatory and profibrogenic mediators play in asbestosis development, researchers have noted other factors may be just as important, because similar mediators are produced with crystalline silica and man-made mineral fibers but very different pathologies result (75). In any case, three factors appear to be critical in determining fibrogenic potential of asbestos: fiber length, biopersistence, and dose (20). D. Beryllium and Chronic Beryllium Disease
Beryllium is a metal that has been used in a wide variety of applications for over 75 years. One of its first applications was in the manufacturing of fluorescent and neon lights; it was in this industry that acute pneumonitis or acute beryllium disease was first identified and coined “Salem sarcoid” by Harriet Hardy and her colleagues, who were studying manufacturing workers exposed to high concentrations of airborne beryllium (82). Because beryllium was critical to the development of military defense weapons during the Cold War, much of its history as it relates to occupational disease [including the development of an occupational exposure limit (OEL)] was derived from the experience of the Department of Energy (DOE). Because of its many unique characteristics, beryllium has been, and continues to be, used by a variety of strategic and vital industries, including aerospace, defense, energy, telecommunications, and medical equipment. There are four primary forms of beryllium: beryl ores, beryllium metal, beryllium oxide, and beryllium alloys. Copper beryllium alloy is most widely used today. It has been estimated that between 26,400 and 134,000 workers are currently exposed to beryllium in the United States (83). Beryllium disease can develop in two forms: acute and chronic. Acute beryllium disease is a form of acute respiratory distress syndrome (ARDS), which is a rapid onset of widespread diffuse or local pulmonary infiltrates that can be caused by fume inhalation. Because acute beryllium disease generally requires exposures on the order of 100 mg/m3, this disease has largely disappeared from occupational settings. Although the occurrence of CBD appears to be dose related, its pathogenesis is complicated by its immunologic basis. CBD is characterized by the formation of noncaseating granulomas, with an appearance and distribution indistinguishable from sarcoidosis. Granulomas can be found along lymphatic routes and in the alveolar interstitium, and may coalesce to form nodular lesions (20). The immunologic basis of CBD was first hypothesized by Sterner and Eisenbud in 1951 (84), and, today, it is largely accepted that individuals with CBD first become sensitized to beryllium before they progress to developing granulomas. At present, persons may be diagnosed as having CBD without any symptoms of
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lung impairment. Medical and diagnostic advances, such as the bronchoalveolar lavage, fiberoptic bronchoscope, and the blood lymphocyte proliferation test (BeLPT) have allowed for CBD diagnosis before any clinical symptoms are detectable. The diagnostic criteria for CBD generally involve two positive blood BeLPTs or one bronchoalveolar lavage BeLPT, and the presence of pulmonary granulomas on lung biopsy (with or without physical symptoms) or detection of chest X ray or pulmonary function changes typically associated with CBD pathology. There are a number of factors that appear to influence the risk of CBD. These factors include particle size, type of operation, chemical form of beryllium, and genetic susceptibility (85–93). While there appears to be a threshold exposure that does not lead to beryllium sensitization or CBD, researchers have had a difficult time identifying the exposure metric (peak, average, cumulative, average) that best predicts the risk of disease (86–89,91,92,94,95). Although the immunologic basis of CBD is not completely understood, there appears to be a strong genetic component associated with the disease, which in turn may explain why some individuals have developed CBD after as little as three months of exposure in an occupational setting (86). The genetic basis of immunologic reactivity to beryllium involves human leukocyte antigens (HLA). Several studies have shown that glutamic acid in the 69th position of the B1 chain of the HLA-DP allele infers an increased risk of CBD; however, studies have also shown that genetic makeup cannot fully account for the incidence of disease. Because the Glu69 marker on HLA-DP has a poor predictive value due to its high prevalence in unaffected populations and the relatively low prevalence of CBD, researchers have investigated other genetic markers for the disease (93). Despite any future potential advancement in identifying persons who might be genetically susceptible to developing CBD, the ethics of genetic testing and potential exclusion of persons from the work environment will likely offer an entirely new set of challenges for this industrial hazard. E.
Engineered Nanoparticles
The emergence of engineered nanoscale materials provides tremendous promise of significant advancements in the fields of imaging, electronics, and therapeutics (96–102). What makes understanding the human health risks associated with engineered nanomaterials particularly challenging is that their potential applications could result in a wide range of plausible chemical exposure scenarios (i.e., worker, environmental, and consumer product), resulting in very different toxicities depending on production method, chemical and physical properties, or ultimate end use. While workers, consumers, or the general public may potentially be exposed to nanoparticles through a number of pathways (e.g., dermal, ingestion, ocular), inhalation, at least from an occupational standpoint, is likely to be one of the most significant routes of exposure. Although the potential biocompatibility and toxicity of engineered nanomaterials has only recently received attention from the scientific community, our understanding of well-studied particles like TiO2 provides a framework for evaluating the characteristics of the engineered nanoparticles that have the greatest influence on biological fate and toxicity. Because TiO2 particles were historically viewed as having low toxicities and were generally classified as a nuisance dust, for many years these particles were used as negative controls in inhalation and intratracheal instillation animal studies investigating
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the toxicity of other particles (103,104). It was not until reports of lung tumors in rodents exposed to extremely high doses of TiO2 particles (i.e., particle overload conditions) (105,106) and different potencies of lung effects for fine versus ultrafine particles that the mechanisms by which these particles induced adverse health effects were further evaluated (107–109). A significant amount of research has been conducted over the past few years to better understand how the different physicochemical forms of TiO2 particles can influence their potential toxicity. Recent investigations of the physicochemical aspects of TiO2 particles show that particle coating, size, agglomeration state, and crystalline form influence pulmonary toxicity; however, particle surface area shows no effect on the cytotoxic responses of pulmonary cells (110,111). In addition, a recent study reported that nano-sized rutile TiO2 could cause severe pulmonary emphysema through activation of inflammatory pathways following acute intratracheal instillation (112). Carbon-based engineered nanoparticles, such as single-walled carbon nanotubes (SWCNTs), multiwalled carbon nanotubes (MWCNTs), and fullerenes, have received notable attention due to their superior electronic, optical, mechanical, chemical, and even biological properties. Recent research efforts on the health effects of engineered nanoparticles have primarily focused on carbon nanotubes (113–120). The majority of these studies were conducted using in vitro methods, while only a few studies have evaluated health effects in vivo. The majority of studies conducted to date have delivered carbon nanotubes to the respiratory tract via intratracheal instillation or pharyngeal aspiration. The type of carbon nanoparticle (i.e., SWCNT, MWCNT, fullerene), method of processing (i.e., refined or unrefined), presence of residual transition metal catalysts, and functionality of different reactive groups are a few of the parameters researchers have tested in cultured cells to better understand which physicochemical characteristics influence toxicity (121–124). Of the in vivo studies conducted on carbon nanotubes, all report inflammation, progressive fibrosis, and granulomas in rodents exposed to carbon nanotubes via intratracheal installation or pharyngeal aspiration. More specifically, as a result of these exposures, acute dose-dependent changes in alveolar wall thickness, immune cell recruitment, and indicators of cellular damage and oxidative stress (measured by levels of inflammatory cells, cytokines, and protein in bronchoalveolar lavage) were observed (116,117,119,120,125). In an attempt to understand how different physical and chemical parameters contribute to these toxicological effects, researchers have evaluated the impact of the method of carbon nanotube production as well as the influence of milling carbon nanotubes or altering the content and type of metal catalyst on the toxicity in animals. Results suggest, however, that carbon nanotubes themselves induce cellular changes, because all of the various formulations of carbon nanotubes produce pulmonary lesions (116).
IV.
Structural Remodeling in the Lungs by Particles in Ambient Air
Particulate air pollutants are complex mixtures derived from a variety of anthropogenic and natural sources. Ambient particulate matter (PM) can cause effects on the respiratory system that are similar to particulates found in occupational settings; however, extrapulmonary changes (e.g., cardiovascular effects) have also been documented from
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peak exposures to ambient PM. While research of the effects of ambient PM over the past few decades has focused on the PM in the coarse- and fine-sized fractions, considerable efforts have recently been made in understanding the role that ultrafine particles may play in either contributing or exacerbating cardiopulmonary disease in normal and susceptible populations. It should be noted, however, that with the wave of enthusiasm about the field of nanotechnology, researchers are now referring to ambient ultrafine particles as nano-sized particles produced by incidental means. The different size fractions of ambient PM have very different physicochemical characteristics, partly because of their emission sources. Coarse particles are generally comprised of natural materials (i.e., minerals, silicates, pollen) derived from weathering and disturbance of earth soils, whereas fine particles usually originate from anthropogenic sources (i.e., combustion processes, industrial emissions) and are comprised of a mixture of elemental and organic carbon, sulfate, nitrate, minerals, and metals. Ultrafine PM is also generated through combustion processes, but can quickly aggregate to form larger-sized particles (20,126,127). Episodes of intense particulate air pollution have been known to cause increased morbidity and mortality for at least a half-century. The Donora death fog in 1948 and the London fog in 1952 were notable air pollution events that led to upwards of a few thousand excess deaths and, more recently, the work from the Harvard Six Cities studies in 1993 showed that peak particulate air pollution events led to increased deaths from lung cancer and cardiopulmonary disease (128). Over the past decade, over 100 studies of more than 35 different cities have investigated the acute effects of ambient PM showing increased hospital admissions and deaths from cardiopulmonary disease (e.g., asthma, chronic obstructive pulmonary disease, arrhythmia, heart attack) (129–131). The effects appear to best correlate with PM2.5, with an increased mortality of 0.5% to 1.5% for every incremental concentration increase of 5 mg/m3 (130). These studies have served as a basis for the National Ambient Air Quality Standard for PM (126). There is considerably less information about the chronic effects of ambient particulate pollution. Although lung tissue remodeling is known to occur from exposure to ambient PM, these changes tend to be less prevalent and severe, compared to those observed in occupational settings. Autopsy studies have provided some means for assessing the potential long-term effects of exposure to ambient PM. Findings from these studies have suggested that long-term exposures to ambient PM can lead to remodeling of the lungs with the most significant changes occurring in the respiratory bronchioles and bronchoalveolar duct junctions or centriacinar regions of the lungs. Pinkerton et al. (2000), for example, evaluated lungs of deceased young males in California’s Central Valley who died of nonrespiratory causes. The individuals had been exposed to ambient conditions consisting primarily of mineral and carbonaceous dusts. Dust deposition was principally observed in tissue sections at the terminal bronchioles and first-generation respiratory bronchioles, with less deposition observed in the upper airways (Fig. 4). There was significant wall thickening via inflammatory cell accumulation (dust-laden macrophages), increased collagen deposition, and smooth muscle-cell hypertrophy, resulting in terminal and first-generation respiratory bronchiole structural remodeling. Changes in lung structure and retention of particles appeared to be inversely proportional to the distance from the center of the lung acinus; particle retention decreased from the first generation, to second generation, to third generation of respiratory bronchioles (Fig. 5). The respiratory bronchiole effects were also observed in the
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Figure 4 Bright field (A) and polarized field (B) images of a first generation respiratory bronchiole from a human lung of a 32-year-old farm laborer. The inset in panel A and shown in panel B contains numerous polarized particles within intraluminal macrophages as well as within the walls of the respiratory bronchiole and adjacent alveoli. Source: From Ref. 132.
lungs of both smokers and nonsmokers, with more severe changes occurring in smokers. It was suggested that there might be synergistic effects between ambient PM and cigarette smoke, but despite any interactions, respiratory bronchiole remodeling as a result of exposure to ambient PM could be detected irrespective of the smoking status (132). The findings from Pinkerton and colleagues have also been observed in other PMexposed populations. Subjects living in high-PM areas in Canada showed increased particle deposition in the respiratory bronchioles and at airway bifurcations that correlated with airway remodeling (133,134). These studies have shown correlations of high ambient PM exposure to particle deposition and airway remodeling in the lungs with indications that fine (PM2.5) and aggregations of ultrafine particles (PM0.1) are likely the particle size fraction contributing to these effects because of their prevalence in lung tissue digests (132,135). Further studies comparing individuals exposed to
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Figure 5 Light micrographs of contiguous first-, second-, and third-generation respiratory bronchioles from the human lung showing the normal structural anatomy (A) and marked alterations (B) due to increased amounts of interstitial collagen, smooth muscle, and visible pigment. These structural changes are most dramatic in the first-generation respiratory bronchiole with a progressive decrease in more distal generations. Source: From Ref. 132.
nonoccupational, high ambient PM have indicated that ultrafine particles retained in the bronchiole airway walls are associated with fibrogenic small airway remodeling and may produce a chronic airflow obstruction (136). Although these results may suggest possible effects from long-term exposures to ambient particulate air pollution, it is important to keep in mind that other confounders (e.g., genetic, lifestyle, smoking, occupational exposures, other ambient pollutants) may have some contribution to these effects and may not be fully accounted for in the individual case history (132,134–138). As a means to understand the potential chronic effects of ambient PM, researchers have utilized tracheal explants as a model system for controlling the administered PM
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dose to a particular region of the lungs. More specifically, it has been shown that particles (collected ambient PM, mineral dusts, and diesel exhaust PM) administered into the airway leads to expression of mediators promoting fibrosis and smooth muscle hyperplasia. The expression occurs without exogenous inflammatory cells and suggests PM may directly cause epithelial cell injury, airway remodeling, and possible obstruction even in the absence of inflammation (139,140). Further, studies of concentrated ambient particles (CAPs) have shown both pulmonary and extrapulmonary effects, including upregulation of proinflammatory genes and markers of oxidative stress in the lungs as well as systemic effects suggesting an increased risk of arthrosclerosis (138,141,142).
V. Experimental Animal Studies in Evaluating Risk of Particle-Induced Disease Because there are so many parameters that may ultimately influence the toxicity or biocompatibility of inhaled particles, researchers have relied on a combination of particle-delivery techniques (intratracheal instillation/aspiration/inhalation or nose-only/ whole-body inhalation) as a means to study the pulmonary and systemic effects of particles. It is important to keep in mind, however, that the method of delivery may have significant effects on the pattern, extent, and timing of lung remodeling. Intratracheal techniques include (i) instillation, which involves injection of particles suspended in saline through a catheter inserted in the trachea of the animal, (ii) aspiration, which entails administration of a suspension as droplets in a puff of air, and (iii) inhalation, which involves cannulating the trachea, attaching the open end of the cannula to a port of an aerosolization system, and ventilating the animal at a known rate and pressure (143–145). During these intratracheal techniques, animals are anesthetized during particle exposures; however, with nose-only or whole-body inhalation exposures, animals are not sedated and are either constrained in a tube or allowed to roam freely within the cage during the exposure. While these methods of particle administration to the respiratory tract have their limitations, they do have unique advantages (Table 1) (145–147). The greatest benefit that intratracheal instillation provides, for example, is delivering a known amount of material so that the effects of different doses and formulations (e.g., particle size, shape, chemistry) can be compared easily and within a reasonable time frame. This method also circumvents the need for the specialized equipment and expertise that are usually required for inhalation studies. When deciding on a method of particle delivery to the lungs, it is important to understand that patterns of particle deposition, translocation, and retention following intratracheal instillation may not accurately reflect the physiologic patterns observed with particle inhalation. Scientists have attempted to address this issue and also to design a system that would allow for delivery of a known amount of test material to the lungs by using intratracheal inhalation. As with intratracheal instillation, intratracheal inhalation bypasses the normal scrubbing mechanisms of the nasal turbinates and, therefore, particles administered in this manner are delivered to a region of the lungs that may not otherwise be accessible. Despite this shortcoming, intratracheal or nose-only inhalation studies offer the advantage over whole-body inhalation studies because particles are delivered by only one route. Recent work evaluating the
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Table 1 Potential Advantages and Limitations of Different Methods of Particles Administration to the Respiratory Tract
Advantages
Intratracheal instillation
Inhalation
Inexpensive
Provides a natural way for delivery of toxicants Deposition and clearance patterns comparable to that in a real world setting Evaluate effects at all levels of the respiratory tract Results in even distribution of delivered toxicant
Actual delivered dose is known
Limitations
Minimal risks to workers administering the material Administration of multiple doses within a short period of time Comparison of responses to different toxicant formulations (size, shape, chemistry) Avoids exposure to animal skin or pelt Localize exposure to specific lung lobes in larger animals Useful as a screening tool for dosing and toxicity ranking Administration of material in a nonphysiologic invasive manner Dose rate is greater than by inhalation Distribution in the lungs will differ compared to inhalation exposures Avoids natural scrubbing mechanisms of nasal passages Bypasses upper respiratory tract, which could normally be a target Suspension may not represent nature of the material in a real world setting Unnatural pattern of deposition may translate to unnatural patterns of clearance and retention Reproducibility of delivery of material dependent on technician experience Clearance mechanisms and kinetics may not be comparable to inhalation Particle clumping, local inflammation, irregular particle retention may be unique to method of administration
Source: Adapted from Refs. 145–147.
Expensive Requires specialized expertise and equipment for system development and design Sufficient test material required for duration of testing Dermal/fur contamination with whole-body exposures Proper handling of hazardous aerosols Delivered dose can be estimated or measured through sophisticated labeling Variability of particle burden can be great
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extrapulmonary transport of ultrafine carbon particles delivered by whole-body inhalation demonstrates that particle accumulation in the liver could not be explained solely by particles deposited in the lungs, and that particle ingestion through external contamination of animal pelt likely contributed to the liver dose (148). Despite the differences in particle fate and transport observed between intratracheal instillation and inhalation studies, general patterns of toxicity, such as inflammatory markers (e.g., immune cell profile, interleukin-1, tumor necrosis factor-a, macrophage inflammatory protein-2) and lung injury (e.g., lactate dehydrogenase, total protein) show similar trends that vary in timing and severity, depending on the administration method (143,144,146,149,150). For this reason, intratracheal instillation studies are generally viewed as useful methods for screening the potential health effects of different materials. Intratracheal instillation also provides an efficient and cost effective way of comparing the relative toxicity of different materials over a range of doses, as well as of evaluating the potential mechanisms by which different materials elicit different biological responses in the lungs and extrapulmonary organs. Instillation methods, though, cannot be used to evaluate particle deposition patterns. Caution should also be taken when interpreting patterns of particle translocation and retention and pulmonary histopathology since particle clumping, local inflammation, and irregular particle retention may be a reflection of the administration method and not of the inherent nature of the administered material. One difficulty to evaluating the toxicology of particles, especially in experimental animal studies, is being able to administer the materials to test species in a manner comparable to that experienced in the real world. In the case of evaluating the hazards of inhaled materials, it is often cost-prohibitive and not feasible to test every material in inhalation studies over the life span of the test species. Thus, researchers have looked to different in vivo and in vitro animal models, efficient dosing methods (e.g., intratracheal instillation), and precursor markers of disease as a means to assess hazards within a testing regimen of reasonable duration and resources as well as a basis for understanding the potential toxicity of new substances (151). There are numerous examples in the scientific literature of pulmonary exposure to respirable-sized particles (e.g., mineral dusts, combustion particles, environmental tobacco smoke) inducing a cascade of inflammatory and oxidative reactions that ultimately lead to pathology and remodeling of the respiratory tract. As a means of predicting whether a substance might pose a risk of disease and cellular restructuring, researchers have utilized various cellular and biochemical markers to detect precursor events to disease. Some pulmonary markers that have found good correlations with pathology include: bronchoalveolar lavage markers (e.g., lactate dehydrogenase, protein, differential profile), cytokine release, macrophage function, cell proliferation, antioxidant capacity (glutathione), collagen formation, and markers for lipid peroxidation (119,151,152). One way that researchers have attempted to bridge responses observed in different model systems or through different routes of exposure is by comparing oxidative (e.g., glutathione, P450, lipid peroxidation), inflammatory (e.g., cell differential, cytokine), and cytotoxicity (e.g., cell viability, permeability, histopathology) marker profiles. While clear toxicological differences may be illustrated for different materials in in vitro cell systems, these same responses are not always seen when administering the same material in vivo (123,153). For example, it has been shown that different in vitro cell lines can exhibit different cytokine profiles, which can also be influenced simply by the
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culture media (154). Additionally, particles have very different physicochemical characteristics depending on whether they exist in a dry aerosolized form or are suspended in a wet media (i.e., water, culture media), all of which can have profound effects on interpreting any potential hazards. As a result, a critical component of “bridging” effects across any series of model systems or testing regimens is a clear understanding of the properties of the test material and the endpoints that best correlate with in vivo conditions.
VI.
Future Research Needs
Future research needs include a better understanding of how various particle characteristics (e.g., size, agglomeration, morphology, solubility, surface chemistry) or exposure/dose metrics (e.g., mass, size, surface area) will influence their biological fate and toxicity and elicit biological responses (Fig. 6). Newly developed engineered nanoparticles (i.e., quantum dots, carbon nanotubes) or model particles (i.e., polystyrene,
Figure 6 Hypothetical mechanisms for particle-induced remodeling of the lung.
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iridium, gold, and manganese oxide) as well as extensively studied particles produced through manufacturing (i.e., ultrafine TiO2 or carbon black) or combustion processes (i.e., vehicle exhaust, air pollution) are being investigated to understand how surface properties of particles delivered via the respiratory tract may influence the disposition, fate and transport, and biological responses in pulmonary and extrapulmonary tissues. Ongoing and future research efforts will benefit from investigators relating exposure and dose metrics administered in experimental studies to those observed in real world settings. Achieving a better understanding of the dynamics at play between particle physicochemistry and transport patterns and cellular responses in the lungs and other organs will provide a future basis for establishing predictive measures of toxicity or biocompatibility for developing testing strategies for evaluating the particle safety through all pertinent routes of exposure and for creating frameworks for assessing overall potential human health risk.
VII.
ACKNOWLEDGEMENT
Background literature research has contributed to conceptual development of original research funded by US EPA Star Grants R831714, R832414, and R82215, NIOSH Grant 0H07550, and Student Fellowship from the University of California Toxic Substances Research & Teaching Program to the University of California, Davis. The authors prepared this chapter under the above noted grants, as well as during the normal course of employment by ChemRisk, LLC (A.M.) and the University of California, Davis (A.M., C.C., K.P.). The authors have sole responsibility for the writing and content of the chapter. The contents of this review may not necessarily reflect the views of the funding organizations.
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11 Effect of Particles on Mucus and Mucociliary Clearance MALCOLM KING University of Alberta, Edmonton, Alberta, Canada
I.
Introduction
This chapter deals with how the mucus reacts to particles and particulate loads, thereby altering its pattern and rate of clearance. Particles can stimulate secretion and modulate ciliary function either due to mechanical factors or irritation or via the chemicals they contain or are capable of releasing. Particles have a direct effect on the physical properties of mucus, even when acting as “neutral filler,” where their contribution to viscoelasticity is a simple function of their volume fraction. Beyond this, particles may interact with the mucous gel network and add multiple cross-link points. Living particles, such as bacteria and leukocytes, can have profound effects on the mucus through the macromolecules and mediators they release. Particles may also disturb the osmotic balance, thereby altering the mucus, and indirectly or directly enhance or depress clearance. The chapter begins with a brief introduction to the mechanical properties of mucus and how these relate to clearance.
II.
Viscoelastic Properties of Airway Mucus
Because of the cross-linking of glycoproteins, mucus rheological behavior is described as viscoelastic, having characteristics of both liquid and solid (1–3). Viscosity is the resistance to flow, reflecting the absorption of energy from an object, such as a solid particle moving through a substance. Elasticity is the recoil energy transmitted back to this object. With ideal fluids, viscosity is independent of the applied stress. With viscoelastic liquids such as mucus, viscosity decreases with increasing stress or rate of strain (shear rate). Mucus responds to stress with an initial solid-like deformation followed by a viscoelastic deformation, and finally by a period of steady flow in which the rate of deformation is constant. Only partial recovery of the strain follows removal of the stress indicating a permanent deformation of its gel structure. This type of viscoelastic behavior is illustrated in Figure 1. Mucus exhibits shear thinning, in that following exposure to high shear forces, it shows a decreased viscosity at low shear rates. Some shear thinning may be permanent with a permanently reduced viscosity (altered molecular structure) while some shear
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Figure 1 Diagram showing the viscoelastic behavior of a mucous gel. The first two panels illustrate stress-strain relationships in idealized materials, namely, an elastic solid, where the displacement or strain is proportional to the applied force or stress, and a viscous liquid, where the rate of strain (displacement/time) is proportional to the stress. Mucus is a viscoelastic semisolid. It responds instantaneously as a solid, with a very rapid displacement in response to an applied force. This is followed by a transition to a liquid-like response, where the rate of strain is constant with time. Finally, a zone of viscous response is reached, where the rate of displacement is constant with time. After release of the applied force, the mucous gel only partially recoils to its initial position.
thinning may be reversible (thixotropy). When sputum is obtained by aspiration under pressure, it undergoes shear thinning, dilution by irrigation fluids, and incorporation of air bubbles. Water can bind to mucus glycoprotein (MGP) macromolecules and influence viscosity. Viscosity can be increased by dehydration of the mucus, as can mucusepithelium adhesion. In purulent sputum the correlation between viscosity and dry weight of solids is poor and may explain why mucous glycoprotein content is a poor index of viscoelasticity in chronic bronchitis, bronchiectasis, and cystic fibrosis. Mucus viscoelasticity increases with acidic pH (4), causing reduced mucociliary clearance. Mucus viscoelasticity is also dependent on the content of low molecular weight electrolytes. These properties reflect the polyelectrolyte nature of mucins (5–7). Changes in mucus viscosity and elasticity are generally interrelated (4,8), although interventions that break or add cross-links can alter the close relationship (9,10). Optimum mucociliary clearance of airway mucus is located at the low end of the normal range of viscoelasticity (1,11,12). A decrease in clearance rate (transport)
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occurs with increasing mucus elasticity and increasing viscosity. Increasing viscosity with constant elasticity causes a pronounced decrease in the mucociliary transport rate (13). Decreasing mucus viscosity alone results in an increased transport rate and may explain the improvement in sputum mobilization following hydration or mucolytic drug therapy. Another measure of elasticity is the spinnability (Spinnbarkeit, filance)—the thread-forming ability of mucus under the influence of large amplitude elastic deformation. Spinnability has been correlated positively with mucociliary clearance (14) and negatively with cough clearance (15). Adhesivity is the ability of mucus to bond to a solid surface measured as the force of separation between one or more solid surfaces and the adhesive material. This is dependent on mucus surface tension, hydration, wettability, and contact (dwell) time. Adhesivity has been found to correlate inversely with both mucociliary clearance and cough clearance (15,16).
III.
Role of Mucus Viscoelasticity in Mucociliary and Cough Clearance
Mucociliary clearance can be measured by visualizing the movement of particles such as Teflon disks, tantalum powder, or charcoal powder placed on airway during bronchoscopy. An inert, radiolabeled tracer can also be placed on the airway and its clearance monitored with scintillation counters or a g-camera. Whole lung clearance is measured by having the subject inhale an aerosol of radiolabeled tracer and then scanning over defined regions in the proximal and distal lung. A decrease in mucus transport velocity occurs with increasing airway generation (17); otherwise stated, the rate of mucus transport accelerates from the peripheral airways to the larynx. For example, in excised dog lungs, the rate of particle clearance was found to increase from 1.6 mm/min in subsegmental bronchi to 8.3 mm/min in mainstem and 12.6 mm/min in trachea (17). Impaired mucociliary transport leads to retained secretions in the airways and increased susceptibility to infection. Accumulation of mucus could increase the risk of destructive, inflammatory, and neoplastic lung disease by prolonging the contact time between inhaled materials and the airway mucosa (18). Since cough clearance is ineffective in peripheral airways (19), mucociliary clearance is relatively more important in cleansing and protecting these lung regions. The structure and function of the epithelial cilia have been the subjects of excellent reviews (20,21). The periciliary fluid appears to be regulated by various mechanisms, the most important of which are active water and ion transport across the airway surface epithelium (22,23). If the depth of the periciliary fluid is too shallow, the cilia are unable to beat effectively and may become entangled in the mucous gel layer. On the other hand, a fluid layer too deep may not allow the cilia to make contact with the mucous gel layer, thus decreasing mucociliary clearance. The depth of the periciliary layer may be regulated by homeostatic mechanisms. The viscoelasticity of the mucus layer contributes to the effectiveness of the mucociliary interaction, but the surface interaction between mucus and cilia, perhaps through the influence of surfactant (24,25), also plays a critical role. Ion and water transport across the surface epithelium appear to be crucial to this interaction. By generating local osmotic gradients, epithelial ion transport processes regulate the depth
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and composition of the periciliary sol layer (22,26). Ion transport is regulated by neurohumoral mechanisms, cholinergic and adrenergic agonists, prostaglandins, substance P, vasoactive intestinal peptide, and bradykinin (27). The transport velocity of mucus simulant gels is directly related to mucus elasticity and the depth of the periciliary fluid, and it is inversely related to mucus viscosity (1). An ideal viscoelastic ratio may exist for optimal mucociliary interaction; an increase in viscosity and/or a decrease in elasticity would result in a reduced transport rate. Transport by cough or airflow interaction depends inversely on viscosity, elasticity (spinnability), and adhesivity (1). Mucus that is elastic rather than viscous is transported well by ciliary action but less well by coughing (2).
IV.
Stimulation of Secretion and Modulation of Mucociliary Function by Particles
Particles can stimulate secretion and modulate ciliary function either due to mechanical factors or irritation or via the chemicals they contain or are capable of releasing. Even neutral particles can represent a burden on the mucociliary system. Although there is some evidence that the completely unloaded mucociliary system may be quiescent, it is apparent over a wide range of loads that mucociliary systems are relatively insensitive to the particulate burden they bear. This has long been recognized for the frog palate mucociliary system (28,29) and appears to be the case for normal mammalian mucociliary clearance as well (30), where particle clearance velocity in healthy dogs was independent of tracheal mucus depth up to apparent depths of about 100 mm. The same is not true for clearance by airflow mechanisms, where increasing the particulate or fluid burden can stimulate cough clearance (31,32).
V. Modulation of Cough Clearance and Aerosolization by Changes in Mucus Viscoelasticity Changes in the viscoelastic properties of mucus can lead to alterations in the efficiency of cough clearance as well as in the aerosolization of mucus due to airflow interactions. In general terms, elasticity in the mucous gel promotes efficient energy transfer between the cilia and the mucus, while elasticity tends to inhibit cough clearance due to recoil (32,33). Hence mucus that is efficient in both forms of clearance must exhibit a balance of viscoelastic properties. This can be tempered by the frequency effect; mucus that is more elastic at low frequency and more viscous at high frequency would be cleared efficiently by both mechanisms. In addition, aerosolization of mucus during coughing is inhibited by cross-links in the mucous gel. Zayas et al. (34) showed that by introducing selective cross-linking with certain agents (mucomodulation), the amount of fine aerosol released during cough simulation could be greatly decreased, while still allowing for bulk flow. Further, this mucomodulation process could be carried out without reducing mucociliary clearance, and even enhancing it (35). These alterations in fine aerosol formation could potentially be utilized to reduce the spread of airborne pathogens, for example, in the control of tuberculosis and influenza.
