Case Studies in NANOTOXICOLOGY AND PARTICLE TOXICOLOGY Edited by ANTONIETTA M. GATTI, PhD Associate Professor, National Research Council, Rome, Italy; Visiting Professor, Institute for Advanced Sciences Convergence, US Department of State, Washington DC, USA; Founder, Nanodiagnostics Srl, Modena, Italy STEFANO MONTANARI, PhD Director, Nanodiagnostics Srl, Modena, Italy Amsterdam • Boston • Heidelberg • London New York • Oxford • Paris • San Diego San Francisco • Singapore • Sydney • Tokyo Academic Press is an imprint of Elsevier Academic Press is an imprint of Elsevier 125, London Wall, EC2Y 5AS, UK 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA 225 Wyman Street, Waltham, MA 02451, USA The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK Copyright © 2015 Elsevier Inc. All rights reserved. Cover Image © 2014: Red cells in a vessel. Photograph by Antonietta M. Gatti and Laura Valentini; elaborated by Daniele Contini. 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British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-801215-4 For information on all Academic Press publications visit our website at http://store.elsevier.com/ Publisher: Christine Minihane Acquisition Editor: Kristine Jones Editorial Project Manager: Molly McLaughlin Production Project Manager: Lucía Pérez Designer: Miles Hitchen Typeset by Thomson Digital DEDICATION Everything has a cause and diseases are no exception. This book does not pretend to be the depository of truth, but is dedicated with due humility to those who want to be put on the trail of truth, even if it implies stepping beyond the frontiers of the present, received knowledge, into no man’s land. This book is also dedicated to humanity and its survival. Acts of faith may not regard science, i.e, knowledge, and believing may not be a syn- onym of knowing. What can be found in these pages comes from direct observation of natural phenomena and should be worthy of serious consideration. Remaining prisoners of convictions of convenience, often not supported by any kind of demonstration besides a sort of more or less vague ipse dixit, threatens the very survival of Homo sapiens, and pretending a priori that this is not true is perhaps the most serious among the dangers we are facing. This is not a millenarian prophecy, but a mathematical, logic extrapolation from what is already happening in the world. FOREWORD Drs. Gatti and Montanari are members of that rare species, scientists whose work is so novel and far-reaching that society is reluctant to believe it in the first instance. However, with time, the ideas and notions put forward make so much sense, they move into the realm of accepted wisdom. The work of these particular authors relates to the origins of hitherto inexplicable but widespread diseases of the modern industrialised world. Evolution has fitted humans to be “at one” with their environment. Disease often occurs when that environment is affected in unnatural ways; for example, the flu pandemic of 1919 was traced back to an abnormal proximity of people and animals, as are present outbreaks of bird flu, and many previous and subsequent plagues can be related to overcrowding and poor sanitation in towns and cities, unnatural situations which human- kind was not evolved to cope with. There is now a crop of new diseases that are besetting society. These diseases and their causes are the focus of this book. Many of these diseases – and their causes – are extremely controversial. Society will often deny what it does not want to admit. But Gatti and Montanari have applied their ex- pertise, experience and professional rigor to uncovering the “key” to this multitude of modern afflictions. And like every new theory, the more it appears to provide the solution to each new problem, the more acceptable it becomes. And the sooner we accept what the authors are telling us, the sooner we can restrict or ban altogether the root causes. Though what they say makes for very uncomfortable reading. So, what are these modern diseases? And what are their causes? The dis- eases, outlined in graphically riveting detail, range from clinical cancers such as mesothelioma and leukemia, to Gulf War syndrome and similar diseases, to diseases associated with proximity to certain industrial plants to work- place diseases; for example, those manifest by some hairdressers, welders, ceramic-tile workers and so on. The authors have made a lifetime’s study of many diseases whose causes have baffled other scientists, and their conclusions form the basis for this book. This publication is not a dry academic treatise; it is an earth-shattering story, written in the finest traditions of detective work, based on uncovering the facts, and then identifying the culprit. For xi xii Foreword scientists and laypeople alike, this book is a fascinating read, not only because it shows science at its best, but also because it communicates its message clearly, and having health as its theme, it touches the fears and hopes of each and every one of us. The critical key to understanding the root cause of these “modern” dis- eases, according to Gatti and Montanari, revolves around the unrecognized exposure to man-made nanoparticles and their subsequent introduction into the body, resulting in damage to surrounding cells, tissues and organs. These novel particles are generally metallic, usually the result of high-temperature combustion, and are often completely new compounds. It is likely this book will engender a great rage that innocent and unknowing individuals in the