Nanopathology: The Health Impact of Nanoparticles

N A N O PAT H O L O G Y

THE HEALTH IMPACT OF NANOPARTICLES

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ANTONIETTA M GATTI

University of Modena & Reggio Emilia, Italy

STEFANO MONTANARI

Laboratory Nanodiagnostics, Italy

N A N O PAT H O L O G Y THE HEALTH IMPACT OF NANOPARTICLES

Published by Pan Stanford Publishing Pte. Ltd. 5 Toh Tuck Link Singapore 596224 Distributed by World Scientific Publishing Co. Pte. Ltd. 5 Toh Tuck Link, Singapore 596224 USA office: 27 Warren Street, Suite 401-402, Hackensack, NJ 07601 UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.

NANOPATHOLOGY The Health Impact of Nanoparticles Copyright © 2008 by Pan Stanford Publishing Pte. Ltd. All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher.

For photocopying of material in this volume, please pay a copying fee through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. In this case permission to photocopy is not required from the publisher.

ISBN-13 978-981-4241-00-7 ISBN-10 981-4241-00-8

Printed in Singapore.

Rhaimie - Nanopathology.pmd

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10/3/2007, 5:24 PM

This book is dedicated to those who are neither young nor old enough to know everything, but know, as we do, that they don’t know anything.

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Acknowledgements

All this research would not have come into fruition if it had not been financed by the European Project NANOPATHOLOGY. Word of thanks to the independence of the referees and the European Commission for its farsightedness. It was due to this initiative that people could now be properly treated and the cause of their contamination could be identified and eradicated. Prof. James Kirkpatrick, Prof. William Bonfield, Prof. Peter Revell and Dr. Diana Boraschi were the first to understand what we meant and to support and encourage us. We are indebted to Dr. Andrea Gambarelli, Dr. Roberta Salvatori, Dr. Federico Capitani, Dr. Daniela Tossini, Dr. Gianluca Sighinolfi for their technical help. Special thanks to Miss Lavinia Nitu, our efficient and patient secretary.

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Preface

Nanotechnologies can represent a real innovation for human society and life. The possibility of “bottom-up” construction makes man look like God, but the wise man knows that every progress can have a negative side and too often, when he realizes that, disasters have already occurred. The primary raison d’être of this book is to help society avoid the repeat of the mistakes made by the Curies and their followers when they discovered radioactivity and started, on the wings of enthusiasm, to use it on people affected by a number of diseases, and did that without being able to anticipate the harmful side effects of those therapies. A new trend has spread all over Europe, meetings after meetings dedicated to radiotherapy were organized, and people have even started to wear necklaces with beads of Cobalt core. It was only a few decades later, after having paid a high price in terms of deaths, that the side effects of radioactivity became evident, but now we can use radioactive materials in a safe way daily, taking advantage from this phenomenon and the technologies it had generated. The new frontier opened by nanotechnology especially in medicine looks extremely exciting. In the future we might see nanodevices equipped with nanomotors inserted in the blood vessels and driven to areas damaged by an infarction to destroy the thrombus or the atheroma and restore circulation, or toward the pneumothorax area to seal the lesion. Or even devices that act like guardians to check the onset of inflammations in precancerous areas. All these may look like a dream, but, it is a dream fast becoming a reality and, before, it becomes real, it is crucial that we verify how organisms, tissues, cells react to the presence of nanoparticles, i.e. foreign bodies whose behaviour is still largely unknown.

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Recent European research projects (Nanosafe1, Nanosafe2, Nanoderm, Nanopathology) have explored the possible risks of nanoparticles on human health, and their results are controversial. Some assert the safety of nanoparticles through in-vitro tests, others are more doubtful and less optimistic, while a few scientists have already presented clinical evidences of the presence of nanoparticles in pathological tissues [A.M. Gatti, 2005]. Besides other pieces of evidence, unintentionally released, nanosized particles were found in soldiers who served in former Yugoslavia during the Balkans War (1993-97). It is widely known that the explosion of Depleted Uranium bombs can develop a temperature exceeding 3000°C [Annual Report, 1978]. The magnitude of this combustion is capable of vaporizing everything. As soon as the vaporized materials cool down, nanosized particles are created and are scattered in the environment. The inhalation or ingestion of those mainly metallic particles by humans and animals can bring about pathological effects [Nemmar A. et al., 2002]. Warfare is not the only one to be blamed for the formation of nanoparticles as a pollutant. Car engines, industry, incineration and high-temperature procedures in general are just a few examples of particulate pollutants producers. It is easy to conclude that in more than one instance, the environment is already contaminated by nanosized particles. It has been proved that 100-nm-sized particles when inhaled can bypass the lung barrier in 60 seconds and reach the liver in 60 minutes. It may not be possible to eliminate nanoparticles from the environment, but, awareness of their possible adverse effects on human health is important. More research are needed to determine the safest procedures to handle them. This book intends to help society change its mind set as in the words of John Steinbeck, Nobel Prize Winner, 1964, “The ability to think differently today than yesterday is what separates the wise from the stubborn.”

Preface

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Bibliography Annual Technical report of the Air Force Armament Laboratory. (1978). Armament development and test Center, Eglin Air Force Base, Florida, USA. Project n° 06CD0101 (From October 1977 to October 1978). Gatti, A.M. Handbook (2005). Risk assessment of micro and nanoparticles and the human health, Handbook of nanostructured biomaterials and their applications. Ed. by American Scientific Publisher USA, cap. 12, pp. 347-369. Nemmar A., Hoet P.H.M., Vanquickenborne B., Dinsdale D., Thomeer M., Hoylaerts M.F., Vanbilloen H., Mortelmans L., Nemery B. (2002). Passage of inhaled particles in to the blood circulation in humans, Circulation, 105 (4), pp. 411-417.

Authors Antonietta M Gatti University of Modena & Reggio Emilia, Italy Stefano Montanari Laboratory Nanodiagnostics, Italy

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Contents

Acknowledgements

vii

Preface

ix

1. How the Whole Thing Began or the Logic Path Towards a Discovery 1 1.1 Introduction...................................................................................... 6 1.2 Bibliography .................................................................................... 10 2. In-vitro and in-vivo Biological Behaviour of Micro and Nanoparticles 2.1 Introduction...................................................................................... 2.2 Nanoparticles and medical devices .................................................. 2.2.1 Dentistry ................................................................................ 2.2.2 Orthopaedics.......................................................................... 2.2.3 Nanostructured surfaces ........................................................ 2.2.4 Drug delivery......................................................................... 2.2.5 Nanomedicine........................................................................ 2.3 The results of the nanopathology project ......................................... 2.3.1 In-vivo experiments ............................................................... 2.4 Bibliography ....................................................................................

11 11 13 13 14 16 17 19 22 28 33

3. Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 3.1 Introduction...................................................................................... 3.2 Lung ................................................................................................. 3.3 Blood................................................................................................ 3.4 Liver................................................................................................. 3.5 Kidney and adrenal gland ................................................................ 3.6 Digestive system .............................................................................. 3.7 Vessels ............................................................................................. 3.8 Sperm ............................................................................................... 3.9 A few considerations on reproduction ............................................. 3.10 Bibliography ....................................................................................

39 39 42 70 80 95 102 114 115 121 131

4. Six “Detective Stories” 4.1 The 1st case....................................................................................... 4.2 The 2nd case...................................................................................... 4.3 The 3rd case ...................................................................................... 4.4 The 4th case ......................................................................................

135 135 142 145 148

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4.5 4.6

The 5th case ...................................................................................... 152 The 6th case ...................................................................................... 155

5. War and Nanoparticles 5.1 Civilians living around a firing ground ............................................ 5.2 A few reflections.............................................................................. 5.3 Bibliography ....................................................................................

161 198 199 200

6. Nanoparticles in the Environment and Working Places 6.1 Introduction...................................................................................... 6.2 Chimneysweeps: a historical case .................................................... 6.3 Welding metals ................................................................................ 6.4 Toner................................................................................................ 6.5 The particulate active filter or FAP.................................................. 6.6 The environment around a foundry.................................................. 6.7 The environment around a power plant............................................ 6.8 The case of a ship............................................................................. 6.9 Incinerators ...................................................................................... 6.10 Tobacco smoke ................................................................................ 6.11 Bibliography ....................................................................................

203 203 205 207 210 212 215 220 227 228 231 237

7. Nanoparticles in Food, Cosmetics and Other Products 239 7.1 Bibliography .................................................................................... 249 8. New York 9/11 251 8.1 Bibliography .................................................................................... 263 9. The Future and Prevention Criteria 9.1 The state of the art............................................................................ 9.2 What is next?.................................................................................... 9.3 The future......................................................................................... 9.4 A few reflections.............................................................................. 9.5 Bibliography ....................................................................................

265 265 267 280 281 284

Appendix

287

Index

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Chapter 1

How the Whole Thing Began or the Logic Path Towards a Discovery ___________

%

ack in 1990, the hospital of Monza, a branch of the University of Milan - Italy, sent to the Laboratory of Biomaterials of the University of Modena a vena cava filter (Montanari, 2000) which had two broken legs and that had been explanted surgically from a 62year-old female patient (Emanuelli, et al., 1995) (Gatti, et al., 2006). The surgeon who had first implanted and then removed the filter just wanted to know why the device had failed and solving his problem was not particularly hard, as it was due to a caudal migration with the two legs trapped in a collateral vein. The filter moved downward, while the two legs could not follow the movement, broke. One of the consequences was that the fracture surfaces remained exposed to the blood and interacted with its components. What remained of the filter and the broken legs was observed under a Scanning Electron Microscope equipped with an X-ray microprobe (Energy Dispersive Spectroscope or EDS). This analysis [A highly energetic electron beam is aimed at the sample, and that yields a number of by-products, among which X-rays. Each chemical element has characteristic energies/wavelengths which can be detected using a solid state energy dispersive spectrometer detector] (See Appendix) on this surface, where the legs had broken, revealed a relatively high concentration of Chlorine, Silicon, Phosphorus and Magnesium; all elements that did not belong either to the filter’s alloy - an AISI 316L stainless steel, whose composition is Chromium 18%, Nickel 14%, Molybdenum 3%, Carbon below 0.03% and Iron to balance - or to the blood, at least not in such quantities. At that time, we could not find any reasonable explanation to what we had seen. 1

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Nanopathology

A couple of years later, the same hospital sent another broken vena cava filter to the Laboratory. This time the device was a temporary one (Bovyn, et al., 1997) removed from a 58-year-old male 23 days after having been implanted. Also that filter had failed, having lost one of its legs which was then retrieved from the patient’s right inferior interlobar renal vein by means of a transvenous noose catheter. The scanningelectron-microscope observation of the fracture showed a few inorganic deposits that could not belong to the product’s composition. As the EDS analysis proved that the filter’s alloy was composed of Cobalt, Chromium, Nickel, Molybdenum, Silicon and Iron (alloy called Phynox), while Calcium and Aluminium were found in one of the deposits, Chlorine, Silicon, Potassium, Calcium, Sulphur, Aluminium, Sodium, Titanium and Magnesium in another, and single particles of Aluminium and Calcium-Sulphur were also detected. Those deposits had obviously formed in vivo, since the specimen had been carefully washed in a Potassium oxide solution, and cleaned in a Hydrogen peroxide ultrasonic bath, a treatment that destroys biofilm and leaves only insoluble precipitates strictly adhered to the surface. Some of the materials detected can be found as trace elements in ionic form or bound to organic molecules in the human organism, but what we saw was particles and the quantities we were confronted with were comparatively high. Again, no satisfactory explanation could be offered then to that phenomenon. In December 1997 a 62-year-old patient (Ballestri, et al., 2001) (Gatti, et al., 2002) was admitted to the University hospital of Modena, where the Laboratory of Biomaterials was then located. He was affected by acute renal failure, hepato-splenomegaly and a mild haemolytic anaemia. In addition to that, for the last eight years he had suffered from a slight increase of the body temperature, a phenomenon that manifested itself in the late afternoon and remitted to a normal temperature in a matter of a few hours, without recurring to any medication. That fever followed periodic cycles, each of which lasted a few months. Among the symptoms the patient suffered from there was also a constant tiredness. Blood and urine culture had been consistently negative and, despite numerous hospitalizations, no aetiology could be determined for such collection of symptoms.

How the Whole Thing Began or the Logic Path Towards a Discovery

3

So, a percutaneous echo-assisted biopsy was performed in the liver and the lower pole of the left kidney, revealing non-caseating granulomatous areas with Langhans-type giant cells in the kidney, while the liver showed a mild fibrosis of the portal tracts filled with mononuclear infiltrates and with the presence of epithelioid giant cells. Scattered polarized light-blue particles either in the kidney and the liver were shown by polarized-light microscopy observation, often inside giant cells located in granulomatous areas, suggesting a foreign-body multi-system granulomatosis. No yeasts or alcohol-acid resistant bacteria could be identified. Clinical and immunological tests excluded tuberculosis, mycotic infection, protozoa infestation, granulomatous vasculitis or sarcoidosis, and the patient’s history was negative for exposure to chemicals, drug abuse or arteriographic procedures. That was indeed a very hard problem to solve through the traditional means of investigation. So, the samples were given to our Laboratory where we studied the debris by means of a scanning electron microscope (SEM XL40 by Philips) and an environmental scanning electron microscope (ESEM XL50 by Fei Company) equipped with an EDS. Both in the liver and kidney specimens, we could identify solid particles whose electronic density was higher than the biological background’s. Their size ranged 6-20 microns in the liver and was smaller than 6 microns in the kidney, and the EDS elemental analyses, identical in both types of specimens, revealed that they were made of Aluminium, Silicon, Oxygen, Sodium, and Potassium or Barium, a composition that suggested that of a ceramic material (and was compatible with feldspars, non-fibrous silicates that are regular components of porcelain). In order to understand the origin of such debris, a specific anamnestic study of the patient was carried out, looking for an internal source of these debris like prostheses, implant, etc. What we learned from the patient, was that nine years before, two ceramic bridges had been placed in his left upper and right lower dental arches. That caused immediately a marked discomfort, an uncontrolled lachrymation of the left eye, and a homolateral earache that could not be solved by the antibiotic treatments the patient had undergone. In addition to that, the patient became a bruxer.