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Effects of Particles as a Mechanical Filler
The effect of cellular debris and other particulate matter on the mechanical properties of mucus depends on the strength of the interaction. It has generally been assumed that cells act as neutral fillers, adding relatively little to the viscoelastic properties of mucus in comparison with other sources of variation in mucous gel viscoelasticity. The contribution of cellular debris to the mechanical properties of mucus has been considered as follows (36): for an ideal gel loaded with noninteracting rigid particles, the elasticity is predicted to increase by the factor (1 f/fm)2.5, where f is the volume fraction of particles (independent of particle size) and (fm is the maximum volume fraction corresponding to close packing (~0.8). For a viscoelastic gel, elasticity G0 and viscosity Z0 are reportedly both increased by about the same factor (8). In simpler terms, the contribution of a neutral filler varies as the volume fraction to the 2.5-power. Thus, a cellular content of 2%, if weakly interacting, would only increase G0 and Z0 by about 6%, and even doubling the number of cells would change these parameters by less than 15%, below the precision level for most rheological methods for mucous gel viscoelasticity. Tracheal mucus samples obtained by scraping, where the cellularity is high, show little variation in viscoelastic properties from samples obtained by more gentle approaches (8). In the referenced study, the cellular contents of the mucus samples were not measured; however, the fraction of cellular debris in the samples obtained by tracheal scraping was probably higher than in those obtained from the cytology brush. In nonpurulent sputum, the nondispersable dry weight fraction, presumably cellular debris, is found to be about 0.3% to 0.4% (37), which would correspond to about 1.5 to 2 vol% cellular debris assuming a cellular water content of ~80%. If the interaction between particles were strong, the particles could act as additional cross-links and increase G0 and Z0 . However, the fact that no significant differences were found between the two sampling techniques suggests that epithelial cells contribute relatively little to mucus cross-linking, other than perhaps their role as noninteracting filler. The same can be said of erythrocytes, which again show little effect on rheology up to 5% to 10% admixture (unpublished results).
VII.
Contributions of Living Particles to Mucus Rheology and Clearance
Some living particles, such as bacteria and leukocytes, can have profound effects on the mucus through the macromolecules and mediators they release. Thus, the above considerations about particles as neutral filler may not extend to leukocytes; however, particularly eosinophils, which are numerous in asthma and bronchopulmonary aspergillosis. The main protein output of eosinophils includes highly basic proteins such as major basic protein and eosinophil cationic protein (38), which are highly charged cations and which could be expected to contribute to cross-linking by interacting with the anionically charged mucin macromolecules. Leukocytes can also contribute to mucus viscoelasticity through the release of DNA and actin proteins, both occurring as inflammatory cells die during infections (39). For example, neutrophil DNA and F-actin are believed to contribute significantly to mucus viscoelasticity in cystic fibrosis lung disease by providing extra cross-linked networks parallel to the basic mucin network
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Figure 2 Diagram illustrating the types of bonding in a mucous gel and the site of interaction
with various mucolytic treatments and of potential interaction with active particles. The mucous glycoproteins (mucins) consist of highly glycosylated subunits of ca. 500 kDa linked by nonglycosylated regions containing cysteines, which stabilize the structure via intramolecular bonds. The oligosaccharide side-chains are composed mainly of N-acetylglucosamine, N-acetylgalactosamine, galactose, fucose, and sialic acid. These glycosidic units contain numerous sites for hydrogen bonds as well as ionic interactions. The blood-group specificity of the terminal saccharide units allows for specific interactions with surface antigens on “particles” such as leukocytes and bacteria. In infection, additional large macromolecules, such as undegraded DNA and actin filaments released from leukocytes and bacteria, participate extensively in the threedimensional structure of the gel. Abbreviations: DTT, dithiothreitol; NAC, N-acetylcysteine; rhDNase, recombinant human Dnase. Source: From Ref. 7.
(40,41). The inadequacy of native levels of DNase and actin-limiting proteins has led to new therapeutic approaches to reduce the hyperviscoelasticity of infected sputum (e.g., rhDNase, gelsolin, thymosin b4) (10,42). Some of these approaches are illustrated in Figure 2. However, neutrophils also release large quantities of proteases, such as elastase and cathepsin G. These proteases are the most potent secretagogues known (43). Not only do they stimulate the output of mucus from secretory cells but are also capable of degrading mucous glycoproteins, particularly when the DNA and actin that accompany the mucins in purulent secretions become degraded (44). Thus, the net effect of the byproducts of infection on mucus rheology is rather unpredictable.
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Effects of Particles as an Osmotic Load
Particles may also disturb the osmotic balance, thereby altering the mucus, and indirectly or directly enhancing or depressing clearance. This possibility is illustrated in Figure 3, indicating the polyelectrolyte nature of mucins. Increasing the ionic content of the mucus could result in reducing the size of the macromolecules, thus reducing entanglement cross-linking (4,45). Several recent studies have indicated a positive effect of hypertonic saline aerosols on mucociliary clearance (46,47), and at least part of the effect appears to occur through altering the cross-link density of the mucus. These
Figure 3 Diagram illustrating the polyelectrolyte nature of mucous glycoproteins (mucins). The
excess of fixed negative charges along the macromolecular chain is indicated by the solid bars. Mobile ions are indicated by þ and symbols. At low ionic strength, the fixed charges along the macromolecule are poorly shielded by the mobile counterions and the polymer is in a highly expanded conformation due to the ionic repulsion. This allows for excess entanglements (crosslinking) between neighboring macromolecules. At higher ionic strength, the excess numbers of mobile ions solvate and shield the fixed charges, reducing the effective size of the mucin macromolecules, resulting in fewer entanglement cross-links.
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effects could be of particular importance in chronic airway diseases, where the mucins become typically more acidic and more highly charged in nature (5,37). Incorporation of charged particles into the mucous gel could also affect its viscoelastic behavior; positively charged particles could interact with the anionic macromolecules, resulting in increased cross-link density. Indeed, negatively charged sulfur colloid is transported consistently faster than albumin (neutral) or anion exchange particles (48). In the normal case, this effect of particle charge on mucociliary clearance appears to be relatively minor, but the effect of particles could become more important in airway disease, where the mucins become more anionic and could interact more strongly with positively charged particles. Further studies are needed in this area.
IX.
Nanoparticles and Mucociliary Clearance
Nanoparticles or ultrafine particles are generally considered as particles of size less than 100 nm. Because of their small size and buoyancy, on inhalation, nanoparticles are less susceptible to deposition through impaction and sedimentation mechanisms, and thus penetrate more deeply in the lung, following ventilation patterns. There have been a number of studies dealing with deposition of nanoparticles but very few have looked at their clearance from the lung. Brown and colleagues (49) carried out a thorough study of ultrafine particle deposition and clearance in the healthy and the obstructed lung. They used Techigas, technetium-99m-labeled carbon particles, and ensured that aggregation was kept to a minimum by continuous generation of aerosol. Because of a largely peripheral deposition, retention fractions at one hour (ca. 90%) and 24 hours (ca. 80%) were much higher than for typical micrometer aerosols. These high retention fractions suggest that the majority of the nanoparticles, even in patients with COPD, overshoot the ciliated airways and are not taken up by the mucociliary system. Presumably, any ultrafine particles caught up in the mucus layer are cleared at rates similar to the mucus itself. Other important considerations regarding nanoparticles and their interaction with mucus are their size range and potential for interactions deviating from those expected of neutral, smooth, spherical particles. Nanoparticles in the 30 to 60 nm range, for example, are sometimes employed as models for viral movement through mucus. In general, such particles, and indeed viruses in this size range, have been found to move through mucus essentially freely, since the effective mucus gel network mesh (20–200 nm in cervical mucus) is greater than that size range. Olmsted and colleagues (50) found that two capsid virus particles, human papilloma virus (55 nm) and Norwalk virus (38 nm), diffused as rapidly in mucus as in saline. In contrast, herpes simplex virus (180 nm) colocalized with strands of mucus, suggesting that the latter virus makes low-affinity bonds with mucins. Even immunoglobulin-M, although much smaller than the mesh spacing between mucins, exhibited retarded diffusion, suggesting that components of the antibody also make low-affinity bonds with the mucus. Interestingly, polystyrene microspheres bonded even more tightly to mucins, bundling them into thick cables. Lai et al. (51) reported that the coating of nanoparticles with polyethylene glycol could greatly increase their diffusion through mucus. Thus, it is clear that specific interactions between mucins and the surface macromolecules on particles can exert significant influence on their mobility in the mucus gel.
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References 1. King M. Mucus, mucociliary clearance and coughing. In: Bates DV. Respiratory Function in Disease. 3rd ed. Philadelphia: Saunders, 1989:69–78. 2. King M, Rubin BK. Rheology of airway mucus: relationship with clearance function. In: Takashima T, Shimura S, eds. Airway Secretion: Physiological Bases for the Control of Mucous Hypersecretion. New York: Marcel Dekker, 1994:283–314. 3. King M. The biophysical properties of mucus. In: Rubin BK, Van der Schans C, eds. Therapy of Mucus-Clearance Disorders. New York: Marcel Dekker, 2004:29–41. 4. Lutz RJ, Litt M, Chakrin LW. Physical-chemical factors in mucus rheology. In: Gabelnick HL, Litt M, eds. Rheology of Biological Systems. Springfield, IL: Charles C Thomas, 1973:158–194. 5. Lamblin G, Aubert JP, Perini JM, et al. Human respiratory mucins. Eur Respir J 1992; 5: 247–256. 6. Bansil R, Stanley E, LaMont JT. Mucin biophysics. Annu Rev Physiol 1995; 57:635–657. 7. Dasgupta B, King M. Molecular basis for mucolytic therapy. Can Respir J 1995; 2:223–230. 8. King M, Macklem PT. Rheological properties of microliter quantities of normal mucus. J Appl Physiol 1977; 42:797–802. 9. Meyer FA, Gelman RA. Mucociliary transference rate and mucus viscoelasticity: dependence on dynamic storage and loss modulus. Am Rev Respir Dis 1979; 120:553–557. 10. Dasgupta B, Tomkiewicz RP, De Sanctis GT, et al. Rheological properties in cystic fibrosis airway secretions with combined rhDNase and gelsolin treatment. In: Singh M, Saxena VP, eds. Advances in Physiological Fluid Dynamics. New Delhi: Narosa, 1996:74–78. 11. Dulfano MJ, Adler KB. Physical properties of sputum. VII. Rheological properties and mucociliary transport. Am Rev Respir Dis 1975; 112:341–347. 12. Shih CK, Litt M, Khan MA, et al. Effect of nondialyzable solids concentration and viscoelasticity on ciliary transport of tracheal mucus. Am Rev Respir Dis 1975; 115:989–995. 13. King M. Relationship between mucus viscoelasticity and ciliary transport in guaran gel/frog palate model system. Biorheology 1980; 17:249–254. 14. Puchelle E, Zahm JM, Duvivier C. Spinnability of bronchial mucus: relationship with viscoelasticity and mucus transport properties. Biorheology 1983; 20:239–249. 15. King M, Zahm JM, Pierrot D, et al. The role of mucus gel viscosity, spinnability, and adhesive properties in clearance by simulated cough. Biorheology 1989; 26:737–745. 16. Puchelle E, Zahm JM, Jacquot J, et al. A simple technique for measuring adhesion tension properties of human bronchial secretions. Eur J Respir Dis 1987; 71(suppl 153):281–282. 17. Asmundsson T, Kilburn KH. Mucociliary clearance rates at various lengths in dog lungs. Arch Environ Health 1970; 29:290–293. 18. Hilding AC. Ciliary streaming in the bronchial tree and the time element in carcinogenesis. N Engl J Med 1957; 256:634–640. 19. Clarke SW. The role of two-phase flow in bronchial clearance. Bull Physiopathol Respir 1973; 9:359–372. 20. Blake JR, Winet H. On the mechanics of mucociliary transport. Biorheology 1980; 17: 125–134. 21. Sleigh MA, Blake JR, Liron N. The propulsion of mucus by cilia. Am Rev Respir Dis 1988; 137:726–741. 22. Boucher RC, Stutts MJ, Bromberg PA, et al. Regional differences in airway surface liquid composition. J Appl Physiol 1981; 50:613–620. 23. Nadel JA, Widdicombe JH, Peatfield AC. Regulation of airway secretions, ion transport, and water movement. In: Fishman AP, Fisher AB, eds. Handbook of Physiology: The Respiratory System I. Bethesda, MD: American Physiological Society, 1985:419–445.
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24. Jacquot J, Hayem A, Galabert C. Functions of proteins and lipids in airway secretions. Eur Respir J 1992; 5:343–358. 25. De Sanctis GT, Tomkiewicz RP, Rubin BK, et al. Exogenous surfactant enhances mucociliary clearance in the anesthetized dog. Eur Respir J 1994; 7:1616–1621. 26. Al-Bazzaz FJ. Regulation of salt and water transport across airway mucosa. Clin Chest Med 1986; 7:259–272. 27. Widdicombe JH. Ion and fluid transport by airway epithelium. In: Takashima T, Shimura S, eds. Airway Secretion: Physiological Bases for the Control of Mucus Hypersecretion. New York: Marcel Dekker, 1994:399–431. 28. Stewart WC. Weight-carrying capacity and excitability of excised ciliated epithelium. Am J Physiol 1948; 152:1–10. 29. Sade´ J, Eliezer N, Silberberg A, et al. The role of mucus in transport by cilia. Am Rev Respir Dis 1970; 102:48–52. 30. King M, Kelly S, Cosio M. Alteration of airway reactivity by mucus. Respir Physiol 1985; 62:47–59. 31. Camner P. Studies on the removal of inhaled particles from the lungs by voluntary coughing. Chest 1981; 80:824–827. 32. King M. Role of mucus viscoelasticity in cough clearance. Biorheology 1987; 24:589–597. 33. King M. Mucus and its role in airway clearance and cytoprotection. In: Hamid Q, Shannon J, Martin J, et al., eds. Physiologic Basis of Respiratory Disease. Hamilton, ON: BC Decker, 2005:409–416. 34. Zayas G, Dimitry J, Zayas A, et al. A new paradigm in respiratory hygiene: increasing the cohesivity of airway secretions to improve cough interaction and reduce aerosol dispersion. BMC Pulm Med 2005; 5:11. 35. Zayas G, Valle JC, Alonso M, et al. A new paradigm in respiratory hygiene: modulating respiratory secretions to contain cough bioaerosol without affecting mucus clearance. BMC Pulm Med 2007; 7:11. 36. Ferry JD. Cross-linked and filled polymers. In: Viscoelastic Properties of Polymers. 2nd ed. New York: Wiley, 1970:455–461. 37. Lopata M, Barton AD, Loureno RV. Biochemical characteristics of bronchial secretions in chronic obstructive pulmonary disease. Am Rev Respir Dis 1974; 110:730–739. 38. Wardlaw AJ, Moqbel R, Kay AB. Eosinophils: biology and role in disease. Adv Immunol 1995; 60:151–266. 39. Lethem MI, James SL, Marriott C, et al. The origin of DNA associated with mucus glycoproteins in cystic fibrosis sputum. Eur Respir J 1980; 3:19–23. 40. Shak S, Capon DJ, Hellmiss R, et al. Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum. Proc Natl Acad Sci U S A 1990; 87:9188–9192. 41. Vasconcellos CA, Allen PG, Wohl ME, et al. Reduction in viscosity of cystic fibrosis sputum in vitro by gelsolin. Science 1994; 263:969–971. 42. Rubin BK, Kater AP, Dian T, et al. Effects of thymosin b4 on cystic fibrosis sputum. Pediatr Pulmonol 1995; 12S:134–135. 43. Schuster A, Ueki I, Nadel JA. Neutrophil elastase stimulates tracheal submucosal gland secretion that is inhibited by ICI 200,355. Am J Physiol Lung Cell Mol Physiol 1992; 262: L86–L91. 44. Lieberman J. Inhibition of protease activity in purulent sputum by DNA. J Lab Clin Med 1967; 70:595–605. 45. McCullagh CM, Jamieson AM, Blackwell J, et al. Viscoelastic properties of human tracheobronchial mucin in aqueous solution. Biopolymers 1996; 35:149–159. 46. Robinson M, Regnis JA, Bailey DL, et al. Effect of hypertonic saline, amiloride, and cough on mucociliary clearance in patients with cystic fibrosis. Am J Respir Crit Care Med 1996; 153:1503–1509.
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47. Tomkiewicz RP, Boyd WA, Feng W, et al. Tracheal clearance and rheology of mucus after aerosolization of 3 and 7% hypertonic saline in healthy dogs. Am J Respir Crit Care Med 1997; 155:A780. 48. Lee TK, Man SFP, Connolly TP, et al. Simultaneous comparison of canine tracheal transport of anion exchange resin particles to albumin macroaggregates and sulfur colloid. Am Rev Respir Dis 1980; 121:487–494. 49. Brown JS, Zeman KL, Bennett WD. Ultrafine particle deposition and clearance in the healthy and obstructed lung. Am J Respir Crit Care Med 2002; 166:1240–1247. 50. Olmsted SS, Padgett JL, Yudin AI, et al. Diffusion of macromolecules and virus-like particles in human cervical mucus. Biophys J 2001; 81:1930–1937. 51. Lai SK, O’Hanlon DE, Harrold S, et al. Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus. PNAS 2007; 104:1482–1487.
12 The Role of Surfactant in Particle Exposure ¨ RCH, and MATTHIAS AMREIN FRANCIS H. Y. GREEN, SAMUEL SCHU University of Calgary, Calgary, Alberta, Canada
PETER J. GERBER University of Berne, Berne, Switzerland
ZOYA LEONENKO University of Waterloo, Waterloo, Ontario, Canada
I.
Introduction
The extracellular fluid lining the respiratory tract and airspaces exists as a continuum from larynx to alveolus. It has a complex composition and a structure that varies from site-to-site that reflects its many functions (1). In the airways, it consists of sol and gel layers surmounted by a surfactant film of unknown composition at the air-liquid interface. In the alveolus, the extracellular fluid consists of a thin hypophase covered by a dipalmitoyl phosphatidylcholine (DPPC)-rich surfactant film. The direct demonstration of a surfactant film in alveoli and airways is relatively recent (2–4), although a surface-active film had been inferred from physiological (5) and electron microscopic studies (6) many years earlier. Such indirect proof for a surfactant film is a low surface tension of the interface. The surface tension in large airways has been measured directly with a bronchoscope from the spreading behavior of oil droplets placed onto the tracheal walls or bronchi of anesthetized sheep and horses (7). A surface tension of approximately 32 mN/m has been recorded at the mucus-air interface in these animals. This suggests the presence of a surfactant film because proteins, surface polymers of blood cells, polysaccharides, and other biopolymers all result in surface tensions between 45 and 60 mN/m (8) when they adsorb to an air-water interface. The surface tension in the alveolar lung is much lower than in the airways, and values close to zero (end-expiration) to 25 mN/m (end-inspiration) have been measured by means of the spreading behavior of oil droplets during the respiratory cycle (9). Thus, both the direct measurement of surface tension and the results of electron microscopy support the concept of a surfactant film existing at the air-liquid interface in the alveoli and airways. These surfactant films confer important mechanical properties, preventing alveolar and airway collapse and maintaining the fluid balance of the lung. They also contribute to innate defense mechanisms (1). Moreover, they are the first point of interaction between inhaled particles and the host. This chapter explores how the properties of this fluid lining with an emphasis on the surfactant film influences deposition, clearance, and toxicity of inhaled particles. We will start with a general introduction of the structure of the fluid lining.
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Structure and Function of the Fluid Lining of the Lung
A. Alveolar Lung
The precise structure of the fluid lining of the airways and the alveolar lung is still in question. Ultrastructural studies by transmission electron microscopy of airway mucus and alveolar fluid lining have been compromised by difficulties of preserving the structure during sample preparation (6,10–12). Sims et al. (13) and others (14) have used a nonaqueous fixative technique (15) to preserve mucus in bovine, guinea pig, and rat trachea. The technique involves dissolving fixative (osmium tetroxide) in a nonaqueous fluorocarbon (FC) solvent with the aim of stabilizing mucous glycoprotein and glycosaminoglycan molecules as well as phospholipids in the surfactant material, before dilution in aqueous fixative can occur. The results obtained this way indicate that the fluid lining of the alveolus is continuous over the alveolar surface and covers the pores of Kohn (12) and varies in thickness from 0.09 mm over protruding features to 0.14 mm over relatively flat areas (12). The hypophase contains tubular myelin (16) and lipid micelles but is otherwise amorphous. It also contains proteins, proteoglycans, and lipoproteins. The surfactant film directly at the air-water interface appears to be continuous and has a variable number of lipid layers at its surface (17,18) as revealed by TEM of the tissue sections (Fig. 1), creating a surfactant reservoir (19). Its structures have also been studied extensively in vitro, after reconstituting films at the air-water interface from extracts. The results are consistent with the observations by TEM but add considerable molecular detail to our understanding of the molecular architecture of the film. This is described in the following sections, after an introduction to its composition and metabolism. Alveolar surfactant consists of approximately 85% to 90% lipids, 10% proteins, and 2% carbohydrates (20,21). In addition to lipids, pulmonary surfactant contains two hydrophobic proteins: surfactant protein-B (SP-B) and SP-C, and two hydrophilic proteins: SP-A and SP-D. SP-A regulates the amount of surfactant secreted through a receptor-mediated process and, together with SP-B, is essential for the formation of tubular myelin in the hypophase. When combined with SP-B and SP-C, SP-A has the capacity of accelerating the adsorption process and enriching the film with DPPC, thereby reducing the compression requirement and stabilizing the film (22–25). SP-B and SP-C are responsible for the low surface tension by facilitating the transfer of surfactant lipids to the surface film. They also contribute to the unique structure of the film associated with the function described below. Surfactant lipids and apoproteins are synthesized by the type-II pneumocytes. The proteins are formed on the rough endoplasmic reticulum whereas the lipids are synthesized inside the Golgi apparatus. The lipids are transported to lamellar bodies where they are stored. The lamellar body contents are secreted from the apices of the type II cells into the alveoli. After their release, the lamellar bodies become hydrated in the aqueous subphase and unravel into a matrix of tubular myelin (Fig. 2). In the alveolus, surfactant is recycled through reuptake into type II cells (major route, about 90%) or degraded in alveolar macrophages after phagocytosis (about 10%). A smaller fraction (about 1%) is lost to the airways (26). The molecular structure of alveolar surfactant has been studied in vitro model systems by others and us (27–38). These studies show that pulmonary surfactants
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segregate into domains of condensed and more fluid lipid areas. Figure 3 (left) shows an atomic force microscopic (AFM) image of this topography on the example of bovine lipid extract surfactant (BLES). The pattern is characteristic for artificial or natural pulmonary surfactants that are functional in the sense that they can reduce the surface tension of an air-water interface close to zero (38,39). The height within stacks increases in increments of about 5 nm or multiples thereof. This is indicative that each layer consists of a lipid bilayer patch. The multilayered areas contain the hydrophobic
Figure 1 (A) Transmission electron micrograph from guinea pig lung following fixation with nonaqueous osmium-fluorocarbon mixture. The surfactant film (arrowheads) is preserved and continuous and overlies a thin hypophase (arrow) above the type I epithelium (magnification: 6000). (B) Higher magnification of the alveolar surface film from a guinea pig showing that it is multilaminated (magnification: 100,000). (C) Close-up of boxed area showing variation in the number of lamellae from two to seven (magnification: 200,000).
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Figure 1 (Continued).
surfactant-associated proteins SP-B and -C (34,36–46). The multilayers are indispensable for the mechanical stability, and films that lack function are devoid of multilayers (39,47–49). Kelvin probe force microscopy (KPFM, an imaging mode of AFM) showed that this molecular arrangement of pulmonary surfactant gives rise to a locally highly
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Figure 2 Transmission electron micrograph showing the formation of tubular myelin (TM) from
a secreted LB in the alveolar space of a guinea pig. The unraveling of the LB to form the tubular myelin can be seen. There is a fuzzy zone within each interstice of the lattices, which has been shown to contain SP-A (magnification: 40,000). Abbreviation: LB, lamellar body.
variable electrical surface potential with a dynamic range of 300 to 400 mV (47,48) (Fig. 3, right). The surface potential, defined as the difference in electrical potential between the film-covered surface and the aqueous subphase, mainly arises from the aligned molecular dipoles of the lipid (50,51) and protein molecules. We have shown that a strong surface potential arises from the a-helical surfactant associated protein C (SP-C) (40,47,48,52). Functional and dysfunctional surfactants (such as in adult respiratory distress) are quite different in surface potential (48). The electric surface potential distribution of the surfactant films has a profound influence on the interaction of inhaled charged or polar particles. This is discussed in section III. B. Conducting Airways
The aqueous layer covering the walls of the airways is thought to consist of two phases, a less viscous sol phase, in which the cilia beat and, above, a more viscous gel phase, the mucous blanket (53). The relatively low viscosity of the sol layer allows the cilia to beat freely. This blanket, containing trapped particles, is moved toward the pharynx by ciliary action. Its total thickness varies from 5 to 30 mm in the trachea (54) to less than 1 mm in the peripheral airways and shows variations in thickness with changes in lung volume (55). Sturgess (56) reported that the extracellular layer of mucus in the human trachea appears as a smooth, cohesive blanket overlaying the tips of ciliated cells.
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Figure 3 (A) Overview of a film of BLES that contains no cholesterol. The topographical image (left, cross-section below) shows a pattern of monolayer and scattered multilayer regions. In the potential map (right and cross-section below), large stacks of bilayer patches are at a potential of up to 200 mV above the monolayer. The arrows in the topographical image and in the potential map point to a region where the topographical height does not change but the potential shows two distinct levels. (B) A BLES film at higher magnification. The outer perimeters of the bilayer stacks from the topological image (left) are overlaid in gray in the potential map (right) to allow for easy correlation between topology and potential. Interestingly, only the larger of the bilayer stacks are at positive potential with respect to the average potential. Stacks with a lateral extension in the range of 200 to 400 nm are either at a more negative or more positive potential than the average potential, and stacks with a lateral extension of about 200 nm or less are up to about 100 mV below average. The larger of the bilayer stacks are at positive potential with respect to the monolayer. Small stacks are up to about 100 mV lower in the electrical potential than average.
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The surfactant film at the air-aqueous interface appears as a continuous multilayered structure (14,54). Biochemical studies have demonstrated the presence of surface-active components, predominantly phospholipids, in airway secretions (57,58). In contrast with alveolar surfactant, the source of airway surfactant is less clear. Since surfactant material is presumed to leave the tracheal surface by mucociliary transport, the surfactant film has to be replenished by local secretion or from the alveolar region. It is likely that airway surfactant is derived from both alveolar and local sources (57–61). The tendency of surfactant to flow from areas of low surface tension (such as exist in the alveoli at the end of expiration) to areas of high surface tension was studied by Grotberg et al. (62), using a mathematical model of fluid dynamics. Alveolar surfactant would be carried along a surface tension gradient by a net force acting to pull the surface film in the direction of the higher surface tension. This motion is resisted by viscous shear stress acting in the liquid layer just beneath the surface. This shear stress, in turn, drags the liquid within the entire liquid layer in the direction of the higher surface tension, producing bulk convection (63). Expansion and contraction of the surface film during respiration will probably assist in the clearance of particles deposited on the airways (64–66).
III.
The Role of Surfactant on Particle Deposition on the Airway and Alveolar Wall
Particle behavior and toxicity is strongly related to size. By convention, inhalable particles range up to 100 mm and are largely deposited in the upper airways, respirable particles, which range from 1 to 10 mm and ultrafine or nanoparticles measuring less than 0.1 mm in aerodynamic diameter readily penetrate all regions of the lung. The movement and impact with the alveolar wall by ultrafine particles has been ascribed to diffusion (67) and chaotic mixing (68,69). However, charged or polar aerosols are bound to also interact electrostatically with the alveolar surface. Bailey et al. (70) have shown that moderately charged particles (q ¼ 200 electrons) with a diameter of 0.5 mm are deposited in the alveolar region of the lung about five times as efficiently as uncharged particles. The electrostatic interaction of particles with the alveolar wall has been ascribed to the attraction of an electrically charged particle and its electrical image induced in the alveolar wall. However, these concepts do not take into account the electrical surface potential of pulmonary surfactant, described previously. With respect to its interaction with charged particles, surfactant at the air-water interface might be described as a thin, charged insulator plate (the lipid film) with a conductive backing (the water phase). For a film of homogenous surface potential, a marked electrostatic force on a charged aerosol may only be expected after the particle has come into close proximity to the interface, on the order of the film thickness. Otherwise, the electrical potential will drop inside the surfactant film from its airside to the water phase and no electrical field will be present in the air above the film. However, for a structured surface potential, the electrically different domains act as poles and give rise to an electrical field in the air above the interfacial film. Local field strength depends on both the lateral distribution and potential difference between the local electrical poles.
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In an initial study (71) using force spectroscopy, we found a strong electrostatic interaction between the sensor tip and surfactant films once the tip was less than about 20 nm from the sample surface. In force spectroscopy, the apex of the atomic force sensor tip is ascribed the role of a particle. The tip is moved toward the interface and the interaction forces measured along this path are sensitive down to a single hydrogen bond (72). Moreover, both strength and range of the interaction strongly depend on whether the film is functional or dysfunctional due to the presence of excess cholesterol (71).
IV.