past have fallen mortally ill from causes that can be traced to industrial and military activity. My wish is that now there will be no excuse for any individual in the future to suffer a similar fate. And no hiding place for governments that do not implement the necessary legislation to stop these preventable diseases in their tracks. Ottilia Saxl Founder, Institute of Nanotechnology and Nano Magazine PREFACE More than a book, this work is a testimony to the uncertain future awaiting humanity if we do not learn how to wisely manage the nanoparticles and the nanoworld of which they are the fundamental constituents. This book, written through the magnifying lens of an electron micro- scope, is meant to present the effects of micro- and, more in particular, nanosized products and by-products on human and animal life and on the environment already affected by them. These incidental particles are gener- ated as unwanted consequences of many human activities involving com- bustion processes, and by those particles we consciously produce through nanotechnologies and through the incineration of nanomaterials at the end of their life cycle. We report our personal investigations and their results, illustrating the fate of those particles in the environment, and more specifically, inside the animal, human body, and their offspring. Through these investigations, we try to offer information that, if duly used, can help to understand the source of some diseases of unknown origin, to have a better view on others that might not have been fully understood, and to protect us and the planet we live in against the dangers inherent in nanoparticles. Quite often, the investigations we present are not systematic and can rightly be considered episodic, but they introduce the new concept of personalized medicine. The work done was supported not only by a few institutions, but also by personal funds. Since our lives are not eternal, we would like to share this knowledge with open-minded scientists, in the hope that they continue our work, understand what we did not, correct our mistakes and achieve posi- tive results. Politicians, industrialists, decision-makers and the general public may also benefit from reading this book. The dream goal of these pages is to save human lives. We are conscious of the fact that not everyone will agree with us, but we intend to continue this work and encourage others to do the same until this dream comes true. xiii ACKNOWLEDGMENTS We are indebted to many collaborators, friends and institutions. We are most grateful to Dr. Federico Capitani and Ms. Lavinia Nitu, patient collaborators of our daily work, for the technical contribution they gave us in writing this book, but also for their loyalty and competence. Our research was supported by the European Commission with two grants (Nanopathology, DIPNA) that gave us the possibility to discover the impact of nanoparticles in living bodies. It was also supported by the Italian Institute of Technology, which funded the nanotoxicology project INESE, and by the Italian Ministry of Defense, which funded our projects BATNAN and VENAM. We want to also thank other occasional collaborators: Professor Pietro Gobbi, Dr. Laura Valentini, Dr. Paola Boi and Dr. Anita Manti of the University of Urbino. We are also grateful to Dr. Claudio Pizzagalli of ARPAM (Pesaro, Italy) for his availability and understanding with regard to our research needs. We thank Professor Nobuo Kazashi of Harvard University for intro- ducing us to the Director of the Hiroshima Peace Memorial Museum, Ms. Anne-Marie Principe, survivor of the collapse of the Twin Towers in New York on September 11, 2001. xv CHAPTER 1 Introduction Contents 1.1 The history 1 1.2 What is Nanopathology? 2 References 5 1.1 THE HISTORY Back in early 2008 we published the book Nanopathology: The Health Impact of Nanoparticles [1]. In those pages we told the circumstances that led us to start our research about how solid, inorganic, and non-biodegradable micro- and nanosized particles, whatever their origin, can interfere with living be- ings, and described the basic principles we abide by. But, on a larger scale, that particular dust also has an impact on the environment of which all living beings are inevitably guests and protago- nists at the same time, by which they are as inevitably influenced and, in some cases, affected. And that topic was also dealt with in the book men- tioned above. Nanopathology is a word that we invented in 2002, which became the title of a European project that Dr. Gatti directed and coordinated (Nanopathology: The role of micro and nanoparticles in inducing human health effect (FP5-QOL-147-2002-05)). At that time it was not much more than an empty box. Today, after little more than a decade, nanopathology is a new philosophy, a novel approach to medicine. As happens with many works in progress, particularly when exploring unknown or little-known territories is the aim, much has been done in the last few years and that work is summed up in this new title. In the lapse of time between the two books, Antonietta M. Gatti had a chance to coordinate a further European project and to be part of a few more research projects sponsored by different agencies; the two of us came across more clinical cases (now amounting to more than 2,000), checked more food, drugs and cos- metics, analyzed more samples collected in polluted environments and, alto- gether, we were lucky enough to learn a great deal. A selection of our cases, some of which will be considered “sentinel” cases, is illustrated in Chapter 4. The technique we used was described in detail in our former book. Case Studies in Nanotoxicology and Particle Toxicology Copyright © 2015 Elsevier Inc. http://dx.doi.org/10.1016/B978-0-12-801215-4.00001-7 All rights reserved. 