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Nanopathology

The prostheses had been milled a little later to try and solve the problem of malocclusion and bruxism, so leaving a highly worn prosthetic surface. So, we started to suspect that the source of the debris found in the patient’s tissues could be those prostheses and had both removed. They were cross-sectioned and subjected to an Rx microanalysis that showed the same elemental composition as the foreign bodies discovered in the tissues. Ceramic particles smaller or as large as 40 microns were found in the patient’s stools before the prostheses were removed, but none one month after. So the patient underwent a 6-month therapy of methylprednisolone and that induced the remission of the fever along with the reversal of hepato-splenomegaly. Inflammation, haemolysis and cholestasis disappeared, while the renal function recovered. After therapy, no steroids were administered for another six month and the clinical picture worsened. A recourse to methylprednisolone and cyclophosphamide, an immunosuppressive therapy, lead to a complete remission. Liver and kidney biopsies performed three years after the first hospitalization showed a marked reduction of the granulomatosis. Now, the question was: how could that debris, whose origin was hard to call into question, have reached the liver and the kidneys? In our opinion, the hypothesis that those particles had been absorbed by the gastrointestinal system looked the likeliest, but no literature existed to support what we suspected. Debris of different sizes had been detected in the stools, the same kind of foreign bodies had been found in the liver and the kidneys, but those in the kidneys were smaller than those in the liver. That could suggest that the particles, undoubtedly present in the bowels, which were absolutely healthy in our patient, had passed through the intestinal mucosa and had been cleared by the liver before entering the general circulation and, from there, the kidneys. Such passage was not described in the then current handbooks of Physiology and sounded hard to believe. Professor Peter Revell of the Free Royal Hospital of London gave us then four bioptic samples of liver granulomatosis whose origin had been declared to be unknown, but viruses, bacteria and parasites had been ruled out as possible causes. In three of them we found evident traces of

How the Whole Thing Began or the Logic Path Towards a Discovery

5

surprisingly enough but undoubtedly environmental dust, while in one we detected particles of a metal that had been used for therapeutic purposes. Gold nanoparticles had been injected in the patient’s knee joint to treat an arthrosis and it was that Gold that we found in the liver tissue. That made us understand that solid environmental pollution could be suspected as being able to penetrate the body and settle in the liver. How, it was far from clear, but the most reasonable ways of entry seemed us to be the digestive system – for which we had already a piece of evidence with the case of the dental prosthesis - and, perhaps - but it was just a guess - the respiratory system. In the meantime, we had received more samples both from our University Hospital and the Istituto Tumori (Cancer Institute) of Milan – Italy, and they regarded cases of colon cancer and Crohn’s disease. In all instances, inorganic dust was present in considerable concentrations and variety. In one case we could even detect 15 different chemical elemental compositions in the same cluster in less than 1 cm2 of tissue. Thanks to our experience in caval filtration and the new availability of filters that could be explanted even after a long time, we started to take into consideration the blood clots that were regularly found stuck to the device when it was removed from the patient and the thrombi that the device had trapped. If inorganic dust, especially composed of chemical elements that should not be present in the organism, had been detected, that would have meant, without any possible doubt, that that dust had an exogenous origin. As a matter of fact, as will be illustrated in detail later in this book, all samples showed that presence. Finding once unexpected inorganic particulate had become a daily experience, but we could say very little or nothing at all about its way of entrance. So, we started to look at some pathological lung sample we got from our University and the University of Siena (Italy), and among them there were also some cases of pneumothorax. Then, we studied cases of cystic fibrosis, sarcoidosis and of different forms of lung cancer. Inorganic micro- and nanodust was to be found with a great frequency. We decided, then, to present a project to the European Community, a project we called “Nanopathology”, by that neologism we had created

6

Nanopathology

meaning the pathologies due to micro- and nanodust, and by that project intending to study if what we had observed for a few years had a scientific basis. 1.1 Introduction Man has only recently landed on what to him was a new planet: Nano. But Nature has always been there. Just to give one example, Nature uses a “nanocode” called genome to preserve all the information about the species each individual, be it a man, a virus or any other living being, belongs to, and that code is extremely complex, sophisticated and efficient. Genome, in its turn, gives all the necessary instructions, many of which we are not aware of, to build the (nanosized) proteins necessary to live, and does that at a nanoscale. We are accustomed to interacting with objects the size we can handle and/or see, and have also been accustomed for a relatively long time to investigating and trying to understand how atoms and molecules behave; but Nano is something different from both worlds. Nanoscale systems are too big to be considered molecules, but definitely too small to be understood in terms of macro, and attempting to extrapolate their behaviour starting either from the smaller or from the larger would lead us astray. Neither quantum nor classic physics can be fully applied. Of this sort of “mesoworld” we know very little, but what has immediately become visible is that the properties of those objects are extremely interesting from a scientific point of view and, in addition to that, offer many prospective possibilities to be exploited to our benefit. Right for that latter reason, an enormous throng of scientists, technicians, industrialists and financiers flung themselves, and are still doing with growing enthusiasm and hopes, headlong into that field, the way pioneers did in the past when a new land was discovered or gold was unexpectedly found somewhere. More than a couple of decades ago, in 1986, Kim Eric Drexler, an American engineer, published Engines of Creation, a book describing nanomachines capable of reproducing both themselves and virtually any material object, extending life duration, curing diseases, in short,

How the Whole Thing Began or the Logic Path Towards a Discovery

7

working all sorts of wonders and, at the same time, reducing pollution. Many scientists ridiculed such outlook, and nevertheless the book exerted a strong influence on many people. And many people means good business. Now, after more than twenty years have elapsed, we are still far from what Drexler we do not know how rightfully anticipated, but it is a matter of fact that nanotechnology is finding more and more applications and the limit seems to be imagination. According to Forbes, (web ref. 1) now the most common applications of nanotechnology are in sports goods, ski wax, tennis racquets and tennis balls being the top three, but some medical applications are already in use and many more are being attempted, cosmetics, photocatalists and computer chips are already available and what are called “smart” surfaces, by that meaning hydrophobic, self-cleaning, anti-bacteria, anti-mould treatments are best sellers. Just as a brief observation, the adjective “smart” sounds very à-la Drexler. So, after having landed in what if not a promised is at least a promising land, it would be fool not to advance and explore it with the purpose of exploiting it. But like any unknown territory, also this can hide unexpected dangers and, for that reason, using prudence and exercising some patience may look advisable. Who carried out exhaustive studies about the impact those technology and, in particular, handling nanodust may have on human and animal organisms? How can we know how long those entities will take to interact with environment and organisms, if ever they will interact in a perceptible way? Is there any long enough experience in that field? No satisfactory answer can be given to those questions. What we can do is extrapolate. Micro- and, much more rarely, nano-scale particles have always been produced by a number of natural sources: belching volcanoes, forest fires, rock erosion, and airborne desert and beach sand, but the greatest quantity of such dust, particularly the smaller, comes from man. Much of it is the undesired by-product of combustion, a process that has started to be used on a large, industrial scale only a couple of centuries ago, i.e. roughly 1/10,000 of the time man has spent on the Earth. Generating heat at temperatures higher than that of burning wood has always been difficult and, when the concept of selling value was introduced,

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expensive. For those reasons, materials such as, for example, glass have been rather precious for a long time. But when we started to exploit fossil combustibles, heat has grown cheaper and cheaper and easy to come by, and now very high temperatures can be attained without any difficulty and at a low cost. All that creates dust, and, as a rule of thumb, the higher the temperature, the tinier the dust. So, foundries, cement plants, incinerators and internal-combustion engines, among an indefinite number of other sources, produce particles, and their quantity is constantly on the increase, particularly as regards nanoparticles, since industrial filters are not efficient enough to trap them and the increase in temperature makes microparticles rarer and nanoparticles more common. Particulate matter is released in the atmosphere and behaves in a way that is very similar, or all but identical in the case of nanosize, to gas. Some of those particles, especially the small ones, tend to form clusters and, for that reason, making a clear distinction between micro and nano is not always possible and often not even meaningful. However, in any case the structure of a nanoparticle remains the same. The dust we deal with is inorganic, most of the times crystalline, insoluble in water or other common, natural solvents, and non biodegradable. Being so small, pressure and thermal gradients and wind carry it virtually everywhere, and when it eventually falls to the ground, a gentle breath of wind can lift it up again, thus restarting the cycle. That way, those particles can travel very long distances and stay with us forever. Besides, when we look at those inorganic particles from the pollution point of view, we must not forget that they are often the carriers of organic pollutants like, for example and among many others, dioxins, furans or polycyclic aromatic hydrocarbons. And we must also remember that in a number of environmental conditions, even inside biological tissues, those particles tend to coalesce and, for that reason, differentiating micro and nanoparticles is not always possible and in some instances could be meaningless. Another important point is that spherical particles generated by combustion, especially the larger ones, are often hollow and very fragile, so, when they break, they create

How the Whole Thing Began or the Logic Path Towards a Discovery

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fragments which are obviously smaller and, as a consequence, microparticles can create nanoparticles. From the compositional point of view, the nanoparticles we deal with are very often alloys fortuitously created by the fact that the chemical elements that make them up were present in the materials burnt and combined to form an alloy that in most cases is not to be found in any metallurgy handbook. So, their elemental chemical composition can be a telltale indication that allows to identify the source. But there are also cases when those particles come from wear and friction of machines or industrial working, and heat is not involved in their formation. In that case, their composition is the same as that of the original material. An important issue regards mass and volume. If, for the sake of simplification, we assume that particles are spherical (which is often the case with the particles we deal with, when created at high temperature), we can easily calculate their volume and their mass, which depends, obviously, on the matter that object is made of. Let’s suppose that we have a sphere whose diameter is 10 µm, i.e. that of a coarse particle. In that case, its volume is 4πr3/3 = 523.3 µm3. Now, let us suppose that we wish to make spheres with a diameter of 2.5 µm, i.e. ¼ of the one of the former ball. In that case, the volume of each sphere will be 8.0593 mµ3, i.e.64 times as small. That means that, using the same amount of matter necessary to make a particle whose diameter is 10 µm, we can create 64 particles whose diameter is 4 times as small. And, continuing, we can create 1,000 particles with a diameter of 1 µm or 1,000,000 with a diameter of 0.1 µm. This is particularly important if we look at this simple geometric fact with the nanopathologist’s eye, since the same amount of matter, if burnt at different temperatures, can give origin to particles whose effects on the organism is very different [see also Chapter 6 – Introduction] On that dust we collected some experience and thanks to that we can try and extrapolate about nanotechnological dust and its biological behaviour. Small particles are classified in a number of different ways, according to the classifier and his discipline. Physicists, chemists and biologists do not seem to be always agreed. We are not much interested in academic

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Nanopathology

classifications and we do not really know where micro ends and nano starts in the organism. It is highly probable that a threshold exists below which natural, physiological barriers are ineffective and even one below which cells do not oppose any resistance to the entrance of foreign bodies. Our studies do not allow us to quantify that barrier nor to say if and, in case, to what extent that threshold is influenced by size, shape, surface/volume ratio, chemistry, state of aggregation or other factors. Ours is a work in progress. 1.2 Bibliography Ballestri, M., Baraldi, A., Gatti, A. M., Furci, L., Bagni, A., Loria, P., Rapanà, R. M., Carulli, N. and Albertazzi, A., (2001), Liver and Kidney Foreign Bodies Granulomatosis in a Patient with Malocclusion, Bruxism and Worn Dental Prosthesis – Gastroenterology,121:1234-38 Bovyn, G., Gory, P., Reynaud, P. and Ricco, J. B., (1997), The Tempofilter: A multicenter study of a new temporary caval filter implantable for up to six weeks – Ann Vasc Surg;11:520-25 Emanuelli, G., Gatti, A. M., Cigada, A. and Brunella, M. F., (1995), Physico-chemical observations on a failed Greenfield vena cava filter – J Cardiovasc Surg;36:121-5 Gatti, A. M. and Montanari, S., (2006), Retrieval Analysis of Clinical Explanted Vena Cava Filters – J Biomed Mat Res Part B: Appl Biomater 77B:307-314 Gatti, A. M., Ballestri, M. and Bagni, A., (2002), Granulomatosis associated to porcelain wear debris – American Journal of Dentistry, Vol. 15, No. 6 Montanari, S., (2000), Malattia tromboembolica e filtri cavali - Ed. C. Rabbia, G. Emanuelli – 90-140 Minerva Medica – Turin Ref.web 1 [http://www.forbes.com/investmentnewsletters/2005/01/12/cz_jw_0112soapbox.html]

Chapter 2

In-vitro and in-vivo Biological Behaviour of Micro and Nanoparticles ___________ 2.1 Introduction

7

he 21st century opened with a revolution due to emerging technologies called nanotechnologies, which are growing exponentially and even if we know that they must have a physical limit, their outlook seems to have no end. “Nano” is a word of Greek origin meaning “very small”, and nanotechnologies are characterized by smaller and smaller technologies, i.e. they deal with items ranging in magnitude between 10-9 and 10-8 m. The first definition was given by Norio Taniguchi, of the Tokyo Science University, in 1971: “Nano-technology is the production technology to get the extra high accuracy and ultrafine dimensions, i.e. the preciseness and fineness of the order of 1 nm (10-9 m in length)”. This definition has been updated by the Europäische Akademie in 2003: “Nanotechnology deals with functional systems based on the use of subunits with specific size-dependent properties of the individual subunits,” in order to include not only the production of nanoparticles, but also the production of systems or processes based on the highest possible miniaturization. Nanotechnologies refer to the technological field concerning the controlled manufacturing of functional nanosystems or the creation of nanostructures, which results in the production of entities with at least one dimension of 100 nm-length scale.

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Nanopathology

To give an idea of the smallness of these things, we can compare them with some biological objects (see Fig. 2.1). A human hair can range between 10 and 60 microns in width, human red cells have a diameter of 5-7 microns, while, at the cell order, ATPsynthase measures 10 nm and a DNA molecule ranges between 0.5 nm and 2 nm.

Fig. 2.1 Scale of dimensions.

The synthesis of such small entities, never seen in the world, as the ones we include in the definition of nano and their (sometimes involuntary) dissemination can represent a new pollution and new stimuli for the animal and human organisms strongly influencing their health. Nobody knows what the impact will be of these new technologies on our society and what side effects are to be expected. It is a novel situation and it is imperative to carry out researches to investigate if and how nanoparticles can represent a possible risk to the environment and who lives in that environment. But our aim is to investigate: - what is considered “nano” for the human body, namely, - what is its reaction against a discrete (not continuous) stimulus, - what is the threshold size below which tissues or cells do not react in a known way since the sensors do not “see” or recognize the stimulus. Nanopathology will try to give an answer.

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13

Applications in the medical field are already possible and a brief overview of the most promising ones is given below. 2.2 Nanoparticles and medical devices Applications of nanotechnologies are being experimented in a number of specific fields of medical interest, but dentistry, by the creation of nanocomposites for dental restorations, is the discipline where they are already commonly available. 2.2.1 Dentistry At present, dental restorations employ amalgams, composites, and Gold or ceramic inlays. There is a real need to dispose of mouldable, more resistant materials, especially for posterior teeth where mastication load is particularly high, capable of replacing amalgam, long suspected to be toxic. Though there are no final scientific demonstrations to support the thesis of its toxicity, a few diseases, and some lethal ones at that, have been associated to the use of amalgams, in particular, amiotrophic lateral sclerosis and Alzheimer’s disease. Amalgam is, in fact, the most common, and inescapable for its carriers, source of human exposure to Mercury, and a chronic low-level exposure to Mercury should be considered as a factor in Alzheimer’s disease or in other diseases of the central nervous system, including multiple sclerosis and Parkinson’s disease (Wenstrup et al., 1990) (Saxe et al., 1999). Some dental materials such as composites, dental filling resins employing nanoparticles made, for instance, of Zirconia (Zirconium oxide), are already available on the market, but other, more sophisticated, materials are being developed. Organo-functionalized nanospheres, containing X-ray absorbing elements such as Zirconium, combined with liquid crystal monomers produce, in fact, composites with minimal polymerization shrinkage and improve mechanical properties (Mitra et al., 2003), (Mayer et al., 1998), (Williams et al., 2003).