The Role of Surfactant on Particle Displacement into the Fluid Layer
Particles deposited onto the alveolar and airway surfaces are coated with surfactant and displaced into the subphase and toward the epithelium (1–4). The extent of particle immersion depends on the surface tension of the surface film. The surface tension force promoting particle immersion depends on the magnitude of the film tension and the wetting contact angle, which, in turn, depends on the film surface tension and the free energies between particle and air and particle and fluid (73,74). Thus, particles having a relatively low surface-free energy (e.g., Teflon) will generally be immersed less than high-energy particles (e.g., glass) for a given film surface tension (4). Small particles, a few micrometers in diameter or less, are displaced to a greater extent than larger ones of the same surface chemistry. This is likely a consequence of line tension acting on the three-phase line between the air, particle, and film during the immersion process (75–77) (Fig. 4). For relatively large particles, this effect is negligible but it plays a significant role for small particles in the micrometer or submicrometer range (23). In addition to particle size and surface chemistry, the shape of particles plays an important role in their wetting and displacement by the surfactant film in the lung. The wetting properties of particles with sharp edges are different from those of smooth, spherical particles. Oliver et al. (78) have shown theoretically and experimentally, using a circular sapphire disk with a 908 edge, that sharp edges inhibit the spreading of liquid. Other experiments reported by Lay et al. (79) have shown that submicron sulfur colloid particles with sharp edges, when instilled into the dog bronchus, were cleared rapidly (within 24 hours) by mucociliary transport rather than by transepithelial adsorption. As might be expected from the effect of line tension on the displacement of small particles, submicron particles should be submersed mainly by line tension and brought into close proximity to the epithelial cells and cleared slowly. The fact that clearance was relatively fast supports the notion that the sharp edges on the sulfur colloid particles resisted wetting. Thus, the particles may not have come into close contact with the epithelium for transepithelial adsorption. Studies on wetting of talc particles in the modified Wilhelmy balance lend support to the concept that sharp edges resist wetting (80). The role of particle shape was the subject of investigations by Gerber et al. (81). Using a model system consisting of a modified Langmuir–Wilhelmy surface balance, they measured the influence of sharp edges (lines) and other highly curved surfaces, including sharp corners or spikes, of different particles on the spreading of a DPPC film. The edges of cylindrical sapphire plates (circular curved edges, 1.65 mm radius) were
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Figure 4 Schematic of particle displacement into the epithelium. The solid spherical particle under consideration has a radius, R. In addition to the interfacial tension g, the line tension contribution to the mechanical equilibrium, s/r (two-dimensional pressure), is considered. Line tension is the one-dimensional analogue of surface tension or the excess free energy density associated with the linear phase (three-phase line) where the phases vapor (1), fluid þ film (3), and solid (2) join. The contact angle y is determined by the three interfacial tensions and the twodimensional pressure s/r, directed toward the center of the three-phase line, s is the line tension and r represents the radius of the three-phase line. F indicates the position of the three-phase line. (A) The particle immediately after deposition onto the surfactant film. Here the particle equator is above the water level and s/r tends to prevent wetting. However, the vertical component of g13 (not shown) is dominant here. (B) The particle is further displaced until contact with the epithelial cell layer is established. The line tension promotes wetting because the particle equator is below the water level. The effect of line tension is to reduce the length of the three-phase line and thereby reduce the contact angle to a smaller value, F2. (C) The surface tension g13, in conjunction with the line tension s, promotes further particle displacement; the cell layer is deformed by the particle. The value of F3 is substantially lower than the original contact angle F1, because of the line tension contribution. (D) The particle is sitting below the surfactant film, which may be considered as an elastic skin keeping the particle submerged. Here, there is no longer an air-particle-water three-phase line and thus no line tension. Note: In B and C only s/r is drawn.
wetted at a surface tension of 10.7 mJ/m2 [standard error (SE) ¼ 0.45, n ¼ 20] compared with that of 13.8 mJ/m2 (SE ¼ 0.20, n ¼ 20) for cubic sapphire plates (straight linear edges, edge length 3 mm) (p < 0.05). The top surfaces of the sapphire plates (cubic and cylindrical) were wetted at 8.4 mJ/m2 (SE ¼ 0.54, n ¼ 20) and 9.1 mJ/m2 (SE ¼ 0.50, n ¼ 20), respectively, but the difference was not significant (p > 0.05). The surfaces of the plates showed significantly higher resistance to spreading compared to that of the edges, as substantially lower surface tensions were required to initiate wetting (p < 0.05). Similar results were found for talc particles, where the edges of macro- and
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microcrystalline particles were wetted at 7.2 mJ/m2 (SE ¼ 0.52, n ¼ 20) and 8.2 mJ/m2 (SE ¼ 0.30, n ¼ 20) (p > 0.05), respectively, whereas the surfaces were wetted at 3.8 mJ/m2 (SE ¼ 0.89, n ¼ 20) and 5.8 mJ/m2 (SE ¼ 0.52, n ¼ 20) (p < 0.05), respectively. Further experiments with pollen of malvaceae and maize (spiky and fine knobbly surfaces) were wetted at 10.0 mJ/m2 (SE ¼ 0.52, n ¼ 10) and 22.75 mJ/m2 (SE ¼ 0.81, n ¼ 10), respectively (p < 0.05). These results show that resistance to spreading of a DPPC film on various surfaces is dependent on the extent these surfaces are curved. This is seen with cubic sapphire plates, which have at their corners, a radius of curvature of about 0.75 mm, spiky malvaceae pollen with an even smaller radius on top of their spikes, or talc with various highly curved surfaces. These highly curved surfaces resisted wetting by the DPPC film to a higher degree than more moderately curved surfaces such as those of cylindrical sapphire plates, maize pollens, or polystyrene spheres, which have a surface-free energy similar to that of talc but a smooth surface. The macroscopic plane surfaces of the particles demonstrated the greatest resistance to spreading. This was explained by the extremely fine grooves in the nanometer range, as revealed by electron microscopy. In summary, to understand the effects of airborne particles retained on the surfaces of the respiratory tract, and ultimately their pathological potential, not only the particle size and surface chemistry but also the particle shape should be taken in consideration. After crossing the surfactant film, the particles may cross the epithelium and exert effects on cell biology. Ultrafine particles may reside in the interstitium (82) or travel into the lung tissue and blood stream (83–87) and account for the well established extrapulmonary effects (85). Fine particles settling in the alveolar lung abide on type I alveolar epithelial cells 95% of the time, because these thin cells cover most of the alveolar surface. A majority of respirable particles (98%) are cleared from the lung over a period of time. Ultrafine particles, on the other hand, do not elicit phagocytosis by macrophages (83), inhibit phagocytosis (88,89), and have the potential to penetrate into the bloodstream. In an animal model, Geiser et al. (90) found no evidence of significant clearance of inhaled TiO2 nanoparticles in rats by lung surface macrophages. There is accumulating evidence that nanoparticles are internalized by passive mechanisms. Red blood cells have been shown to incorporate fine particles (0.2 mm) (91,92) even though they are unable to phagocytose and are devoid of clathrin and caveolin, associated with active uptake. Passive uptake of particles in lung epithelial culture models has been demonstrated (82,91,93). Irrespective of whether ultrafine particles enter cells passively or actively, uptake efficiency should depend on the adhesive interaction and be governed by the particles’ surface properties. However, red blood cells take up a range of different particles (TiO, gold, or polystyrene) equally well (93). This may be explained, by our recent observation, using force spectroscopy, that particles exposed to culture media or serum immediately become coated by proteins and so lose their specific binding properties (94). Thermodynamically, any specific or nonspecific interaction between a particle and a cell will be reflected in a global way by the adhesive interaction. We recently established the measurement of the adhesive interaction (work of adhesion) between the apex of an AFM sensor tip (acting as the particle) and a primary type II cell culture (95). We found that the work of adhesion developed over the first 100 seconds after contact. During this time, the AFM tip appeared to be taken up by and moved into the cell. This
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study demonstrates that the thermodynamic parameters governing the first steps of particle uptake and their time course can be studied quantitatively with force spectroscopy.
V. Role of Surfactant in Particle Clearance Changes in surface tension caused by expansion and contraction of the surface film during respiration and surface tension gradients along the airways may enhance particle clearance (56,65,66). Surfactant also enhances particle clearance by accelerating ciliary beat frequency (96–98) and by conditioning the viscosity of the mucus (99). These latter effects might be mediated by the stimulatory effects of surfactant on chloride ion transport across airway epithelium (100).
VI.
Role of Surfactant in Particle Toxicity
Upon the initial contact between inhaled particles and the host at the air-liquid interface of the conducting airways and alveoli, the particles may become coated with surfactant. This process may change their toxicology or pharmacokinetics or even affect surfactant function directly (101). The amount of a surfactant adsorbed to the surface of particles of quartz or kaolin after mild saline rinsing amounted to the equivalent of a bilayer in which one monolayer coats the particle with the zwitterionic head groups toward the dust’s surface and the ionic phosphorylcholine heads of the second layer orientate toward the aqueous phase (102). These in vitro observations are in keeping with the observations of deposited particles studied by electron microscopy, which show coating with a bilamellar film (2). In vitro studies with DPPC, a primary component of surfactant, adsorbed to mineral and organic particles, have shown a marked suppression of their cytotoxic activity (103,104). This effect was demonstrated as long ago as 1957 (105), when it was demonstrated that quartz cytotoxicity was reduced following the adsorption of surfactant. In vivo, most inhaled mineral dust particles are rapidly phagocytosed into macrophages and incorporated into secondary lysosomes; surfactant coating enhances this process (106). In the intracellular environment, particles are exposed to hydrolytic lysosomal enzymes, including phospholipase A2. In vitro studies modeling this process have shown that removal of the lipid coating by phospholipase A2 restores the cytotoxicity of mineral dusts (102). The rate of removal of the surfactant coating by macrophages is different for kaolin and quartz; the coating being removed more slowly from kaolin than from quartz (107). Thus, the strength of adsorption of surfactant to the particle surface and the kinetics of desorption in the lysosome may account for the differences in cytotoxicity between these dusts (108). These models are consistent with the prompt neutralization of particle toxicity by adsorbed surfactant and a gradual retoxification within the macrophage (109). The reduced cytotoxicity of surfactant-coated particles may be related to a suppression of surface-free radical activity (110) by surfactant. Natural surfactant is capable of scavenging hydrogen peroxide (H2O2), the oxygen free radicals (O˙ and OH˙), and radical species derived from peroxynitrite (111). In the process, peroxidation of
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unsaturated lipids in surfactant and degradation of SPs may occur with impairment of surfactant function (112–114) and type II cell metabolism (115). Alveolar lining fluid (112,116,117) and airway mucus (118,119) also contain potent antioxidants, including catalase, superoxide dismutase, peroxidases, and glutathione. These enzymes are physically associated with airway and alveolar surfactant and may serve to protect surfactant from oxidative injury. Prolonged inhalation of dusts by humans (120), rodents (121,122), and other species (123,124) is associated with an increase in the number of type II cells and increased secretion of surfactant. The stimulation of surfactant appears to be directly related to the toxicity of the dust. It may be so florid, as in acute silicosis, that flooding of the alveolar spaces with surfactant lipids and associated proteins may occur, a condition known as alveolar lipoproteinosis (120). In experimental lipoproteinosis in the rat, the major lipid component is disaturated phosphatidylcholine (121), but all lipid fractions are increased. In the sheep model of experimental silicosis, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylinositol showed the greatest increases following silica exposure (123). The excess production of surfactant in response to silica dust may be an adaptive response, perhaps to reduce particle cytotoxicity or to compensate for oxidant-induced lipid peroxidation (125,126). The adsorption of surfactant to particles could also affect the surface tension in the conducting airways or alveoli (127–129). A significant biological effect would probably be seen in vivo only for fine and ultrafine particles, such as fumes and smoke (130), which have high surface areas relative to their mass. Abnormalities of surfactant function have also been reported in bronchoalveolar lavage (BAL) fluid from cigarette smokers (130–135). The adsorption of surfactant to porous particles, such as diesel exhaust soot containing absorbed mutagens (136), might affect their genotoxic activity. Particle-free surfactant extracts of diesel soot are not mutagenic in vitro (136,137).
VII.
Implications for Surfactant in Disease States
A. Particle-Induced Acute Lung Injury
Adverse effects on pulmonary surfactant have been demonstrated after experimental exposures to wood smoke (130); hydrochloric acid (138); hypochlorous acid (139); and toxic gases such as hydrogen sulfide (140), nitrogen dioxide (141), nitric oxide (125), ozone (142), and other particles (143). The impaired ability of surfactant to lower surface tension may be a direct consequence of the particle-surfactant interaction (101) or could be mediated by inflammatory cells or their products. Inhaled particles, such as asbestos and silica, stimulate macrophages to release reactive oxygen metabolites (144,145). Activated neutrophils also release proteolytic enzymes, such as elastase and lysozyme (146,147). Activated polymorphonuclear leukocytes impair surfactant function through a process that involves proteolysis of SPs (114,148). SP-A also has antioxidant properties of its own (148,149). Karagiorga et al. (150) reported an increase of cholesterol in surfactant from acute respiratory distress syndrome (ARDS) patients over control (8.5 5% w/w) from 15.6 2% w/w up to 43 22% w/w. Markart et al. (151) also found cholesterol levels
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enhanced in clinical ARDS samples. Cholesterol in surfactant is also increased to 20% w/w in animal models of lung injury (152). We recently analyzed the effect of an elevated level of cholesterol in surfactant. At cholesterol levels of 20%, the lowest surface tension observed in vitro using captive bubble surfactometry (CBS) was 16 mN/m (35). In our more recent studies using atomic force microscopy, we show the mechanism by which cholesterol inhibits surfactant (48,153). A comparison of the currently proposed mechanisms of surfactant inhibition in our laboratory singles out increased levels of cholesterol as having the most pronounced inhibitory effect (154). Hence, particle-induced lung injury may impair surfactant by causing inflammation, which in turn results in increased cholesterol levels in surfactant. Many of the adverse effects of surfactant deficiency can be reversed in the experimental model by intratracheal instillations of natural and artificial surfactants (155–157) and surfactant-associated proteins (158); it may be appropriate to treat acute lung injury with these preparations. The effect of particle-induced injury on airway surfactant has been studied in the guinea pig. Animals were exposed to sulfuric acid aerosol at high concentration (43 mg/m3) for four hours (14). The aqueous lining of the airways was preserved using an osmium FC fixative (13). Following exposure, the surface of the mucous layer was covered with a granular protein-like material. TEM revealed irregular thickening of the osmiophilic film at the air-mucus interface. The surface tension of the acid-treated tracheas (~32 mN/m) was similar to that of the control animals, and the ability of the mucus to submerge particles was not compromised, indicating that the surface film was functionally normal. It is possible that the known inhibitory effects of tissue injury and protein leakage on surfactant activity were mitigated by an excess of surfactant flowing from the alveolus. The morphological data were consistent with this, for the osmiophilic film at the air-mucus interface was greatly thickened in the acid-exposed animals (14). B. Asthma
By definition, asthma is a recurrent reversible obstruction of the airways accompanied by bronchial hyperreactivity and inflammation. It may be triggered by exogenous or endogenous stimuli. Asthma has increased during the past decades, mainly in industrialized countries and, in epidemiological studies, is strongly associated with exposure to fine (PM2.5) airborne particles (159) and specific allergens. Immune mechanisms as well as nonspecific stimuli can trigger an asthma attack. How inhaled allergens and irritant particulates are processed following deposition in the airways may have profound implications for the genesis of asthma and for the perpetuation of the asthmatic response. There are many reasons to consider that surfactant may be important in the pathogenesis of asthma. Surfactant’s role in promoting displacement and retention of particles in the airways may be important in determining the site and amount of antigen presentation. Alveolar and airway surfactant, by lowering surface tension, are important for maintaining small airway patency (160,161) and prevent liquid accumulation in the lumen (56). Surfactant dysfunction has been demonstrated in a guinea pig model of allergic asthma (162) and, in this model, the airway narrowing can be alleviated by surfactant inhalation (163). SP-A is deficient in the bronchoalveolar fluid of some asthmatics (164) and surfactant inhalation may alleviate the symptoms of asthma (165). Enhanced oxygen
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free radical release by alveolar macrophages from asthmatic patients (166) might be influenced by the hydrophilic proteins in surfactant (149,167,168) or be suppressed by their lipid components (116,146,156). The immunosuppressive effects of the lipid component of surfactant (169,170), particularly its effects on interleukin (IL)-2 release and adhesion molecules (171), may also be important in dampening inflammatory reactions that occur at the surface of the airways. Finally, a surfactant layer may have important implications for airway responses to inhaled drugs (172,173).
VIII.
Summary
Retention of particles by surface, line tension, and electrostatic forces exerted on inhaled particles by surfactant situated at the aqueous phase–air phase interface is the initial step of a complex cascade of defense mechanisms in the lungs. Surfactant coating of particles renders them more palatable to phagocytic cells, reduces their cytotoxicity, and enhances mucociliary clearance. Surfactant has important antimicrobial and antioxidant properties. Its role in particle-associated toxicity appears to be related to the surface properties of the particle. Given the complexity of the system and the multitude of interactions between inhaled particles and surfactant, it is necessary to examine the role of surfactant in diseases associated with particle exposures, such as some forms of acute alveolar injury and asthma. A knowledge of the mechanisms involved and, in particular, the role of surfactant will greatly enhance our ability to understand these diseases and to develop appropriate therapeutic strategies.
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13 Nanoparticle–Cell Membrane Interactions ¨ HLFELD*, BARBARA ROTHEN-RUTISHAUSER, FABIAN BLANK, CHRISTIAN MU and PETER GEHR University of Bern, Bern, Switzerland
I.
Introduction
During the past years, there has been a substantial increase in the debate on the potential harmful effects of nanomaterials (1–4), defined as materials with a diameter below 100 nm, and nanotubes, which have two dimensions below 100 nm while the third dimension can be much larger (5). Among these nanomaterials, specific concern is expressed about adverse health effects of nanoparticles (NP), since recent studies indicate a specific toxicological effect of inhaled combustion-derived ultrafine particles (UFP) (6–11). Although there are obvious differences between UFP, which are polydispersed and have a chemically complex nature, and NP, which are in contrast monodispersed with precise chemically engineered characteristics, the same toxicological principles have been assumed (3). An important basis for the current concerns about the possible adverse health effects of NP and nanotubes has been provided by research in the field of inhalation toxicology (12). Rodent inhalation studies provided evidence that NP induce considerably stronger pulmonary toxicity when compared at equal mass dose with larger particles. For instance, inhaled or intratracheally instilled poorly soluble particles of low toxicity such as carbon black and titanium dioxide induce pulmonary inflammation in proportion to their surface area (3,13,14). These findings indicate that a large surface area deposited in the lung may be sufficient to initiate inflammation. The in vivo studies are supported by in vitro studies where UFP show stronger inflammatory or toxic responses than larger-sized particles of the same chemical composition (15). In vitro and in vivo studies with UFP, such as diesel exhaust particles, carbon black, and welding fumes, have also strongly contributed to the findings that UFP are the most harmful constituent of environmental particulate air pollution (1). Support for the relevance of environmental particles in the nano-size range also comes from selected human studies showing associations between ambient exposure to UFP and adverse health effects (7,16). Various aspects of NP toxicity have been described in a number of recent reviews (1,3,5,17,18). Such studies also include the issues of the material composition and exposure sources, routes of exposure and administration (e.g., inhalation, oral, dermal, intravenous application), or aspects of distribution and translocation [e.g., air-blood *
Current affiliation: University of Giessen, Giessen, Germany.
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tissue barrier, blood-brain barrier, olfactory nerve], and clearance from the body. Most important however, detailed mechanism whereby NP may interact with their target cells and how this can impact on toxicity are still poorly understood. The principal interaction of NP with their biological environment occurs at the cellular level, that is, by interaction with structural and functional cell compartments (nucleus, organelles) (18). Reactive oxygen species generation is described in many studies as a hallmark of the biological effects of NP that have entered the cells (17,19). Besides the cell membrane, interactions with and effects in mitochondria and in components and compartments of the cell nucleus are considered as in view of their potential relevance for NP-induced toxicity. In this article, we discuss, on the basis of existing literature, the potential uptake and entering mechanism of NP across tissue and cell membranes as well as their possible interaction with intracellular compartments.
II.
Membranes
A. Tissue Membranes
Despite the existence of structural and functional barriers in the lung, respiratory diseases are frequent and increasing (7,9,20), and more attention has been directed toward elucidating how and when the antigens overcome these barriers. Insoluble particles deposited in the airways are largely cleared by the mucociliary action but not all deposited particles are removed by this mechanism. The lung barriers may not be effective to protect the body from particles less than 100 nm in size. Deposition as well as the subsequent fate of inhaled UFP and NP is different from larger-sized particles. Diffusion is the main mechanism for deposition of UFP and NP in the respiratory tract. On the basis of predictive mathematical models, it has been shown that of all deposited particles, 90% of inhaled 1 nm particles are deposited in the nasopharyngeal compartment whereas 20 nm particles have the highest deposition rate in the alveolar region (50%) (3). Most of the translocation studies described have been done for ambient UFP. Once deposited, a fraction of UFP appear to move rapidly to extrapulmonary sites via blood vessels and reach other organs like the heart or the brain, where they may penetrate into or through cells (12,21–23). The structural barriers protect the respiratory system against harmful and innocuous particulate material (24). This is important as the internal surface area of the lungs is vast [alveoli plus airways approximately 150 m2 (25)], facilitating easy access to the lung tissue, including cells of the defense system, such as dendritic cells and macrophages. The series of structural barriers are summarized in Table 1. The fate of inhaled and deposited particles depends on their physical-chemical characteristics (e.g., chemical composition, size/geometry, surface charge, coating, and aggregation status), the exposed cell type (professional phagocytes (marcophages), professional antigen-presenting cells (dendritic cells), epithelial or endothelial cells), as well as on their microenvironment (like the liquid-lining layer or the aqueous hypophase, the lung surfactant at the interface of those microcompartments with the air; the blood plasma; the basement membrane). A first line of structural barriers includes the surfactant system (26–28). All inhaled particles (PM10: particulate matter with a diameter equal or smaller than 10 mm) deposited in the airways are displaced into the subphase below the surfactant film by
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Table 1 The Structural Barriers of the Tissue Membranes
Structural barriers
References
The surfactant film The aqueous surface lining layers including the mucociliary escalator A population of macrophages (professional phagocytes) in the airways and in the alveoli The epithelial cell layer endowed with tight junctions between the cells The dendritic cells that are located within the epithelium making small cytplasmic processes toward the luminal side The basal lamina and extracellular matrix The capillary endothelium
26–28 29 30,31 32,33 34–38 39,40 41,42
surface- and line-tension forces exerted on it by the surfactant film and may be modified by surfactant components or coated with surfactant or surfactant components during the displacement process (26,28,43,44). As a result of the displacement, particles come into contact with the lung epithelium endowed with tight junctions (32), whose effectiveness is greatly enhanced by the aqueous surface lining layer and the mucociliary escalator (29). A second line of defense comprises cells of the innate and the adaptive immune system positioned throughout the respiratory tree. These cells are located above and below the respiratory epithelium and their activities are tightly regulated to protect the integrity of the airways and the vital gas exchange region (45). The innate response is largely governed by macrophages (46,47) whereas respiratory tract dendritic cells are responsible for the adaptive immune response (48,49). In vivo, alveolar macrophages occupy the luminal aspect of the epithelium (50,51) while immature dendritic cells occupy the basal aspect of the epithelium (52,53). In an in vitro study, it has been shown that macrophages form small cytoplasmic processes through the epithelium toward the basal side (34). Dendritic cells are located in the epithelium and the lamina propria. Those in the epithelium access the luminal side with fine cytoplasmic processes pushed between the epithelial cells (35,54,55). Transport of the particles to the dendritic cells presupposes their passage across the epithelium either through the epithelial cells or between the epithelial cells, through the tight junctions. It has been shown in gut mucosa that subepithelial dendritic cells were capable of capturing antigens outside the epithelium by extending fine cytoplasmic processes through the tight junctions (35–38). We have obtained evidence from in vitro studies that dendritic cells collect particles (1 mm in diameter) on the luminal side of the epithelium (Fig. 1) and that dendritic cells and macrophages collaborate as sentinels against fine particles by building a transepithelial interdigitating network of cell processes (34). In further studies, by using a triple-cell coculture model simulating an airway epithelial barrier, the entering and translocation of fine particles and NP into cells was compared. The model included epithelial cells, macrophages, and dendritic cells. The interaction of fluorescently labeled polystyrene particles of two different sizes (1 mm, and 50 nm) with the cells of the triple-cell coculture system was studied by laser scanning microscopy in combination with immunofluorescence methods and digital image restoration (56). After 24 hours, macrophages and dendritic cells were found filled with 1 mm particles and only few of these particles were detected in epithelial cells (Fig. 2). By applying a
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Figure 1 Laser scanning micrograph of a particle exposed triple cell coculture. A dendritic cell
residing underneath the insert membrane pushed processes between the epithelial cells upward into the “luminal space” to take up 1 mm particles (arrow).
deconvolution algorithm, even NP, that is, 50 nm polystyrene particles, could be visualized and many particles were found in macrophages as well as in dendritic cells, and again, only few particles in epithelial cells (Fig. 2). By a quantitative analysis, using a contingency table analysis (57) to compare the localization of fine particles with NP in different cell types, we found evidence for different translocation characteristics (56). Although we only found few particles of all sizes in epithelial cells in the triple-cell coculture system, an important role of the epithelial cells for particle translocation in vivo has to be considered. Particulate antigens less than 0.1 mm are able to cross the air-blood barrier of the lung and thus can enter the circulatory system (58). In the human lung, however, there is only one study that describes a rapid and significant translocation of inhaled carbonaceous NP to the systemic circulation and other extrapulmonary organs (58), whereas most other studies only detected a low degree of translocation for iridium (21) or carbonaceous NP (59,60). The studies of Nemmar et al. (58) and Mills et al. (59) had a very similar design, and Mills et al. have provided a convincing discussion that the strong translocation observed by Nemmar et al. (58) was mainly related to the translocation of soluble pertechnate, which was cleaved from the carbonaceous particles. It is therefore currently accepted that the degree to which inhaled UFP and NP translocate to
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Figure 2 Intracellular particle localization in triple-cell cocultures visualized by laser scanning microscopy. Fluorescent labeled polystyrene particles (1 mm, and 50 nm) were added to the cells for 24 hours. After fixation cells at the upper side of the insert (upper row) were stained for CD14 (macrophages) and F-actin (all cells); cells at the lower side (lower row) for CD86 (dendritic cells) and F-actin (all cells). Macrophages and dendritic cells were filled with particles (arrows), considerably fewer particles were found in epithelial cells (arrowhead). All images represent xzprojections. Source: Modified from Ref. 56.
the circulation is rather small, however, knowledge about cumulative effects of this translocation are lacking so far. After antigens have passed through the epithelial barrier, they may pass through the basement membrane and subsequently through the subepithelial connective tissue layer, and eventually come into contact with endothelial cells lining the capillaries. Since endothelial cells play an important role in inflammation processes (61), particles might affect endothelial cell function and viability inducing proinflammatory stimuli. It has been proposed that the permeability of the lung tissue barrier to NP is controlled at the epithelial and the endothelial levels (62). Antigens that have passed through the epithelial and endothelial barrier are transported by the blood circulation and reach other organs like the liver or the heart (21,63). There is also evidence that inhaled UFP can reach the brain (64) by translocation along or in neurons from the nasal epithelium to the brain. Recently it has also been shown that ultrafine manganese oxide particles are able to translocate to the olfactory bulb and other regions of the central nervous system in a whole-body exposure model (65), although the discussion whether entire particles were translocated or solubilized manganese was transported to the central nervous system (66) needs further research. B. Cellular Membranes
The surfactant film at the air-liquid interface in airways and alveoli and the phospholipid bilayers of cells with different types of proteins shaping the cellular membranes are considered actual membranes although their composition is completely different. The
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membranes also include the nuclear membrane and the membranes of the different organelles. Pulmonary surfactant is produced in the lung to decrease surface tension of the fluid lining. It is a complex mixture of phospholipids, neutral lipids (particularly cholesterol), and proteins. So far, four surfactant-associated proteins have been described, surfactant proteins-A, -B, -C, and -D, which are essential for the formation and structural integrity of surfactant components (67). Upon deposition, the first structure in the alveolus NP are interacting with is surfactant; however, there is only very limited knowledge about this interaction. For larger-sized particles, it has been shown that once the particles get into contact with the surfactant film, they are displaced from the airspace to the hypophase due to wetting forces (26,28,68), where they may be interacting with proteins of the hypophase (e.g., surfactant protein-A and -D or glycoproteins) (69). For NP, this interaction with surfactant has not been shown so far but it is likely to be similar. Surfactant lipids and proteins may adsorb to the surface of PM2.5 particles, thereby eventually modulating the function of pulmonary surfactant (70). Similarly, Bakshi and colleagues have demonstrated that gold NP sequester lung surfactant and may interfere with its normal function (71). After displacement of NP into the hypophase, the particles come into close contact with the membranes of the cells. All cell membranes have a common structure: They consist of a very thin film of lipid and protein molecules, held together mainly by noncovalent interactions (70,72). The lipid molecules are arranged as a continuous double layer about 5 nm thick in cell membranes. This lipid bilayer serves as a relatively impermeable barrier to the passage of most water-soluble molecules. The transmembrane protein molecules mediate specific functions as transporting proteins across the bilayer or catalyzing membrane-associated reactions. Some proteins serve as structural links that connect the cytoskeleton through the lipid bilayer to the extracellular matrix or an adjacent cell by integrins and cadherins, while others serve as receptors to detect and transduce chemical signals into the cell’s environment (73). How NP may enter cells across the cell membrane is described in detail in the next section “Mechanisms of nanoparticle penetration.” All eukaryotic cells contain functionally distinct, membrane-enclosed compartments. The main types are the nucleus and the organelles mitochondria, endoplasmic reticulum, Golgi apparatus, peroxisomes, lysosomes, and endosomes. Nucleus and organelles are enclosed by a double lipid bilayer containing distinct proteins (74). NP can cross the membranes of organelles since they have been localized in lysosomes, mitochondria, and the nucleus. This is described in the section “Nanoparticle localizations inside cells.”
III.
Mechanisms of Nanoparticle Penetration
The plasma membrane of the cells is a dynamic structure and segregates the chemically distinct intracellular milieu (the cytoplasm) from the extracellular environment by coordinating the entry and exit of small and large molecules. While essentially small molecules are able to traverse the plasma membrane through the action of integral membrane protein pumps or channels, macromolecules must be carried into the cells in membrane-bound vesicles derived from the invagination and pinching-off
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of pieces of the plasma membrane to form endocytic vesicles. This process is termed endocytosis and two types of endocytosis are distinguished on the basis of the size of endocytic vesicles formed: pinocytosis (“cellular drinking”) involves the ingestion of fluid and molecules via small vesicles (<0.15 mm in diameter) whereas phagocytosis involves the ingestion of large particles such as microorganisms and cell debris, at the formation of large vesicles called phagosomes (generally >0.25 mm in diameter). Whereas all eukaryotic cells are continuously ingesting fluid and molecules by pinocytosis, large particles are ingested mainly by specialized phagocytic cells. The mechanistic complexities that govern endocytosis suggest that great evolutionary effort has been expended to control entry into the cell, and thereby to control cellular responses to the environment (75). The different mechanisms of cellular entering and intracellular trafficking have been discussed in Ref. 76 and are summarized in Figure 3.
Figure 3 Summary of possible entering mechanisms of nano-sized particles. Particles may
actively be taken up by cells via phagocytosis (A), macropinocytosis (B), clathrin-mediated endocytosis (C), clathrin- and caveolae-independent endocytosis (D) or by caveolae-mediated endocytosis (E). In most cases of active uptake, particles will be transported via vesicular structures to form phagolysosomes or endosomes (A–D) but they may also be transported to the endoplasmic reticulum, cytosol or through the cell as part of transcytotic processes (E). Apart from these mechanisms, a passive movement through the plasma membrane with subsequent access to all subcellular compartments, including nucleus and mitochondria, has been proposed (F). The significance of particular intracellular localizations and entering mechanisms for specific cellular responses awaits further study. Source: Modified from Refs. 17 and 76.
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A. Phagocytosis
By contrast to pinocytosis, phagocytosis involves the uptake of large particles (>0.25 mm) into cells. It is an actin-based, mostly receptor-mediated mechanism, and is usually independent of clathrin (77). Phagocytosis in mammals is conducted primarily by specialized cells, including macrophages, dendritic cells, monocytes, and neutrophils that function mainly to clear large pathogens, such as bacteria, or large debris such as dead cells or the remnants of dead cells. Monocytes/macrophages and neutrophils have been referred to as professional phagocytes. They are very efficient at internalizing particles. On the other hand, most cells have some phagocytic capacity. For example, thyroid and bladder epithelial cells phagocytose erythrocytes in vivo (77), and numerous cell types have been induced to phagocytose particles in culture. In particular, there are a number of studies demonstrating the uptake of fine and UFP by lung epithelial cells in vitro (78–80) and in vivo (81). Particle internalization is initiated by the interaction of specific receptors on the surface of the phagocyte with ligands on the surface of the particle. This leads to the polymerization of actin at the site of ingestion and the internalization of the particle via an actin-based mechanism. After internalization, the phagosome matures by a series of fusion and fission events with components of the endocytic pathway, culminating in the formation of the mature phagolysosome. Since endosome-lysosome trafficking occurs primarily in association with microtubules, phagosome maturation requires the coordinated interaction of the actin- and tubulin-based cytoskeletons (75). Particle uptake by professional phagocytes serves as defense mechanism to remove foreign material from the body. However, for inhaled NP it is discussed that they are recognized to a lesser extent by alveolar macrophages than fine particles (82), leading to an increased contact with epithelial cells, penetration into deeper regions of the lung, and eventually, translocation into the circulation (19). In cultured macrophages, the entering of NP (0.078 mm) after cytochalasin D treatment could not be blocked completely, contrary to fine particles (1 mm in size), therefore particle uptake by any actin-based mechanism can be excluded (83). The kinetics of such processes are considered to depend largely on NP surface properties as well as in in vivo surface modifications, for example, by interactions with endogenous proteins (3). Contrasting uptake mechanism of NP in professional phagocytes versus other cell types in relation to different behavior with the cell membrane structures (e.g., receptors) are indicated in a number of studies. For instance, silver NP were observed on cellular membrane surfaces and free in the cytoplasm but also trapped by phagocytic or endocytic vesicles. This suggests that internalization of silver NP occurred through two distinct mechanisms, that is, by phagocyosis and/or endocytosis and perhaps by diffusion (3,84). B. Macropinocytosis
Like phagocytosis, the signaling cascades that induce macropinocytosis involve an actin-driven formation of membrane protrusions. However, instead of invaginating a ligand-coated particle, they collapse onto and fuse with the plasma membrane to generate large endocytic vesicles called macropinosomes that sample large volumes of extracellular milieu. Macropinocytosis fulfils diverse functions. It can be transiently induced in most cells and might play a role in down regulation of activated signaling
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molecules (75). Activation of antigen-presenting dendritic cells triggers extensive and prolonged macropinocytotic activity, enabling these cellular sentinels to sample large volumes of the extracellular milieu and to fulfill their role in immune surveillance (85). However, little is known about the nature of the whole uptake process (75) and there is no evidence so far that NP are taken up by macropinocytosis. C. Caveolae-Mediated Endocytosis
Caveolae are flask-shaped invaginations of the plasma membrane of a diameter of 50 to 100 nm observed in several cell types including capillary endothelium, type I alveolar epithelial cells, smooth muscle cells, and fibroblasts. They were also identified in macrophages (86,87) and dendritic cells (88). They are known to demarcate cholesterol and sphingolipid-rich microdomains of the plasma membrane in which many diverse signaling molecules and membrane transporters are concentrated (89). The shape and structural organization of caveolae are conferred by caveolin, a dimeric protein that binds cholesterol, inserts as a loop into the inner leaflet of the plasma membrane (90), and self associates to form a striated caveolin coat on the surface of the membrane invaginations. In most cells, even after activation, caveolae are only slowly internalized (half time:>20minutes) and the small vesicles carry little fluid volume. Thus, it is unlikely that this process contributes significantly to bulk fluid uptake (75). Whether this process is important for the endocytosis of NP needs further research. D. Clathrin-Mediated Endocytosis
This uptake mechanism is very well studied and constitutively occurs in all mammalian cells and carries out the continuous uptake of essential nutrients such as the cholesterolladen low-density lipoprotein particles that bind to the low-density lipoprotein receptor, and iron-laden transferrin that binds to transferrin receptors (91,92). Therefore it is, like most pinocytic pathways, a receptor-mediated endocytosis. Clathrin-mediated endocytosis involves the concentration of high-affinity transmembrane receptors and their bound ligands into “coated pits” on the plasma membrane, which are formed by the assembly of cytosolic proteins, the main assembly protein being clathrin. Coated pits invaginate and pinch of to form endocytic vesicles, clathrin-coated vesicles, which are encapsulated by a polygonal clathrin coat and carry concentrated receptor-ligand complexes into the cell (75). Clathrin is a three-legged structure, called triskelion, formed by three clathrin heavy chains, each with a tightly associated clathrin light chain (92,93). Again, whether clathrin-mediated endocytosis is important for NP needs to be studied in more detail. E.