1 2 Case Studies in Nanotoxicology and Particle Toxicology Though it is not always possible, here we will try as much as we can to avoid repeating what has already been written and keep referring our readers to Nanopathology: The Health Impact of Nanoparticles for what they will not find in these chapters. The book describes also the methods and protocols used to detect nanoparticles in biological matrices performed by means of an Environmental Scanning Electron Microscope (ESEM) and of Field Emission Gun Environmental Scanning Electron Microscope (FEG-ESEM); the equipment is also described. 1.2 WHAT IS NANOPATHOLOGY? It is understood that the particles discussed here are both engineered, i.e., fabricated in a laboratory, and non-engineered, i.e., incidental. Among the incidental, are those produced by natural phenomena, those coming from high-temperature industrial processes and those generated as by-products of a fair number of activities carried out at low temperature. The others are those intentionally manufactured by nanotechnology industries and, in this case, their fate at their usable-life end will be discussed. Natural inorganic nanoparticles do exist in nature: sea-water aerosols, some structures of snow and dust erupted by volcanoes are a few of them and with them man has lived all along. The nanosized by-products are the nanoparticles mostly discussed in this book, since, in our opinion, they are responsible for a non-negligible number of diseases affecting people. They are mostly generated by acci- dental combustions and are freely dispersed in air, in water and in soil, thus exposing their effects to people who inhale or ingest that particulate matter (PM), sequestering it, at least in part, in their organism. In general, we take into consideration solid, inorganic and non-biodegradable, i.e., biopersistent, PM. In this book we do not consider polymeric particles or carbon nanotubes. Even if the ISO standard [2] defines nanoparticles – but their interest is limited to the engineered ones, those obtained with nanotechnological processes and ranging from just above 0 to 100 nm – in our opinion, for the focus on possible risks to human life, we would rather refer to a functional definition of nanoparticles, i.e., we consider nanoparticles all the particles able to escape the physiological barriers including the cell and its nuclear membrane, so capable of inducing a nanoeffect. This is a definition made of nanoobjects, but what is nano when biol- ogy is what matters? What is the minimum size range of foreign bodies that Introduction 3 can induce a nanoeffect, i.e., an effect that the same material, when in bulk form, is unable to induce? A more specific definition of nanoeffect can be: Whatever damages, changes or causes a modification to a cell that can alter its physiological metabolism. For this reason, we consider “nano” to be what can be inter- nalized by a cell: to give an order of magnitude, that which is below one micron. We know some “bureaucrats” of science will turn up their nose at the way we use the word, but words are nothing more than a means to com- municate concepts in the easiest possible way, and we claim the right to use the word nano as it is more convenient for us. The adjective “submicronic” will also be used throughout the book as a synonym, meaning a particle whose size is smaller than one micron. Until now, there has been no real definition of nanoeffect, but by that term we mean any interaction of nanoparticles with cellular organelles, proteins, enzymes, DNA, etc., interactions that can give origin to stable compounds that are not recognized or only partially recognized by the bio- logical environment where they have been somehow introduced and can no longer participate in the local physiological metabolism. It is a “partial” foreign body that the cell probably tolerates, but this unwanted coexis- tence can physically alter/condition the normal cellular functions. It must be considered that a foreign body occupies a volume and, at the cell level, can block the in-and-out-flow of nutrients/metabolites or can disturb the chromatin strands during mitosis (see Chapter 10). But it is not only nanoparticles, whatever their definition, that are respon- sible for health effects. Particles with a size up to some tens of microns, when they manage to enter the organism, interfere negatively with tissues and organs. In the last few years many studies have been performed by a great num- ber of authors on engineered nanoparticles matched with cell cultures or organs. In contrast, the reader will find in this book, a focus mostly on hu- man cases, something largely missing in today’s literature and very often seen as premature. But, premature or not, what we found in our research was a very interesting new world. Micro- and nanoparticles are produced in quantities that are rapidly increasing and, in spite of pointless discussions, man is actually reacting to them in a very visible and, to be sure, worrisome way. Unfortunately, we may not infer the behavior in humans of particles generated by the multitude of sources characterizing our world from how a cell reacts when matched for a short time and in laboratory conditions with very simple (and, for the time being, very uncommon in the environment)