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Nanopathology

The improvement of these properties is only partial, since the wear due to mastication cycles is not decreased and the risk of wear debris ingestion continues (Ballestri et al., 2001). Until a few years ago, this event did not cause any alarm, since it was believed that that debris was gotten rid of with the faeces like all the non degradable, small things people, and babies in particular, ingest involuntarily. Unfortunately, it is not so. It is growing clearer and clearer that biocompatible materials can lose their biocompatibilty when they pass from a bulk form to a fragmented one. The nanosized form is responsible for until-now hardly suspected direct interactions with single cells, and those interactions can induce unknown, unexpected reactions (Peters et al., 2004), (Lucarelli, et al 2004). Toothpastes with either Silver or Gold or hydroxypatite nanoparticles and used as nanofillers are already on the market, as well as the bristles of some toothbrushes coated with nanoSilver (see Chapter 7). 2.2.2 Orthopaedics Besides dental implantology, another field of application of nanotechnology is orthopaedics. Orthopaedic devices, especially hip and knee joint prostheses, present two main problems that show over time: wear of the matching surfaces in the joint and loosening of the stem in the femoral channel. The former causes the failure of the device because of the granulomatous reaction induced by the wear debris, the loss of the three-dimensional movement due to the loss of sphericity of the head, and, finally, the rupture of the acetabulum with the ensuing migration of the head. To overcome these problems, new nanocomposite-based, wearresistant and self-lubricating PVD (plasma vapour deposition) coatings (Stueber et al. 2002) are being studied. The wear of the hip joint prostheses and the health problem due to the production of wear debris induce researchers to develop new coatings more resistant to wear and,

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possibly, self-lubricating. A possible solution can be the development of a metastable hard matrix incorporating homogenously distributed nanoclusters of MoS2. The use of cross-linked polyethylene does not seem to solve the problems, also because wear produces sub-micronic particles that can trigger a tissue reaction and be widely disseminated in the tissues because of their ultrafine size (Urban et al., 2000), (Revell et al., 1997). The creation of nanosized-morphology surfaces enhances biocompatibility and is supposed to ameliorate the interaction between the device and specific cellular components. In biomaterial research, it has been found that even though a bulk material may be well tolerated by the body, finely divided particles of the same material can often lead to severe, even carcinogenic, complications. Differences in particle size influence histological reactions and cytokine production. Macrophage response to particulate debris appears, for example, to be dependent on particle size, composition, and dose as given by surface area ratio. Several studies have also been carried out to determine the relationship between cell and particle size with respect to cytotoxicity. (Goodman et al., 1990), (Shanbagh et al., 1994), (Tamura, 2003). Carbon nanofibres have been proposed as a possible, new orthopaedic implant material because of their unique mechanical, electrical and cytocompatibility properties. These fibres have a size close to hydroxyapatite (Calcium phosphate) crystals and collagen fibres found in the bone. Moreover, “in vitro” studies provided evidence that nanophase Carbon fibres enhance osteoblast function over conventional Carbon fibres and current orthopaedic implant materials (such as titanium). To determine the influence of Carbon nanofibre wear debris on osteoblast viability, direct contact toxicity studies were performed. The results proved that nanophase Carbon fibres were less detrimental to osteoblast viability than larger-diameter, conventional Carbon fibres. In other words, they provide evidence of the promise of nanophase materials in improving orthopaedic implant efficiency (Price et al., 2004).

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Nanopathology

2.2.3 Nanostructured surfaces Implantable medical devices are bound to employ biocompatible materials, i.e. materials that must be not only accepted by the biological environment, but also promote an appropriate and favourable biological interaction. The basic concept is that biological activity depends on the structure of the matching matter and, at the same time, much of the matter properties depends on size (Dobson et al., 1999). So the biocompatibility of a material must have an “appropriate” cell adhesion, that is preceded by a specific protein adsorption from the extracellular matrix. The adsorption of a specific monolayer of proteins will be of the utmost importance for the success of an implant. In fact, once the proper protein has been adsorbed by the implanted surface, the appropriate cells need to adhere and proliferate. The target of achieving proper cell adhesion and proliferation is every biomaterial engineer’s challenge. (Whiteside et al., 1991). There are many cellular processes which are triggered by the type of protein adsorbed, its conformation and its biological activity. If the presence of a certain protein is requested to guarantee a “proper” interaction of an implant with the biological environment, it may be possible to manipulate the implant surface in order to induce in advance that situation. The so-called “biomimetic” surfaces base their activity in the human body upon this concept and, typically, biomimetics aims at copying nature’s way of organizing and ordering complex molecules at surface, to inhibit non-specific surface reactions and, finally, to create “smart surfaces” that undergo rapid shifts in properties with small external change (Castner et al., 2002). The ability to design such a system is greatly supported by biotechnologies and nanotechnologies. Examples of that are the molecular self–assembly as a strategy for nanofabrication that involves designing molecules and supramolecular entities so that shapecomplementarity causes them to aggregate into desired structures. Just as an example, the Self-Assembly Monolayers (SAMs) are considerably important in developing arterial prostheses, where it is absolutely essential that the interior of the walls gets quickly coated by endothelial cells. The efforts to attach heparin molecules to the surface of vascular

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catheters in order to avoid blood clotting was not particularly successful, especially because thrombosis cannot be reduced to a simple surface problem, but the new strategy of SAMs can certainly improve and stabilize these surfaces, and help to achieve better results. (Zhang et al., 2002). Nanotechnologies can create functionalised nanosurfaces attaching polipetides or dendrimers (repeatedly branched molecules) that can increase the biocompatibility of the implantable devices. Suitable candidates for dendrimers synthesis and study include monomers currently used for the preparation of medical-grade linear polymers, and natural metabolites. For example, monomers from synthetic polymers such as polyethylene-glycol, polytrimethylene-carbonate and natural amino-acids represent classes of monomers suitable for use. Incorporation of one or more of these building blocks in a dendritic structure provides new opportunities to create well-defined polymers for tissue engineering applications. The versatility of dendrimer technology provides also unlimited possibilities in the synthesis of novel nanomaterials and unprecedented control over these systems (Eppel et al., 2002), (Bellingham et al., 2003), (Carnahan et al., 2001), (Balogh et al., 1999). 2.2.4 Drug delivery Other emerging and especially promising applications are those in the drug-delivery field and gene therapy. Those are made possible by the versatility of nanotechnologies and, particularly, by the extremely reduced size of those devices, equal to that of their targets. In the targeted delivery of drugs there are attempts to use dendrimers because drug molecules can be loaded both in the interior of the dendrimers and attached to the surface groups in order to control the rate of drug’s release into the body. ( Kilts, 2003), (Orive et al., 2003), (Buxton, 2003). One of the most promising application of nanoparticles is their use in pharmaceuticals as a drug-releasing support for neural diseases, capable of negotiating the blood-brain barrier. The existence of this barrier is in fact a severe limitation for the delivery of potentially useful drugs such as cytostatics and central nervous system in-vitro active agents.

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Nanopathology

Nevertheless, it has been demonstrated that different drugs bound to nanoparticles can be transported across the blood-brain barrier and achieve pharmacological effects in the brain (i.e. brain tumour treatment) (Li et al., 2003), (Lockman et al., 2002), (Koziara et al., 2003), (Rantioa et al., 2004), (Schroeder et al., 1998). Nanosized materials can be the substrate where viruses or DNA molecules can be encapsulated or sorted. The interaction of these biomolecules with the nanoparticle, nanotube or nanosized surface can serve to detect specific proteins or viruses and can guarantee that the molecules are carried to the specific target. The integration of biological molecules with Carbon nanotubes (i.e. fullerene molecules with a cylindrical or toroidal shape) has potential applications in gene- and drug-delivery technology and enables the use of hybrid systems like biomolecular sensors. In addition, peptide functionalized nanoparticles were created as a new class of therapeutic and diagnostic agents with the purpose of specifically recognizing and binding the characteristic molecular signature of cancer and other pathological conditions. (Salem et al, 2003), (Zheng et al, 2003), (Sando et al,2003), (Chakrabarti et al., 2003), (Lehmann-Horn et al., 2003), (Ai et al. 2003), (Gao et al., 2003). But, besides its visible advantages, the possibility to interact with the smallest components of the human body makes nanoparticles potentially dangerous, and verifying the results of this interaction is an awkward matter (Pereira et al., 1999), (Saebo, 2004). Many researchers and organizations (web ref. 1), (Tran et al., 2005), (web ref. 2), (Greenpeace Environment Trust Report, 2003), (US EPA Report, 2005), (SCHENIR Report, 2005), (web ref. 3), (Proceedings of St. Gallen, 2006), (web ref. 4), (web ref. 5), (web ref. 6) are busy discussing the possible risks related to these new technologies and the possibility to manipulate in such a small-scale way the cell response. They can actually enter the caveolar openings in cell membranes and the perturbations produced can cause the death of the cell. Nanoparticles have also a high surface reactivity and can interact with enzymes or proteins, disturbing biological processes and causing impairments to structural or metabolic processes. (Schler et al., 2003), (Colvin, 2003), (Warheit, 2004), (Borm et al., 2004), (Brook et al., 2004), (Maynard et al., 2004), (Hett, 2004).

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2.2.5 Nanomedicine Nanomedicine (Editorial, 2003), (web ref. 7), (web ref. 8) is a new field of research that deals with all the applications of nanoparticles or nanodevices for medical uses. It refers to highly specific medical intervention at the molecular scale that involves the use of engineered nanodevices and nanostructures to monitor, repair, construct and control the human biological system. The most elementary of nanomedical devices will be used in the diagnosis of illness. A more advanced use of nanotechnology might involve, for example, implanted devices to dispense drugs or hormones as needed in people with chronic imbalance or deficiency states. Lastly, the most advanced nanomedicine involves the use of nano-robots as miniature surgeon. Such machines might repair damaged cells or even get inside cells and replace or assist damaged intracellular structures (heart defibrillators and pacemakers are likely to be replaced by more sophisticated devices that can act on the individual cells). And in a possibly not so far future, nanomachines might replicate themselves or correct genetic deficiencies by replacing DNA molecules. Nanospheres are already employed in humans, though only experimentally, in diagnostics. Inhalable nanoparticles can sense, for example, the local ventilatory status, releasing drugs at appropriate local dosage in response to physiological stimuli and lipid-based nanoparticles are being considered for site-specific delivery of antifungal therapy to the pulmonary epithelium. Moreover, it was observed that highly lymphotrophic super-paramagnetic nanoparticles (monocrystalline Iron oxide) can easily gain access to lymph-nodes by means of interstitiallymphatic transport in patients affected by prostate cancer. Their presence in lymph-nodes can be detected by MRI (Magnetic Resonance Imaging) and their concentration can indicate a metastasis (Harishingani et al., 2003), (Babes et al., 1999). Scientists have also shown that Iron oxide nanoparticles as small as a virus can outline not only brain tumours under MRI, but also other cerebral lesions caused by multiple sclerosis, stroke and neurological disorders, that may otherwise go unnoticed. Iron oxide nanoparticles can

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Nanopathology

be delivered across the blood-brain barrier and stay in the brain lesions at least for days. For this reason, Iron oxide has some advantages over Gadolinium, which must be administered just before surgery and which doesn’t enter cells. Advances in magnetic resonance imaging using Iron oxide are showing to be particularly promising to improve diagnoses also in liver and neck tumours (Mack et al., 2002), (Schultz et al., 1999). The same nanoparticles can be used also for cancer therapy. Once delivered in the tumour they are involved in a field of radiofrequency waves that induce a heating of the Iron particles and of the cells that produces the death of the cells and the necrosis of the tumour. (Brigge et al., 2002), (Fahlvik et al., 1990), (Boomemain et al., 2001), (Hogemann et al., 2001), (Moghimi et al., 2001), (Allport et al., 2001), (Wunderbaldinger, 2001), (Wilhelm et al., 2002), (Moore et al., 2001). This new possible therapy is under evaluation with in-vitro and in-vivo experiments. The given references can be the base for a deepening of the matter. From our point of view, we look with a critical eye such an invasive diagnosis/therapy that releases intentionally not biodegradable particles inside the organism, particles which are inevitably sensed as foreign bodies. The reaction against that unusual, unexpected invasion is not the one produced against, for example, foreign proteins: we do not have specific enzymes, specific antibodies and there is no anaphylactic shock. Those particles represent something unknown to the human body, but when they are inside the blood circulation they are immediately sensed and interact with the blood’s components and they do it physically, chemically and biologically. This interaction is the key-point of modifications that can have systemic effects, not immediately the way it happens with foreign proteins, but after a more or less long while. Nanoparticles have a size that is roughly the same as the tri-dimensional size of proteins. The attachment of a nanoparticle to a protein (for physical adhesion, for electrostatic attraction, etc.) can cause a stretching of chemical bonds, a stress of the protein’s morphology, and the denaturation of the proteinic structure, causing the non recognition of the compound as something rightfully belonging to the organism. That can be the foundation of an immunological disease.

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But what is very critical, is the fact that after the imaging or the therapy have been performed, these foreign bodies cannot be disposed of, remain inside the body as foreign particulate matter, and their long- term effects are mostly unknown. Something about those effects is described in the following paragraph. Also the application of quantum dots in Medicine may be seen as a possible cause of unknown effects. Quantum dots (QDs) are semiconductive crystals that do not exist in nature, with dimensions ranging from 1 to 10 nm, and that can be fluorescent. They can emit light throughout the visible spectrum from the infrared to the ultraviolet. What makes our dots unique is that their luminescence can be tuned to any wavelength over a broad spectral range and be stable under ambient conditions (Bruchez Jr M et al., 1998), (Bruchez Jr M, 2005). (web ref. 9). Examples of QDs are Cadmium selenide, Lead selenide, and Lead Sulphur. Cadmium selenide can be the core of a QD with an outer shell of Zinc. Their peculiarity is the fluorescence emitted after having been excited. That fluorescence is more perceptible and more prolonged as to that of the normal fluorescent agents used in Medicine (fluorescin, etc.) They can be combined with antibodies in an easy way and can be driven inside a cell. Their fluorescence can be detected and be used to verify its state. Studies to use them in vivo seem to demonstrate their capacity to be attracted by tumour cells and show their borders in a body. But the main problem that remains beyond the technological beauty of these items is “what is the fate of them after use inside the body?” They can enter easily in the cells but they are inorganic matter masked by organic one.