Clathrin- and Caveolae-Independent Endocytosis
This mechanism is just poorly understood. It is generally referred to as cholesterol-rich microdomains, called rafts, with a diameter of 40 to 50 nm. Their unique lipid composition provides a physical basis for specific sorting of membrane proteins, glycoproteins, and/or glycolipids based on their transmembrane regions (94,95). These small rafts can presumably be captured by and internalized within any endocytic vesicle.
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Other Possible Penetrating Mechanisms
All of the previously presented endocytic pathways have in common, that the particle, which is taken up, is finally located in an intracellular vesicle. However, there are studies that reported the intracellular localization of UFP and NP of different materials, which were not membrane bound, indicating alternative pathways for particles to enter the cells (83,84,96). Furthermore, the localization of NP in cells that were treated with cytochalasin D in these studies excluded the possibility that the particles were taken up by an actin-driven process (83). In another study, red blood cells that do not have any phagocytic receptors on their surface nor the necessary intracellular structures for phagocytosis or endocytosis were used, and 1 mm polystyrene particles were found attached to the cell surface whereas 0.078 mm nanoparticles could be detected inside the cells, thus they must have entered the cells by nonphagocytic or nonendocytic mechanisms (83,96). Besides other possible mechanisms, passive and active (receptor-mediated) diffusion through membrane pores and passive uptake by Van der Waals or steric interactions (subsumed as adhesive interactions) (97) are suggested by the authors of these studies. However, it remains to be determined which chemical and physical properties of membranes and particles are responsible for the translocation of UFP/NP into cells, the nucleus, and organelles in vitro and in vivo.
IV.
Nanoparticle Localization in Cells
In many studies, NP have been found as aggregates in vesicles. In vivo inhaled gold NP have been detected only as aggregates inside small vesicles in macrophages of the rat lung (98). Also, in in vitro studies, vesicles containing NP aggregates have been described, a finding that might be explained by the fact that NP in suspensions have the tendency to aggregate. Polystyrene particles with a diameter of 50 nm were also phagocytosed by cells when they formed bigger agglomerates in media, and these aggregates were found to be membrane-bound inside the cells. Additionally, single polystyrene particles were identified and they could be detected freely inside the cytoplasm (data not shown). Other studies have shown vesicles containing mostly large aggregates of titanium dioxide in A549 cells (80) or membrane-bound ceria NP agglomerates in human lung fibroblasts (99). However, in all these studies, only conventional transmission electron microscopy was used to detect the particles, and it might well be that single NP or small aggregates of few particles could not be identified. Sophisticated microscopic methods are needed to detect and localize different NP inside cells or tissues (100). One example was the application of electron energy loss spectroscopy to identify titanium dioxide or silverenhanced gold NP inside cells. The particles were localized inside different cell types, for example, epithelial cells, macrophages, and dendritic cells as membrane-bound larger aggregates and free as smaller aggregates or individual particles in the cytoplasm (56) (Fig. 4). NP penetration in vitro into cells does not exclusively occur by any of the expected endocytic processes but also by other yet unknown mechanisms that had been called adhesive interactions, as for instance, electrostatic, Van der Waals, or steric interactions (83,97,101). Particles within cells have been found not to be membranebound (56,83,102) (Fig. 4). Hence, they have direct access to cytoplasmic proteins,
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Figure 4 Micrographs of energy-filtering transmission electron microscopy by means of electron
energy loss spectroscopy. They show cells of the triple-cell cocultures containing titanium dioxide and silver-enhanced gold particles. Titanium dioxide particles (A) were found inside cells as aggregates in vesicles (A, left panel), and as single particles or as small aggregates free in the cytoplasm (A, right panel). Silver-enhanced gold particles (B) were found in cells as single particles or as small aggregates, always free in the cytoplasm and also in the nucleus (B, arrow). The circles mark the region where the element analysis was performed. Source: Modified from Ref. 56.
important biochemical molecules in organelles like the respiratory chain in the mitochondria and the DNA in the nucleus, which may greatly enhance their hazardous potential. It is generally accepted that besides the cell membrane, mitochondria and cell nucleus are the major cell compartments relevant for possible nanoparticle-induced toxicity (17). A recent study shows that carbon-based NP, such as Buckminster fullerene (C60), can enter macrophages and is distributed within the cytoplasm and found in lysosomes as well as in the nucleus (103). Other in vitro experiments revealed penetration of UFP into mitochondria of macrophages and epithelial cells, associated with oxidative stress and mitochondrial damage (104). This penetration of UFP led to a loss of cristae in the mitochondria. The inner mitochondrial membrane was destroyed by UFP. Regardless of the mechanisms causing this mitochondrial damage, there is evidence that it is the organic substances attached to the particle that are responsible for this
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(105). The inner mitochondrial membrane is the structure carrying the respiratory chain, which converts molecular oxygen into energy stored as ATP. Penetration of particles into the nucleus has been shown in a number of in vivo studies for inhaled titanium dioxide NP in rats (83,106) and in vitro studies in different cell types for nano wires (107), gold NP (56) (Fig. 4), polystyrene NP, and C60 (103). Tsoli and colleagues (108) have shown that Au55 clusters interact with DNA of different cell lines in a way that may be the reason for the strong toxicity of these tiny 1.4 nm particles.
V. Conclusion Besides a series of structural and functional barriers, which protect the respiratory system from inhaled particulate antigen, respiratory diseases are frequent and increasing. Many epidemiologic studies have shown a direct relationship between ambient air pollution and increased health effects. Additionally, recent studies indicate that engineered NP are specifically toxic. It is well known that environmental UFP and artificial NP have the capacity to enter different cell types. After particle deposition in the lung, NP may cross the tissue and cellular barriers and reach different tissue compartments with various cell types. They may also enter capillaries and ultimately end up in many other organs. NP of various materials can cross any cellular membrane, although the mechanisms are just poorly understood. Once inside the cells, they may move freely in the cytoplasm or enter organelles like the mitochondria or nuclei, causing severe problems for the cells, the tissue, and the organ, which may have consequences for health. The distribution of the particles (particle trafficking) inside the cells as single particles or as aggregates is of interest to correlate the penetration mechanism and the intracellular translocation with the induction of cellular responses.
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14 Mechanisms and Processes Underlying Toxicological Responses to Particles VICKI STONE Edinburgh Napier University, Edinburgh, U.K.
KEN DONALDSON University of Edinburgh, Edinburgh, U.K.
I.
Overview of Particle Toxicity
The aim of this chapter is to provide an overview of the multiple mechanisms of particle-induced toxicity leading to a number of diverse disease endpoints. It lays out the notion of dose as it relates to particle effects—the principal pathogenic processes, cellular events, and diseases caused by particles—and then summarizes the toxic mechanisms for a range of common pathogenic particles. Figure 1 provides an overview of those processes and diseases that arise from exposure to a range of particle types. In addition, in writing this chapter it became apparent that the literature currently available and reviewed here provides the basis for initiating a matrix to relate physicochemical characteristics to biologically effective dose (BED) and toxicity of all respirable particles. A. Adverse and Pathogenic Effects Caused by Particles
Table 1 provides a list of the more common diseases caused by particles and the setting in which they commonly arise—occupational or environmental. Occupational Exposure
The history of occupational health documents that exposure to particles is associated with disease development. The highest exposures to particles occur in occupational settings, and the best-documented particle-related diseases have historically been the pneumoconioses. The pneumoconioses are fibrosing lung diseases specifically caused by particle exposure and include silicosis, asbestosis, and coal workers’ pneumoconiosis (1). There are many documented pneumoconioses arising from other types of dust but the above three formed the main occupational disease legacy of the industrial revolution. All these are still prevalent diseases and have been studied in detail by particle toxicologists. Classically, the pneumoconioses arise in manual workers (e.g., miners, quarriers) as a result of high exposures to airborne particles. The disease is found in the lungs, when there is a high lung burden (dose) of particles, often measurable at autopsy,
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Figure 1 A general diagram of the pathogenic processes induced by relatively low concentrations
of respirable air pollution particles in the general population (top half) and to relatively high concentrations of industrial dusts in occupational groups (lower half).
Table 1 Diseases Associated with Particle Exposure in Occupational (Occ) and Environmental
(Env) Settings Site
Setting
Disease
Exemplar particles
Airways
Occ/Env
Bronchitis
Occ Occ Occ/Env
Small airways disease Sensitization/asthma Lung cancer
Env
Exacerbations of airways disease—COPD and asthma Emphysema Silicosis (pneumoconiosis)— nodular fibrosis Asbestosis (pneumoconiosis)— interstitial fibrosis Progressive massive fibrosis Silicosis (pneumoconiosis)— nodular fibrosis Mesothelioma Stroke/heart attack
Coalmine dust, organic dust, PM10 Coalmine dust, quartz Metals, organic dusts Quartz, metals, asbestos, PM10 PM2.5
Parenchyma
Occ Occ
Airways
Occ Occ
Pleura, peritoneum Cardiovascular system
Occ Env
Coalmine dust, quartz Quartz, coalmine dust Fibers Coalmine dust
Fibers PM2.5
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due to a high workplace exposure. There is no known susceptibility in these workers, except for occasional unusual proclivities such as Caplan’s syndrome (2), and indeed most workers in such industries would normally be anticipated to have good pulmonary health at the outset of their employment, as they were employed in physically demanding manual jobs. Because of the expansion of nanotechnology, exposure to a variety of engineered nanoparticles generated in large volumes (e.g., nanotubes, fullerenes, nanoclays, silicates) is also anticipated (3). Almost no data are currently available to quantify exposure in these work environments, and no data exist that attempt to quantify health effects due to the short history of these exposures. Whether or not these exposures will result in adverse health effects (acute or chronic) remains to be seen, and forms the driving force behind the expanding field of nanotoxicology. Environmental Exposure
There is also a public burden of ill health due to environmental/ambient particles. The epidemics of ill health associated with sulfurous smog episodes resulting from domestic coal burning in the early parts of the 20th century (4) are now a thing of the past. However, there remains a consistently present burden of ill health as a consequence of ambient particles (5). Particulate matter (PM) represents a part of the air pollution cocktail present in ambient air, which also comprises gases such as ozone, nitrogen dioxide, and so on. PM is measured as the mass of particles collected using the PM10 or PM2.5 sampling conventions (6). The adverse health effects of PM10 are seen at the levels found in modern cities today and there is often no threshold (5). The diverse adverse effects that PM cause are shown in Table 1 marked with “Env” in the second column and include deaths and exacerbations of airways disease, and deaths from, and exacerbations of cardiovascular disease. These adverse health effects of air pollution have been measured in epidemiological studies, and there is good coherence between the acute effects seen in time series and panel studies and the chronic effects seen in environmental studies. The exposure to PM is very low—in the low tens of mg/m3 compared to workplace exposures that are in the mg/m3 region. The toxicity caused by such low doses of PM may be explained by the fact that the populations that are affected are susceptible and already have diseases characterized by inflammation and oxidative stress, the same effects that are caused by particles, so providing the opportunity for interaction and worsening of their disease. B. Toxic and Pathogenic Processes
A wide spectrum of pathobiological processes are seen in cells and tissues exposed to particles, and these can be understood in terms of the properties of the particles, for example, ability to cause oxidative stress and cell death, fiber length exceeding the phagocytic ability of the cells, and so on, and these are discussed below. Inflammation is a common finding following particle exposure and there is a clear link between inflammation and fibrosis, cancer, cardiovascular disease, and airways disease, the pathological outcomes associated with particle exposures. Central to understanding toxic response is defining and understanding the “dose” for different particles, and this is a unifying theme throughout this review.
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II.
Dose
As in all toxicology, defining the dose is central to understanding the biological response to particles.
A. Defining the Biologically Effective Dose for Particles and a General Scheme for Particle Responses at the Cellular Level
The toxicological responses to particles are diverse, occurring primarily in the lungs but there is also increasing data showing that other tissues can be affected by particle inhalation exposures. In toxicology, response follows dose and the dose-response relationship is key to understanding the outcome of any exposure. The concept of the BED is a key one in mechanistic toxicology. Rather than the total dose expressed as mass of toxin per unit mass of tissue, the common expression of dose, the BED, is the fraction of the mass dose that actually delivers a specific toxic damage. Thus, while the mass of a classical toxin like dimethylbenzanthracene (DMBA) in tissue constitutes the total dose, it is only the fraction of DMBA that is biotransformed to the diol epoxide that is adductforming; therefore, the BED for adduct formation is the dose of diol epoxide, which is much less than the total DMBA mass. To date, for inhaled particles there is little evidence for any biotransformation, and the BED of any particle is in fact some physicochemical entity or combination of physicochemical entities for example, surface area, surface area times surface reactivity, fiber length, and so on (Table 2). Exposure is frequently used as a surrogate for dose, but exposure measured by the common metric of mass is often not closely related to the BED, since there has been no modification by entry and residence in the target tissue. In the case of particles, this complexity increases, since the elutriating effect of negotiating the airways prior to deposition “size-fractionates” the exposure and macrophage clearance further operates on the particles.
Table 2 BED for Different Particles and the Mismatch Between the Current Metric and the BED
Particle
Biologically effective dose
Current metric
Quartz
Area of reactive (unblocked or unpassivated) surface Biopersistent fibers longer than 2.5 mm
Respirable mass
Asbestos
Number of fibers >5 mm length, <3 mm diameter l Aspect ratio >3 Mass by PM10 convention Respirable mass Nuisance dust standard of respirable mass l l
PM10 Welding fume (NP) Carbon black (NP)
Organics/metals/surfaces Soluble transition metals Surface area
Abbreviation: BED, biologically effective dose.
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Figure 2 Diagram to demonstrate the relationship between factors involved in the response to
particle deposition in lung tissue.
Furthermore, even the total deposited mass of particles does not represent the BED as research has shown that, for all particle studied so far, the BED is a physicochemical characteristic such as surface area, transition metal content, fiber length, etc. (Table 2). The BED then acts on cells and tissues to produce a stress of some sort (Fig. 2), typically oxidative stress but also stress caused by macrophage production of proteases or complement activation, for example. This causes the induction of defenses such as antioxidant enzymes, anti-proteases, etc. If the dose and therefore the stress is low level, then the threshold may not be exceeded, and the stress is ameliorated by the stress-defense response, for example, antioxidants are able to deal with the oxidative stress and return the tissue to normal redox balance. However, if the BED is large and the stress is severe enough to exceed the threshold, then damage ensues with oxidation of proteins, induction of inflammation, and generation of oxidative adducts; while at very high levels of stress, cell death may be provoked (7) (Fig. 2). Being able to define the BED for particles is important as the BED could ideally be used as the exposure metric, which would produce optimal risk management; additionally, in epidemiological studies relating endpoints to the BED would greatly improve correlation of exposure to response. Defining the BED for particles also enhances our understanding of how adverse effects are produced at the cellular/molecular level since the BED, by definition, is the particle characteristic(s) that “drives toxicity.” Conversely, interpreting the cellular/molecular events caused in cells by particles and their variants and subfractions can also provide the data that allows the BED to be defined. Recently, the authors have suggested that oxidative stress potential (see below) might be considered as an improved metric for PM10 exposure, since toxicological studies have identified it as a key BED for PM10 (8). Considerable particle toxicology research has gone into defining the BED for the principal pathogenic particles, and these are shown in
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Table 2. Currently, for the main pathological particles, there is a considerable mismatch between the exposure metric and the BED as determined from research (9) (Table 2). The toxicological mechanism, including the BED, is described below for some categories of particles.
III.
Common Processes and Mechanisms Involved in Particle-Induced Responses
A number of processes and mechanisms are outlined below, which are relevant to particle-induced responses. The cardiovascular effects of particles, although relevant, are not included as they form the basis of chapter 7. In addition, the mechanisms by which particles might cross epithelial barriers could also be considered under this heading, so the reader is referred to chapter 13. A. Oxidative Stress
Most organisms contain antioxidants that protect the cells and molecules of that organism from the damaging effects of oxidants. Oxidants include free radicals and reactive oxygen species (ROS) that are made naturally (e.g., by normal metabolism), and also from endogenous sources (e.g., UV radiation, xenobiotic metabolism). ROS are oxygen-containing molecules that possess an unpaired electron, making them highly reactive and damaging to endogenous molecules. Oxidative stress occurs when there is an imbalance between oxidants and antioxidants, favoring the presence of oxidants either due to excessive production of oxidants or depletion of antioxidants (10). Because of the depletion of antioxidants, the potential for ROS-induced oxidation of DNA, proteins, and lipids are enhanced, which usually leads to loss of function and, ultimately, cytotoxicity (11). Depletion of antioxidants can be caused by excessive exposure to ROS but is also more likely in an individual who is compromised, for example, due to poor diet or inflammatory disease. It is also very important to note that sublethal levels of oxidative stress can activate cellular signaling processes including calcium-signaling, transcription factor activation leading to either protective mechanisms (e.g., upregulation of antioxidant gene defense mechanisms), or proinflammatory signaling (e.g., cytokine gene expression). The latter can be associated with disease (see below). There is much evidence to demonstrate that inhaled particles result in at least two potential sources of ROS. The first is generated by phagocytic cells, which on ingestion of particles generate ROS and reactive nitrogen species as part of their antimicrobial activities. This is particularly important for high aspect ratio (fiber-like) particles such as asbestos, which due to complete uptake by macrophages result in frustrated phagocytosis and continual superoxide anion production (12,13). The second source includes ROS and free radicals generated directly at the particle surface (10). Many pathogenic particles have been shown to generate ROS, and this oxidative ability has been considered a key mechanism of toxicity. For example, Fubini (14) identified a variety of radical types including superoxide anions and the peroxyradical at the surface of a number of standard quartz samples. The production of ROS by phagocytic cells exposed to quartz particles was demonstrated by Castranova et al. (15), while Vallyathan et al. (16)
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demonstrated that quartz was able to induce oxidative stress in rats (10 mg/rat), associated with upregulation of the antioxidant enzymes superoxide dismutase and glutathione peroxidase, two and eight days following instillation. Oxidative damage was evidenced by lipid peroxidation, which increased with time. Albrecht et al. (17) also investigated the ability of quartz particles to induce oxidative damage in the rat lung. This study indicated that pulmonary oxidants such as hydrogen peroxide and nitrite were elevated seven days following silica exposure, which were also associated with increased antioxidant levels of the bronchoalveolar lavage fluid, suggesting that antioxidant defenses had been switched on in response to the particle exposure. Coating the particle surface prevented this oxidant generation and antioxidant response, demonstrating the importance of the quartz surface in generating ROS. Interestingly, the oxidant effects of the silica particles were not associated with DNA damage detected as 8-hydroxy-20 -deoxyguanosine at day 7. Asbestos-induced disease has also been linked to ROS and oxidative stress. Gilmour et al. (18) demonstrated that asbestos and synthetic vitreous fibers were able to generate free radicals using the cell-free DNA plasmid assay in which free radicals cause scission. The ability of fibers to induce ROS production was found to be associated with their ability to activate the transcription factor nuclear factor kappa B (NF-kB) in epithelial cells in vitro; NF-kB is a key transcription factor in the control of genes that regulate inflammation (19,20). In vivo, Brown et al. (21) also demonstrated that pathogenic fibers can induce oxidative stress in the rat lung, as indicated by glutathione and ascorbate depletion. In addition to the results with asbestos, the plasmid assay has also been used to demonstrate the ability of PM10 (22), carbon nanoparticles (23), and a panel of metal nanoparticles (24) to induce ROS production. Additional support for the production of ROS by nanoparticle carbon black (25) and polystyrene beads (26) has been published using the dye 2,7-dichlorofluorescin-diacetate that fluoresces under oxidizing conditions (27). Dichlorofluorescin diacetate (DCFH-DA) can also be used to measure intracellular ROS (28); for example, Wilson et al. (25) demonstrated that carbon nanoparticles (14 nm diameter), but not larger carbon particles (260 nm diameter), stimulated an increase in intracellular ROS in the macrophage cell line Monomac6. The role of ROS production by nanoparticles in inducing cytotoxicity was demonstrated by Stone et al. (23) when the carbon black nanoparticle caused depletion of glutathione in epithelial cells, and that decreased cell viability could be prevented by antioxidants. More recently, Sayes et al. (29) have also demonstrated that C60 NPs elicit oxidative stressmediated toxicity in vitro. Instillation of nanoparticle carbon black into the rat lung is also associated with a depletion of the antioxidant glutathione (11). These studies therefore provide evidence that nanoparticles induce oxidative stress, suggesting that the particle-generated ROS impact upon cells in the body. B. Inflammation
The role of the immune system is to defend the body against foreign material, including infectious agents and particles. With respect to inhaled particles, the defensive response is led by the macrophage, as this cell is responsible for identification and clearance of foreign particles that deposit on the surface of the airspaces. The influx of inflammatory
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cells such as macrophages and neutrophils into a tissue following some sort of insult is the hallmark of inflammation. In the case of particle deposition beyond the ciliated airways, clearance revolves around uptake of the particles by these professional phagocytes (30). Macrophages can take up relatively large materials (>0.5 mm) such as bacteria or cell debris, but the natural elutriation that is caused as the airstream negotiates the bends and bifurcations on the bronchial tree means that only small particles, less than about 5 mm, reach centriacinar regions, where the alveoli are present (31). During phagocytosis, the cell recognizes ligands via cell surface receptors, but the receptors involved in particle uptake are ill understood. Receptor binding then triggers the polymerization and rearrangement of the actin cytoskeleton to form membrane extensions so that the plasma membrane surrounds the material to be internalized (31–33). The phagosome that is formed then fuses with lysosomes, so that the cargo can be degraded if possible (33). This process appears to be relatively successful for finesized (>100 nm, but <2.5 mm diameter) particles made from low-toxicity materials (e.g., carbon, TiO2, and polystyrene beads), allowing clearance of particles from the lung surface via migration to the lymph nodes or by movement out of the respiratory system via the mucociliary escalator. However, for pathogenic particles, phagocytosis appears to either be ineffective or cause sufficient stress to macrophages, following uptake, that inflammation results. Inflammation is required for normal health. Without inflammation humans would die from a simple infection. However, when inflammation that is inappropriate in duration or amplitude occurs, at a sensitive site like the gas-exchange regions of the lungs, or if it occurs in a susceptible individual with a preexisting disease, then inflammation can either cause new disease or worsen the preexisting disease. Inflammation is clearly implicated in the disease or toxic mechanisms of many particles including PM10, asbestos, silica, and nanoparticles. For example, elevated PM10 is associated with increased hospital admissions and mortality via both respiratory and cardiovascular disease. In each case, the individuals affected suffer from a preexisting inflammatory disease such as asthma, bronchitis, or cardiovascular disease (34). In these individuals, it is hypothesized that PM10-induced inflammation provides the driving force to exacerbate their disease symptoms (35,36). In fact, PM10 has been shown to induce inflammation in humans (37) and animal models (38) and to activate proinflammatory signaling in vitro (39). There are a number of components within PM10 that contribute to the ability to induce inflammation, including metals (40) and combustion-derived ultrafine or nanoparticles (11,41). In fact, a number of nanoparticles have been shown to induce inflammation in the rat lung to a greater extent than larger particles of the same composition. On the whole, these particles include low toxicity, low solubility materials such as carbon (11), TiO2 (42,43), and polystyrene beads (26). For such particles, there are a number of studies, which suggest that the ability to induce inflammation is related to the particle surface area dose (26,44–46) (described in more detail below). Some studies suggest that exposure to relatively low concentrations of TiO2 and carbon black nanoparticles increases the phagocytic ability of macrophages (47,48), while at higher concentrations, the phagocytic function of these cells is diminished (48,49). The mechanism or the cause of this inhibition remains unknown. Macrophages have the ability to ingest large volumes of particles, and therefore inhibition of phagocytosis could be as a result of filling of the cell volume or a decrease in membrane area
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to allow continued uptake or a general impairment in function. One research group has suggested that NP uptake into cells does not require membrane invagination, phagocytosis, or pinocytosis (50), suggesting uptake via diffusion across the cell membrane. Further studies are required to verify this result with other types of nanoparticles and cell types. Inhalation of asbestos also induces an influx of inflammatory cells into the lung as would be required for clearance. However, as mentioned previously, asbestos uptake by macrophages results in frustrated phagocytosis if the fiber is longer than the cell size (approximately 15 mm). The incomplete uptake of the fibers results in superoxide anion production (51) associated with cytotoxicity and upregulation of the proinflammatory cytokine tumor necrosis factor alpha (TNFa) (52). Recent data from our own group suggest that long (10–50 mm) straight multiwalled carbon nanotubes also stimulate superoxide and TNFa production by primary macrophages in vitro to a greater extent than short or entangled nanotubes (13). Donaldson et al. (53) demonstrated that the ability of asbestos fibers to induce inflammation in the mouse peritoneal cavity was a function of the fiber length, with longer fibers being more potent. The ability of asbestos to induce oxidative stress and inflammation is thought to underlie the ability of this fiber to induce fibrosis (asbestosis) and cancer (e.g., mesothelioma) (12,54). Recent studies provide comparable data for carbon nanotubes, with long rigid nanotubes providing a greater inflammatory response in the mouse peritoneum than entangled multiwalled carbon nanotubes (55). In this study, the inflammation induced by the long rigid multiwalled carbon nanotubes advanced to granuloma formation on the peritoneal surface of the diaphragm, suggesting that these nanotubes possess asbestos-like properties. In the lung, crystalline silica (a-quartz) has also been shown to induce inflammation in animal models (56), with freshly fractured surfaces being more inflammogenic than aged silica particles. Driscoll et al. (57) demonstrated that the silica exposure was associated with upregulation of proinflammatory cytokine gene expression in both macrophages and epithelial cells stimulating cytokine production [macrophage inflammatory protein 2 (MIP-2)]. The key role of the silica surface in inducing this inflammatory response has been tested by coating the particles with aluminum salts (58,59) that prevent silica-induced inflammation in the rat lung, which was associated with a prevention of MIP-2 upregulation and NF-kB activation. The inflammation and oxidative stress induced by silica is hypothesized to drive the fibro- and carcinogenicity of this particle (60). Therefore, inflammation is a clear, unifying process in the mechanism of particleinduced disease. C. Fibrosis
Fibrosis is a common response to particle exposure. Pulmonary fibrosis is scarring, which includes replacement of the normal architecture of the lungs with mesenchymal cells and extracellular matrix, resulting in a net loss of normal tissue. Scarring of the alveoli can reduce the ability of the lungs to transfer oxygen, producing restrictive lung disease. Scarring of the airway walls diminishes their function and narrows their caliber, reducing flow and causing obstructive lung disease. Interstitial fibrosis involves the presence of the fibrotic lesions in the interstitium and is commonly seen in asbestosis, while a more nodular form of fibrosis is seen in silicosis (2). Small airways fibrosis is
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also seen in silica exposure and progressive massive fibrosis of coal workers’ pneumoconiosis (2). Fibrosis caused by particles is normally consequent to ongoing chronic inflammation with cell activation, epithelial injury, and remodeling of the normal architecture of the lung. The cellular and molecular mechanism of fibrosis development after particle exposure has been especially well studied for the chrysotile asbestos model by Brody and co-workers over a 20-year period. This elegant body of work stands as a useful model for pulmonary fibrosis that is probably generically applicable to particles. Brody and co-workers first demonstrated preferential fiber deposition at the ridges of the bifurcations sites at the transition between airways and alveoli following high-dose, short-term asbestos exposure (61). Given the relationship between dose and response, the responses would be expected to be greatest at these anatomical positions, where the dose is greatest; this has indeed turned out to be the case. At these sites “prototypic lesions of fibrosis” were found to be evolving; containing increased cell numbers, ongoing proliferation, and increased expression of proinflammatory and growth factor cytokines that were presumably driving the development of the lesion. These cytokines included PDGF (62), TGFb (63), and TNFa (64). Increased procollagen gene expression has also been documented in the lesion, making a direct link to the increased accumulation of collagen that is typical of fibrosis (65). Brody and co-workers have also shown an anatomically based dose response in the centriacinar region in terms of rate of gene expression for cytokines and procollagen (65). Following chrysotile inhalation, the greatest response in terms of gene expression for the various proteins was present at the bronchoalveolar duct junction, followed by the first alveolar duct bifurcation, then the second alveolar duct bifurcation. This is entirely consistent with dose driving the response and anatomy driving the dose that these regions receive. The lungs are a blind-ended tube and movement of air finally stops at the terminal bronchioles. At this point, since airflow is zero, there is high deposition as particles deposit largely by diffusion and there is diffusion out into the ducts and their bifurcations in a gradually decreasing gradient. This was reflected in the rates of gene expression, greatest centrally at the terminal bronchiole and diminishing out through the alveolar duct bifurcations. It seems likely that these events, or some of their variants, are typical for the fibrotic response to other particle types, modified by their anatomical position, intrinsic toxicity of the particle, and so on. D. Genotoxicity and Carcinogenesis
Cancer is a toxicity outcome that is seen following occupational exposures to classically studied carcinogenic particles like asbestos and quartz (1), and is also associated with environmental exposure to ambient particles (66). Asbestos produces an unusual type of tumor, mesothelioma, which forms on the outside lubricated lining of the lungs and chest wall, the pleural tissues (2). Animal studies have demonstrated the link between particle exposures and cancer. For example, in the case of occupational dusts, there are well-documented studies showing that rats exposed to high airborne levels of quartz develop lung cancer (67) and that rats exposed to long, but not short, amosite asbestos were found to develop lung cancer and mesothelioma (68). High levels of low solubility, low toxicity nuisance dusts also cause lung cancer in rats, due to the phenomenon of rat lung overload (69). Rat lung
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overload presents potential problems in terms of using rat studies for particle risk assessment, but dosimetry studies have been helpful in allowing the “dose” to be identified as surface area (70). Armed with knowledge of the threshold surface area burden for overload of a low toxicity, the low solubility dust (see below) (70), and the surface area lung burden under consideration, it is possible to separate overload from nonoverload responses. Inflammation is the common denominator for carcinogenesis caused by such diverse particle types as quartz and nuisance dusts. However, the surface area dose that is necessary to elicit the inflammatory response is much less in the case of quartz than with a low toxicity dust, highlighting the increased reactivity of the quartz surface (46) and allowing overload to be excluded as the cause. There are two modes of genotoxic action by particles—primary and secondary genotoxicity. Primary genotoxicity is characterized by genetic damage in the absence of inflammation, whereas secondary genotoxicity is genetic damage resulting from oxidative DNA attack as a consequence of particle-induced inflammation. Reactive oxygen and nitrogen species (ROS/RNS) generated by the phagocytic burst during particleelicited inflammation form DNA adducts that shape the genotoxic insult. Primary genotoxicity results from the actions of ROS generated at the particle surface forming adducts or results from adducts produced by reactive metabolites of particle-associated organics such as polycyclic aromatic hydrocarbons or transition metals. Clearly, the inflammatory pathways of indirect genotoxicity are more difficult to study, but experiments have shown that leukocytes from particle-induced inflammation can cause genotoxicity (71,72). There is also evidence that particles can cause direct genotoxic effects to cells in culture. Quartz can cause oxidative DNA adducts of 8-hydroxy-20 deoxyguanosine in epithelial cells (73) and passivating the quartz surface to reduce its oxidative activity reduces this response (74). Long fibers of asbestos (75) and glass fiber (76), but not the shorter versions, are genotoxic by interfering with chromosome migration at cell division, producing aberrations and aneuploidy. Ambient particles are also capable of causing oxidative genotoxic damage via the action of metals (77), which may be linked to the increased cancer levels seen in areas of high particulate air pollution. Some types of nanoparticles, because of their small size, are able to preferentially enter the nuclear compartment, which normally excludes particles, providing potential for direct genotoxic interaction with the DNA (78). Figure 3 provides a diagrammatic overview of the mechanisms of particle genotoxicity.
Figure 3 Processes involved in particle-mediated genotoxicity.
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Clearly, the genotoxicity of particles, evident in culture needs to be balanced against DNA repair mechanisms. A recent study showed that concomitantly with DNA damage by asbestos to cells in culture, DNA repair was switched on (79), and exposure to PM10 and nanoparticles is also associated with switching on of DNA repair (80).
IV.
Toxic Action and BED of Exemplar Pathogenic Particles
A. Low-Toxicity, Low-Solubility (Nuisance) Particles
A focus on the problem of “nuisance dusts” and the phenomenon of rat lung overload in the past (1980s) focused research attention on low-toxicity, low-solubility (LTLS) particles. Research demonstrated that, for LTLS particles, the surface area constituted the BED. Initial work concentrated on LTLS particles in the micron size. In these studies, Tran et al. (70) compared the inflammatory response to two very different LTLS particle types (TiO2 and BaSO4) of micron size. Rats were exposed to similar airborne mass concentrations of TiO2 and BaSO4 particles to achieve similar lung burdens. At equal lung mass burden, the TiO2 particles produced more inflammation than the BaSO4, but the two LTLS types were equally inflammogenic when the lung burden was expressed as particle surface area (Fig. 4) (70). For a fixed mass of particles, the surface area increases as particle size becomes smaller, and so the authors predicted that a dose-dependence related to surface area might produce a greater inflammogenicity in nanoparticles compared to the same mass of fine particles of the same material (81). This hypothesis was tested by instilling a range of LTLS panel, ranging from micron-sized to nanoparticles, into rat lungs and
Figure 4 Dependence of lung inflammation, as assessed by number of PMN in lung lavage, on
surface area for two low toxicity low solubility micron-sized particle types from following inhalation exposure. Left: when the lung burden is expressed as mass the two particles show different response curves; right: when the lung burden is re-expressed as surface area dose, the response to the two dusts falls on a common line. Source: Redrawn from Ref. 70.
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Figure 5 Dependence of lung inflammation on surface area dose of LTLS panel of both micro-
and nano-size instilled into rat lungs. Left: the lung burden instilled is expressed as mass and shows no clear relationship. Right: when the lung burdens are re-expressed as surface area, they all fall on one line. Source: Redrawn from Ref. 46.
assessing the inflammation at 24 hours (Fig. 5). As can be seen from Figure 5, different masses showed no pattern in relation to inflammation. However, when the surface area dose was recalculated as surface area dose and replotted, there was a direct relationship between surface area and inflammation, with nanoparticles being the most inflammogenic since they had the greatest surface area dose (Fig. 5). Thus, the BED for LTLS particles is the surface area. The mechanism whereby large pulmonary burdens of LTLS surface elicit inflammation is not fully understood. However, nanoparticles can provide a large surface area dose of low toxicity surface to lung cells. As described previously, for such LTLS there are a number of studies that suggest that the ability to induce inflammation is related to the particle surface area dose (26,44–46,82,83). This is important since the surface area is proportional to the number of atoms at the particle surface available for interaction with the surrounding environment. Of course, as particle size decreases, the surface area per unit mass increases. In addition, for nanoparticles below 10 nm diameter, the bond angles between atoms become less than optimum, providing strains that may increase reactivity (84). B. Quartz (Crystalline Silica)
In the case of LTLS surfaces, the total surface area dose is the BED for inflammation. The line in Figure 5 passes through zero for inflammation demonstrating that only surface area was involved. However, it is clear from Table 2 that there are particles whose surface area alone is not the BED. For compact particles, this means that when a more reactive surface is applied to the target cells, there is more response than for the same surface area dose of low toxicity surface. This is exemplified by quartz, where previous research has shown that quartz has a very reactive surface that can, for example, lyse red cells at low dose (85). This was addressed using the same instillation
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Figure 6 Demonstration of the high inflammogenicity of the quartz surface compared to LTLS
panel in causing inflammation. Source: Redrawn from Ref. 46.
approach as used in Figure 5, and both quartz and the LTLS panel were instilled. In Figure 6, the slope for surface area versus inflammation for quartz is much steeper than the LTLS panel slope (same as the slope in Fig. 5 with points omitted), indicating that there is something in addition to surface area that drives inflammation in the case of quartz. In trying to determine the nature of the quartz surface “reactivity,” many studies have implicated oxidative stress in the cellular action of quartz, and certainly quartz induces oxidative stress in target cells (60,83). However, the quartz surface silanol groups also interact with cell membranes (86), and so membrane-perturbing effects of quartz may be another “reactivity” factor that explains the greater toxicity of quartz per unit surface area. Therefore, this suggests that surface reactivity has a role in the effective dose of quartz for the inflammatory response and that the BED for quartz is “surface area surface reactivity.” The latter is a variable entity that depends on age, surface contamination, and so on, and this makes the identification of the quartz hazard, and hence its risk, a variable and difficult entity to study (60). C. Fibers
It is clear (Table 1) that fibers, typified by asbestos, present an unusual hazard in that they cause fibrosis (asbestosis), lung cancer, and a very unusual tumor, mesothelioma. The World Health Organization defines a fiber as a particle longer than 5 mm, less than 3 mm in width, and having an aspect ratio of more than 3:1. The current paradigm for the fiber BED has been found to be effective for asbestos, vitreous fibers, ceramic fibers, and one organic fiber. The BED paradigm for fibers suggests that length, width, and aspect ratio all play a role in defining pathogenicity. Width or diameter is important since fiber diameter plays a central role in dictating aerodynamic diameter (Dae), and pulmonary deposition depends on Dae. Only very thin fibers (<1 or 2 mm in actual diameter) present a sufficiently small Dae to penetrate beyond the ciliated airways to the most fragile, slow clearing region of the lungs.
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The evidence implicating length as a key factor in pathogenicity of fibers comes from a number of toxicological sources, many of which have been reviewed above. In animal and cell studies, unlike human epidemiology studies, it is possible to isolate length categories, expose animals or cells and to assess the length effects. Davis et al. (68) used this approach with a long and a short amosite sample obtained from the same sample by ball-milling and exposed rats to the long and the short amosite at the same airborne mass concentration. After lifetime exposure, there was substantial tumor and fibrosis response in the rats exposed to the long amosite and very little effect at all in rats exposed to the short amosite. The peritoneal cavity was utilized as a means of directly exposing a mesothelium to a controlled dose of fibers to produce mesothelioma. A given intraperitoneal mass dose of the short amosite caused only one mesothelioma in 36 rats while the long sample produced mesothelioma in 95% of rats (68). As described previously, macrophages in vitro produce much more TNFa and oxidative burst (87) on exposure to long fibers of amosite than to short fibers (52). Long glass fibers were also more effective in stimulating TNFa release in vitro than short fibers of the same type (88); the long fibers also activated the inflammatory transcription factor NF-kB to a greater extent than was seen with the short fibers (88). Length also impacts on genotoxic endpoints and Code 100/475 glass fibers that contain a proportion of long fibers (>20 mm) were found to cause dose-dependent transformation of SHE cells. However, when these were milled to short fibers, the dose response was abolished (76). The long and short amosite fibers were also used in a study that showed greater chromosomal abnormalities and aneuploidy in epithelial cells treated with longer fibers (75). In addition to long fibers being the BED for stimulating pro-pathogenic effects in a range of cells, there is also evidence that long fibers are more slowly cleared. Clearance is a consequence of two factors: 1. The biopersistence of the fiber, that is, the potential of the fibers/particles to dissolve away, in whole or part—this can result in breakage of long fibers into shorter fibers or complete dissolution 2. Mechanical clearance in the macrophages or on the mucociliary escalator These two combine to clear particles/fibers from the lungs. Studies into the clearance of the different size fractions of a long asbestos sample one year after it was instilled into rat lung is shown in Figure 7 (89). It shows the failure to clear the long fibers while the shorter fibers are effectively cleared. However, even a long fiber is cleared if it is nonbiopersistent, breaks, and is cleared from the short fiber compartment as shown in the diagram in Figure 8, which summarizes the clearance process leading to the accumulation of long biopersistent fibers, the BED for respirable fibers. D. Nanoparticles
The BED for engineered nanoparticles has largely been described above in that some engineered nanoparticles are clearly LTLS particles (e.g., carbon, TiO2), others possess fiber-like dimensions (e.g., carbon nanotubes) and therefore are likely to exhibit the BED of asbestos, while others possess a highly reactive surface and therefore BED is a function of surface area and surface reactivity (e.g., quantum dots). It might therefore be more useful in future to classify nanoparticles on the basis of their physicochemical characteristics, which are likely to determine their BED as well as their toxicity.
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Figure 7 Clearance of nonbiopersistent glass fibers from the lungs of rats (90). Figure shows
percentage of the fibers of any length category present in the lung one year after instillation. Note that long fiber dissolution and breakage causes it to decrease but that the expected production of short fibers, as the long fiber break up, means that the short size classes do not clear rapidly.
E.
PM10 and Combustion-Derived Nanoparticles
As described previously, the toxicity of PM10 is associated with a number of the components within this mixed particulate exposure. Of these, the combustion-derived nanoparticles (CDNP) are a likely source of oxidative stressing activity containing, as it does, large surface area, organics, and metals (90). PM10 BED relates closely to the BED of the CDNP derived from combustion sources. The BED of PM10, and possibly other particles that are heterogeneous, is made complex by potential interactions between these different components. For example, Wilson et al. demonstrated that in the rat lung, instillation of nanoparticle carbon black induced inflammation while instillation of a solution of iron chloride (FeCl3; 100 mM) did not. Combining the metal salt with the carbon nanoparticle led to a potentiation of the acute inflammatory response (25). In vitro, the combination also potentiated ROS production in a cell-free environment but not in macrophages. More recent data with zinc chloride demonstrated that while zinc chloride could not enhance ROS production by carbon particles in a cell-free system, it could synergistically enhance TNFa production by macrophages in vitro (91). Therefore, the BED for PM10 may vary according to the composition and is likely to include a complex interaction between the components.
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Figure 8 A model for the role of length and biopersistence in the fiber paradigm.
V. Conclusion We conclude that pathogenic particles induce toxic effects via common mechanisms including oxidative stress, inflammation, fibrogenesis, genotoxicity, and carcinogenesis. As a consequence, particle exposures are associated with a spectrum of adverse effects, from exacerbation of asthma (PM10), through to debilitating diseases such as fibrosis and cancer (e.g., asbestos and silica). In each case, the consequence is related to specific physicochemical characteristics of the particles including size, shape, biopersistence, contaminants, and surface reactivity, that is, there is a structure/activity relationship analogous to that seen with conventional chemical toxins. In the case of particles, the key “structures” are likely to differ from those that are understood to drive chemical toxicity. However, as a consequence of the revolution in particle toxicology that has accompanied the nanoparticles debate, improvements in capacity and methodology for characterizing particles are being seen. As regards “activities,” as described in detail in this review, the cellular and molecular mechanisms that drive particle effects are well understood (Fig. 9). We anticipate that the BED for various nanoparticle types will become evident in the next few years, as current effort is directed to assessing toxicology of the thousands of engineered nanoparticles currently in use and under development. It is clear from this review that much useful data is already available from conventional particle toxicology that is generalizable to nanoparticles. We believe that while the numbers of nanoparticles
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Figure 9 Diagram summarizing the effects of particles on pathogenic processes at the cellular
level.
that require testing appears daunting, particle toxicologists can step up to this challenge. As shown above, we are already in a position where a preliminary matrix that describes the properties that drive particle pathogenicity could be put together for some nanoparticles.
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15 Aeroallergen-Lung Interactions CARSTEN SCHLEH and JENS M. HOHLFELD Fraunhofer Institute of Toxicology and Experimental Medicine, and Hannover Medical School, Hannover, Germany
I.
Introduction
With every breath, the human lung is exposed to a myriad of particles. While some of the inhaled particles are harmless, a large proportion of these particles (the exact amount depends on the current environment) has allergen potential. In susceptible individuals, allergens induce sensitization and a chronic inflammatory response in target organs (airways, skin), which typically includes the production of antigen-specific immunoglobulin E (IgE). Allergens recognized by human IgE sera of more than 50% of patients in a clinically sensitive group are called major allergens. In contrast, allergens not important for the major population, but on individual basis, are called minor allergens. Over the past 25 years, an increase in the prevalence of aeroallergen sensitization has occurred in all age groups, affecting over 25% of the population (1). Majority of the allergens are proteins or glycoproteins with a molecular weight of 5 to 60 kD (2). Allergens possess biochemical properties that can potentially influence the biology of a range of cell types and noncellular components in the lung (3). The biochemical properties of allergens include hydrolytic enzymes (e.g., proteases, carbohydrases, ribonucleases) and nonhydrolytic enzymes, enzyme inhibitory activity, transport protein homology, and regulatory protein function. According to the allergen nomenclature, purified allergens (e.g., Lol p 1) are described by the first three letters of their genus (i.e., Lolium) and one or two letters of the species (i.e., perenne) followed by a number reflecting either the order in which the allergen was isolated or its clinical importance, or both. In case of sequence identity, allergens of different species within a genus or across phylogenetically related genera, the same number arrangement is used [e.g., the house dust mite (HDM) cysteine protease allergens from the mites Dermatophagoides pteronyssinus, Der p 1, and D. farinae, Der f 1, are members of the group 1 mite allergens]. In the environment, allergens do not appear as single pure allergens but are usually derived from mixtures of (glyco)proteins within a particular source. If they can become airborne, they are defined as aeroallergens. Aeroallergens enter the body mainly via the respiratory tract, where they deposit depending on their aerodynamic diameter. Although allergens like latex can get airborne (4,5), typically aeroallergens are derived from plants (wind pollinated), fungi, mites, cockroaches, and mammals like cats, dogs, and rodents.
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In this chapter, we describe relevant aeroallergens, focus on their interactions with noncellular and cellular elements in the lung, and highlight the subsequent biological responses.
II.
Aeroallergen Description
Aeroallergens are derived from wind-pollinated plants (grasses, trees, and weeds), fungi, bugs, or mammalian sources. Their primary size can vary considerably from less than 5 mm to more than 50 mm. A. Plants
The main aeroallergen sources of plants are pollen. Region and season determine the actual composition, concentration, and resulting exposure to pollen allergens. The most prevalent sources of pollen allergens are grasses, trees, weeds, and, because of an increased use during the past years, ornamental plants. Whole pollen range between 15 and 40 mm in size and therefore cannot enter the lower airways. Pollen grains deposit in the nasal and pharyngeal region where they interact with noncellular and cellular elements of the local environment (6,7). In addition to the entire grain, pollens contain small allergen-loaded particles, which are between 0.5 and 2.5 mm in size (Fig. 1). These particles are called pollen starch granules (PSG) or subpollen particles (SPP) and are released upon contact with water (Fig. 2) or even high humidity (8–10). Their release is assumed to account for a thunderstorm-related increase of asthma symptoms (10). Because of their smaller size, SPP are able to reach the lower airways where they can
Figure 1 Scanning electron microscopic picture of subpollen particles released from Phleum
pratense on a filter grid.
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Figure 2 Release of subpollen particles from whole pollen (Phleum pratense) upon contact with
water.
trigger reactions in the bronchial and alveolar regions. In addition to released SPP, ruptured fragments of whole pollen, nonpollen plant parts, and nonplant particulate matter, which is loaded with transferred allergens derived from pollen grains, also represent particles with allergen potential (11). Moreover, pollens contain bioactive lipids that can potentially modify immune responses (12). Purified plant allergens are divided into several groups. Some of the clinically most relevant allergens are, for example, the group 1 (e.g., Lol p 1) and group 5 (e.g., Lol p 5) allergens from grass. Group 1 grass allergens have a molecular weight of approximately 30 kD and display a biochemical function of b-expansins. Group 5 allergens have a molecular weight of 29 to 31 kD with a biochemical function as ribonucleases (Table 1). For a systematic review, the reader is referred to standard allergy textbooks (13). B. Fungi
Spores from fungi are widespread aeroallergens. They were even found on the Mir Space station (14). Fungal spores differ in size and hence deposit in the upper and lower airways (15). Geiser and colleagues investigated the retention and deposition of inhaled puffball spores from Calvatia excipuliformis with an aerodynamic diameter of 3.1 mm in Syrian gold hamsters. They found that most of the spores were retained by the alveoli. Admittedly, in this experimental setup, the nasopharyngeal region was bypassed by an intratracheal cannula, and normally by nose breathing, the nasopharyngeal region is a good particle collector (16). In addition to spores, fungal fragments, which are derived from intracellular or extracellular fungal structure, also represent allergen containing submicron or larger particles and seem to deposit at a high rate in the human airways (17). Important major allergens from fungi are, for example, Alt a 1 (from Alternaria alternata) with a molecular weight of 14 kD (the biochemical function is unknown) and Pen ch 13 (from Penicillium chrysogenum), an alkaline serine protease with a molecular weight of 34 kD (Table 1).
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Table 1 Description of Selected Aeroallergens
Source
Scientific name
Allergen
Grasses
Lolium perenne, Phleum pratense
Group 1 (e.g., Lol p 1) Group 5 (e.g., Lol p 5) Bet v 1 Bet v 2 Bet v 3
Trees
Betula verrucosa
Weeds
Ambrosia artemisiifolia
Fungi
Aspergillus fumigatus Alternaria alternata Penicillium chrysogenum/ notatum Dermatophagoides pteronyssinus, Dermatophagoides farinae
House dust mite
Cockroach
Mammals
Blatella germanica
Felis domesticus Canis familiaris Mus musculus Bos domesticus
MW (kD)
Allergen description
30
b-Expansin
29–31
Ribonuclease
17 14 23
Ribonuclease Profilin Calcium binding protein Pecate lyase Unknown function Ribonuclease Unknown function Alkaline serine protease Cysteine protease
Amb a 1 Amb a 5 Asp f 1 Alt a 1 Pen ch 13
40 5 17 14 34
Group 1 (e.g., Der p 1) Group 2 (e.g., Der p 2) Group 3 (e.g., Der p 3) Group 6 (e.g., Der p 6) Group 9 (e.g., Der p 9) Bla g 2
25
Bla g 5
23
Fel d 1 Can f 1 Mus m 1 Bos d 2
33–39 19–25 19 18
14 25
Unknown biological function Trypsin
25
Chymotrypsin
28
Collagenase-like serine protease Inactive aspartic proteinase Gluthatione transferase Unknown function Lipocalin Lipocalin Lipocalin
36
C. Mite and Cockroach
Although HDM themselves and parts of the pounded mite body can become airborne, the most important allergen source are their feces (18,19). Similarly, aeroallergens from cockroaches are feces particles. A lot of the mite allergens are enzymes involved in digestion. For example, group 1 allergens like Der p 1, Der f 1, and Eur m 1 (from Euroglyphus maynei) are cysteine proteases, group 3 allergens (e.g., Der p 3) are trypsins, group 6 allergens (e.g., Der p 6) are chymotrypsins, and group 9 allergens (e.g., Der p 9) are collagenase-like serine proteases (Table 1).
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Allergens derived from mammalians exhibit various origins. The major sources are dander, urine, saliva, hair, and feathers, and depend strongly on the animal’s race. The most important allergens are derived from cats and dogs, whereas others (e.g., rabbits, mice, and rats) are also, but to a lower extent, sources of allergens (20). Except for cat allergens, the major mammalian dander allergens belong to the lipocalin superfamily (21), which is important for binding and transport of small hydrophobic molecules. Examples for allergens from the lipocalin group are Equ c 1 (from Equus caballus), Bos d 2 (from Bos domesticus), Can f 1 (from Canis familiaris), Mus m 1 (from Mus musculus), and Rat n 1 (from Rattus norvegicus) (Table 1).
III.
Interaction with Noncellular Components
A. Airway Lining Layer and Alveolar Hypophase—Pulmonary Surfactant
By inhalation, aeroallergens enter the airways, and depending on their size, deposit throughout the respiratory tract. Deposition represents the initial event that allows for an aeroallergen-lung interaction when particles impinge in the airway lining layer. In the peripheral airways, the respiratory bronchioles, and in the alveoli, this airway lining layer is mostly composed of an aqueous hypophase covered with a surfactant film at the air-liquid interface. The surfactant layer is the first barrier upon particle deposition where surface forces affect particle-hypophase interaction (22,23). Thereby particles are displaced into the liquid layer and subsequently can interact with the liquid and with epithelial cells or cells of the defense system. Pulmonary surfactant mainly consists of phospholipids (*90%), and four specific surfactant proteins (SP)-A, -B, -C, and -D. SP-B (*8 kD) and SP-C (*4 kD) are small hydrophobic proteins. Their main function in conjunction with phospholipids is to decrease surface tension during the breathing cycle and thereby to prevent alveolar collapse. SP-A (*28–36 kD) and SP-D (*43 kD) are large hydrophilic proteins. They belong to the family of the collectins (collagen-containing lectins) and participate primarily in immune responses. Both SP-A and SP-D assemble from monomers into their quaternary structure. SP-A forms an octadecamer with a typical flower bouquet structure while SP-D as a dodecamer has a typical crucifix structure. Each monomer has four distinct regions: (i) a cysteine-rich region at the N-terminus, (ii) a collagen-like domain, (iii) an a-helical neck region, and (iv) a carbohydrate recognition domain (CRD). Preferentially, via the CRD, SP-A and SP-D bind to various pathogens and thereby modulate the immune response. Importantly, besides interacting with bacteria and viruses (24–26), SP are able to bind to and interact with aeroallergens (Fig. 3). SP-A and SP-D are found to bind to whole mite extracts (D. pteronyssinus) and the purified allergen Der p 1. This binding is carbohydrate specific and calcium dependent, suggesting that the CRD of the proteins binds to carbohydrate residues on the surface of the allergen (27). Malhotra et al. were the first to show direct binding of SP-A to pollen grains (28). Furthermore, direct binding of SP-D to SPP from Phleum pratense and Dactylis glomerata was demonstrated (29). This binding led to aggregation of the SPP, and sensitivity to calcium and sugars indicated that the CRD of
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Figure 3 Schematic structure of SP-A and -D that are assembled from monomers and trimeric units into octadecamers (SP-A) and dodecamers (SP-D). The potential interaction sites between allergens and SP are circled. References are given in brackets. Abbreviation: SP, surfactant protein.
SP-D was involved in SPP aggregation. In addition, SP-D increased phagocytosis of the SPP by alveolar macrophages (AMs), indicating an essential role in cellular clearance processes (29). Moreover, SP-A and SP-D were demonstrated to inhibit binding of aspergillus allergen to allergen-specific IgE from aspergillosis patients (30). SP-D but not SP-A reduced the release of b-hexosaminidase from passively sensitized peritoneal mast cells after allergen stimulation (31). Finally, SP-D is also able to inhibit allergen-induced nitric oxide (NO) production by macrophages (32). While surfactant components can affect allergen particle properties and subsequent cellular responses, there is also evidence that allergens alter surfactant structure and function. For example, binding of major HDM allergens Der p 1 and Der f 1 to SP-D and SP-A results in degradation of both SP under physiological conditions with multiple sites of cleavage. This degradation was mediated by the cysteine protease activity of the two allergens and was enhanced in the absence of calcium (33). Furthermore, allergens are able to induce an increase in surface tension at the airliquid interface in the lung. This was first shown in guinea pigs by using the model allergen ovalbumin (34). Most likely, the reduced surface activity was caused indirectly by an increase in the protein concentration in bronchoalveolar lavage (BAL) upon allergen challenge. These results were confirmed in humans (35). Here, surfactant dysfunction was found in BAL fluid from patients with mild allergic asthma but not in healthy control subjects after segmental allergen challenge. Besides an infiltration of inhibiting proteins, Haczku and coworkers suggested that allergen-induced surfactant dysfunction can be due to decreased levels of SP-B, possibly mediated by
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interleukin (IL)-4 (36). Schmiedl and colleagues found an altered structure of intraalveolar surfactant subtypes in ovalbumin-sensitized mice that exhibited a significantly increased fraction of inactive unilamellar surfactant vesicles (37). Finally, Hite and colleagues demonstrated an alteration of phospholipids, particularly depletion of phosphatidylglycerol in the large aggregates of surfactant as a potential mechanism in asthma-associated surfactant dysfunction (38). Further, indirect interactions between allergens and surfactant components exist. It is, for example, known that airway inflammation induced by inhaled allergens can alter the levels of SP in the lung (39–43). It is also suggested that pulmonary surfactant might be involved in the development of allergic reactions caused by aeroallergens. For example, the lung function of guinea pigs improves if the animals receive a calf lung surfactant extract instillation prior to allergen challenge compared to control animals (44). Furthermore, SP-D and SP-A were able to modulate and reduce allergen-mediated airway responses. This was shown for Aspergillus fumigatus (43–48), HDM allergens (49–51) and the model allergen ovalbumin (52,53).
IV.
Interaction with Cellular Components
A. Epithelial Cells
For several years, the role of the epithelial cell layer in the lung was strongly misconceived as a simple physical barrier. However, recent studies highlight that epithelial cells modulate the immune system upon contact with aeroallergens in manifold ways. Epithelial cells produce a variety of proinflammatory cytokines, chemokines, mediators such as NO and prostanoids, and other proteins such as protease inhibitors or matrix metalloproteases (54). For example, the epithelial expression and activity of matrix metalloproteinases (MMPs) are affected by allergens. MMPs are important for cell migration, invasion, proliferation, and apoptosis. They regulate many developmental processes, including branching morphogenesis, angiogenesis, wound healing, and extracellular matrix degradation. They are synthesized as inactive proenzymes and are being activated by removal of an amino-terminal propeptide (55). It is suggested that MMPs are also involved in the remodeling of lung architecture, which is a hallmark of many lung diseases (56). Interestingly, expression of matrix metalloproteinase-9 (MMP-9) by airway epithelial cells can be stimulated by allergens (57). Since MMP-9 is secreted as pro-MMP-9, it has to become activated by cleavage at the N-terminus. Importantly, serine proteases from German cockroach frass are able to activate MMP-9, and therefore, it is speculated that they could influence airway remodeling (58). Cytokines and Chemokines
Upon contact with allergens, epithelial cells can secrete a huge variety of cytokines and chemokines. For example, chemotaxis of eosinophils (RANTES, Eotaxin), neutrophils (IL-8), or monocytes (MCP-1) can be induced. Furthermore, the activation and maturation of lymphocytes (IL-1, IL-6) or the maturation of dendritic cells (DCs) [granulocyte-macrophage colony-stimulating factor (GM-CSF)] can be modulated (Table 2). In particular, chronic exposure to HDM aeroallergens during infancy
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Table 2 Main Functions of Selected Cytokines/Chemokines Produced by Relevant Lung Cells
Upon Contact with Aeroallergens Cell type
Chemokines
Epithelial cells
RANTES Eotaxin IL-8 MCP-1
Cytokines
GM-CSF IL-6 IL-1 Macrophages
MCP-1 MIP-1-a IL-8 IL-1 IL-6 TNF-a IFN-g
Dendritic cells
IL-8 MCP-1 MIP-1-b IL-6 IL-10 IL-12 TNF-a TNF-a IL-4
Mast cells
IL-3 GM-CSF IL-5 IL-8 IL-16
Main functions Eosinophil chemotaxis Eosinophil chemotaxis Neutrophil chemotaxis Monocyte/Basophil chemotaxis Maturation of DCs Maturation of B lymphocytes Lymphocyte-activating factor Basophil chemotaxis Eosinophil chemotaxis Neutrophil chemotaxis Lymphocyte-activating factor Maturation of B Lymphocytes Proinflammatory action Inhibition of IL-4 mediated expression of low-affinity IgE receptors Neutrophil chemotaxis Monocyte/Basophil chemotaxis Proinflammatory action Stimulation of B lymphocyte maturation Inhibition of IFN-g production Activation and proliferation of NK cells Proinflammatory action Proinflammatory action Associated with Th2 cell differentiation and IgE synthesis Involved in eosinophil development Maturation of DCs Involved in eosinophil development Neutrophil chemotaxis Lymphocyte chemotaxis
Abbreviations: IL, interleukin; MCP, monocyte chemoattractant protein; GM-CSF, granulocytemacrophage colony-stimulating factor; TNF-a, tumor necrosis factor-alpha; IFN-g, interferon gamma; DCs, dendritic cells.
enhances localized eotaxin-3 expression in the airway epithelium, which might explain recruitment of eosinophils to the airway mucosa (59). In cell cultures, IL-6 and IL-8, among other cytokines and chemokines, are released by airway epithelial cells after incubation with mite allergens, pollen allergens, fungal allergens, and cockroach allergens (60–68). In addition, HDM allergens are able to increase TGF-b gene expression and protein release from the RPMI 2650 human nasal epithelial cell line whereas grass pollen are without effect (69). Since transforming growth factor alpha (TGF-a) stimulates fibroblast proliferation and goblet cell differentiation, the increased release of TGF-a by tumor necrosis factor TNF-a and combinations of IL-4, IL-13, and Der p in epithelial cell cultures from asthmatic subjects could be a matter of particular
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interest (68). This finding provides a potential link between allergen exposure, Th2 cytokines, and airway remodeling in asthma. Cytokine/chemokine secretion is, at least partly, dependent on intracellular calcium. It was shown that Der p 1, Der p 3, and Der p 9 stimulate changes in the intracellular calcium ion concentration in epithelial cell lines (63,70). In addition, Sohn et al. have shown that HDM- as well as Pen c 13-induced IL-8 production is at least partly regulated by Ca2þ signaling (67,71). In contrast, no intracellular calcium mobilization could be found for Der p 1 but for Der p 5 in A549 cells indicating different activation pathways for the cytokine/chemokine secretion by these cells (62). German cockroach extract (GCE) is also able to mobilize calcium. A baseline type of intracellular calcium oscillations could be induced in a dose-dependent manner. The oscillations persisted for long periods of time in the absence of calcium entry across the plasma membrane, suggesting that the observed increase of intracellular calcium was due to calcium release from intracellular stores. Accordingly, after depleting endoplasmic reticulum calcium, the GCE-mediated intracellular calcium signals were abolished (72). Transcription Factors
Little is known about the transcription factors involved in allergen responses, but nuclear factor-kappa B (NF-kB) seems to be very important. Der p 1 promotes NF-kB nuclear translocation and Stacey et al. showed that this translocation is increased through phosphorylation and degradation of Ik-Ba, the cytoplasmic inhibitor of NF-kB (73). The involvement of NF-kB in IL-6 and IL-8 expression after allergen incubation (A. fumigatus) was also suggested by Borger and colleagues. Additionally, they found enhanced DNA binding of nuclear factor-IL-6 (NF-IL-6) whereas activator protein (AP)-1 was not involved (74). Adam et al. showed that Der p 1-mediated IL-8 expression was mainly dependent on NF-kB whereas Der p 3 regulated IL-8 expression through the activation of NF-kB and AP-1. Furthermore, Der p 1 induced IL-8release exclusively via the MAP kinase ERK1/2 signaling pathway whereas Der p 3 involved the three MAP kinases, ERK1/2, p38, and C-Jun N-terminal kinase (JNK) (75). It was further shown that ERK plays an important role in signal transmission for efficient activation of the HDM-induced IL-8 signaling pathway in lung epithelial H292 cells (71). Moreover, Pen c 13-induced IL-8 expression via activation of ERK 1/2, and not of p38 and JNK (67). In nasal epithelial cells from healthy donors, HDM incubation led to an increased expression of the two transcription factors NF-kB and AP-1 as well as their regulatory molecules. Interestingly, the authors found that gene levels from allergic epithelium are already in an activated condition without in vitro allergen incubation. In contrast to the results obtained from the healthy material, it was found that in cells from allergic patients, NF-kB regulatory pathway remained unchanged upon exposure to HDM allergens whereas the AP-1 pathway was downregulated (76). In addition, expression of MMP-9 in human bronchial epithelial cells (16HBE14o) and primary normal human bronchial epithelial cells by German cockroach feces also involves ERK and AP-1 (57). Taken together, several transcription factors are regulated in epithelial cells upon aeroallergen contact. The respective transcription factor seems to be dependent on the type of epithelial cell and the kind of allergen (Table 3).