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Nanopathology

2.3 The results of the nanopathology project In 2001, we coined a new word: “nanopathology”, meaning by that the collection of pathologies due to micro- and nanoparticles. At that time, this was a void concept, since a comparatively small number of pathologies were recognized worldwide as triggered by particulate matter. Silica particles and asbestos fibres are recognized to be able to induce lung diseases as silicosis and asbestosis. Inhalation of dust containing particles of quartz or fibres of a silicate is known to trigger lethal pathologies of the lungs. It is a well-known fact that the inhalation of mine dust or of cigarette smoke are risk factors for the onset of lung cancer. But a possible correlation with nanoscaled particulate matter was completely unknown. In 2001, submicronic matter was recognized to be dispersed in the environment, though very few studies existed, though none of them was exhaustive, on its behaviour, but nothing had been done for nanosized particles. No evidence identified nanodust as responsible for pathologies. Just micronic and, in very few instances, submicronic particles were considered to be the cause of pathologies (e.g. pneumoconioses). Now that nanotechnologies are a rapidly growing discipline, society is scared by possible side effects of the production, manipulation and use of nanoparticles, and nanosafety has become by necessity a priority. Many researchers are investigating the toxicity of nanoparticles towards cells, while our project has already gone further by investigating their invivo interaction and the impact they have to humans. Among the most abundant air pollutants in urban areas is particulate matter with a mean diameter ≤10 µm (also called PM10 by environmental toxicologists, defined as particulate matter with an aerodynamic size of 10 µm or smaller). Over the years, it has become clear that particles with a very low size (especially ≤100 nm) are more significant health-wise than larger particles, since they have been shown to be capable of inducing far more severe effects. (Oberdörster, 2001). The surface/size-ratio increases exponentially with the decreasing of particle sizes, leading to enhanced surface reactivity. This enhanced

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surface reactivity might lead to greater biological activity per given mass, compared to larger particles, which, in turn, might have effects, for example, on the internalization of particles into tissues, cells and organelles, or on the induction of oxidative stress (Oberdörster et al., 2005). By definition, particles with a size below 100 nm are called nanoscaled particles (short: nanoparticles) or ultrafine particles by toxicologists. Because of the minute size of nanoparticles, the internalization into the body’s tissues appears to be extremely easy. This was shown by experiments in human volunteers with radioactive-labelled Carbon nanoparticles (i.e. `Technegaz`) that were shown to pass rapidly into the systemic circulation after inhalation. Radioactivity could already be detected in the blood one minute after inhalation (Nemmar A. et al., 2002). Furthermore, animal studies revealed that inhaled nanoparticles were relocated into the liver (Oberdörster G. et al., 2002) and the brain (Oberdörster G. et al., 2004). Thus, nanoparticles seem to be able to circumvent the tight blood-brain-barrier and possibly cross the bloodplacenta barrier (Reichrtova E. et al., 1998), (Kaiglova A. et al., 2001). Moreover, it has been suggested, and we offered further evidence, that nanoparticles are involved in thrombus formation (Nemmar A. et al. 2002), (Gatti A.M. et al., 2004) and today we know that particulate air pollution is associated with enhanced mortality from respiratory and cardiovascular diseases (Pope C.A., 2000). As the sources of internalized nanoparticles (food, air, etc.) and the location of particle detection are generally far apart, a distribution via the blood stream must have occurred. Thus, endothelial cells, which line the inner surface of blood vessels, will have direct contact with the particles. Those cells are important in inflammation mechanisms and wound healing. Upon pro-inflammatory stimulation of the endothelium, adhesion molecules are expressed on the cell surface, thus mediating leukocyte attachment (e.g. E-selectin and intercellular adhesion molecule-1/ICAM-1). Besides, endothelial cells can release cytokines, such as interleukin-8 (IL-8, a key factor in neutrophil chemotaxis). Thus, these features contribute to the pro-inflammatory endothelial phenotype that permits the transmigration of leukocytes from the blood into the perivascular space (Cook-Mills J.M. et al., 2005).

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The activation of IL-8, E-selectin and ICAM-1 is regulated by the same transcription factors NF-κB (nuclear factor-κB) and AP-1 (activator protein-1) (Montgomery K.F. et al., 1991), (Roebuck K.A., et al., 1995), (Mukaida N. et al., 1994). The in-vitro tests we carried out within the scope of the European project considered the interaction of 5 different engineered nanoparticles: Titanium oxide: titania (TiO2), Silicon oxide: silica (SiO2), Polyvynilchoride (PVC), Cobalt and Nickel with cells (human endothelial, gut epithelial, liver epithelial and monocytic cells) and organs (in-vivo tests with rats). Those nanoparticles were examined with respect to the cellular internalisation and their influence on the cell viability, proliferative activity, and the pro-inflammatory endothelial phenotype. Titanium oxide (titania) was selected since it is employed in many beauty and sun-screen creams and in wall paints, Silicon oxide for its use in chewing gum and coatings, PVC because it is used in a bulk form for medical devices, Cobalt because of its toxicity and Nickel because it is allergenic1. One of the main characteristics of nanoparticles is their clustering, namely their capability to form microsized aggregates that provoke their sinking in the medium. So, a real interaction with cells is not allowed if proper procedures to disperse them in the medium are not adopted. Some authors claim good non-cytotoxic results, but they did not verify if a real cell-nano interaction was set-up. The nanoparticles mentioned were added to macrophages/monocytes, gut and liver epithelial cell culture medium and the cytotoxicity assays verified the cell survival, the production of inflammatory and defence mediators as the cytokine production, and the modulation of TLR expression. These are parameters that can give an idea if the presence of nanoparticles weakened the “healthy” state of the cells . The cytotoxicity studies carried out by Dr. D. Boraschi (CNR Pisa – Italy) showed that Cobalt nanoparticles induced a lesser expression of

1 SiO2 and TiO2 particles were produced by flame spray pyrolysis. The size range of SiO2 was 4-40 nm with 14 nm mean particle size; of TiO2 20-160 nm (mean size 70 nm), of Co particles (Sigma) 50-200 nm (mean size 120 nm), of Ni was about 62 nm (Nanoamor), of PVC (polyvinylcloride without phthalate) 100 nm.

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TLR4, TLR7, TLR8, TLR5 TLR10 and CD14. The other materials have different behaviours (Lucarelli M. et al., 2004). The tests verified that: 1- Nano-particles do not affect cell survival or proliferation. Only Co nano-particles are toxic at >100 mg/106 cells 2- Co nano-particles could have an M2 biasing inhibiting effect of TLR4/CD14-mediated responses. It was noted a down-regulation of TLR4 and CD14 expression, inhibition of reactivity to LPS in macrophages. 3- Nano-particles, in particular ZrO2, induce increase expression of viral TLR. There is a possible impact on cell reactivity to viral infections 4- SiO2 nano-particles have M1 biasing effect on macrophages. There is the induction of M1 markers in naive cells, amplification in M1 cells 5- Nano-particles, in particular TiO2, amplify IL-18 activity (role in initiation of autoimmunity?). That means that there is a differential expression of IL-18 regulatory molecules in macrophages and hepatocytes. So there is not an evident and significant cytotoxic effects at subtoxic concentration, but an impairment of cellular viability was observed at different levels (i.e. decrease of cell number, protein expression of the proliferation marker Ki67, and the metabolic activity). According to studies carried out by J. Kirkpatrick (University of Mainz – Germany) a pro-inflammatory effect in HDMEC (Human Dermal Microvascular Endothelial Cells) occurred after exposure to SiO2, Co, and Ni particles and was apparent by an enhanced release of IL-8. Only higher particle concentrations (25 and 50 µg/ml) induced this increase in IL-8 release. The E-selectin protein expression was enhanced by high amounts of Co-particles whereas Ni-particles induced no protein expression of E-selectin. In contrast to the particles, divalent Co and Ni ions induced the expression of all pro-inflammatory markers tested (i.e. IL-8, E-selectin, ICAM-1) (Peters K. et al., 2004). That means that the normal cell defences are weakened in presence of nanosized foreign bodies In order to evaluate and quantify of pro- and anti-angiogenic characteristics of the nanoparticles we selected another model system

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Nanopathology

that leads to the formation of in-vitro capillaries within a three-dimensional extracellular matrix (fibrin and type I collagen). The addition of the different particles (TiO2, SiO2, Ni, Co, PVC) into the three-dimensional matrix revealed that only the presence of Co particles led to changes in the in-vitro capillary phenotype. A number of abortive sprouts were formed and the developing sprouts were not so pronounced as in the positive control. However, cell viability appeared unaffected. Software-supported quantification of these Co-particleinduced changes revealed a significant reduction in the degree of angiogenesis in vitro. Also the tests employing the co-cultivation of endothelial cells together with the monocytic cell line U937 in the angiogenesis model system verified the hypothesis according to which the addition of PMAstimulated U937 cells to the culture could lead to a distinct decrease of the length of in-vitro-capillaries and also to a reduction of endothelial cell number. In detail the tests with endothelial cells verified: a- The nanoparticles investigated do not affect cell survival or proliferation. Only Co nanoparticles are toxic at >100 mg/106 cells. They inhibit the expression of LPS receptors TLR4 and CD14 and induce a down-regulation of mRNA expression for TLR4 and CD14 (the two receptor chains that recognise bacterial lipopolysaccharide and activate macrophage defence functions). An interesting result is that Co nanoparticles impair macrophages activation by bacterial LPS, in fact a negligible cytokine production was detected. These results mean that in presence of these metallic nanoparticles the macrophages become unable to mount appropriate defences to bacterial challenge. That means that danger exists of increased susceptibility to infections. These very interesting results were not obtained with Ni nanoparticles since, due to their forming micrometric aggregates and sinking in the medium, they did not interact with the cells. b- The Cobalt nanoparticles with endothelial cells showed an angiogenic behaviour. Our study has shown that human endothelial cells possess a large capability to internalize nanoparticles. All nanoparticles tested were taken up by the endothelial cells and to a major extent into vacuoles.

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The Cobalt nanoparticles showed an angiogenic behaviour, that can trigger a carcinogenic reaction. The results induced to put forward the hypothesis that the observed pro-inflammatory activation after Co particle exposure may be attributed to a release of divalent Co ions by the particles [Cobalt nanoparticles inside the cell corrode and release Cobalt ions], since the exposure of endothelial cells to these ions leads to the impaired endothelial viability and pro-inflammatory stimulation. This contrasts with the effects of the Ni particles. Here, the suggestion of a Ni ion release by the particles resulting in an induced pro-inflammatory stimulation is not congruent with the pro-inflammatory effects induced by the respective ions, since Ni ions induced both an increase in the release of IL-8 and the protein expression of endothelial cells adhesion molecules (i.e. E-selectin and ICAM-1), whereas Ni particles induced only an increased IL-8 release and the expression of adhesion molecules was not initiated. This indicates an activation mechanism for the Ni-particles that deviates from the Ni-ion-induced activation that is shown to occur via a cooperation of the above-mentioned transcription factors NF-κB and AP-1. Since oxidative stress is also a relevant aspect in the mechanisms of (Ni-) particulate matter-induced effects (Koziara et. al., 2003) this mechanism of differential activation of pro-inflammatory gene promoters might play a role. Thus, it can be suggested that the Ni-ion release by the nanoparticles remains under the critical limit for pro-inflammatory activation but further Ni nanoparticle induced effects (possibly oxidative stress) are responsible for the enhanced IL-8 release. Also the tests with the co-cultures (macrophage-endothelial cells) indicate a deep interaction of Co nanoparticles with the cells. The addition of PMA-stimulated U937 cells to the culture led to a distinct decrease in the length of invitro capillaries and also to a reduction of endothelial cell number. In addition to that, the number of U937 colonies within the 3-dimensional extracellular matrix rose. These in-vitro results indicate some mechanisms of actions of nanoparticles with cells and explain the in-vivo results and the clinical evidence.

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2.3.1 In-vivo experiments The 5 different materials mentioned above (Cobalt, Nickel, silica, titania, PVC) were implanted in rats in two forms (bulk samples as discs, and nanoparticles). That experiment was performed in order to verify if the size of these debris is an important factor in triggering pathological reaction. Each rat (see Fig. 2.3.1) was implanted in the dorsal muscle with two similar implants: an amount of 5 cc of nanoparticles and a 10-mm diameter, 0.8 mm height disc of the same chemical composition. For each group of materials, 2 rats were sacrificed after 6 months (short term), and 3 rats were sacrificed after 12 months (long term). In addition, 5 control rats were implanted with a reference material. Two of them were sacrificed after 6 months and 3 of them were sacrificed after 12 months. Five rats were fed by gavage with a mixture of nanoparticles (50% Ni and 50% ZrO2 with two different ranges) (about 0.030 – 60 mg by day): 2 were sampled after 6 months and 3 were sampled after 12 months, in order to see if the small-size particles can negotiate the bowel barrier and contribute to the pathogenesis of some diseases. The animals were sacrificed after 6 and 12 months. After the short- and long-term implantation, samples were explanted and the gross necropsy of the animals performed. All samples were formalin-fixed and paraffin-embedded, microtome-sliced and HematoxylinEosin-stained. The sections were analyzed histopathologically and under ESEM in order to verify the interface of the bulk and nanosized materials after the interaction. Our animal studies verified that the metallic nanoparticles were cancerogenic, while the bulk material induced just a fibrotic capsule with a granulomatosic reaction.

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Fig. 2.3.1 Cobalt and Nickel nanoparticles induced rhabdomyosarcoma in the rats’ back. The metallic disks induced only fibrotic capsules. (Courtesy of Biomatech-France)

The other materials (PVC, silica, and titania) developed only a fibrotic capsule or a granulomatous tissue. The reason could be the agglomeration of the nanoparticles that transforms them to micrometric debris. In this case, the body reacts to the size of particulate matter, as the materials are chemically inert. A summary of macroscopic and microscopic findings are presented in Tab. 2.3.1.

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Nanopathology Tab. 2.3.1 Synopsis of the histological findings. Material

6-month period

12-month period

TiO2: NP TiO2: B SiO2: NP SiO2: B

Granuloma Inflammation Inflammation Inflammation

PVC: NP

Granuloma

PVC: B Co: NP

Inflammation Preneoplasia Inflammation + granuloma Neoplasia Neoplasia

Granuloma Inflammation Inflammation Inflammation Granuloma + fibroblastic proliferation Inflammation Neoplasia*

Co: B Ni: NP Ni:B

Preneoplasia* # #

Note: B= bulk, NP= Nanoparticles; * Animals were euthanized after 8 months. # No analysis - animals were euthanized after 6 months.