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Table 3 Transcription Factors That Are Regulated in Epithelial Cells Upon Contact with
Aeroallergens Involved transcription factor
Effect
Cell system
Allergens
Reference
NF-kB
IL-6 and IL-8 induction
Human primary bronchial and nasal epithelial cells, A549
73,74,75,76
AP-1
IL-8 upregulation, MMP-9 stimulation
Primary human bronchial and nasal epithelial cells from healthy donors, 16HBE14o, A549 Primary human nasal epithelial cells from allergic donors A549
Der p 1, Aspergillus fumigatus filtrates, HDM extract Der p 3, HDM extract, German cockroach feces
HDM extract
76
Aspergillus fumigatus filtrates
74
Downregulation of AP-1 pathway NF-IL-6
IL-6 and IL-8 induction
57,75,76
Abbreviations: HDM, house dust mite; IL, interleukin; NF-kB, nuclear factor kappa B; MMP, matrix metalloproteinase; AP, activator protein.
Aeroallergens Harm Epithelial Cells
Cell-cell contacts between adjacent respiratory epithelial cells are sealed by tight junctions. Several proteins have been identified as components of the tight junctions: claudin 1 and 2 as well as occludin are transmembrane molecules. These proteins are associated with proteins of the zonula occludens family: ZO-1, ZO-2, and ZO-3. It is well known that several allergens are able to damage tight junctions, which is assumed to be a critical step for the allergens to reach the underlying tissue. Importantly, this could be an initial event to enable an easier antigen collection for the DCs at the luminal side. In addition to tight junction cleavage, whole cell detachment, shrinking, and desquamation were detected after incubation of epithelial cells with allergens (60,61,77) (Table 4). A direct cleavage of the extracellular domain of occludin and the adhesion protein claudin-1 was described for Der p 1. It was also shown that this cleavage nonspecifically increased epithelial permeability (78). Further studies with 16HBE14o-human bronchial epithelial cells showed a reduction in the tight junction content of the protein ZO-1 after incubation with Der p 1 (79). Besides Der p 1 cysteine proteinase also serine proteinases of HDM allergens impair the epithelial barrier function. Again, proteolysis of occludin and ZO-1 was involved in this process (80). In addition to HDM allergens, mold and
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Table 4 Effects of Aeroallergens on Epithelial Cells
Aeroallergen Cellular target
Effective
Cleavage of occludin
Der p 1, HDM feces, Pen ch 13, diffusates from stored pollen of giant ragweed, white birch and Kentucky blue grass, fresh pollen from easter lily, aminopeptidase from Parietaria judaica pollen Der p 1, diffusates from stored pollen of giant ragweed, white birch and Kentucky blue grass, and fresh pollen from easter lily Der p 1, HDM feces, diffusates from stored pollen of giant ragweed, white birch and Kentucky blue grass, and fresh pollen from easter lily Isolated aminopeptidase from Parietaria judaica pollen Der p 1
66,71,77,78, 79,80,81,82
Der p 1, HDM extracts, diffusates from pollens of Lolium perenne, Poa pratensis, Acacia longifolia, or Casuarina distyla Der p 1, Der p 5, HDM extract, Alternaria alternata extract, Aspergillus fumigatus extract Der p 1, HDM extract, Alternaria alternata extract, Aspergillus fumigatus extract Fel d 1
60,83,84
Cleavage of claudin-1
Cleavage of ZO-1
Cleavage of Ecadherin Tight junction degradation Cellular detachment
Cellular shrinking
Cellular desquamation
Degradation of denatured collagens and cleavage of plasma fibronectin
Abbreviation: HDM, house dust mite
Noneffective
Reference
78,82
79,80,82
77 83
Der p 2, allergen extracts from Cladosporium herbarum
61,62
Der p 2, allergen extracts from Cladosporium herbarum
61,62
85
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pollen allergens are able to degrade tight junction proteins. Incubation of 16HBE14o cells with the alkaline serine protease Pen ch 13 allergen from P. chrysogenum led to cleavage within the second extracellular domain of occludin (66,81). Diffusates from various allergens caused loss of tight junction proteins after incubation with confluent monolayers of Calu-3 cells, a human bronchial epithelial cell line (82). An isolated aminopeptidase from Parietaria judaica pollen degrades occludin and E-cadherin and causes cellular detachment of A549 cells (77). Several studies showed that allergen extracts from D. pteronyssinus and Lepidoglyphus destructor (60), pure Der p 1 (83), and diffusates from pollen of Lolium perenne, Poa pratensis, Acacia longifolia, or Casuarina distyla (84) are able to detach airway epithelial cells completely. In A549 cells, Der p 5 induced cell shrinking whereas Der p 1 and HDM extracts induced shrinking and desquamation. Interestingly, Der p 2 showed no effect (62). Morphological changes of A549 cells like cell shrinking, cell desquamation, or both, were also shown after incubation with fungal extracts from Altermaria alternata or A. fumigatus. Both, shrinking and desquamation were protease dependent. In contrast, neither cell shrinking nor desquamation was induced by allergen extracts from Cladosporium herbaraum (61). In addition, it was shown that Fel d 1 is able to degrade denatured collagens and cleave plasma fibronectin (85). Involvement of Protease-Activated Receptors
Protease-activated receptors (PARs) are G-protein-coupled receptors that signal in response to extracellular proteases. Four mammalian PARs are known: PAR1, PAR2, PAR3, and PAR4. PARs play an important role in hemostasis and thrombosis and also trigger inflammatory responses (86). In detail, an N-terminal peptide bond is cleaved, which leads to the formation of a new N-terminus that acts as a tethered ligand and binds intramolecularly to the receptor to trigger transmembrane signaling (87). Recent studies showed that PAR can interact with aeroallergens and thereby play an important role in modulating allergen protease effects. PAR2-deficient mice, for example, have a lower airway inflammation after allergen challenge compared to wild type mice. In contrast, mice overexpressing PAR2 showed a higher airway inflammation compared to wild type mice (88). However, a recent study demonstrated an anti-inflammatory effect upon PAR2 activation (89). This conflicting data could be explained by a dual response upon PAR2 activation, which is determined by the subsequent coupling of the receptor to either an inhibitory or stimulating G protein. A direct interaction of PAR with mite allergens Der p 3 and Der p 9 was demonstrated in bronchial epithelial cells. Both allergens cleaved the peptide corresponding to the N-terminus of PAR2 at the activation site suggesting that Der p 3- and Der p 9modulated release of proinflammatory cytokines from bronchial epithelial cells is at least partly mediated by PAR2 (70). Similarly, Asokananthan and colleagues found that the cytokine release of respiratory epithelial cells after incubation with Der p 1 is partly mediated by activation of PAR2, while PAR1 does not seem to be involved (90). In contrast, a recent study indicated that Der p 1-induced IL-8 production in A549 cells is independent of PAR2 activation (75). An explanation for these different results could be due to a putative contamination of the allergens. Cleavage of PAR2 is also shown for cockroach proteases, and it is suggested that MMP-9 stimulation by cockroach feces in human bronchial epithelial cells (16HBE14o) and primary normal human bronchial
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epithelial cells is mediated by a mechanism involving PAR2 (57,91). Furthermore, the generation of intracellular calcium oscillations by GCE through Ca2þ release from thapsigargin-sensitive calcium stores is also accomplished through activation of PAR2 (72). Finally, PARs are not restricted to epithelial cells. For example, PAR2 is also present on eosinophil granulocytes. Miike and Kita have shown that these cells can be activated by Der f 1 and they suggest the participation of PAR2 (92). B. Monocytes and Macrophages
Macrophages are derived from monocytic cells, which originate from bone marrow precursors. After entering the circulation, monocytes are guided by chemokines, cytokines, and cell adhesion molecules to migrate to their target organ where they differentiate to special organ macrophages like AMs. Besides AM, other kinds of lung macrophages are known: airway, connective tissue, pleural, and intravascular macrophages (93). The major tasks of AM are antigen uptake (phagocytosis), antigen cleavage, and participation in clearance processes. It is, for example, known that macrophages are able to take up SPP derived from whole grass pollen (29,94) or puffball spores from C. excipuliformis (16). While cleavage can lead to degradation and inactivation of allergens, the peptides can also be presented on the major histocompatibility complex I or II on the surface of the macrophages and hence stimulate the immune system. Importantly, various receptors can be found on the surface of macrophages and it is known that macrophages can alter their phenotype after stimulation with allergens. For example, an increased expression of CD11b and CD14 was detected upon contact with aeroallergens, which resemble an activated phenotype (95,96). In addition, AM can modulate the immune response by secretion of various cytokines and chemokines like IL-1, interferon (IFN)-g, TNF-a, IL-6, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1-a, and IL-8 (97,98). The secretion of these cytokines results in recruitment and activation of other inflammatory cells (99). Andersson Lundell and colleagues investigated the direct influence of HDM- and catallergen extract, and the recombinant allergen Fel d 1 on monocytes and monocytederived macrophages (100). According to this study, cat allergen extract induced production of TNF-a and IL-6 by CD14þ macrophages. Additionally, cat allergen extract and HDM extract stimulated CD14þ monocytes to release TNF-a and IL-6. Interestingly, blocking the CD14 receptor on monocytes led to an approximately 60% decrease of Fel d 1–induced cytokine production whereas HDM stimulated monocytes independently from the CD14 receptor. Importantly, an LPS contamination was ruled out. D. farinae can induce an activation of NF-kB in AMs and an upregulation of IL-6, TNF-a, and NO (101). In addition, antioxidants were down regulated. Furthermore, Der f-stimulated macrophages expressed enhanced levels of costimulatory B7 molecules and supported T cell proliferation. (101). It is suggested that this mite-induced activation of AMs is mediated by CD14/TLR4 receptors (32). C. Dendritic Cells
Several DC populations have been identified in the upper and lower respiratory tract. Beside the populations within alveolar septal walls and on the alveolar surface, DCs within the conducting airways exist (102–109). They collect antigens and migrate to
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secondary lymphoid tissue where they present antigen on their surface to T-lymphocytes. This is a crucial step for the initiation and maintenance of T cell–mediated responses. DCs of the intrapulmonary airways are predominantly located in an immature state beneath the airway epithelial basement membrane (110). To come into contact with antigens, DCs have to pass the tight junctions between the epithelial cells. It is known that intestinal DCs can send up dendrites through tight junctions without compromising the barrier function (111,112), and a recent study from Jahnsen and colleagues demonstrated this “snorkeling” behavior for lung DCs also (113). These findings were confirmed by an in vitro study from Blank and coworkers, who used blood-derived DCs in an epithelial airway model (114). In this in vitro system, the DCs migrated to the luminal space. Antigen collection by DCs could be facilitated, if the antigen has protease activity and so cleaves single tight junction proteins. In allergic rhinitis, DCs have been demonstrated to penetrate beyond the mucosal epithelium if the epithelial barrier function is affected (115). Furthermore, a study with a triple-cell culture model composed of macrophages, DCs, and epithelial cells showed a higher DC migration rate toward the epithelial cells, when the transepithelial electric resistance (TEER) was lower compared to cultures with a high TEER value (114). In this study, the authors also showed that macrophages collect fine polystyrene particles and extent processes between the epithelial cells toward the basal side where the DCs reside. This might indicate that macrophages are seeking for an interaction with DCs and illustrates a possibility for DCs to receive antigens. By collecting antigens, DCs undergo a maturation process, which facilitates their migration to lymph nodes where they present processed antigens to T cells. Deslee and colleagues investigated the uptake of fluorescent-labeled Der p 1 in monocyte-derived DCs and they found that Der p 1 was taken up in a dose-, time-, and temperature dependent manner. Uptake was at least partly dependent on the mannose receptor (116). In contrast, class A scavenger receptor (SR-A) known for its participation in uptake of particles in macrophages was not involved in allergen uptake by DCs but in DC migration. In SR-A-deficient, ovalbuminsensitized mice, ovalbumin challenge led to an increased migration of ovalbumin-loaded DCs compared to respective wild type control. Additionally, the increased migration rate of DCs in SR-A-deficient mice was accompanied by an enhanced proliferation of ovalbumin-specific T cells in thoracic lymph nodes, which was not due to an increased ovalbumin uptake/single cell (117). DCs express a variety of distinct surface markers, which are important for interaction with antigens and the resulting DC behavior. Comparable expression profiles of various surface markers (HLA-DR, CD80, CD86, CD83, CD1a, and CD11c) were observed in monocyte-derived DCs from allergic or healthy individuals. Cells from healthy donors stimulated with both, cat and birch allergen, showed a similar activation of the CD80 and CD86 costimulatory molecules compared to nonstimulated cells. After incubation with cells from allergic individuals, birch allergen was unable to produce the same increased expression of CD80 alone or in combination with CD86, compared to cells stimulated with cat allergen and LPS. In addition, secretion of various chemokines and cytokines by nonstimulated DCs (e.g., IL-6, IL-8) were similar for both groups of subjects. By contrast, the spontaneous secretion of IL-12p70 and TNF-a was higher for DCs from healthy subjects when compared with that from allergic individuals (118). An interesting study was accomplished by Lamhamedi-Cherradi and coworkers, who explored the mechanisms by which DCs treated with Aspergillus proteases can initiate
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and maintain a Th2 immune response. They found that human DCs derived from CD14þ monocytes from healthy donors underwent partial maturation when incubated with Aspergillus proteases (119). In addition, an enhanced production of IL-4 and reduced expression of IFN-g were found by naive allogeneic T cells primed with Aspergillus protease-activated DCs as compared with naive T cells primed with LPS-activated DCs. This denotes a Th2 immune response. Importantly, DCs revealed low expression of IL-12p40 in Aspergillus-activated DCs as compared with LPS-activated DCs. Hence, the authors concluded that the induced Th2 responses are initiated by limited maturation of DCs with reduced production of IL-12 (119). Taken together, DCs collect antigens, which can lead to an activated DC phenotype and to a release of chemokines and cytokines. This could lead to a significant modulation of the immune response. D. Mast Cells
Acute allergic responses, especially in the upper airways, are characterized by the release of preformed mediators, especially histamine from mast cells. This mast cell stimulation includes allergen binding and cross-linking of allergen-specific IgE. Besides IgE-dependent responses, mast cells are able to degranulate in a non-IgE-dependent manner. For example, Der p 9 can induce mast cell degranulation (120). However, the majority of aeroallergen-mast cell interaction is IgE-dependent. In addition, mast cells secrete a variety of cytokines like TNF-a, and it was shown in a mouse asthma model that TNF-a plays an important role in allergic inflammation and airway hyperreactivity (121,122). Furthermore, upon allergen stimulation mast cells can produce a variety of cytokines and chemokines that are involved in various processes like Th2 cell differentiation, chemotaxis of neutrophils and lymphocytes, eosinophil development, or the maturation of DCs (123).
V. Summary Aeroallergens are airborne particles including pollen grains, fungal spores, and microparticles from anthropogenic and natural emissions, which can cause an allergic response. Dependent on their size, aeroallergens can reach all regions of the respiratory tract—from the nasal mucosa down to the alveoli. They can affect various noncellular and cellular elements (Fig. 4) by distinct biochemical functions. For example, the proteolytic activity of Der p 1 can cleave the tight junction protein occluding, leading to increased epithelial permeability. Although DCs are able to sample antigens from the periphery even if tight junctions are functional, cleaved tight junction proteins might facilitate antigen sampling by airway DCs. In addition, the cysteine protease activities of Der p 1 and Der f 1 degrade the SP-A and -D. Since both SP-A and -D participate in innate immune functions, this could lead to an attenuation of the allergic inflammation and impaired defense properties against pathogens. Furthermore, epithelial cells, macrophages as well as DCs can become activated upon contact with aeroallergens and secrete various cytokines and chemokines. Hitherto, a definite relationship between exposure, sensitization, airway inflammation, and allergic symptoms has not been made for any of the aeroallergens. Therefore, much remains to be elucidated regarding aeroallergen-lung interactions in allergic disease.
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Figure 4 Diagram of aeroallergen-lung interactions. Arrows with solid lines represent inter-
actions of aeroallergens with the respective target (arrows). Dotted arrows display the resulting effect.
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90. Asokananthan N, Graham PT, Stewart DJ, et al. House dust mite allergens induce proinflammatory cytokines from respiratory epithelial cells: the cysteine protease allergen, Der p 1, activates protease-activated receptor (PAR)-2 and inactivates PAR-1. J Immunol 2002; 169:4572–4578. 91. Page K, Strunk VS, Hershenson MB. Cockroach proteases increase IL-8 expression in human bronchial epithelial cells via activation of protease-activated receptor (PAR)-2 and extracellular-signal-regulated kinase. J Allergy Clin Immunol 2003; 112:1112–1118. 92. Miike S, Kita H. Human eosinophils are activated by cysteine proteases and release inflammatory mediators. J Allergy Clin Immunol 2003; 111:704–713. 93. Brain JD. Lung macrophages: how many kinds are there? What do they do? Am Rev Respir Dis 1988; 137:507–509. 94. Currie AJ, Stewart GA, McWilliam AS. Alveolar macrophages bind and phagocytose allergen-containing pollen starch granules via C-type lectin and integrin receptors: implications for airway inflammatory disease. J Immunol 2000; 164:3878–3886. 95. Lensmar C, Prieto J, Dahlen B, et al. Airway inflammation and altered alveolar macrophage phenotype pattern after repeated low-dose allergen exposure of atopic asthmatic subjects. Clin Exp Allergy 1999; 29:1632–1640. 96. Viksman MY, Bochner BS, Peebles RS, et al. Expression of activation markers on alveolar macrophages in allergic asthmatics after endobronchial or whole-lung allergen challenge. Clin Immunol 2002; 104:77–85. 97. Gosset P, Tillie-Leblond I, Oudin S, et al. Production of chemokines and proinflammatory and antiinflammatory cytokines by human alveolar macrophages activated by IgE receptors. J Allergy Clin Immunol 1999; 103:289–297. 98. Rosenwasser LJ. New immunopharmacologic approaches to asthma: role of cytokine antagonism. J Allergy Clin Immunol 2000; 105:S586–S591. 99. Vissers JL, van Esch BC, Hofman GA, et al. Macrophages induce an allergen-specific and long-term suppression in a mouse asthma model. Eur Respir J 2005; 26:1040–1046. 100. Andersson Lundell AC, Grindebacke H, Karlsson H, et al. Cat allergen induces proinflammatory responses by human monocyte-derived macrophages but not by dendritic cells. Allergy 2005; 60:1184–1191. 101. Chen CL, Lee CT, Liu YC, et al. House dust mite Dermatophagoides farinae augments proinflammatory mediator productions and accessory function of alveolar macrophages: implications for allergic sensitization and inflammation. J Immunol 2003; 170:528–536. 102. Holt PG, Schon-Hegrad MA, Phillips MJ, et al. Ia-positive dendritic cells form a tightly meshed network within the human airway epithelium. Clin Exp Allergy 1989; 19:597–601. 103. Hammad H, Lambrecht BN. Recent progress in the biology of airway dendritic cells and implications for understanding the regulation of asthmatic inflammation. J Allergy Clin Immunol 2006; 118:331–336. 104. Schon-Hegrad MA, Oliver J, McMenamin PG, et al. Studies on the density, distribution, and surface phenotype of intraepithelial class II major histocompatibility complex antigen (Ia)-bearing dendritic cells (DC) in the conducting airways. J Exp Med 1991; 173: 1345–1356. 105. Holt PG, Schon-Hegrad MA, Oliver J. MHC class II antigen-bearing dendritic cells in pulmonary tissues of the rat. Regulation of antigen presentation activity by endogenous macrophage populations. J Exp Med 1988; 167:262–274. 106. Gong JL, McCarthy KM, Telford J, et al. Intraepithelial airway dendritic cells: a distinct subset of pulmonary dendritic cells obtained by microdissection. J Exp Med 1992; 175: 797–807. 107. Casolaro MA, Bernaudin JF, Saltini C, et al. Accumulation of Langerhans’ cells on the epithelial surface of the lower respiratory tract in normal subjects in association with cigarette smoking. Am Rev Respir Dis 1988; 137:406–411.
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16 Dose-Response Relationships TOBIAS STOEGER and OTMAR SCHMID Helmholtz Zentrum Mu¨nchen—German Research Center for Environmental Health, Institute for Lung Biology and Disease (iLBD) and Focus Network Nanoparticles and Health, Neuherberg, Germany
I.
Introduction
Inhalation of high concentrations of airborne, so-called “low toxicity,” particles can cause adverse health effects. Epidemiological studies have repeatedly described correlations between the level of ambient particulate matter (PM) and increased morbidity and mortality in adults and children (1,2). Ambient PM has been recognized as important and even independent risk factor for both respiratory diseases like chronic obstructive pulmonary disease, asthma, and lung cancer, and also for cardiovascular disorders such as coronary artery disease, atherosclerosis, and stroke (3). The quantitative relationship between the amount of exposure (dose) to a stressor (e.g., PM) and the resulting changes in body function or health (response) is typically referred to as the dose-response relationship. The dose-response relationship—seen as causal link between a dose parameter and a toxicological effect—is often used to try to identify the driver of toxicity and to establish regulatory measures. Once the relevant dose parameter for particle toxicity is identified, dose-response relationships can be used to determine a threshold dose, which is a key issue in risk assessment and the development of (legislative) guidelines. While in numerous countries, maximum exposure levels (based on mass as dose parameter) for various types of PM are already established in workplace safety regulations (e.g., for coal or asbestos particles), similar measures have been established for ambient PM in some countries, and discussions about regulatory measures are currently intensifying for nanoparticles due to the increasing significance of nanotechnology. In this chapter, we will review numerous dose-response studies for various particle types with particular emphasis on nanoparticles and draw conclusions on the most relevant dose parameter [particle mass, surface area (SA), or number], the existence of a threshold dose, and the underlying mechanisms of biological response (here inflammatory response).
II.
Methodology
A. Response Endpoints
The response (toxicity) of an organism to PM is likely to depend on numerous aspects including particle solubility (in body fluids), biopersistence, and reactivity (e.g.,
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oxidative capacity). While the former two are mainly related to the lifetime and fate of the particles within the organism, the latter is typically referred to as its toxicity, which can be characterized by dose-response curves. For non-water-soluble PM, as considered here, the dose can be given in various ways including particle number, SA, and mass. Ideally, one chooses the dose parameter such that particles of similar chemical and structural nature have similar dose-response curves. While historically, particle mass has been used as dose metric, we will show below that particle SA is the single relevant dose metric for PM exposure. Although numerous biological response parameters (endpoints) exist, lung inflammation is seen as the key response to increased levels of particulate air pollution (4). As the toxicology of inhaled particles becomes increasingly better understood on a molecular level, oxidative stress-mediated inflammation is now generally regarded as common pathway underlying the various forms of adverse health effects independent of the particle type (4–6). Since it is believed that pathogenic effects due to particle exposure are initiated by inflammation (7), the following discussion will focus on acute inflammation as the central biological effect. A commonly used and effective method for rapid screening for pulmonary inflammation in laboratory animals is based on the analysis of bronchoalveolar lavage (BAL) fluid, a saline wash (lavage) of the airways (broncho) and air sacs (alveolar) for recovery of inflammatory mediators and cells. BAL is a medical procedure typically performed to diagnose human lung disease. In particular, the occurrence of inflammatory, polymorphonuclear cells (PMNs) in BAL is most frequently used as indicator for an inflammatory response, since infiltration by these neutrophilic granulocytes is a general hallmark of inflammation (8). Moreover, their abundance has a high dynamic range between about 2% (no inflammation) and 70% of all the lavagable cells. Using the number of PMNs normalized to total number of lavagable cells (%PMN) instead of the absolute number of PMNs has the advantage of not being affected by variations in BAL cell counts due to species-dependent and methodological variations in lavage efficiency. B. Exposure Scenario
While inhalation is the natural route of exposure to airborne PM, numerous doseresponse studies utilize intratracheal instillation (IT) as exposure route, that is, they administer a small volume of an aqueous PM suspension via the airways directly into the lower respiratory tract. Both inhalation and IT of PM evoke inflammatory responses in humans (9,10) and in animal models like dogs (11), rats (12–19), and mice (20,21). Since the data pool on humans is scarce, we will only consider dose-response relationships from controlled animal exposure for this following analysis. Most of the particle exposure experiments addressed here have been conducted utilizing IT. However, IT-based dose-response curves may deviate from the more physiological inhalation-based response due to several methodological limitations including liquid-dependent particle agglomeration (in the suspension to be instilled) and subsequent changes in particle size distribution as well as differences in clearance and response pattern between inhalation and IT of PM (22). However, IT is experimentally much simpler, and it avoids inhalation-related dose uncertainties due to poorly known particle deposition efficiencies in the lungs, uncertain inhaled air volume, and inadvertent deposition in the nasal passages. Thus, for dose-response measurements, the
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disadvantages of IT are outweighed by the fact that IT is a relatively inexpensive and accurate method of administering known amounts of PM directly and instantaneously into the lower respiratory tract. Since the PMN influx is time dependent and reaches a maximum at about 24 hours after the challenge with a particulate stressor, it is essential for comparative toxicological studies to consider the PMN influx in a narrow time window (18–24 hours) after exposure. For the identification of the most relevant dose parameter we provide a synopsis of toxicological in vivo rat and mouse data for different types of particles (different material, varying size) using PMN influx (18–24 hours after IT; acute inflammation) as toxicological response parameter. To allow conversion into different dose parameters, we only included data sets that provided information on particle SA (SABET, i.e., measured by gas adsorbtion as described by Brunauer, Emmet, and Teller), size, and mass. In toxicology and pharmacology, exposure doses are traditionally expressed in terms of the gravimetric mass of an agent. However, for non- or poorly soluble PM (as considered here), it is well known that other aspects such as particle size also play an important role. For instance, the small size of so-called nanoparticles (at least one dimension is below 0.1 mm) reduces the efficiency of phagocytotic clearance from the alveolar regime of the lungs (longer lifetime in organism), increases the probability for endocytotic uptake into cells, and possibly enhances transcellular processes, that is, transcytosis across epithelial-endothelial barriers (frequently referred to as translocation) into the blood and lymph circulation from where they might reach sensitive target cells of the vasculature, lymph nodes, bone marrow, and heart. Moreover, nanoparticles may pose a higher health risk than larger particles of the same material and mass dose, since nanoparticles may induce a more pronounced biological response than larger particles (on a per mass basis) due to their higher surface-to-volume (mass) ratio (23–25 and reviewed by 26,27). On the other hand, it is also conceivable that the biological response is mainly governed by the number of particles entering the organism rather than by their specific properties. This appears particularly plausible if one considers that the rate of biochemical reactions depends more on the number of “indivisible reactants” like molecules than on their mass (“Law of Mass Action”). Since for a given mass of spherical particles (PM), a change in particle diameter (d) will affect particle number (scales with d3) and SA (scales with d1) differently, we will show below that it is possible to decide which of the available dose metrics is more adequate for describing the biological response of particles.
III.
Identification of the Most Relevant Dose Parameter
A. Particle Mass
Figure 1 depicts the mass-based dose-response relationship after intratracheal particle instillation for five categories of particles (carbon black, polystyrene, manganese oxide, titanium dioxide, and quartz) in mice or rats using the observed PMN fraction in the BAL 24 hours after IT as indicator of inflammation. The most relevant information for each of the five particle categories is listed in Table 1.
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Figure 1 Mass-based dose-response relationship for the five different particle types listed in
Table 1. To combine mouse and rat data, the instilled particle mass was normalized to the (average) mass of the lungs of mice and rats, respectively. *Lung weights: 0.18 g/mouse and 1.5 g/rat. Table 1 The Characteristic Parameters of the Five Different Particle Types That Were Included
in the Comparative Study Particle composition
Size range [nm]
Dose range [mg]
SABET range [m2/g]
Species
Reference no.
Carbon black Carbonaceous MnO2 Polystyrene Quartz Quartz TiO2 TiO2 TiO2 TiO2 TiO2
14 9–51 N.A. 65–535 12–50 300–1500 20 20–250 20–250 25–180 300
125 5–50 37–2500 125–1000 275–1375 275–1375 125 5–400 32–2000 1000–6000 275–1375
254 43–800 0.2–62 11–89 31–91 4–5 50 7–50 7–50 10–50 6
Rat Mouse Mouse Rat Rat Rat Rat Mouse Rat Rat Rat
13 21 28 12 19 19 13 26 26 16 18
All studies applied the particles directly into the lungs of mice and rats (with body weights of about 22 g and 275 g, respectively) via intratracheal instillation. Abbreviation: SABET, surface area measurement developed by Brunauer, Emmett, and Teller.
As previously suggested (26), both rat and mouse data can be presented in the same figure, if the administered particle dose is normalized to the species-specific mass of the lungs (an average value of 0.18 g and 1.5 g was used for mice and rats, respectively) and presented in units of “mg” particle per “g” lung. This normalization approach is feasible as all experiments have been conduced with animals of comparable age and the interstrain variation in lung weights is within a range of 30% (e.g., female mice at three months of age, BALBc/J: 0.16 g, DBA/2J: 0.17 g, and NMRI: 0.20 g).
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The wide dose range from 20 to 14,000 mg/g lung (or from 0.13–90 mg particles/g animal) seen in Figure 1 suggests that the mass-based toxicity of the particles differed substantially. To facilitate the presentation of such a wide dose range, the abscissa appears on a logarithmic scale, as commonly chosen for pharmacological doseresponse graphs. Obviously, there is a large variability in mass-specific biological response, which not only depends on particle categories but even within the same category. For instance, for a mass dose of 183 mg/g lung quartz particles, the observed PMN levels vary between 14% and 43%, and even more obvious for the 13.9 mg manganese oxide dose, the PMN response ranges from 2% to 60%. Hence, it is impossible to provide a category-specific toxicity rating using particle mass as dose metric. B. Particle Size
Several investigations identified the size of the particles as an important factor not only affecting the cellular uptake of particles and thus phagocytotic clearance from the lungs but also the inflammatory response mechanisms. Expressing the data from Figure 1 as mass-specific PMN response (ratio of PMN and PM mass) versus particle diameter, it is evident that all particle types except for quartz show a negative correlation between mass-specific PMN levels and particle size (Fig. 2). MnO2 particles were omitted due to a lack of size information. Consequently, it is now generally accepted that for a given particle type (material), ultrafine or nano-sized particles (diameter <0.1 mm) have a higher mass-specific toxicity level than larger respirable particles (so-called fine particles; 0.1 mm < diameter < 2.5 mm). However, it is evident from Figure 2 that quartz does not adhere to this rule for reasons discussed below.