TiO2 – In detail: a thin, fibrous, moderately capillarized pocket surrounding the bulk material implanted subcutaneously was observed in all cases, but no granulomas were present. SiO2 – In detail: in all cases, an adherent thin fibrous pocket surrounding the bulk material implanted subcutaneously could be seen, like what happened with TiO2, without any evidence of local intolerance. The inflammatory infiltrate consisted mainly of lymphocytes and monocytes, but not of mast cells. At the site of the intramuscular implantation, neither particles nor macroscopic abnormality could be seen. Histological examination of the specimens with implanted particles revealed inflammatory infiltrates which were localised subcutaneously and intramuscularly. An increased numbers of mast cells, typically localized perivascularly, could be detected in most cases. PVC – Also in all these cases, a thin fibrous pocket around the bulk material implanted subcutaneously was observed, without any visible, local lesion. And also in these cases, the bulk material itself did not reveal anything abnormal. Histological evaluation of implanted PVC

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bulk showed discrete chronic inflammatory infiltrates. No particles could be observed. The sites implanted intramuscularly with nanoparticles did not show anything macroscopically remarkable. In 8 cases out of 10, particles were easily visible in the subcutaneous tissue. On histology, all cases of implanted PVC nanoparticles revealed intramuscular foreign body-type granuloma. Macrophages and multinucleated foreign-body giant cells could be seen around amorphous, unstained polygonal material that did not exhibit birefringence under polarized light. However, the particles revealed intense cherry-red colour by staining with the Oil Red-reaction. A slight perigranulomatous fibrosis was also observed. An interesting find is the observation of foci of fibroblastic proliferation in three animals. The cells were spindle-shaped and showed some nuclear pleomorphism. On immuno-histochemistry, they showed an enhanced nuclear expression of PCNA (Proliferating Cell Nuclear Antigen). Cobalt – Clinical examination revealed the formation of nodules in three animals occurred within the 6-month observation period. Then, all remaining animals implanted with Co developed nodules at the sites where nanoparticles had been implanted. One animal of the 12-month observation group died 8 months after implantation. So, for ethical reasons because of the development of these handicapping tumors, all remaining animals were sacrificed at 8 months. In three out of the four cases belonging to the 6-month group and in six cases of the 8-month group, nodules developed at the site where nanoparticles had been implanted. Through histological observation of the specimens with implanted nanoparticles, malignant, intramuscular, mesenchymal tumors at the original implantation site was demonstrated in one out of four animals of the 6-month observation period and in five out of six cases of the 8month period. Particles were observed in three of six animals belonging to the 8-month group. No such tumors were not observed around the bulk material, where a discrete inflammatory infiltrate composed of mononuclear cells and lymphocytes was present. In the subcutaneous area, a discrete fibrosis had developed, but not granulomas.

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In two of the four animals of the 6-month observation period (at the nanoparticulate site) and in two of the six animals of the 8-month observation period, a capsule with fibroblastic proliferations was visible. The cells showed an increased pleomorphism of the nuclei and mitotic rate. In addition, they displayed strong expression of the proliferation marker PCNA. These lesions are classified as pre-neoplasia. Grouped together, these results show the presence of a malignant mesenchymal tumor in five of the six cases of implanted Co nanoparticle sites and two further cases (one around bulk material and one at the nanoparticulate site) with pre-neoplasia. Nickel – Three of the animals from the Ni-implanted group died respectively 4, 4.5 and 5.5 months after implantation. Because of such a high mortality rate and the development of tumors, we decided to sacrifice all the remaining animals belonging to that group at 6 months. Both subcutaneous and intramuscular implantation sites had developed visible nodules in all cases and in all cases necrosis was detected inside the nodule associated with deposits and exudate seen in the implanted subcutaneous pocket. Both Ni bulk and nanoparticles of implanted Ni showed a malignant mesenchymal tumor surrounded by a fibrous capsule localized subcutaneously and intramuscularly. In general, abundant areas of central necrosis, dystrophic calcification and ossification were also observed. In contrast with that, tumors in tissue specimens implanted with bulk material showed a central cystic component, suggestive of the implant site. In addition to that, necrosis looked less important, and ossification was more rarely seen. Particles could not be observed histologically. The findings indicate a malignant mesenchymal tumor, which is classified on the basis of the morphology and the immuno-histochemical marker profile as rhabdomyosarcoma. The results obtained, though preliminary and in need to be confirmed by further experiments, show that size is a decisive factor in determining the compatibility of a material with the organism. As to materials, metals seem to be much more reactive in a pathogenic way then ceramics and polymers. These results must be reminded for all the following chapters.

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2.4 Bibliography Ai, H., Meng, H., Ichinose, I., Jones, S.A., Mills, D.K., Lvov, Y.M. and Qiao, X. (2003). Biocompatibility of layer-by–layer self-assembled nanofilm on silicon rubber for neurons, J Neurosci Methods. 128 (1-2), 1-8 Babes, L., Denizot, B., Tanguy. G., Le Jeune, J.J. and Jallet, P. (1999). Synthesis of Iron oxide nanoparticles used as MRI contrast agent: a parametric study, J. of Coll and Interface Science 212, 474-482 Ballestri, M., Baraldi, A., Gatti, A.M., Furci, L., Bagni, A., Loria, P., Rapanà, R., Carulli, N. and Albertazzi, A. (2001). Liver and kidney foreign bodies granulomatosis in a patient with maloocclusion, bruxism, and worn dental prostheses Gastroenterology. 121, 1234-38 Balogh, L., Valluzzi, R., Laverdure, K., Gido, S.P., Hagnauer, G. and Tamalia, D.A. (1999). Formation of Silver and gold dendrimers nanocomposites, J. of Nanoparticle Research 1, 335-368 Bellingham, C.M., Lillie, M.A., Gosline, J.M., Wright, G.M., Starcher, B.C., Bailey, A.J., Woodhouse, K.A. and Keeley, F.W. (2003). Recombinant human elastin polypeptides self-assemble into biomaterials with elastin-like properties, Biopolymers. 70 (4), 445 Borm, P.J.A. and Kreyling, J. (2004). Toxicological hazards of inhaled nanopartciles: potential implication for drug delivery, Journal of Nanoscience and Nanotechnology, 4, 1-11 Brook, R.D., Franklin, B., Cascio, W., Hong, Y., Howard, G., Lipsett, M., Leupker, K., Mittleman, M., Samet, J., S.C. Smith and Tager, (2004). Air pollution and cardiovascular diseases, Circulation, 109, 2655-2671 Bruchez Jr M, Moronne M, Gin P, Weiss S, Alivisatos AP., (1998) Semiconductor nanocrystals as fluorescent biological labels. Science, 281, 2013 Bruchez Jr M, Turnig A, (2005) All the light on: quantum dots in cellular assays, Curr.Opin.Chem.Biol, 9 (5), 533-7. Buxton, D., Lee, S. C., Wickline, S., and Ferrari, M. (2003). Recommendations of the National Heart, Lung and Blood Institute Nanotechnology Working Group, Circulation,108 (22), 2737-42 Carnahan, M. A. and Grinstaff, M.W. (2001). Synthesis and characterization of poly (glycerol-succinic acid) dendrymers, Macromol. 34, 7648-55 Castner, D. G. and Ratner, B.D. (2002). Biomedical surface science: foundation to frontiers, Surface Science, 500, 28-60 Chakrabarti, R. and Klibanov, A. M. (2003), Nanocrystals modified by peptide nucleic acids (PNAs) for selective self-assembly and DNA detection. J Am Chem Soc, 125, 12531-12540 Colvin, V. L. (2003). The potential environmental impact of engineered nanomaterials, Nat. Biotechnol., 21(10), 1166-70 Cook-Mills, J. M., and Deem, T. L., (2005) Active participation of endothelial cells in inflammation. J Leukoc Biol, 77, 487-495 Dobson, J. (1999). Nanoparticles and nanocomposites, Commercial and Industrial Applications of Microengineering and Nanotechnology, London, 26th April, 1-11

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Editorial, (2003). Nanomedicine: ground for optimism, and a call for papers, Lancet. 362 (9385), 673 Eppel, S.J. and Tong, W. (2002). Nanobiomaterial Workshop, Ed. Soc. of Biomaterials, Hawaii, April Gao, H., Kong, Y., Cui, D. and Ozkan, C.S. (2003). Spontaneous insertion of DNA oligonucleotides into carbon nanotubes, Nanoletters, 3, 471-473 Gatti, A.M. and Rivasi, F. (2002). Biocompatibility of micro- and nanoparticles Part I in liver and kidney.” Biomaterials, 23 (11), 2381-2387 Gatti, A.M. (2004). Biocompatibility of micro- and nanoparticles in the colon (part II) Biomaterials 25 ( 3), 385-392 Gatti, A.M. (2005). Handbook of Nanostructured biomaterials and their applications. Ed. by American Scientific Publisher USA cap. 12, 347-369. Gatti, A.M. (2005). Symposium Keynote Presentation “Risk Assessment of NanoParticles and Nano-Technologies for Human Health. 7th World Biomaterials Congress, 748-749 Gatti, A. M., Montanari, S., Monari, E., Gambarelli, A., Capitani, F., and Parisini, B., (2004) Detection of micro- and nano-sized biocompatible particles in the blood. J Mater Sci Mater Med, 15, 469-472. Goodman, S.B., Fornasier, V.L., Lee, J. and Kei, J. (1990). Histological effects of implantation of different size nanoparticles, J.Biomed, Mat.Res. 24, 517-524 Harishingani, M.G., Barentsz., J., Hahn, P.F., Deserno, W.M., Tabatabaei, S. and Hulsbergen, (2003), Noninvasive detection of clinically occult lymph-node metastases in prostate cancer Eng J Med. 19, 348(25), 2491-9. Hett, A. (2004). Nanotechnology: from the insurers’ perspectives, Nanotechnologies: A preliminary risk analysis 103, (www.europa.eu.int/com/health/ph_risk/events_risk_en.htm) Kaiglova, A., Reichrtova, E., Adamcakova, A., and Wsolova, L., (2001) Lactate dehydrogenase activity in human placenta following exposure to environmental pollutants. Physiol Res, 50, 525-528. Kilts, C.D. (2003). Potential new drug delivery systems for antidepressants: an overview, Clin.Psychiatry. 64(18), 31-33 Koziara, J.M., Lockman, P.R., Allen, D.D. and Mumper, R.J. (2003). In-situ blood brain barrier transport of nanoparticles, Pharm.Res. 20(11), 1772 Lehmann-Horn, F. and Jurkat-Rott, K. (2003). Nanotechnology for neuronal ion channels, J Neurol. Neurosurg Psychiatry. 74(11), 1466 Li, X.W., Lee, D.K., Chan, A.S. and Alpar, H.O. (2003). Sustained expression in mammalian cells with DNA complexed with chitosan nanopartciles, Biochim. Biophys Acta. 1630(1), 7-18 Lockman, P.R., Mumper, R.J., Khan, M.A. and Allen, D.D. (2002). Brain uptake of thiamine-coated nanoparticles, Drug Dev Ind Pharm. 28(1), 1 Lucarelli, M., Gatti, A.M., Savarino, G., Quattrini, P., Martinelli, L., Monari, E. and Boraschi, D. (2004). Innate defence function of macrophages can be biased by nano-sized ceramic and metallic particles, Cytokin Network, 15 (4), 1-8

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Mack, M.G., Balzer, J.O., Straub, R., Eichler, K. and Vogl, T.J. (2002). Superparamagnetic Iron oxide.enhanced MR imaging of head and neck lymph nodes, Head and Neck Imaging, 222(1), 239-244 Mayer, A.B. (1998). Formation of noble metal nanoparticle within polymeric matrix: nanoparticle features and overall morphologies Mat. Sci. and Engineering. C6, 155-66 Maynard, A.D., Boron, P.A., Foley, M., Shvedova, A.A., Kisin and Castranova, E.R. (2004). Exposure to carbon naotubes, Journal of Toxicology and Environmental Health 67A,87-107 Mitra, S.B., Wu, D. and Holmes, B.N. (2003). An application of nanotechnologies in advanced dental materials, J.Am. Dent. Ass. 134(10), 1382 Montgomery, K. F., Osborn, L., Hession, C., Tizard, R., Goff, D., Vassallo, C., Tarr, P. I., Bomsztyk, K., Lobb, R., Harlan, J. M., and et al., (1991), Activation of endothelial-leukocyte adhesion molecule 1 (ELAM-1) gene transcription. Proc Natl Acad Sci U S A, 88, 6523-6527. Mukaida, N., Okamoto, S., Ishikawa, Y., and Matsushima, K., Molecular mechanism of interleukin-8 gene expression. J Leukoc Biol 1994, 56, 554-558. Nemmar, A., Hoylaerts, M. F., Hoet, P. H., Dinsdale, D., Smith, T., Xu, H., Vermylen, J., and Nemery, B., (2002), Ultrafine particles affect experimental thrombosis in an in vivo hamster model. Am J Respir Crit Care Med 166, 998-1004. Nemmar, A., Hoet, P.H.M., Vanquickenborne, B., Dinsdale, D., Thomeer, M., Hoylaerts, M.F., Vanbilloen, H., Mortelmans, L. and Nemery, B. (2002). Passage of inhaled particles in to the blood circulation in humans, Circulation, 105 (4), 411-417 Oberdorster, G., Sharp, Z., Atudorei, V., Elder, A., Gelein, R., Lunts, A., Kreyling, W., and Cox, C., (2002) Extrapulmonary translocation of ultrafine carbon particles following whole-body inhalation exposure of rats. J Toxicol Environ Health A, 65, 1531-1543. Oberdorster, G., Sharp, Z., Atudorei, V., Elder, A., Gelein, R., Kreyling, W., and Cox, C., (2004) Translocation of inhaled ultrafine particles to the brain. Inhal Toxicol, 16, 437-445. Oberdorster, G., Oberdorster, E., and Oberdorster, J., (2005) Nanotoxicology: An emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect. 113(7), 823-39. Orive, G., Hernandez, R.M., Rodriguez Gasco, A., Dominguez-Gil, A. and Pedraz, J.L. (2003). Drug delivery in biotechnology: present and future, Curr. Opin Biotechnol. 14(6), 659 Pereira, M.C., Pereira, M.L. and Sousa, J.P. (1999). Histological effects of Iron accumulation on mice liver and spleen after administration of metallic solution, Biomaterials 20, 2193-2198 Peters, K., Unger, R., Gatti, A.M., Monari, E. and Kirkpatrick, J. (2004). Effects of nanoscaled particles on endothelial cell function in vitro:Studies on viability, proliferation and inflammation, J. of Material Science: Mat. in Medicine 15 (4), 321-325