Figure 2 Mass-specific inflammatory response of four particle types presented in Figure 1 (PMN
response divided by instilled particle mass) versus particle diameter, where only PMN data within the dynamic response range (10–35%) are included. All particle types (solid line) show increasing mass-specific toxicity with decreasing particle size, except for quartz, which shows the opposite trend (dashed line). MnO2 particles are not included since no size information was available.
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C. Particle Number
For a given mass of PM, the particle number will increase with decreasing particle diameter (d) (number of spherical particles scales with d3), that is, decreasing the particle size by a factor of 10 will enhance the particle number by a factor of 1000. It is evident from Figure 3, which depicts the number-based dose response of the particles from Figure 1 (again except for MnO2 due to a lack of size information) that the numberbased toxicity appears to increase with particle size relatively independent of the material of the particles, as illustrated by the trend lines (power law fits) for five different size ranges, namely, 12 to 25 nm, 50 to 65 nm, 180 to 300 nm, 534 nm, and 1500 nm. Increasing the particle size from 12 to 1500 nm changes the number-based dose by about seven orders of magnitude (107–1014 particles/g lung), while differences in material for a narrow size range only results in a variability of about one order of magnitude. If particle number were the governing parameter for particle toxicity, one would expect a close correlation between biological response and particle number independent of particle size. Since this is not the case, particle number is not directly related to the mechanisms of particle toxicity and hence not the most relevant dose parameter. It will be shown below that the apparent increase in number-based toxicity with particle size is a result of the increasing surface-to-number ratio with increasing particle diameter.
Figure 3 Number-based dose-response for four of the five particle types presented in Figure 1,
for which sufficient information was available to derive particle number values [polystyrene, carbon black (CB), titanium oxide (TiO2) and quartz]. Obviously, particle size (not material) dominates the number-based particle toxicity, since particles with similar size appear in a narrow dose-response range (factor of 10 to 100). We refrained from fitting the data with the theoretically expected sigmoidal curves due to a lack of data in the high-concentration (saturation) regime.
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For (spherical) particles, the geometric SA scales with d1, that is, for a given mass of particles, a decrease in particle diameter by a factor of 10 will result in a 10-fold increase in particle SA. Similar to particle number, one could convert the data from Figure 1 into (geometric) SA using the information on particle mass, density, and size. However, we will use SABET instead of geometric SA as dose metric (Fig. 4), where “BET” refers to a method for determining the SA accessible to gas molecules as described by Brunauer, Emmett, and Teller (29), since the SABET can be measured directly (not derived from mass, density, and microscopically determined size) and—for reasons discussed below—is more closely related to particle-induced toxicity (30). It is evident from Figure 4 that using SABET as dose metric allows the identification of three different toxicity classes, where the fine quartz and MnO2 particles represent the high- and lowtoxicity class, respectively, and all other nonsoluble particle types presented here belong to the intermediate toxicity class. As mentioned above, expressing dose and response on a logarithmic (X axis) and linear (Y axis) scale, respectively, results in the typical sigmoidal dose-response curves that are characterized by three domains: baseline or zero response (low dose), dynamic response (domain where a small increase in dose causes a large increase in response), and saturated (maximum) response regime (high dose). While the dose range of most of the dose-response curves shown in Figure 4 is too limited for a reliable identification of the saturation levels (>40% for all particle types), the dynamic response regime for the low, intermediate, and high-toxicity particles is in the range of 200 to 2000, 20 to 200, and 2 to 20 cm2/g lung, respectively. Compared to particle mass and number (Figs. 1 and 3), SABET is clearly the most relevant dose parameter, since most of the diverse data presented in Figure 4 (five different materials and several particle sizes for each material) fall into a narrow doseresponse regime (intermediate toxicity class) with the exception of fine quartz and
Figure 4 BET surface area (SABET)-based dose response for the data presented in Figure 1.
Clearly, three different toxicity regimes can be distinguished, where the onset of the biological response curves ranges from about 2 to 20, 20 to 200, and 200 to 2000 cm2/g lung for the low-, intermediate-, and high-toxicity regime, respectively.
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MnO2 particles, which show a significantly higher and lower toxicity, respectively. This implies that there may be a causal link between SABET and pulmonary inflammation. However, the distinctly higher toxicity of fine (1500 nm) quartz compared to nano-sized (12–50 nm) quartz indicates that other parameters such as crystalline structure and surface charge may also play a role at least for some of the particle types presented here (15,31). A more detailed discussion of the underlying toxicological mechanisms will be provided below. E.
Mechanisms of Particle Toxicity and Implications for the Most Relevant Dose Parameter
The underlying mechanism for particle-induced inflammation is believed to be linked to the generation of reactive oxygen species (ROS) as mediator of cellular oxidative stress, which triggers intracellular signaling processes such as the activation of redox-sensitive transcription factors (32,33). For nanoparticles, two main pathways of particle-induced ROS formation have been identified: (i) catalytic reactivity of the SA of the inert particle matrix (including bioavailability of redox cycling catalysts like transition metals or quinones) and (ii) metabolic generation of reactive by-products during the cellular detoxification of organic compounds. In particular for larger-sized particles, a further mechanism is related to the enzymatic production of ROS upon particle phagocytosis (34,35). Radical formation during phagocytosis is crucial for bacterial (~1 mm) killing and correspondingly this pathway is probably of minor relevance for smaller particles (<500 nm). Each of these mechanisms can be related to specific particle properties. While the first two pathways are linked to the particle SA, the latter is triggered by individual particles (particle number), whereas SA or mass plays a minor part. Hence, the relevance of a specific dose metric provides indirect evidence for which of the pathways mentioned above are most relevant for particle toxicity. Clearly, the first pathway depends on the particle SA, since the catalytic activity is driven by the molecules that are in direct contact with the biological system. However, not all particles have the same catalytic activity due to differences in the type of bioavailable surface atoms (molecules). Among the most reactive compounds are redox catalysts such as quinones and transition metals, which can produce highly toxic free OH radicals species from hydrogen peroxide. In addition to the chemical composition, the surface reactivity also depends on the crystalline structure and, in particular, the ratio of edge-site to plane-site atoms, which increases with the number of structural defects. For instance, curved fullerenoid carbon structures are considerably more reactive than planar graphite-like carbon particles (36,37). In contrast to surface catalytic processes (pathway i), the second pathway for ROS formation (pathway ii) requires metabolic activity, namely, the bioactivation (detoxification side effect) of certain organic compounds like polycyclic aromatic hydrocarbon (PAH) by xenobiotics metabolizing enzymes. In particular, the synthesis of the PAH-inducible phase I enzyme cytochrome P450 1A1 (CYP1A1) is known for its catalytic production of ROS, if phase I is not tightly coupled with and balanced by the phase II detoxification reaction (38,39). Since many organic compounds are condensed onto some inert particle core, the amount of available bioactive compounds depends on the particle SA available for condensation and subsequent representation, which is well characterized by the SABET.
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While the phagocytosis-related pathway is likely to also contribute to the overall particle-induced ROS formation, pathways i and ii are the main pathways for nanoparticles, as indirectly evidenced by the strong correlation between biological response and SABET (not particle number or mass) as shown in Figures 1, 3, and 4. In fact, the strong increase of number-based toxicity with particle size (Fig. 3) can now be explained by the increasing SA of larger particles (SA ~ d2). Particle mass fails to adequately describe particle toxicity (Fig. 1), since for a given particle mass, particles can have vastly different SA depending on particle size. This can be seen from Figure 2, which shows an increasing biological response (per mass) with decreasing particles size for all particle types except quartz. The “anomalous” behavior of quartz may be explained by the quartz-related increased ROS formation during phagocytosis (34,40). Regarding the role of transition metals, there is evidence from rat instillation experiments that reactive transition metals such as cobalt and nickel inducing 30% PMN at about 30 cm2/g belong to the “high-toxicity class” (13, data not shown). Finally, we note that compared to the geometric particle SA (derived from geometric particle diameter), the enhanced toxicity due to surface reactivity is likely to be better characterized by the SABET, since this parameter depends on the adsorption of gaseous molecules onto the SA, which is sensitive to more reactive corner/edge-site atoms and micropores. This is supported by the fact that the absorption energy has been suggested as characteristic parameter for the catalytic activity of single crystal surfaces (41). The latter also explains the empirical finding that SABET works better as dose metric than does the geometric SA (30). In summary, over the past decade, many studies reported that especially SABET is directly related to particle toxicity (12,15–17,21,28,39,42,43). Since the SA to volume (mass) ratio increases with decreasing particle size, small or even nano-sized particles are typically considered to be more dangerous (on a per mass basis) than larger particles. We have shown here that the SABET dose-response curves for a diverse set of particles both in terms of material (carbon black, polystyrene, TiO2, and nano-quartz) and size (12–250 nm) fall into a narrow regime (onset between 20 and 200 cm2/g lung), here referred to as “intermediate” toxicity regime. However, both high-toxicity (fine quartz) and low-toxicity particles (MnO2) have also been observed. This indicates that additional aggravating or mitigating factors are relevant such as surface charges, protein binding, and oxidative burst activity associated with phagocytosis. F.
The Question of a Dose-Response Threshold
Only few animal exposure studies investigated the existence of a particle SA threshold dose, below which no pulmonary inflammation is observed. The reliability of the available threshold estimates is limited by the insufficiently characterized shape of the dose-response curve due to a lack of sufficiently detailed data. For TiO2 and BaSO4 particles, a threshold dose of 130 to 200 cm2/g lung for PMN recruitment in rats was estimated (44). Using the same data, Dankovic et al. (45) suggested a pulmonary inflammation threshold dose of 90 cm2/g lung. For TiO2 particles, Cullen et al. (46) derived a threshold of 290 cm2/g lung for rats. To mitigate the methodological difficulties with deriving a threshold from the shape of the dose-response curve, we utilize the MinED (minimal effective dose), LOAEL (lowest adverse effect level) and NOAEL (no observed adverse effect level),
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where the MinED is defined as the limiting dose above which only statistically significant adverse effect levels are observed, the LOAEL indicates the lowest dose tested with a statistical significant effect, and the NOAEL refers to the limiting dose below which no statistically significant adverse effect levels are observed (47). Using our inflammatory response data for six different types of carbonaceous nanoparticles (CNP) (Fig. 5), including spark discharge generated carbon, carbon black, flame soot, and
Figure 5 The dose-response curve for pulmonary inflammation caused by IT of six types of CNP,
UfCP, carbon black (PrintexG, Printex90), and flame soot which high (SootH) and low (SootL) organic content (SootH and SootL, respectively), and DEP suggests a limiting dose of about 30 cm2/g lung, below which no adverse effect can be detected, since the NOAEL (dotted vertical line) and LOAEL (dotted vertical line) correspond to 27 and 30 cm2/g lung, respectively. An effective dose response is seen only for doses higher than 107 cm2/g lung (MinED, dashed line), which is supported also by the UfCP dose response, the only data set for which five doses (18.8, 75.0, 188, 750, and 1875 cm2/g lung) were investigated (right insert), because the two lowest doses did not cause any adverse response. Abbreviations: IT, intratracheal instillation; CNP, carbonaceous nanoparticles; DEP, diesel exhaust particles; NOAEL, no observed adverse effect level; LOAEL, lowest adverse effect level; MinED, minimal effective dose, UfCP, spark discharge generated carbon particles.
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diesel exhaust particles, we have previously suggested an SA threshold value of about 20 cm2 for mice (~110 cm2/g lung) when using PMN cell number as response metric (21). However, for the less error prone %PMNs, the NOAEL and LOAEL corresponded to 27 cm2/g lung and 30 cm2/g lung, respectively (see vertical dotted lines, left insert, Fig. 5). In accordance with the MinED of 107 cm2/g (right insert Fig. 5), the transition from NOAEL and LOAEL occurred for the dose-response curve with the highest dose resolution [spark discharge generated carbon particles (UfCPs); five dose levels of about 20, 80, 200, 800, and 2000 cm2/g lung] between 80 and 200 cm2/g lung (insert, Fig. 5). Hence, our CNP data would suggest a range from 30 and 100 cm2/g lung for an existing threshold. By comparison with Figure 4, it is evident that the range of estimated threshold values for CNP, TiO2, and BaSO4 particles (90–290 cm2/g lung) falls into the expected range for particles from our intermediate toxicity class (Fig. 4). However, it needs to be reemphasized that it is not unambiguously clear whether these values represent true threshold values (below which no biological effect occurs) or just the result of the limited detection efficiency near the noise level. G. Toward Quantification of the Different Pathways of Toxicity
Although SABET is a useful exposure dose parameter for particle toxicity, the surfacespecific toxicity depends at least to some degree on the particle type (not all doseresponse curves in Figure 4 fall into a narrow range), and it does not allow an assessment of the contribution of the two different toxicological pathways described above. Thus, the use of animal models is still unavoidable for reliable toxicity tests. To minimize the need for animal (in vivo) testing, the development of screening strategies for particle toxicity using cellular and acellular in vitro assays is of particular interest (30,45,46). In addition, this approach allows insights into the contributions of surface reactivity (pathway i) as well as bioavailability of organic contaminants (e.g., on diesel exhaust particles) to the overall particle toxicity. Nanoparticle surface-derived free radical activity has been shown in cells and in a cell-free system (1,6,31,48). In a recent review (49), a variety of cell-free and cellular test methods have been discussed including measurements for antioxidant depletion (e.g., glutathione, vitamin C, or dithiothreitol) and ROS formation (hydroxyl radical formation by electron paramagnetic resonance, intracellular oxidation of fluorescent dyes, quantification of cellular oxidized fatty acids). In addition, analysis of genomic or proteomic expression markers can be used for the detection of (i) cellular antioxidant response (glutathione S-transferase A1, heme oxygenase-1 etc.) or (ii) bioavailability of organic compounds (phase I enzyme: CYP1A1). Application of this type of measurement techniques may eventually lead to a more pathway-specific, quantitative toxicity model for nanoparticles. As an example for this type of studies, we summarize the results from Stoeger et al. (39), who investigated the toxicological pathways for six types of CNP. The formation of free radicals at the surface of particles (not contaminated with biodegradable organic or metallic compounds) can be interpreted as the particles’ innate “oxidative potency.” Using the depletion of ascorbate (vitamin C) in an aqueous particle suspension as a measure of the oxidative potency, the oxidative potency of the six types of CNP was obtained by Stoeger et al. (39). This relatively simple, cell-free in vitro test proved to be linear over a wide dose range and revealed large differences in the oxidative potency of the six CNP
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Table 2 Particle Characteristics of the Six Different Particle Types Studied
Particle
SABET (m2/g)
Organic carbon content (%) (NIOSH 5040a)
Inflammatory efficacy (%PMN/mg)b
Oxidative potency (nmol/mg)c
UfCP SootL SootH Ptx90 DEP PtxG
800 441 268 272 108 43
<4e 7 19 2 20g 1
4.8 2.5 1.7 1.2 0.5 0.4
0.839 0.617 0.057 0.278 0.026 0.024
R2
d
1.00f 1.00f 0.99 1.00 0.27 0.88
UfCPs are generated by spark-discharge from graphite electrodes (GFG1000, Palas, Germany) (20,21). SootH (high organic content, high OC), and SootL (low OC) particles are produced by a well-controlled diffusion flame (CAST burner, Matter Engineering AG, Wohlen, Switzerland) with low and high oxygento-fuel ratio, respectively, resulting in soot particles with different amounts (and types) of organic compounds (50). DEP (diesel exhaust particles) represents standard reference material SRM-1650a, purchased from NIST (Gaithersburg, Maryland, U.S.), and PrintexG and Printex90 (pigment black) from Degussa (Frankfurt, Germany). a The organic carbon fraction was determined with a thermo-optical method according to the NIOSH 5040 standard protocol (51). b Inflammatory efficacy (in vivo) is defined as the 20% PMN effect level divided by the respective particle mass causing this effect level. c Particle oxidative potency is calculated from the slope of the consumed ascorbate versus the three doses (0.2, 1, and 5 mg) (see Fig. 1). d 2 R gives the significance (goodness-of-fit) of the regression. e Two recent studies suggest that the UfCPs contained <4% of organic matter (50,52). f This result is trivial, since only two data points were available for UfCP and SootL due to saturation issues (see Fig. 1). g This value was not obtained according to the NIOSH protocol, it refers to the solvent (Soxhlet) extractable mass fraction as reported by NIST.
(Table 2). As expected from the mechanistic analysis above, the oxidative potency is well correlated to the specific SABET (Fig. 6A) for those particles with a “clean” surface (no significant organic contamination), namely, PrintexG, Printex90, SootL, and UfCP. Hence, for different types of pure carbon particles, their mass-specific oxidative potency varies linearly with the mass-specific SABET, similar to the surface reactivity of catalysts. However, the two particle types with relatively high organic (in particular PAH) content, namely, DEP and SootH (Table 2), show reduced oxidative potency relative to the available SABET (Fig. 6A). On the other hand, the in vivo inflammatory efficacy (Table 2) not only correlates well with the SABET of the low organics particles but there is also no reduced inflammatory efficacy for DEP and SootH as might have been expected from their lower innate oxidative potency (Fig. 6B). Hence, a different toxicological pathway must have compensated for the lack of oxidative potency. Since only DEP and SootH particles (and to a much lesser extent PrintexG) induced overexpression of the PAH-inducible enzyme CYP1A1 (39), the most likely explanation for both the reduced innate oxidative potency and nonreduced inflammatory efficacy of those particles is the presence of bioactive organic compounds. While on the one
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Figure 6 Six different carbonaceous particles were assessed for the oxidative properties in a cell-
free in vitro system (39). Particle oxidative potency was calculated from the consumed ascorbate normalized to the applied particle mass. The good linear correlation between SABET and oxidative potency for the low organics particles (all but SootH and DEP) indicates that noncontaminated carbon surfaces have similar surface toxicity (A). On the other hand, (B) shows excellent linear correlation between (in vivo) inflammatory efficacy and SABET for all particle types, which indicates that the reduced (surface-related) oxidative potency of the high organics particles (SootH and DEP) is compensated by another pathway of toxicity, namely, detoxification of PAHs.
hand, the organic coating apparently shields the reactive carbon structures and mitigates their oxidative potency (measured by an in vitro assay), the induction of CYP1A1 shows that the available PAHs are metabolized in a living organism, which on the other hand leads to additional ROS formation and enhanced inflammatory efficacy. Thus, the excellent correlation between SABET and in vivo inflammation response (Fig. 6B) is in part the result of a direct relationship between surface reactivity and inflammation (pathway i) for pure carbon particles (UfCP, Printex90, PrintexG, SootL) and the competing effects of reduced surface reactivity (pathway i) and additional ROS formation due to detoxification of PAHs (pathway ii) for SootH and DEP.
IV.
Summary
We investigated the dose-response curves (PMN fraction in the BAL 18–24 hours after IT) for five types of particles (carbon black, polystyrene, manganese oxide, titanium oxide, and quartz) with varying particle size (9–1500 nm; Table 1) in terms of particle number, SABET, and mass. We showed that the adverse inflammatory effects of inhaled particles shows by far the best correlation with SABET, which implies that surfacerelated pathways of inflammation are likely the most prevalent pathways. Those are the generation of ROS via the catalytic oxidative activity of the molecules at the surface of the inert particle matrix (including bioavailability of redox cycling catalysts like transition metals or quinones) (pathway i) and the metabolic generation of reactive byproducts during the cellular detoxification of (adsorbed) organic compounds (pathway ii). While most of the investigated particle types fell into a narrow dose-response regime with an onset of toxicity between 20 and 200 cm2/g lung (Fig. 4), the existence of
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particles with higher and lower toxicity attests to the fact that at least for some particle types, other pathways may play a more pronounced role such as the number of crystalline defects, surface charges, passivation/toxification due to specific protein-binding properties, and oxidative burst activity associated with phagocytosis of micron-sized particles. In conclusion, we state that for particle toxicity, considerations not mass (or number) but SABET should be used as dose metric.
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40. Albrecht C, Knaapen AM, Becker A, et al. The crucial role of particle surface reactivity in respirable quartz-induced reactive oxygen/nitrogen species formation and APE/Ref-1 induction in rat lung. Respir Res 2005; 6:129. 41. Janssens TVW, Clausen BS, Larsen B, et al. Insights into the reactivity of supported Au nanoparticles: combining theory and experiments. Top Catal 2007; 44(12):15–26. 42. Oberdo¨rster G, Oberdo¨rster E, Oberdo¨rster J. Concepts of nanoparticle dose metric and response metric. Environ Health Perspect 2007; 115(6):A290. 43. Oberdo¨rster G. Significance of particle parameters in the evaluation of exposure-doseresponse relationships of inhaled particles. Inhal Toxicol 1996; 8(S):73–89. 44. Tran CL, Buchanan D, Cullen RT, et al. Inhalation of poorly soluble particles. II. Influence of particle surface area on inflammation and clearance. Inhal Toxicol 2000; 12(12):1113–1126. 45. Dankovic D, Kuempel E, Wheeler M. An approach to risk assessment for TiO2. Inhal Toxicol 2007; 19(S1):205–212. 46. Cullen RT, Jones AD, Miller BG, et al. Toxicity of volcanic ash from Montserrat. IOM Research Report TM/02/01. Edinburgh: Institute of Occupational Medicine, 2002. 47. Faustman EM, Omenn GS. Risk assessment. In: Klassen CD, ed. Casarett and Doull’s Toxicology the Basic Science of Poisons. 7th ed. New York: Mc Graw Hill Medical, 2007:107–125. 48. Foucaud L, Wilson MR, Brown DM, et al. Measurement of reactive species production by nanoparticles prepared in biologically relevant media. Toxicol Lett 2007; 174(1–3):1–9. 49. Ayres JG, Borm P, Cassee FR, et al. Evaluating the toxicity of airborne particulate matter and nanoparticles by measuring oxidative stress potential—a workshop report and consensus statement. Inhal Toxicol 2008; 20(1):75–99. 50. Matuschek G, Karg E, Schro¨ppel A, et al. Chemical investigation of eight different types of carbonaceous particles using thermoanalytical techniques. Environ Sci Technol 2007; 41(24): 8406–8411. 51. Cassinelli ME, O’Connor PF (eds). NIOSH method 5040. In: NIOSH Manual of Analytical Methods (NMAM), Second Supplement to NMAM. 4th ed. DHHS (NIOSH) Publication No. 94–113, 1998; Cincinnati, OH:National Institute for Occupational Safety and Health. Available at: http://www.cdc.gov/niosh/nmam/pdfs/5040.pdf Accessed on 22 December 2005. 52. Frampton MW, Utell MJ, Zareba W, et al. Effects of exposure to ultrafine carbon particles in healthy subjects and subjects with asthma. Res Rep Health Eff Inst 2004; 126:1–147.