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Pope, C. A., 3rd, (2000), Epidemiology of fine particulate air pollution and human health: biologic mechanisms and who's at risk?, Environ Health Perspect, 108 Suppl 4, 713-723. Price, R.L., Haberstroh, K., and Webster, T. (2004) Improved osteoblasts viability in the presence of smaller nanometre dimensioned carbon fibres, Nanotechnology, 8, 101 Rantioa, J. and Chickhale, P.J. (2004) Drug delivery systems for brain tumor therapy, Current Pharmaceutical Design, 10(12) Reichrtova, E., Dorociak, F., and Palkovicova, L., (1998), Sites of lead and nickel accumulation in the placental tissue. Hum Exp Toxicol, 17, 176-181 Revell, PA., Al Saffar, N. and Kobayashi, A. (1997). Biological reaction to debris in relation to joint prostheses, Proc. Inst. Mech. Eng. 211(H), 187-197 Roebuck, K. A., Rahman, A., Lakshminarayanan, V., Janakidevi, K., and Malik, A. B., (1995) H2O2 and tumor necrosis factor-alpha activate intercellular adhesion molecule 1 (ICAM-1) gene transcription through distinct cis-regulatory elements within the ICAM-1 promoter. J Biol Chem, 270, 18966-18974 Saebo, K.B. (2004). Degradation, metabolism and relaxation properties of Iron oxide particles of MRI, Acta Universitatis Upsaliensis, Uppsala Salem, A.K., Searson, P.C. and Leong, K.W. (2003). Multifunctional nanorods for gene delivery, Mat. Mater. 2(10), 668 Sando, S., Sasaki, T. and Aoyama, Y. (2003). Encapsulation of DNA with neutral glycocuster nanoparticles. A step toward artificial viruses, Nuclei Acids Res Suppl. 3, 289-90 Saxe, S.R., Wekstein, M.W., Kryscio, R.J., Henry, R.G., Cornett, C.R., Snowdon, D.A., Grant, F.T, Schmitt, F.A., Donegan, S.J., Wekstein, D.R., Ehmann, W.D. and Markesbery, W.R. (1999). Alzheimer disease, dental amalgam and mercury, J.Am.Dent.Assoc. 130(2), 191-199 Schler, E. (2003). The public has its own view of what is a risk, Nature, 424(6946), 237 Schroeder, U., Sommerfeld, P., Ulrich, S., Sabel, BA., (1998) Nanoparticle technology for delivery of drugs across the blood-brain barrier, J Pharm Sci. 87(11), 1305-7 Schultz, J.F., Bell, J.D., Goldstein, R.M., Khun, J.A. and McCarty, T.M. (1999). Hepatic tumor imaging using Iron oxide MRI: comparison with computed tomography, clinical impact, and cost analysis, Annals of Surgical Oncology, 6(7), 691-698 Shanbagh, A.S., Jacobs, J.J., Black, J., Galante, J.O. and Grant, T.T. (1994). Macrophages/particle interactions : effect of size, J.Biomed.Mat.Res. 28, 81-90 Stueber, M., Holleck, H. and Haefke, H. (2002). New nanocomposite-based wearresistant and self-lubricating PVD coating for future applications in tools and components (nanocomp) Proceed. of the 3rd joint EC-NSF workshop on Nanotechnology, (Lecce), 154 Tamura, K., Takashi, N., Akasaka, T., Roska, I.D., Uo, M., Tostuka, Y. and Watari, F. (2003). Effect of micro/nanoparticle size on self function and morphology, Key Engineering Materials, 254-56 Technical report (Annual) of the Air Force Armament Laboratory (1978) – Armament development and test Center, Eglin Air Force Base, Florida, USA. Project n° 06CD0101 (From October 1977 to October 1978)

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Urban, R., Jacobs, J., Tomlinson, M. et al. (2000). Dissemination of wear particles to the liver, spleen and abdominal lymph nodes in patients with hip or knee replacement. J Bone Jt Surg. 82-a(4), 455-477 Warheit, D.B. (2004). Nanoparticles and health impacts, The Dupont Company Materials Today, 2. 32-35, Wenstrup, D., Ehmann, W.D. and Markesbery, W.R. (1990). Trace elements in balances in isolated subcellular fractions of Alzheimer’s disease brains, Brain Research 533,125-130 Williams, D. (2003). nanocrystalline metals:another opportunity for medical devices, Med Device Technol 14(9):12, 16-7 Whiteside, G., Mathia, J.P., John, P. and Seto, C.T. (1991). Molecular self-assembly and nanochemistry: a chemical strategy for the synthesis of nanostrucures, Science. 254, 1312 Zhang, S. (2002). Emerging biological materials through molecular self-assembly, Biotechnol. Adv. 20(5,6), 321 Zheng, M., Jagota, A., Strano, M.S., Santos, A.P., Barone, P., Chou, S.G. and Diner, B.A. (2003). Structure-based carebon nanotube sorting a sequence-dependent DNA assembly, Science. 28:302(5650), 1545-8 Web Ref. 1: http://www.defra.gov.uk/environment/nanotech/research/pdf/nanoparticlesrisk report.pdf Web Ref. 2: http://www.tenk.fi/nechelsinki/Presentations/Wybo%20Dondorp%20text.pdf Web Ref. 3: ftp://ftp.cordis.europa.eu/pub/nanotechnology/docs/inputs_nanoecotox.pdf Web Ref. 4: http://www.nanoforum.org/ Web Ref. 5: http://unesdoc.unesco.org/images/0014/001459/145951e.pdf and http://portal.unesco.org/shs/en/ev.php-URL_ID=9648&URL_DO=DO_TOPIC& URL_SECTION=201.html Web Ref. 6: www.nanotechproject.org/file_download/164 Web Ref. 7: ftp://ftp.cordis.europa.eu/pub/nanotechnology/docs/nanomedicine_bat_en.pdf Web Ref. 8: http://cordis.europa.eu/nanotechnology/nanomedicine.htm Web Ref. 9: http://www.llnl.gov/str/Lee.html

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Chapter 3

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm ___________ 3.1 Introduction

7

his chapter contains a number of clinical cases studied on bioptic or autoptic samples. No homogeneity or statistical meaning is to be expected or looked for here, as this is just the start of a collection of cases that will hopefully keep coming and being added to the ones illustrated here. Anyhow, what is described in this part of the book represents answers given to patients, or their relatives or doctors when the patients had died, regarding the exposure those patients had undergone. In many instances, we had no advice to give as to a possible treatment, but when somebody knows he is dying, knowing why can be important to him. In any case, the experience we are collecting can be useful to prevent the repetition of similar cases or to find an explanation to cryptogenic diseases. It was hard, when not impossible at all, to guess how particles actually behave once they have entered the organism. So, one of the problems was to select the organs that make up a favourite mark for those particles, if such a thing as favourite organs exists. Inflammatory pathologies of unknown origin had already proved to be a very interesting source of knowledge, but in our then limited experience we had seen that dust might possibly be blamed for other forms of adverse reactions like, for example, the formation of blood

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thrombi or - but in that case the thing was far more complicated - for some neurological diseases or pathologies whose classification is controversial like chronic fatigue. We studied more than 600 aggregate cases of vein thrombosis, chronic fatigue, lymphomas, solid cancers of various organs, etc. Reference tissues were obtained from cadavers of young, presumably healthy, subjects, died in car accidents. All the pathological specimens we had the chance to examine showed the presence of inorganic, non biocompatible and non biodegradable debris, ranging 10 nm - 10 microns. Some of those debris (10-100 nm) were also photographed inside cellular nuclei. The observation and study of the pathological samples we came across and are described here showed a few interesting data. In the cases of biological reactions typical of some diseases like cryptogenic granulomatosis, sarcoidosis and Crohn’s disease, foreign bodies were always present inside the granulomatous tissue. In some cases, the chemical composition of the particulate detected made it possible to identify the kind of exposure the patient had undergone. Micro and nanoparticles were always present in cancerous tissues, but it must be emphasized that this constant presence was due to a pre-selection of the cases we observed. As a matter of fact, in a number of patients suffering from cancer we had the chance to check outside the scope of this project, no particles were found. This is only natural, as predisposing and causing factors exist, such as genetics, radiations, exposure to organic solvents and many other pollutants. In the cases investigated, mainly concerning primary cancers, the inorganic foreign bodies were concentrated at the interface between cancer and healthy tissue. This specific location, never discovered before, seems to be meaningful in understanding the mechanism regulating the onset of cancerogenicity. This specific location could also explain why in some cases we can’t find anything: The planar section we analyzed was part of a bulk, tri-dimensional sample and it is likely that the interface was not included in that particular section. Once, we could not use the tissue harvested from one of the “healthy” cadavers we used as reference, as it contained Calcium carbonate. It turned out that that boy had been drug addicted and the mineral was used

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to blend the drug. After that, considering the difficulties that carrying out an anamnestic study implies with relatives or friends about the health state of somebody who has died, our attention was addressed to people we had reasons enough to consider “non contaminated”. After the 3-year project sponsored by the European Community we carried out, the reference samples, i.e. “the standards”, are represented by the internal organs of feti of induced abortions. Their tissues are “clean”, since we believed they had not been exposed to environmental pollution. That was an extreme choice to obtain reference samples with “zero contamination”, but that choice reserved some surprise. In fact, the study mentioned above involved the verification of animal and human malformed feti. In a few instances, dust was detected in their tissues, and that showed that nano-contamination can be shared between mother and child through the umbilical circulation.

Fig. 3.1 Schematic view of the entrance ways of particles in the organism.

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Fig. 3.1 serves to understand the paragraphs that follow, as it describes graphically how particles, be they inhaled or ingested or, as a matter of fact, introduced in any other way into the organism, reach any tissue. The lung is the first structure to be interested, followed by the blood, the liver and the kidney. After that, the digestive system has been treated, but the contaminations found in the liver and the kidney could have originated from inhalation as well as from ingestion. Sperm closes the chapter. We did not describe anything regarding the brain, as we could examine only two cases, in both of which particulate matter could be seen. All the images below are electron-microscope picture of pathological tissues. They are presented together with their relative chemical EDS spectrum (see Appendix). All spectra show the peaks of Carbon and Oxygen that belong to the biological substrate, to the Carbon support where the specimen is laid, but may as well be part of the spot aimed at, which is in all cases an inorganic particle. We cannot be certain if Carbon and Oxygen are real components of the particle or just an artifact. For that reason, Carbon and Oxygen are not listed in the elements detected. 3.2 Lung We may say that we inaugurate our life with an inspiration of air. In fact, neither our entire body nor any of the cells that make it up could survive without Oxygen, since much of our metabolism is based upon a series of oxidations. That is the reason why the inhalation of air, a blend of gases where Oxygen represents about one fifth, is necessary. But the air we breath is not so pure as we would desire it to be. Among many other pollutants, it contains all the by-products of the combustion processes related to fires, heating, and industrial and automotive processes. So it contains gases like Carbon dioxide and Carbon monoxide, furans, dioxins, hydrocarbons, etc. And it contains dust which for size and chemical composition is different from the natural one.

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Medical Geology is a new discipline of investigation that studies the impact of natural dust on human health. (Centeno, 2005). Who, for instance, lives close to a volcano has a higher probability to suffer from lung diseases, since, even if that volcano is not active, the erosion of lava streams by rain, winds and temperature changes pollutes the environment in a constant way. But the industrial and/or combustive processes release dusts with chemical compositions that have never been present on this planet and, therefore, are completely unknown to the human body and its metabolism. Because of its morphology and size, inhaled dust can interfere physically with the passage of Oxygen through the alveoli, since it creates a hard-to-negotiate barrier to the exchanges of the gases (Oxygen against Carbon dioxide). But dust, perceived as a foreign body in the alveoli, can also create a different local problem consisting in an inflammatory reaction which can degenerate. For those very well-known reasons, many environment-protection agencies, among which that of the European Community, set limits to the concentration of particulate pollution in the atmosphere. Nowadays those limits are based on the mass of PM10 in a fixed volume, but a more scientific determination, like number of particles evaluated according to the reciprocal of their size is certainly hoped for. (Armaroli, N. et al., 2003), (L.M. Brown et al., 2000), (J. Ruuskanen et al., 2001), (S. Ebelt et al. 2001), (R.M. Harrison et al., 2000). The European Community is preparing a new regulation based on PM2.5, unfortunately still considering just mass. For these reasons, we started to investigate samples of lungs affected from pathologies with different levels of seriousness. Tab. 3.2.1 reports the list of the pathologies investigated and all the images that follow are related to these cases. As can be seen, there are some cases seemingly not related in a direct way to lung pathologies. For instance: Hodgkin’s and non-Hodgkin’s lymphoma, skin pesudolymphoma, Wegener’s granulomatosis, nephrocalcinosis, and Gulf War Syndrome. All these pathologies are multi-organ-diseases and we analyzed many samples coming from the same subject, included the lung, even if that was not the organ that looked mainly interested by the pathology.

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We selected the particles shown according to peculiarity of morphology, size or chemistry. The lungs, as well as the mouth, the stomach or the colon have a surface that has been exposed for a long time to pollution, so we find much dust that remained trapped for a certain period of time. In the case of a half-mm2 sample of the mouth mucosa we found as many as 600 sub-micronic particles of 10 different chemical compositions. Tab. 3.2.1 List of the investigated samples of the lung. Pathology

No.

Fibrosis Silicosis Granulomatosis Wegener’s granulomatosis Pulmonary Emphysema Sarcoidosis Gulf War Syndrome Pneumothorax Pleural Coniosis Nephrocalcinosis Racemose ossification Hodgkin’s Lymphoma Non-Hodgkin’s Lymphoma Skin Pseudo-lymphoma Pleural Mesothelioma Cancer Not diagnosed Reference Total

8 1 3 1 1 3 2 14 1 1 1 1 1 1 32 17 4 20 112

First of all, a basic concept must be clear: most gross particles remain on the inner surface of the alveoli, while the smaller ones, and the more so the smaller they are, can negotiate that anatomical barrier and enter the bloody stream. Fig. 3.2.1a shows the surface of a lung alveolus with large particles of surely combustive origin. That can be inferred by their high porosity, something typical of combustion processes. Fig. 3.2.1b, instead, shows the section of a blood vessel with red cells in its lumen stuck together,

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and white particles with a size smaller than 2 µm. They look whiter than the biologic matter around them because of their atomic density, higher than the biological surroundings’. In this context they can express their possible non-biocompatibility by a thrombogenic effect and thus induce the formation of a thrombus. Pulmonary thromboembolism may be the consequence of that phenomenon in the venous circulation, while stroke and infarction may be what the phenomenon triggers in the arteries. What we documented in the blood vessels suggests also another possible effect. Particles are much harder than any blood components and of the tissue making up the vessels. They are transported by the flow, so they can hit and “scratch” the endothelium, causing a vasculitis. As a matter of fact, many of the pathologies investigated present also that particular symptom.

Fig. 3.2.1a – Fig. 3.2.1b ESEM images of 2 types of particles. The former is deposited on the surface of an alveolus, while the latter is trapped inside a blood vessel.1

Much literature exists describing an increase of lethal episodes in cardiopaths in relation with an increase of PM2.5 in the atmosphere, and the fact becomes easy to explain in the light of our observations (Courter

1 This photo was published in Handbook of Nanostructured Biomaterials and Their Applications, Volume 2, Gatti A.M., Montanari S. “Risk assessment of micro and nanoparticles and the human health”, cap. 12, 347-369 ed American Scientific Publisher USA 2005.

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L. A. et al., 2007), (Schwarze, P. E. et al., 2006), (Newby, J. A. et al., 2006). For a long time, silica and silicates particles have been known as the responsible to a form of pneumoconiosis called silicosis, while asbestos has been blamed as the cause of mesothelioma (asbestos can be considered nanoparticle, since one of its dimensions is in the nanosize). Pleural Malignant Mesothelioma, a malignant tumour that spreads from the parietal pleura in the chest cavity and sometimes involves the lung, is a kind of tumour strictly correlated with inflammatory reactions and is due to environmental contamination. Though not the only one, asbestos exposure is the most common risk factor (involving more or less 80 percent of mesothelioma patients), but other promoting factors are known, including chronic lung infections, tuberculous pleuritis, radiation and exposure to the simian virus 40 (SV40) or other mineral fibres. Of that highly malignant tumour very little is known to date about its molecular and metabolic features and awareness is very recently growing about the relevance of cell-to-cell signalling involved in the modulation of its cell growth and differentiation, e.g. the presence of NOX and other signal molecules (Soini et al., 2000) recently found. Of course it is impossible to see gaseous compounds like NOx, COx, SOx, etc., so only their effects are detectable, like the production of cytokines or others compounds of the cell down to gene expression. Our studies started from a pragmatic vision: if particulate matter originating from environmental pollution is to be blamed for some human diseases, we must find it where the biological tissue shows its main lesions. Fig. 3.2.2 shows asbestos fibres (a silicatic compound containing, according to their species, Calcium, Silicon, Magnesium and Iron) inside a pathological human lung tissue affected by mesothelioma. Around a fibre, formations called “pearls of asbestos” can be seen. Those are precipitates of Iron protein induced by the chemical reaction of the silicatic fibre with the extracellular matrix, rich in Iron. The periodicity of those pearls is due to the diffusion of ions that rules the phenomenon. This aspect is well known among biomaterialists expert of bioactive glasses (a compound of Silicon-Sodium-Calcium and Phosphorus),

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materials commonly used for bone defect repair. (Gatti, et al, 1998) In fact, these materials are not inert in the human organism, but degrade releasing ions that create a concentration gradient activating diffusive processes. In this specific case, the glass releases immediately Sodium ions and there is a diffusion from the core toward the surface of the glass of the Phosphorus and Calcium ions, leaving a matrix rich in Silicon (a Silicon hydrated gel). The surface grows now rich in Calcium and Phosphorus that precipitate as Calcium phosphate, the matrix for the colonization of the osteocytes. This way, new bone is rapidly generated.

Fig. 3.2.2 ESEM Images of asbestos fibres with its chemical composition, found inside a pleural mesothelioma. “Pearls of asbestos” (arrow) are visible(marker 5 µm), (marker 10 µm).2

Pleural mesothelioma has been described in literature for a long time as related to the inhalation of asbestos nanoparticles with their peculiar form of needles. But it also a fact that some patients who developed that 2 This photo was published in Handbook of Nanostructured Biomaterials and Their Applications, Volume 2, Gatti A.M., Montanari S. “Risk assessment of micro and nanoparticles and the human health”, cap. 12, 347-369, ed American Scientific Publisher USA 2005.

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disease were never exposed to that pollutant. It is a well-known fact that there is a correlation between environmental pollution and some diseases, so we looked for environmental contaminants in the pathological lung tissues. Fig. 3.2.3 shows another example of asbestos fibres embedded in the lung tissue. Iron protein pearls are clearly visible.

Fig. 3.2.3 Asbestos fibre in a pleural mesothelioma. (marker 10 µm).

Asbestos is evident in the latter picture, with the typical pearls of Iron protein. It is important to observe the pointed shape of asbestos crystals, a shape that makes penetration into tissue easier as compared to other, bulkier shapes. Racemose ossification, the disease related to Fig. 3.2.4 is a, rare, diffuse pulmonary ossification of unknown origin in which mature bone is found in the pulmonary parenchyma. It affects in general middle-aged males and is asymptomatic. In the vast majority of cases, the condition is discovered incidentally at autopsy. The arrow in Fig. 3.2.4 shows a particle of Iron embedded in the new “bone”. (Wells et al. 1943), (Fried et al. 1992) (Ikeda et al. 1998), (Trejo et al. 2002).

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Fig. 3.2.4 Image of a racemose ossification. The arrow indicates a particle entrapped in the part of lung that modified its chemical content into a Calcium-phosphate (marker 50 µm).

Fig. 3.2.5 shows a case of round atelectasis (Stark, 1982) (Menzies et al., 1987). That disease is a rare lung pseudotumour that in most cases is associated with asbestos-related pleural disease but can result from a variety of other chronic pleural diseases like, for example, an acute pleuropulmonary illness caused by Legionella pneumophila. The disease presents often as an asymptomatic radiographic finding associated with chronic pleural disease, usually related to asbestos exposure. In the case we studied, we found silicatic particles containing Silicon-AluminiumMagnesium-Iron-Sodium-Phosphorus-Sulphur-Chlorine-PotassiumCalcium-Titanium-Iron inside the round-shaped whiter areas. (Stark P., 1982), (Menzies R et al., 1987).

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Fig. 3.2.5 Image of a lung sample affected by round atelectasis. Inside the lung fibrotic tissue with chronic inflammation we found “round” areas full of silicatic nanoparticles.

Like all sarcoidoses, lung sarcoidosis is characterized by the presence of small granulomas. In the case of the lung, granulomas can appear on the walls of the alveoli or on those of the bronchioles. Its origin is still unknown, but most scientists think it is caused by a disorder of the immune system (Heffner DK, 2007). The case to which Fig. 3.2.6 refers shows Mercury-Sulphur nanoparticles in the reaction cutaneous lymphoid infiltrate. Granulomas can contain foreign bodies. In this case the nanoparticles detected are chemically toxic.

Fig. 3.2.6 Lung sarcoidosis. Inside a non necrotizing dermal granulomas, chemically toxic nanoparticles of Mercury-Sulphur-Silicon-Chlorine were found. (marker 5 µm).

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Fig. 3.2.7 concerns a case affected from tubercolo-silicosis. At optical microscopic observation, the lung parenchyma showed silicotic nodules with concentric bands of hyalinized collagen surrounded by fibroblasts and histiocytes with a blackish pigment . We report images of wide lung calcific areas (white dots). Their chemical analyses reveal that that is not a normal calcification but the biological tissue turned into Calcium phosphate. This tissue is hard, not elastic and can compromise the elasticity of the lung and of breathing.

Fig. 3.2.7 Low-magnification image of tubercolo-silicosis with wide Calcium phosphate areas (marker 300 µm).

Fig. 3.2.8 High-magnification image of tubercolo-silicosis. At such magnification, SiliconTitanium-Aluminium-Iron particles become visible. (marker 50 µm).

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At higher magnification (Fig. 3.2.8), it can be seen that the white dots are micro and nanoparticles of a compound made of Titanium-SiliconAluminium-Iron-Magnesium-Sodium-Chorine-Potassium-CalciumSulphur-Phosphorus. It is hard to tell the origin of that pollution, but wide areas of the lung are interested by these foreign bodies. The lung tissue is a composite material with altered morphology and physical properties.

Fig 3.2.9 Image of a squamocellular carcinoma sample containing Tungsten–based particles (marker 20 µm).

The patient whom the biopsy relative to a squamocellular carcinoma (Fig. 3.2.9 and Fig. 3.2.10) was taken from worked at the milling and rough-hewing of metal bushings for oil-pressure pumps where mineral oils derived from hydrocarbons were used. Tungsten-Aluminium-IronTitanium nanoparticles were present in his pathological tissue. At histopathological observation, the squamocellular carcinoma was hardly differentiated, and differentiation areas were in an adeno-squamous direction. Wide necrotic foci and marked atypias were also present.

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Fig. 3.2.10 Squamous cellular carcinoma with Titanium-Silicon-Aluminium-PhosphorusChlorine-Potassium-Iron particles (marker 20 µm).

Fig. 3.2.11 Iron nanoparticles found in the lung of a soldier suffering from Gulf War Syndrome (marker 5 µm).

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Fig. 3.2.12 Solitary Gold-Silver-Copper particle found in the lung of a soldier suffering from Gulf War Syndrome (marker 10 µm).

The images of Fig. 3.2.11 and 3.2.12 show nanoparticulate matter of different compositions in the lung tissue of a veteran from the First Gulf War, suffering from the so-called “Gulf War Syndrome” The samples come from an officer active in the Canadian Intelligence who died in 1999 as a victim of the so-called Gulf War, during which he served as a mine clearer. Natural Uranium (90%) and depleted Uranium (9-10%) were found in his urine by another laboratory when the patient was still alive. No such element, though, was found in particulate form in our investigations. In the samples studied we found nanoparticles of Titanium compounds and of Iron, along with particulate matter of Silver and precious alloys like Gold-Copper. Finding such small particles inside that patient’s lungs was strange indeed, as he had lived most of his life lived in the Canadian countryside, and had never worked in a nanotechnology laboratory where particles that size are common. He grew ill after a 6-month mission in Iraq and, in fact, had been exposed to the war pollution created by bombing. (See Chapter 5: War and nanoparticles) In another case, this time of a patient affected from the Gitelman’s syndrome, we found many different foreign bodies in the same sample. Gitelman’s syndrome is a rare, often asymptomatic, autosomalrecessive disorder affecting the renal tubules, causing them to pass Sodium, Magnesium, Potassium and Chloride into the urine. The patient,

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suffering from chronic interstitial pneumonia with allergic fibrosing alveolitis, had been in touch with depleted Uranium. Inactivating mutation in the SLC12A3 gene was found in the patient, which is compatible with a diagnosis of Gitelman’s syndrome. (O’Shaughnessy KM et al., 2004), (Naesens M. et al., 2004). Nanoparticles of a rather complex composition are visible in the tissue. The different chemical composition of the particles found indicates an origin from different sources.

Fig. 3.2.13 Low-magnification image of a lung tissue affected by Gitelman’s Syndrome. Arrows show Silicon-based pollution (marker 300 µm).

The first image (Fig. 3.2.13) shows a common silicate particle (normal dust) and the second one (Fig. 3.2.14) is a round-shaped particle of a compound of Cerium-Lanthanum-Neodymium. In our experience it can have been originated by smoking, as in some tobacco leaves we found the presence of this strange environmental, weakly radioactive composition. The third image (Fig. 3.2.15) is related to the presence of another unusual compound containing Copper-Chlorine-SiliconAluminium-Sulphur-Phosphorus-Iron.

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Fig. 3.2.14 Images of a single Cerium-Neodymium-Iron particle in a lung sample affected by Gitelman’s Syndrome (marker 20 µm).

Fig. 3.2.15 Images of a lung sample affected by Gitelman’s Syndrome. Note the nano-scale Chlorine-Copper-Silicon-Sulphur particulate matter (marker 2 µm).

The case shown in Fig. 3.2.16 concerns a 42-year-old woman who died of malignant pleural mesothelioma without having been exposed to asbestos. In fact, no such mineral could be found in her pathological samples, but we detected Iron nanoparticles and fibres of AluminiumSilicon-Iron, whose long-shaped morphology is similar to asbestos. The only anamnestic detail of this patient is that she underwent a five-year chemotherapy treatment because of a breast cancer, before falling ill of mesothelioma (Melato M et al., 2001). Another case of the same disease (Fig. 3.2.17) shows the presence of Iron micro and nanoparticulate.

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Fig. 3.2.16 Images of malignant pleural mesothelioma’s sample. A fibre of Aluminium silicate is clearly visible (marker 20 µm).

Fig. 3.2.17 Images of malignant pleural mesothelioma’s sample with Iron micro and nanoparticles. (marker 10 µm).

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Fig. 3.2.18 Iron-based particles in the sample of a patient with a multi-visceral granulomatosis (marker 100 µm).

Fig. 3.2.18 concerns the case of a patient for whom no clear diagnosis was issued. He suffered from a multi-visceral granulomatosis and what we found in the lung and spleen samples were wide areas where IronSilicon-Aluminium-Magnesium-Sulphur-Phosphorus were present. The presence of Silicon, Aluminium and Magnesium with Iron induces to think that Iron is not endogenous but comes from outside the body. The case of Fig. 3.2.19 is about a young lady of 22 who suffered from talcosis and pneumothorax. The histological study revealed the presence of blebs, fibrosis, lymphocytic phlogosis and giant cells with particles. The particles we detected were composed mainly of Copper (Fig. 3.2.20) and of Silicon-Magnesium (talc).

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Fig. 3.2.19 Talc particles contained cells in a pneumothorax (talcosis) (marker 10 µm).

Fig. 3.2.20 Solitary Copper particle in a pneumothorax area (marker 10 µm).

The round-shaped Copper particle shows that the patient had been exposed to a toxic material coming from a combustion occurred at about 1,100°C, since the melting temperature of Copper is 1,083°C. That element has been known for a long time to be toxic both in vitro and in vivo causing tissue necrosis. If the particles are located outside of the lung, in touch with the pleura, they can induce a necrosis, a rupture and the onset of a pneumothorax.

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Fig. 3.2.21 Case of pneumotorax with the presence of a Titanium needle (marker 10 µm).

Fig. 3.2.22 Case of pneumotorax with the presence of macrophages full of Iron-SiliconAluminium-Calcium particles (marker 20 µm).

In a 65-year-old workman a pneumoconiosis was diagnosed. The histology showed blebs, fibrosis, lymphocytic inflammation and giant cells with particles we showed to be composed of Iron-Silicon-SulphurCalcium-Aluminium (Fig. 3.2.22). The pricking needles of Titanium (Fig. 3.2.21) can be responsible of the pneumotorax, but the damage created by the Iron particles may have also contributed.

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Fig. 3.2.23 Cells full of Iron-Silicon-Aluminium-Phosphorus-Sulphur-Calcium-TitaniumChromium particles surrounding a pneumothorax area (marker 20 µm).

Fig. 3.2.24 Particular of Fig. 3.2.24 (marker 10 µm).

A further case of pneumothorax, this time of a 42-year-old solderer came to our observation. Coniotic materials in giant cells (at different magnifications) were easily visible, composed of Iron-Silicon-TitaniumCalcium-Aluminium-Phosphorus-Sulphur-Chromium (Fig. 3.2.23 and Fig. 3.2.24). White, round shaped particles are clearly visible inside the cells. This evidence is compatible with a working-place exposure.

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Fig. 3.2.25 Area around pneumothorax. Many silicatic particles are visible (marker 20 µm).

Fig. 3.2.26 Particular of the lung sample around the area of the pneumothorax above. Cerium-Iron particles are contained in cells (marker 10 µm).

A businessman of 42 suffered from a pneumothorax. Blebs with fibrosis, chronic lymphocytic infiltration and giant cells containing nanoparticles whose main component was Silicon were present. (Fig. 3.2.25). Also in this case, in the sample of the lung lesion, the tissue was full of micro and nanosized foreign bodies. Rather peculiar is the probably weakly radioactive debris containing Cerium-Iron-AluminiumSilicon-Phosphorus and Sulphur (Fig. 3.2.26).

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Fig. 3.2.27 Image of a lung sample in the area of the pneumothorax. Silicon-Aluminium based particles are visible (marker 100 µm).

Fig. 3.2.28 Particular of Fig. 3.2.27. At higher magnification, numerous needle-shaped Silicon-Aluminium based particles can be seen (marker 10 µm).

A case similar to the one illustrated in Fig. 3.2.25 is shown in Fig. 3.2.27. In this instance the patient was a 71-year-old workman. The coniotic nodule and the giant cells contained particles whose principal component was a composition of Silicon-Aluminium-PotassiumTitanium-Iron-Magnesium-Sodium-Phosphorus-Sulphur, i.e. a ceramic dust. Fig. 3.2.28 regards the same patient as above, but in this portion of the lung the particles had the same composition but a different morphology.

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Fig. 3.2.29 Cluster of Silver nanoparticles in a lung sample affected by allergic pneumonia (marker 5 µm).