Index
A549 cells, 70 ACS. See American Cancer Society (ACS) Actinolite, 174 Acute beryllium disease, 175. See also Chronic beryllium disease (CBD) Acute respiratory distress syndrome (ARDS), 175, 215–216 Acute silicosis, 172–173 AD. See Aerodynamic diameter (AD) Adenovirus, 151 Adhesive interactions, 235 Aeroallergen-lung interactions, 266 aeroallergen sources fungi, 268–269 mammals, 270 mite and cockroach, 269 plants, 267–268 with cellular components dendritic cells, 278–280 epithelial cells, 272–278 mast cells, 280 monocytes and macrophages, 278 with noncellular components airway lining layer and alveolar hypophase—pulmonary surfactant, 270–272 Aeroallergens harm epithelial cells, 275–277 Aerodynamic diameter (AD), 17 Aerodynamic Particle Sizer (APS), 24 Aerosol bolus inhalation technique COPD patients, 88 Aerosol drug, 60 treatment of asthma, 64 treatment of COPD, 64 Aerosol inhalation, 87
Aerosolization, changes in mucus viscoelasticity and, 196 Aerosol Mass Spectrometer (AMS), 24 Aerosols particles hygroscopic properties, 87 Aerosol therapy, 82 Afferent innervation, 8 AFRESA, 62 AI. See Alveolar interstitial (AI) region Air-blood barrier design of, 2 Airborne particulate matter (PM) comparison of, 30 Airborne PM, respiratory virus infections and diesel exhaust and, 154–155 epidemiologic evidence, 152–153 experimental studies, 153 indoor air pollution and, 156–157 potential mechanisms dendritic cells, 160–161 epithelial cells, 158–160 macrophages, 158 oxidative stress, 157–158 tobacco smoke and, 155–156 Air pollution index (API), 152–153 Airway lining layer and alveolar hypophase—pulmonary surfactant, 270–272 Airways, surfactant conducting, 208–210 role on particle deposition, 210–211 AKITA inhalation device, 87 Allergens, defined, 266. See also Aeroallergen-lung interactions Alveolar epithelium, 4–5 Alveolar interstitial (AI) region, 85 Alveolarization of lungs, infant, 87
305
306 Alveolar lipoproteinosis. See Acute silicosis Alveolar lung surface tension in, 204 surfactant, 205–208 role, on particle deposition on alveolar wall, 210–211 Alveolar macrophages (AM), 81 in mouse lung, 11 AM. See Alveolar macrophages (AM) Ambient particulate matter (PM), structural remodeling in lungs by, 177–181 dust deposition, 178 Ambient tropospheric particles characteristics of, 25–26 general issues, 21–23 overview, 17–21 American Cancer Society (ACS), 110 American Heart Association, 121 Amosite, 174 AMS. See Aerosol Mass Spectrometer (AMS) Anthophyllite, 174 Anti-infectives lipid microspheres, 67 liposomes, 67 for treatment of tuberculosis, 66 Antioxidant depletion, measurements for, 298 Anti-tubercular drugs (ATD), 67 API. See Air pollution index (API) APS. See Aerodynamic Particle Sizer (APS) ARDS. See Acute respiratory distress syndrome (ARDS) Arrhythmias, 131 Asbestos, 174–175, 248 BED for, 256–257 Asbestos-induced small airway disease, 174 Asbestosis, 174–175 Aspergillus fumigatus, 272 Asthma, 151 defined, 216 surfactant and, 216–217 ATD. See Anti-tubercular drugs (ATD) Atenolol, 144 Atherosclerosis, development of, 128–129
Index Atmospheric particulate matter (PM), 17 filter media for chemical characterization, 23 measurement methods, 23–25 physical properties, 23, 24 Autonomic nervous system, PM effects on, 132–133
Bacteria, effects on mucus, 197–198 BADJ. See Bronchiole-alveolar duct junction (BADJ) BAL. See Bronchoalveolar lavage (BAL) BALF inflammatory cells. See Bronchoalveolar lavage fluid (BALF) inflammatory cells BALT. See Bronchoalveolar lymphoid tissue (BALT) BB. See Bronchial (BB) conducting airways Bb. See Bronchiolar (bb) conducting airways BBB. See Blood-brain barrier (BBB) BED. See Biologically effective dose (BED) BeLPT. See Blood lymphocyte proliferation test (BeLPT) Benign asbestos effusion, 174 Benign asbestos-induced pleural disease, 174 Beryllium, CBD and, 175–176 BET surface area, particle, 83 Biologically effective dose (BED), 243 of exemplar pathogenic particles, toxic action and fibers, 256–257 low-toxicity, low-solubility (nuisance) particles, 254–255 nanoparticles, 257–258 PM10 and combustion-derived nanoparticles, 258–259 quartz (crystalline silica), 255–256 and particle responses at cellular level, scheme for, 246–248 Biomass fuels, 156 Black fibrotic lung lesions, 171 BLES. See Bovine lipid extract surfactant (BLES)
Index Blood-brain barrier (BBB), 95 Blood lymphocyte proliferation test (BeLPT), 176 Blood pressure changes airborne particle, 115 Boston CAPs, 137 Bovine lipid extract surfactant (BLES), 206, 209. See also Surfactant British Standards Institution, 38 Bronchial (BB) conducting airways, 85 Bronchiolar (bb) conducting airways, 85 Bronchiole-alveolar duct junction (BADJ), 170 Bronchiolitis, 151 Bronchitis, 151 Bronchoalveolar lavage (BAL), 3, 93 BeLPT, 176 markers, 183 protein concentration in, 271 Bronchoalveolar lavage fluid (BALF), 176, 249, 289 inflammatory cells, 154, 156 Bronchoalveolar lymphoid tissue (BALT), 66 Bronchoscopy, 195 Brownian motion, 43, 47 Buckminster fullerene (C60), 236, 237
Calvatia excipuliformis, 268 Capillary endothelium, 6 Caplan’s syndrome, 245 Capreomycin, 67 treatment of multidrug-resistant tuberculosis, 67 CAPs. See Concentrated ambient particles (CAPs) Capsazepine (CPZ), 145 Captive bubble surfactometry (CBS), 216 CAR. See Coxsackievirus and adenovirus (CAR) Carbohydrate recognition domain (CRD), 270 Carbonaceous nanoparticles (CNP), 297, 298 Carbon black (CB) particles, 153, 177, 226, 250
307 Carcinogenesis, particle-induced responses and, 252–254 Cardiovascular diseases, PM effects and, 120 atherosclerosis, development of, 128–129 epidemiological studies findings of, 121–126 populations at risk, 127–128 mechanisms autonomic nervous system and, 132–133 endothelial function, 131 particles direct effects, on cardiovascular system, 129 pulmonary inflammation, role of, 129–131 vascular function and, 131–132 neural hypothesis, 140–141 heart, neural input on, 142 lung, neural signals from, 143–144 PM-induced sympathovagal imbalances and cardiac ROS, 144 pulmonary reflexes, 145–146 ROS, as central mediators of cardiac dysfunction by PM, 144–145 oxidant stress, after particle inhalation, 139 particle exposures and, 127 mechanistic pathways for, 130 pathogenesis, 128–129 Cathepsin G, 198 Cationic lipids, 68 Caveolae-mediated endocytosis, 234 Caveolin, defined, 234 CBD. See Chronic beryllium disease (CBD) CB particles. See Carbon black (CB) particles CBS. See Captive bubble surfactometry (CBS) CDNP. See Combustion-derived nanoparticles (CDNP) Cellular components of immune system, 10–12 CF. See Cystic fibrosis (CF) Chemical mechanical polishing (CMP), 39, 40–41 Chemical Speciation Network (CSN), 23 Chronic beryllium disease (CBD), 169, 175–176
308 Chronic bronchitis, 172 Chronic obstructive pulmonary disease (COPD), 60, 151–152 Chronic silicosis, 172 Chrysotile, 174 Cigarette smoke, 155 emphysema and, 172 side-stream (SS), 156 Cigarette smoke condensate (CSC), 158–159 CK. See Creatine kinase (CK) Clathrin, 233 Clathrin- and caveolae-independent endocytosis, 234 Clathrin-mediated endocytosis, 234 Clean Air Act, 151 CLIJ. See Confined liquid impinging jets (CLIJ) CMP. See Chemical mechanical polishing (CMP) CNP. See Carbonaceous nanoparticles (CNP) Coal dust macules, 171 nodules, 171 Coal workers’ pneumoconiosis (CWP), 169 coal and, 171–172 Coarse particles, 29–31 Cohort mortality studies particles, health effects of, 110–113 Combustion-derived nanoparticles (CDNP) BED for, 258 Complicated silicosis. See Chronic silicosis Concentrated ambient particles (CAPs), 137–138, 139, 142, 143, 144, 181 Condensation particle counter (CPC), 24 Conducting airways, 208, 210 bronchial (BB), 85 bronchiolar (bb), 85 particle clearance in, 89, 90 surface tension in, 215 thoracic, 86 Confined liquid impinging jets (CLIJ), 65 Controlled loco-regional delivery, lungs, 64–65 asthma, 64 COPD, 64 immunosuppressives, 65 pulmonary arterial hypertension, 65
Index COPD. See Chronic obstructive pulmonary disease (COPD) Copper beryllium alloy, 175 Coronavirus, 151 Cough clearance mucus viscoelasticity and changes in, 196 role of, 195–196 Coxsackievirus and adenovirus (CAR) receptor for, 159 CPC. See Condensation particle counter (CPC) CPZ. See Capsazepine (CPZ) CRD. See Carbohydrate recognition domain (CRD) C-reactive protein (CRP), 124 Creatine kinase (CK), 139 Cristobalite, 172 Criteria Document, 108, 121, 123 Crocidolite, 174, 175 CRP. See C-reactive protein (CRP) Crystalline Silica. See Quartz CSC. See Cigarette smoke condensate (CSC) CSN. See Chemical Speciation Network (CSN) Cuboidal type II cells, 5 CWP. See Coal workers’ pneumoconiosis (CWP) Cyclosporine A, 65 CYP1A1. See Cytochrome P450 1A1 (CYP1A1) Cystic fibrosis (CF), 67 lung disease, 197 Cytochrome P450 1A1 (CYP1A1), 295
Dactylis glomerata, 270 DCFH-DA. See Dichlorofluorescin diacetate (DCFH-DA) DCs. See Dendritic cells (DCs) DE. See Diesel exhaust (DE) Dendritic cells (DCs), 11, 89, 156, 228 aeroallergen-lung interactions with, 278–280 and airway epithelium, interactions between, 160 particles transport to, 228–229 respiratory viral infections and, airborne particles and, 160–161
Index DEP. See DE particles (DEP) DE particles (DEP), 160, 299 Department of Energy (DOE), 175 Deposition, particles. See Total particle deposition Deposition probability, particles in the human airways, 86 Dermatophagoides pteronyssinus, 266 Dextran, 62 Dichlorofluorescin diacetate (DCFH-DA), 249 Diesel exhaust (DE), virus infections and, 154–155 Diffuse pleural fibrosis, 174 Diffusion, 43–44 length scale for nanoparticles, 45 UFP and NP deposition and, 227 Dihydroergotamine mesylate for treatment migraine, 63 Dilauroylphosphatidylcholine (DLPC), 63 Dimethyl-b-cyclodextrin, 63 Dimethylbenzanthracene (DMBA), 246 Dipalmitoylphosphatidylcholine (DPPC), 63 Dipalmitoyl phosphatidylcholine (DPPC)-rich surfactant film, 204, 211, 212, 214 DLPC. See Dilauroylphosphatidylcholine (DLPC) DMBA. See Dimethylbenzanthracene (DMBA) DOE. See Department of Energy (DOE) Donora death fog, 178 Dose-response relationships defined, 288 dose parameter, identification of particle mass, 290–292 particle number, 293 particle size, 292 particle surface area, 294–295 dose-response threshold, 296–298 methodology exposure scenario, 289–290 response endpoints, 288–289 particle toxicity different pathways of, quantification of, 298–300 mechanisms of, and implications for dose parameter, 295–296
309 Dose-response threshold, 296–298 DOTAP, 70 Doxorubicin, 139 DPPC. See Dipalmitoyl phosphatidylcholine (DPPC)
EC. See Elemental carbon (EC) ECG. See Electrocardiogram (ECG) Efferent innervation, 7 Einstein–Stokes equation, 43 Elafin, 159 Elastase, 198 Elasticity, defined, 193 Electrocardiogram (ECG), 125, 144 Elemental carbon (EC), 25, 130, 138 Eli Lilly, 62 Emphysema cigarette smoke and, 172 in coal workers, 172 Endocytosis, 232 caveolae-mediated, 234 clathrin- and caveolae-independent, 234 clathrin-mediated, 234 Endosome-lysosome trafficking, 233 Endothelial dysfunction, cardiovascular effects of PM and, 131 Endothelin-1, 131 Engineered nanoparticles, 176–177. See also Nanoparticles (NP) ENO. See Exhaled NO (eNO) Environmental (Env) setting, particle exposure in, 244, 245 Environmental Protection Agency’s Criteria Document for Particulate Matter, 121 Environmental tobacco smoke (ETS), 155–156 Eosinophil cationic protein, 197 EPA. See United States Environmental Protection Agency (EPA) EPA Aerometric Information Retrieval System, 127 EPA’s Federal Reference Monitoring (FRM) network, 23 Epiglottitis, 151
310 Epithelial cells, 4, 228 aeroallergen-lung interactions with aeroallergens harm epithelial cells, 275–277 cytokines and chemokines, 272–274 protease-activated receptors (PARs), 277–278 transcription factors, 274–275 respiratory viral infections and, airborne particles and, 158–160 ET. See Extrathoracic region (ET) ETS. See Environmental tobacco smoke (ETS) European Commission, 151 Exhaled NO (eNO), 129–130 Exposure-dose-response relationships, 98 Extrathoracic region (ET), 85 Exubera, 61
F-actin, 197 1968 Farmington mine disaster, 171 FC. See Fluorocarbon (FC) FDA. See United States Food and Drug Administration (FDA) FDMS-TEOM. See Filter Dynamics Measurement System-Tapered Element Oscillating Microbalance (FDMS-TEOM) Federal Coal Mine Health and Safety Act, 171 Federal Reference Monitoring (FRM), 23 FEV1. See Forced expiratory volume in one second (FEV1) Fiberoptic bronchoscope, 176 Fibers, BED for, 256–257 Fibroblast cells, 44 Fibrosis, particle-induced responses and, 251–252 Filter Dynamics Measurement System-Tapered Element Oscillating Microbalance (FDMS-TEOM), 24 Fine particles, 28–29 chemical components of, 29 long-range transport of, 29 timescale measurements of, 28
Index Fluid lining of lungs, structure and function of alveolar lung, 205–208 conducting airways, 208–210 Fluorocarbon (FC) solvent, 205 Forced expiratory volume in one second (FEV1), 109 Forced vital capacity (FVC), 89 Force spectroscopy, 211, 213, 214 FRM. See Federal Reference Monitoring (FRM) Fullerenes, 177 Fungi, allergens from, 268–269 FVC. See Forced vital capacity (FVC)
Gas-phase methods nanoparticles, 42–43 Gas-phase processing for nanoparticles, 48 Gastrointestinal (GI) tract, 88 Genotoxicity, particle-induced responses and, 252–254 Geometric standard deviation (GSD), 17 GI. See Gastrointestinal (GI) tract Glu69 marker, 176 Glutathione (GSH), 158 Glycopyrrolate, 144 GM-CSF. See Granulocyte-macrophage colony-stimulating factor (GM-CSF) Granulocyte-macrophage colony-stimulating factor (GM-CSF), 160 Granulocytes, 12, 278, 289 Granulomas, 175 GSD. See Geometric standard deviation (GSD) GSH. See Glutathione (GSH)
Harvard Ambient Particle Concentrator, 138 Harvard Six Cities Study, 121, 178 Hazard potential classification, nanoparticles activity for, 50–51 degradable materials, 53
Index [Hazard potential classification, nanoparticles] degradation rate, 50 hazard grid, 51 persistent, 51–52 semi-persistent, 52 Hazard ratio (HR), 113 HDM. See House dust mite (HDM) Heart, neural input on, 142 Heart CL, 139 CAPs effects on, 142, 145 and HRV oxidant stress, 144. See also Oxidative stress PM exposure and, 144 PM effects on, 143 Heart rate variability (HRV), 113, 124, 140. See also Cardiovascular diseases, PM effects and PM exposure and, 132 heart CL and, 144 Hemoxygenase 1 (HO-1), 158 Heparin, 12, 61 Herpes simplex virus, 200 Heterogeneous catalysis, 39–40 HFE gene, 115 Histamine, 12 HLA. See Human leukocyte antigens (HLA) HLA-DP allele, 176 HO-1. See Hemoxygenase 1 (HO-1) House dust mite (HDM) cysteine protease allergens, 266 HR. See Hazard ratio (HR) HRV. See Heart rate variability (HRV) Human leukocyte antigens (HLA), 176 Human papilloma virus, 200 Hydrogen peroxide (H2O2), 214 8-Hydroxy-20 -deoxyguanosine, 249
IARC. See International Agency for Research on Cancer (IARC) ICRP. See International Commission on Radiological Protection (ICRP) model
311 ICRP Human Respiratory Tract Model for inhaled radiolabeled aerosols, 91 ICRP-66 particle clearance model of particle clearance, 89 schematic view of, 90 IFN. See Interferon (IFN) IFNg, 156 IL-12. See Interleukin (IL)-12 ILO. See International Labour Organization (ILO) Immune system, 9–12 cellular components, 10–12 flow chart of, 9 noncellular components, 9–10 and particles, 12 role of, defined, 249 Immunosuppressives for lung transplant, 65 IMPROVE. See Interagency Monitoring of Protected Visual Environments (IMPROVE) Inflammation process particle-induced responses and, 249–251 Influenza, 151 virus, 159 Inhalation therapy for drug delivery system, 64 Inhaled particles deposited mass for dose assessment, 80 pulmonary structures of capillary endothelium, 6 immune system, 9–12 nervous system, 6–9 Insulin, 61 Interagency Monitoring of Protected Visual Environments (IMPROVE), 23 Interferon (IFN), 154 Interleukin (IL)-12, 156 Intermediary biomarkers particles, health effects of, 114–115 International Agency for Research on Cancer (IARC), 173 International Commission on Radiological Protection (ICRP) model, 85 International Labour Organization (ILO), 171
312 Interstitial fibrosis, diffuse, 172 Intratracheal instillation (IT), 183 of PM, 289–290 Intravenous cancer therapy, 67 IT. See Intratracheal instillation (IT)
Kaolin, 214 Kelvin probe force microscopy (KPFM), 207 KPFM. See Kelvin probe force microscopy (KPFM)
Lactate dehydrogenase (LDH), 139 Langerhans cells, 11 Langmuir–Wilhelmy surface balance, 211 L-arginine, 131 Laryngotracheitis (croup), 151 LDH. See Lactate dehydrogenase (LDH) Leukocytes, effects on mucus, 197–198 Liposomes, 63 Liquid-phase methods nanoparticles, 42 LMWH. See Low molecular weight heparin (LMWH) LOAEL. See Lowest adverse effect level (LOAEL) London fog, 178 Lower respiratory infections (LRIs), 151–152, 156 Lowest adverse effect level (LOAEL), 296–298 Low molecular weight heparin (LMWH), 62 Low-toxicity, low-solubility (LTLS) particles BED for, 254–255 Low toxicity particles, 288 LRIs. See Lower respiratory infections (LRIs) LTLS particles. See Low-toxicity, low-solubility (LTLS) particles Lung cancer therapy, 69–70 Lung-heart link, 145–146
Index Lungs cancer, 174 controlled loco-regional delivery, 64–65 fluid lining of, structure and function of alveolar lung, 205–208 conducting airways, 208–210 mucus cell metaplasia, 154 neural signals from, 143–144 organization of, 1–2 pain relief in, 63 squamous cell carcinoma, 69 systemic drug delivery in, 61–63 Lungs, structural remodeling of, 167 fiber length and, 168 hypothetical mechanisms for, 184 macules, 169 particle-induced disease risk evaluation, animal studies in, 181–184 by particles in ambient air, 177–181 by particles in occupational settings asbestos and asbestosis, 174–175 beryllium and chronic beryllium disease, 175–176 coal and coal workers’ pneumoconiosis, 171–172 engineered nanoparticles, 176–177 silica and silicosis, 172–174 site-specific particle effects in, 168–171 Lymphocytes, 12
Macrophage inflammatory protein 2 (MIP-2), 251 Macrophages, 228, 249–250 aeroallergen-lung interactions with, 278 airborne particles and, respiratory viral infections and, 158 Macropinocytosis, 233–234 Macropinosomes, defined, 233 Macules, 169 Magnetic nanoparticles, 41 Magnetic resonance imaging (MRI), 41 Mammals, allergens from, 270 Manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), 139 MannKind Corporation, 62
Index Mass-based dose-response relationship, particle, 290–292 Mast cells, 12 aeroallergen-lung interactions with, 280 Mechanical fillers, effects of particles as, 197 Membranes, nanoparticles (NP) interactions with cell membrane, 230–231 tissue membrane, 227–230 Mesothelioma, 174 Metabolic syndrome (MS), 127 Metal metabolism, 114–115 MGP. See Mucus glycoprotein (MGP) Micro-Orifice Uniform Deposit Impactors (MOUDI), 24 Migraine therapy, 63 MinED. See Minimal effective dose (MinED) Minimal effective dose (MinED), 296–298 MIP-2. See Macrophage inflammatory protein 2 (MIP-2) MIP-3a, 160 Mir Space station, 268 Mite and cockroach, allergens from, 269 MnO2 particles, 292, 293, 294 MnTBAP. See Manganese (III) tetrakis (4-benzoic acid)porphyrin (MnTBAP) Monocytes, aeroallergen-lung interactions with, 278 Monodisperse particles, 85 Monomac6, 249 MOUDI. See Micro-Orifice Uniform Deposit Impactors (MOUDI) MPEG-DSPE. See Poly-(ethylene oxide)block-distearoyl phosphatidyl-ethanolamine polymer (mPEG-DSPE) MPPD. See Multiple Path Particle Deposition (MPPD) MRI. See Magnetic resonance imaging (MRI) MS. See Metabolic syndrome (MS) Mucociliary escalator, 10
313 Mucociliary system clearance, 10 mucus viscoelasticity, role of, 195–196 nanoparticles and, 200 function particles, secretion and modulation stimulation by, 196 Mucus, 196 effects of particles as mechanical filler, 197 osmotic load, 199–200 gel, types of bonding in, 198 rheology and clearance, living particles and, 197–198 shear thinning in, 193–194 viscoelasticity of, 193–195 changes in, cough clearance and aerosolization and, 196 mucociliary and cough clearance, role in, 195–196 Mucus glycoprotein (MGP), 194 polyelectrolyte nature of, 199 Multimedia box models, 48 Multiple Path Particle Deposition (MPPD), 86 Multiwalled carbon nanotubes (MWCNTs), 177 MWCNTs. See Multiwalled carbon nanotubes (MWCNTs) Mycobacterium tuberculosis, 66, 167
NAAQS. See National Ambient Air Quality Standards (NAAQS) N-acetyl cysteine, 131, 139, 144 Nanobiomaterials, 41 Nanocomposites, 41 Nanoparticles (NP), 80, 226, 290 accumulation in secondary target organs, 97–98 advantages of, 38–42 applications of, 38–42 BED for, 257–258 cell membrane interactions cellular membranes, 230–231 tissue membranes, 227–230
314 [Nanoparticles (NP)] combustion-derived (CDNP), BED for, 258 commodity applications, 41–42 commodity products containing, 49 defined, 38 deposition, diffusion and, 227 dispersion stability of, 47–48 end-of-life scenarios of, 49 engineered, 176–177 hazard potential for, 49–50 classification, 50–53 industrial manufacture, 42–43 localization, in cells, 235–237 mucociliary clearance and, 200 overview, 38 penetration, mechanisms of, 231–232, 235 caveolae-mediated endocytosis, 234 clathrin- and caveolae-independent endocytosis, 234 clathrin-mediated endocytosis, 234 macropinocytosis, 233–234 phagocytosis, 233 physical behavior of, 43–48 polarization of, 57 products with bound, 49 relocation in epithelium, 92–94 risk of exposure, 48–50 translocation in secondary target organs, 95–97 Nano-sized particles (NSP), 1, 79. See also Nanoparticles Nanotechnology industrial development of, 53 NARSTO PM assessment, 23 National Ambient Air Quality Standards (NAAQS), 18, 151, 178 National Institute for Occupational Safety and Health (NIOSH), 171 National Morbidity, Mortality, and Air Pollution Study (NMMAPS), 122 NEB. See Neuroepithelial bodies (NEB) Nebulized fentanyl for pain relief, 63 Nervous system, 6–9 afferent innervation, 8 efferent innervation, 7 and particles, 8–9
Index Neuroepithelial bodies (NEB), 6 Neutrophil DNA, 197–198 NF-kB. See Nuclear factor kappa B (NF-kB) NHANES-III, 127 NIOSH. See National Institute for Occupational Safety and Health (NIOSH) NMMAPS. See National Morbidity, Mortality, and Air Pollution Study (NMMAPS) NOAEL. See No observed adverse effect level (NOAEL) Noncellular components of pulmonary defense system, 9–10 Nonspherical particles, 81 Nonviral vectors, 68 No observed adverse effect level (NOAEL), 296–298 Norwalk virus, 200 NOS. See NO synthases (NOS) NO synthases (NOS), 131 Novo Nordisk, 62 NP. See Nano-sized particles (NP) NSP. See Nano-sized particles (NSP) N-trimethyl chitosan, 62 Nuclear factor kappa B (NF-kB), 249 Nuisance dust, 176, 254 Number-based dose-response relationship, particle, 293
Obstructive sleep apnea syndrome, 127 Occupational exposure limit (OEL), 175 Occupational (Occ) setting, particle exposure in, 243–245 OEL. See Occupational exposure limit (OEL) Oligonucleotide 20-O-methyl-RNA (2-OMR), 69 2-OMR. See Oligonucleotide 20-O-methylRNA (2-OMR) Organ chemiluminescence (CL), 138 as predictor of oxidative damage, 139–140 Organ CL. See Organ chemiluminescence (CL)
Index Osmotic balance mucus and, particles effects on, 199–200 O-stearylamylopectin, 67 Ovalbumin-induced allergic airway inflammation, 154 Oxidative potency, 299 Oxidative stress, 114 airborne particles and, respiratory viral infections and, 157–158 cardiac, after particle inhalation, 139 heart CL, as marker of, 144 in vivo measurements of, 138–139 particle-induced responses and, 248–249
PAH. See Polycyclic aromatic hydrocarbon (PAH) Pain relief in lungs, 63 Parainfluenza, 151 PARs. See Protease-activated receptors (PARs) Particle(s) clearance, 88–94 role of surfactant, 214 composition, 82–83 density, 81–82 deposition on airway and alveolar wall, surfactant and, 210–211 in respiratory tract, 84–86 health effects of cohort mortality studies, 110–113 intermediary biomarkers, 114–115 overview, 108 pulmonary effects, 108–110 traffic exposure, 113–114 hygroscopicity, 82–83 internalization, 233 modeling in, respiratory tract, 84–86 shape, 81 size, 80–81 surface parameters, 83 surface structure and, 5–6 surface structures lining, 3–6 alveolar epithelium, 4–5 surfactant, 3–4
315 [Particle(s)] transformation during inhalation, 83–84 transport in air, 82 van der Waals interactions in, 45 velocity of aggregation, 46 Particle dosimetry particle composition, 82–83 particle density, 81–82 particle hygroscopicity, 82–83 particle shape, 81 particle size, 80–81 particle surface parameters, 83 Particle-induced acute lung injury, surfactant and, 215–216 Particle-induced disease evaluating risk of, animal studies in, 181–184 lung remodeling. See Lungs, structural remodeling of Particle-induced responses, processes and mechanisms in fibrosis, 251–252 genotoxicity and carcinogenesis, 252–254 inflammation, 249–251 oxidative stress, 248–249 Particles clearance, 88–94 from airways, 88–90 from lung periphery, 91 nanoparticle relocation in epithelium, 92–94 in patients with lung diseases, 91–92 Particles retention. See Particles clearance Particle toxicity adverse and pathogenic effects environmental exposure, 245 occupational exposure, 243–245 and biologically effective dose (BED), 243 of exemplar pathogenic particles, toxic action and, 254–259 and particle responses at cellular level, scheme for, 246–248 different pathways of, quantification of, 298–300 mechanisms of, implications for dose parameter, 295–296 particle-induced responses, processes and mechanisms in
316 [Particle toxicity particle-induced responses, processes and mechanisms in] fibrosis, 251–252 genotoxicity and carcinogenesis, 252–254 inflammation, 249–251 oxidative stress, 248–249 pathobiological processes, 245 surfactant, role in, 214–215 Particle transformation biomechanical reactions, 92 in epithelial lung lining fluid, 92 Particle uptake, 233 Particulate air pollution, 122 Particulate matter (PM), 17, 79, 121, 122, 151, 245 Peroxynitrite, 131 Pfizer, 62 Phagocytic cells, 89 Phagocytosis, 233 Phagosomes, defined, 232 Phleum pratense, 270 Phospholipase A2, 214 Phthisis, defined, 167 Pinocytosis, 232 macropinocytosis, 233–234 Plants, allergens from, 267–268 Plasma cells, 12 Plasma membrane, defined, 231 Pleural plaques, 174 PM. See Particulate matter (PM) PM10, BED for, 258 PMNs. See Polymorphonuclear cells (PMNs) Pneumoconioses, 243 Pollen starch granules (PSG), 267 Polycyclic aromatic hydrocarbon (PAH), 295 Poly-(ethylene oxide)-block-distearoyl phosphatidyl-ethanolamine polymer (mPEG-DSPE), 64 Polymers mechanical properties of, 41 Polymorphonuclear cells (PMNs), 289, 290, 296 Polystyrene particles, 235 Printex90, 299 PrintexG, 299 Professional phagocytes, defined, 233
Index Progenitor cells, 5 Progressive massive fibrosis. See Chronic silicosis Protease-activated receptors (PARs), 277–278 PSG. See Pollen starch granules (PSG) Pulmonary arterial hypertension, 65 Pulmonary drug delivery controlled loco-regional delivery, lungs, 64–65 future prospects, 70, 73 overview, 60–61 of different particle types for, 71–72 in respiratory tract, 65–70 systemic drug delivery, lungs, 61–63 Pulmonary effects particles, health effects of, 108–110 Pulmonary gene therapy, 68–69 for nebulization of naked plasmid DNA, 68 for treatment of CF, 68 Pulmonary inflammation, cardiovascular effects of, 129–131 Pulmonary macrophages, 10 Pulmonary surfactant, 231
Quartz, 172, 214, 253, 296 BED for, 255–256
Radiopneumography, 89 Rapidly adapting stretch receptors (RAR), 6 RAR. See Rapidly adapting stretch receptors (RAR) Reactive nitrogen species (RNS), 173 Reactive oxygen species (ROS), 139, 172, 173, 248–249, 295–296, 298 cardiac, PM-induced sympathovagal imbalances and, 144 as central mediators of cardiac dysfunction by PM, 144–145 Receptor locations estimating source contributions, 31 Redox-sensitive transcription factors, 295 Regional particle deposition. See Total particle deposition
Index Respiratory syncytial virus (RSV), 151, 153, 154 Respiratory tract insoluble particles in, transport pathways of, 169 Respiratory tract, targeted delivery of drugs in, 65–70 anti-infectives, 66–68 lung cancer therapy, 69–70 pulmonary gene therapy, 68–69 vaccines, 66 Respiratory virus infections, airborne PM and, 151 diesel exhaust and, 154–155 epidemiologic evidence, 152–153 experimental studies, 153 indoor air pollution and, 156–157 potential mechanisms dendritic cells, 160–161 epithelial cells, 158–160 macrophages, 158 oxidative stress, 157–158 tobacco smoke and, 155–156 Rheumatoid nodules, 172 Rhinovirus, 151 RNS. See Reactive nitrogen species (RNS) ROS. See Reactive oxygen species (ROS) Rounded atelectasis, 174 RSV. See Respiratory syncytial virus (RSV)
SA. See Surface area (SA) Salem sarcoid, 175 SAPALDIA. See Swiss Cohort Study on Air Pollution and Lung Diseases in Adults (SAPALDIA) SAR. See Slowly adapting stretch receptors (SAR) SARS. See Severe acute respiratory syndrome (SARS) Scanning mobility particle sizer (SMPS), 24 Secondary target organs nanoparticle accumulation in, 97–98 nanoparticle translocation in, 95–97 Secondhand tobacco smoke (SHS), 151
317 Secretory leukocyte protease inhibitor (SLPI), 159 Sedimentation processes, 44 Semivolatile organic species (SVOC), 23 Severe acute respiratory syndrome (SARS), 152–153 Shear thinning, in mucus, 193–194 SHS. See Secondhand tobacco smoke (SHS) Side-stream (SS) cigarette smoke, 156 Silica-induced carcinogenesis, 173 Silicon dioxide (SiO2), 172 Silicosis, 172–174 acute, 172–173 chronic, 172 Single-walled carbon nanotubes (SWCNTs), 177 Site-specific particles, lung remodeling and, 168–171 Six City Study, 111 Size, of particle dose-response relationships and, 292 SLM. See Solid lipid microparticles (SLM) Slowly adapting stretch receptors (SAR), 6 SLPI. See Secretory leukocyte protease inhibitor (SLPI) SMPS. See Scanning mobility particle sizer (SMPS) Solid lipid microparticles (SLM), 64 SootH particles, 299 SootL, 299 SP. See Surfactant proteins (SP) SP-A, 160, 205, 215, 216, 231, 270–271 SP-B, 205, 207, 231 SP-C, 205, 207, 208, 231 SP-D, 160, 205, 231, 270–271 Spinnability, of mucus, 195 SPP. See Subpollen particles (SPP) Squamous cell carcinoma, lungs, 69 Squamous type I cells, 5 SS cigarette smoke. See Side-stream (SS) cigarette smoke Subpollen particles (SPP), 267–268 Superoxide, 131, 158, 173, 248, 249, 251 Supersites Program to measure UF PM mass, 24 Surface area (SA), dose-response relationships and, 294–295
318 Surface potential, defined, 208 Surface tension in alveolar lung, 204 Surfactant proteins (SP), 160, 231 Surfactants, 3–4 alveolar, 205 in disease states, implications for asthma, 216–217 particle-induced acute lung injury, 215–216 fluid lining of lungs, structure and function of alveolar lung, 205–208 conducting airways, 208–210 metabolism of, 4 morphological complexity of, 3 particle clearance, role in, 214 particle deposition, role on on airway and alveolar wall, role in, 210–211 into fluid layer, 211–214 particle toxicity, role in, 214–215 pulmonary, 231 SVOC. See Semivolatile organic species (SVOC) SWCNTs. See Single-walled carbon nanotubes (SWCNTs) Swiss Cohort Study on Air Pollution and Lung Diseases in Adults (SAPALDIA), 109 Systemic drug delivery, lungs, 61–63 large molecules, 61–63 small molecules, 63
TAT-PEG-PEI conjugates, 68 T cells, 66 Techigas, technetium-99m-labeled carbon particles, 200 Technosphere, 62 Teflon filters, 23 Telomerase, cancer therapy in human, 69 Tetradecyl-b-maltoside, 62, 63 Thoracic particles, defined, 21 Thrombosis, 115 Th1-type immune response, 160, 161
Index Thymic stromal lymphopoietin (TSLP), 160 Titanium dioxide (TiO2) particles, 176–177, 226, 235 TNFa. See Tumor necrosis factor alpha (TNFa) Tobacco smoke, virus infections and, 155–156 Tobramycin, 67–68 Total particle deposition, 83–88 hygroscopic properties of, 87 mechanisms of particle deposition in, 84–86 mechanisms of particle modeling in, 84–86 patients with lung diseases, 87–88 of pharmaceutical, 87 for salt particles, 88 for Sebacate oil, 88 in susceptible individuals, 87–88 transformation during inhalation, 83–84 Traffic exposure particles, health effects of, 113–114 Transient receptor potential (TRP) vanilloid receptor family, 145 Tremolite, 174 Tridymite, 172 TRP vanilloid receptor family. See Transient receptor potential (TRP) vanilloid receptor family TRPV1 receptors, 145 Trypsin-like serine proteases (tryptases), 159 TSLP. See Thymic stromal lymphopoietin (TSLP) Tubular myelin, 3 Tumor necrosis factor alpha (TNF), 251 Type II alveolar epithelial cells, 3
UfCP, 299 UFP. See Ultrafine particles (UFP) Ultrafine particles (UFP), 26–27, 124, 146, 226. See also Nanoparticles (NP) carbon black (CB), 153 deposition, diffusion and, 227 diurnal behavior of, 27 exposure, cardiorespiratory mortality and, 125 measurements of, 26
Index United States Environmental Protection Agency (U.S. EPA), 21, 151 United States Food and Drug Administration (FDA), 60 Upper respiratory infections (URIs), 151, 156 Urban air particles (UAP), 143–144 URIs. See Upper respiratory infections (URIs) U.S. EPA. See United States Environmental Protection Agency (U.S. EPA)
Vaccines for infectious disease, 66 pulmonary delivery of, 66 van der Waals interactions, 235 Vanilloid (capsaicin) receptors, 145 Vascular function, PM effects on, 131–132 Virus infections and airborne PM diesel exhaust and, 154–155 epidemiologic evidence, 152–153
319 [Virus infections and airborne PM] experimental studies, 153 indoor air pollution and, 156–157 tobacco smoke and, 155–156 Viscoelasticity, of mucus, 193–195 changes in aerosolization and, 196 cough clearance and, 196 mucociliary and cough clearance, role in, 195–196 Viscosity, defined, 193
Wheat germ agglutinin, 67 WHO. See World Health Organization (WHO) Women’s Health Initiative Observational Study, 128 Work-Related Lung Disease Surveillance Report, 171 World Health Organization (WHO), 151
Pulmonary Medicine about the book… Written by an experienced team of leading international scientists, this timely Second Edition presents pulmonologists, particle research scientists, and toxicologists with a thorough investigation of the latest research and therapies for managing the adverse physiological effects of air-borne particles on the respiratory tract, lungs, and other organs. Key benefits include: s up-to-date information—nine new chapters cover recent developments and research, with several chapters focusing solely on the fast-developing areas of man-made and naturally occurring nanoparticles, to keep readers aware of advances in the field s multidisciplinary coverage—of the lung as a gateway for particle damage to organs outside the respiratory system, gives clinicians and scientists the information they need to understand and combat the numerous and varied ailments caused by inhaled particles s expert contributorship—a distinguished team of over 40 key opinion leaders in the field provide both established information and new observations on particle-lung interactions, giving readers a comprehensive overview of this important health concern s promising treatment advances—often neglected in other texts, such as the beneficial developments and uses of inhaled man-made particles and reduced side-effects of alternate therapies, provide clinicians with an armamentarium for managing diseases caused by airborne particles about the editors... PETER GEHR, Ph.D., is Professor and Chairman, the Institute of Anatomy, University of Bern, Bern, Switzerland. He received his Ph.D. in Biology from the Institute of Anatomy and the Institute of Zoology, University of Bern, Bern, Switzerland. His area of research is the particle-lung interaction, paying particular attention to particle-cell interaction, nanoparticles, and nanotoxicology. Dr. Gehr is a member of the International Society for Aerosols in Medicine, the American Thoracic Society, the European Respiratory Society, and the Swiss Society for Anatomy, Histology and Embryology, among others. CHRISTIAN MÜHLFELD, M.D. is Assistant Professor, the Institute of Anatomy and Cell Biology, University of Giessen, Giessen, Germany. He received his M.D. from the University of Göttingen, Göttingen, Germany. His area of research is the structure-function relationship of the cardiopulmonary system. Dr. Mühlfeld is a member of the American Thoracic Society, the German Anatomical Society, the German Transplantation Society, and the German Society of Molecular Biology and Biochemistry, among others. BARBARA ROTHEN-RUTISHAUSER, Ph.D., is Associate Professor, the Institute of Anatomy, University of Bern, Bern, Switzerland. She received her Ph.D. from the Department of Cell Biology, the Swiss Federal Institute of Technology Zürich, Zürich, Switzerland. Her area of research is particle-cell interactions studied with cell models by microscopic means. Dr. Rothen-Rutishauser is a member of the International Society for Aerosols in Medicine, the European Respiratory Society, the Swiss Tissue Culture Society, and the Swiss Society for Anatomy, Histology and Embryology, among others. FABIAN BLANK, Ph.D., is Research Associate, the Department of Clinical Research, University of Bern, Bern, Switzerland. He received his Ph.D. from the Institute of Anatomy, University of Bern, Bern, Switzerland. His area of research is the interaction of biocompatible nanoparticles with the cellular immune system of the lung. Dr. Blank is a member of Swiss Society for Optics and Microscopy, among others. Printed in the United States of America
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