The picture above (Fig. 3.2.29) shows a case of chronic, allergic pneumonia of exogenous origin with lung interstitial fibrosis and secondary leiomyomatosis, a rare, benign tumour that generally develops in the uterus, but can affect organs with smooth muscle cells. In this case, clusters of Silver nanoparticles were clearly visible in the pathological tissue.

Fig. 3.2.30 Unusual morphology of pulmonary cells (adenocarcinoma) containing IronSulphur-based particles (marker 50 µm).

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Fig. 3.2.31 Same sample as the one of Fig. 3.2.29. Normal red cells are inside the ellipse, while lighter red cells are indicated by arrows (marker 20 µm).

Fig. 3.2.32 Same sample as the one of Fig. 3.2.29 and Fig. 3.2.30. What look like Iron phosphate precipitates on a needle-shaped Iron particle (marker 10 µm).

Figs. 3.2.30-31 and 32 show different types of particles found in a small lung sample affected by adenocarcinoma. The first image show round-shaped organic bodies with Iron nanoparticles inside. The second one shows metallic particles (Chromium-Iron-Nickel) embedded in red cells (lighter in colour and indicated by arrows), while the third one is an Iron needle with endogenous Iron-phosphatic crystals. A surgical metal clip was detected at histology. It could be responsible for the debris found. A few cases of pleural mesothelioma are shown below.

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Fig. 3.2.33 Images of Iron-Sulphur and Iron-Zinc-Sulphur-Silicon-Phosphorus nanoparticles of in a lung tissue affected by mesothelioma (markers 10 µm and 20 µm respectively).

In the same case of mesothelioma, two different types of nanoparticles were detected. One was made of Iron-Sulphur, while the other was composed of Iron, Zinc, Silicon, Sulphur and Phosphorus (Fig. 3.2.33). Among the particles found in the case illustrated in Fig. 3.2.34 and Fig. 3.2.35, we focused our attention to some debris with unusual compositions. The former contained Uranium-Phosphorus-Potassium particles, (a radioactive composition) the latter contained Tungsten, Tantalum, Niobium, Phosphorus and Sulphur.

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Fig. 3.2.34 Pleural mesothelioma with particles containing Uranium (marker 50 µm).

Fig. 3.2.35 Lung tissue affected by mesothelioma with Tungsten-Tantalum nanoparticles (marker 20 µm).

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Fig. 3.2.36 The same sample of a tissue affected by mesothelioma are shown at different magnifications (marker 200, 50 and 5 µm respectively).

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This case of mesothelioma is rather peculiar. In fact, macrophages were found in the tissue full of small, round-shaped particles of Aluminium-Magnesium. Fig. 3.2.36 shows part of the tissue at increasing magnifications. The second image shows a blood vessel surrounded by whiter cells. They are full of sub-micronic globular entities containing Aluminium. The patient had worked for a long time in a mechanic industry and he was exposed to vapours exhaled by what is called “Aluminothermy process”. Also this case confirms that what was found inside is the expression of the environmental exposure the patient underwent. The inorganic matter we inhale can remain trapped in the lung at different tissue levels. In the case of needle-shaped particles, once inside the lung, because of the respiratory acts, they are pushed toward the more external part of the organ, i.e. the pleura. When they are there, they have the time to express their toxicity and induce a tissue or cell reaction. The biocompatibility of a material is also a function of time: a short time interaction is less detrimental then a long term contact. But in the analysis of the possible damage due to environmental pollution we need to take into account other phenomena that were observed by the School of Leuven (Belgium). The main work is due to Dr. Nemmar who had some 100 nm-sized Technetium-radiolabelled Carbon nanoparticles inhaled by 5 volunteers. The experiment showed that those particles negotiate the lung barrier in 60 seconds and their dissemination in internal organs, the liver in this particular case was the organ observed, occurs in 60 minutes. (Nemmar et al., 2002) That simple experiment simulated what probably happens every day. It is well-known that 100nm-sized particles are released, of course unintentionally, by car engines, industrial fumes, incinerators, etc. and the paper mentioned offers an explanation to what we find in many tissues other than hepatic (Tonomori et al., 2004). The next paragraphs show that contamination can be very deep, and no organ is free from danger. It is only a matter of size, chemical composition, affinity for some function of the organ and chemistry of the particle.

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3.3 Blood The presence of the inorganic particles found in the internal organs was explained putting forward the hypothesis that they are carried by the blood circulation. In order to demonstrate this presence inside the blood, we wanted to take pictures of circulating particles, but also of possible problems induced by their interaction with the blood components. To this aim, we tackled the problem from three different points of view. Firstly we looked for inorganic debris in the blood of clinically healthy people living in polluted areas. Then, we checked thrombi formed in-vivo, either in people at risk of pulmonary thromboembolism and in patients who had a myocardial infarction. In the former case, we analyzed venous clots trapped by caval filters, and in the latter the thrombi removed from the coronary arteries by means of a new procedure making use of a thrombectomy equipment. And finally we analyzed samples of patients suffering from vasculitis, a cryptogenic inflammation of the blood vessels. The list of Tab. 3.3.1 shows the pathologies considered. Fig. 3.3.1 shows the image of red cells smeared on an acetate sheet. Small particles are visible, but it is hard to tell if they are immersed in the plasma, attached to the cell or embedded inside it.

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Tab. 3.3.1 List of the pathologies analysed. Pathologies Thromboembolic disease Aneurism Idiopathic Thrombocytosis Diabetes Parassitosis Cerebral angioma Hemangioma - Angiolipoma non-Hodgkin’s Lymphoma Hodgkin’s Lymphoma Lymphadenopathy Not diagnosed cases Reference samples Total

No. 65 1 1 1 2 1 1 1 1 2 6 2 84

This analysis itself is simple to perform and particles are easy to detect, but that is not enough to allow us to say that the presence of foreign bodies means that a pathology is starting. More interesting proved the study of thrombi formed in-vivo, since particles found inside them can mean that they acted as nucleating agents, or their non biocompatibility triggered the coagulation cascade.

Fig. 3.3.1 Image of an Antimony–Cobalt nanoparticle and red cells (marker 5 µm).

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In order to demonstrate the interaction particle-blood in vivo, we took patients treated with caval filters. Those are medical devices that are implanted in the inferior vena cava either as a preventive means before surgery in patients who were deemed as being at risk of developing a deep-vein thrombosis (DVT) with a consequent pulmonary embolism (PE), or to prevent PE episodes in potentially relapsing patients. In those cases, a metallic structure is inserted in the inferior vena cava and left there to capture the thrombi formed in the lower limbs and pelvis.

Fig. 3.3.2 Image of a vena cava filter by ALN (France).

The analyses of explanted temporary vena cava filters (ALN Implants Chirurgicaux, - France) (Fig. 3.3.2) offered the possibility to analyse the thrombus entrapped, without influencing the system. Those filters, usually implanted in the lumen of the inferior vena cava, are the most common mechanical device for the prevention of pulmonary thromboembolism and their use is growing more and more widespread, especially in the USA. The observation of the filters explanted allowed to analyze the small thrombi captured in the human venous circulation. (Gatti, A. M. et al., 2006) (Montanari S., 2000). The vena-cava filters examined are composed of nine AISI-316L stainless steel, 0.3-mm diameter, wires

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gathered together inside an ogival capsule made of the same material, from where they branch out, forming a conical skeleton. Each of the shorter 6 of the 9 prongs that make up the device has a distal hook that penetrates the venous wall and serves to keep the filter anchored in place, while the longer three are meant as stabilizers to keep the device lined up with the vascular axis, and have no hooks. One of the long prongs has a small ring at its distal end. The overall height of the device is 5 cm and, being it elastic, its diameter adjusts itself to the section of the vessel up to 32 mm. In two different studies, we analyzed respectively 14 and 20 temporary filters explanted from patients who had had this device implanted for different reasons and for different periods (from 18 to 384 days). The main indications were: 1. Anti-thrombosis prophylaxis in bone fractures or implant of hip joint prosthesis or cancer; 2. Deep Vein Thrombosis (DVT); 3. Protection in loco-regional thrombolysis treatment. The particles found in the thrombi trapped in the filters were characterized with a size ranging from 10 down to 0.1 micron and for a wide variety of chemical compositions. Some of the compositions found are listed below. The first element considered is that expressed in the spectrum with the highest peak: - Si; - Si; Mg; - Cr, Fe, Si, S, Na; - S, O, Ba ,Na; - Fe, Sb, S, P, Si, Na; - Sb, S; - W, O, S, P, Fe; - Bi ,S, Cl, Si, Na Ca; - Pb, Si, Al ,Cr, Fe, Mg, Ca; - Ag, Si, Mg, S, Bi, Cl, O, Ca, Cu; - Co, S, Ag, Ca, Cl, P, W, Al, Zn, Fe; - S, Ba, Ca, Cl, Na; - Fe, Cr, O, S, Ca, Cl, Cu; - S, Cl, Ca, Bi, P, Na, Mg;

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- Ag, O, S, Ca, P, Si, Mg, Ni; - Au, Cl, S, O, Ca, Ag, Na, Si, Mg, Cu, Fe. It is interesting to see how all the thrombi come to our observation had foreign bodies trapped. One case was particularly interesting from the nanopathological point of view, as the thrombus contained micro- and nano-sized foreign bodies composed of as many as 6 different chemical compositions (Co-W, AlSi, Si, Fe, Ag-S, Fe-Cr-Ni). Fig. 3.3.3 and 3.3.4 show particles of Barium Sulphate and Silver dispersed in the thrombus. (Gatti, A. M. et al., 2006)

Fig. 3.3.3 Image of red cells in a thrombus trapping nanoparticles of Barium-Sulphate (marker 10 µm).

Fig. 3.3.4 Image of a particle composed of Silver and Calcium found in a thrombus (marker 10 µm).

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Fig. 3.3.5 Sulphur-Barium-Sodium-Aluminium-Silicon-Phosphorus-Calcium-Iron particles in an fibrotic tissue adherent to a caval filter (marker 10 µm).

The presence and quantity of thrombotic material found in the filters did not seem to depend on the time the device had stayed implanted in the organism, but on the extent of contamination of the blood, i.e. how many particles per volume unit were present. That means that the higher the patient’s exposure was, the greater was the concentration in the blood and the higher was their probability to induce a thrombotic reaction. It has been demonstrated that foreign bodies the size of 100 nanometres, once inhaled, can leave the alveoli and enter the blood circulation in 60 seconds. (Nemmar et al. 2002). In Fig. 3.3.5 Sulphur-Barium-SodiumAluminium-Silicon-Phosphorus-Calcium-Iron nanoparticles are shown trapped in the fibrotic tissue adherent to a caval filter.

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Fig. 3.3.6 Cluster of nanoparticles composed of Titanium-Sulphur–Barium-CalciumChlorine (marker 10 µm).

Fig. 3.3.6 and Fig. 3.3.7 show metallic debris found in thrombi trapped by two caval filters. The former is a cluster of TitaniumSulphur–Barium-Calcium-Chlorine nanodebris, while the latter is a single Iron-Chromium-Nickel needle-shaped particle.

Fig. 3.3.7 Image of stainless-steel debris found inside a thrombus (marker 20 µm).

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In the cases we had the chance to observe, particles much larger than 100-nanometer were detected in the blood. Whether they are the cause of the thromboses or they are just mechanically trapped by the blood during its pathologic coagulation may be discussed, but it is not unreasonable to suppose that a foreign body can be thrombogenic, especially, as has always been the case in this study, when those foreign bodies are neither biodegradable nor biocompatible, and being not biocompatible means, in the vast majority of cases, being also thrombogenic. Recently it was demonstrated that when the environmental concentration of PM2.5 increases, the mortality rate of cardiopaths, or patients suffering from cardiovascular diseases increases as well. The images shown can confirm these data. The size and composition of the debris found is rather typical of industrial pollution, in particular of high-temperature processes involving inorganic materials, such as occurs in a foundry, in a cement plant, in an incinerator or in engine fumes. All patients were considered to be at risk of developing a deep-vein thrombosis (DVT) or, in some cases, were already diagnosed to be affected by that disease. This is one of reasons that make us suspect that the thrombi detected or, at least, part of them, embolized from the DVT foci. Some of them, though, could have been originated by inorganic particulate circulating in the blood and become actually thrombogenic once the organism has been stimulated by particular, well-known, conditions like, for example, surgery, bone fractures, cancer or a relatively long stay in bed. A number of the many pulmonary embolism episodes observed worldwide are classified as idiopathic, since no focus can be identified nor an explanation regarding their origin is possible according to the socalled Virchow’s Triad, that states that pathological clotting of the blood in the vessels occurs when the blood flow is somehow obstructed or slowed down, when the vascular endothelium is damaged or when the chemistry of the blood is disturbed or incompetent. In many instances, it is a combination of the three factors that is responsible for the thrombosis, as one single factor may not always be enough. The presence of particulate matter in the blood, added to the Triad as a fourth factor, may explain in part or totally the not infrequent cases

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when none of those classical three causes exist, yet a pulmonary thromboembolism occurs. That particulate air pollutants are responsible for or, at least, have some form of relationship with the onset of cardiovascular and pulmonary diseases has been amply demonstrated (Peters A. et al., 1997), (Samet J. M. et al., 2000) and is supported by clinical evidence, though much remains still to be explained as to the patho-mechanisms they follow. At present, for instance, we are not in a position to say if the blood reacts to the presence of clusters of nanoparticles and of isolated microparticles in a different way. Also analyzing the material that occluded a coronary vessel we found foreign bodies. Myocardial infarction is caused by an acute obstruction of the coronary lumen and in many cases that obstruction is caused by a thrombus or, at least, contains thrombotic material together with atheromatous plaque. A few years ago, a mechanical technique aimed at aspiring that material grew popular in interventional cardiology. (Junichi K. et al., 2002), (Andrew, M. W. et al., 2006). We had the chance to investigate on a sample taken from a myocardial infarction thanks to the technique of mechanical coronary thrombectomy and found micro and nanoparticulate in that thrombus as well. Fig. 3.3.8 and the ones that follow (3.3.9 and 3.3.10) show inorganic particulate inside coronary thrombi. Such unexpected finding may rouse

Fig. 3.3.8 Coronary thrombus with an agglomeration of many nanoparticles with one microparticle of a Titanium-Bismuth-Sulphur-Phosphorus compound (marker 10 µm).

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the suspicions that those foreign bodies can have triggered the pathological coagulation. This could explain why a higher cardiogenic mortality is reported when PM2.5 concentration in the atmosphere grows higher (Pope, C. A. et al., 2006), (Pope, C. A. et al., 2006).

Fig. 3.3.9 Small Platinum-Tungsten-Copper-Sodium debris found in a coronary thrombus (marker 10 µm).

Fig. 3.3.10 Cluster of nanoparticles composed of Antimony-Cobalt Sulphur-Sodium embedded in a fibrin reticulum (marker 10 µm).

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3.4 Liver Granulomatous hepatitis is a lesion found in a wide spectrum of liver diseases (Denk, H. et al., 1994). TBC and sarcoidosis infection have been most frequently incriminated, while other causes are represented by viral infections, immunological disorders, drug-induced injuries, Crohn’s disease and foreign-body reaction (Anderson, D. S. et al., 1998). A minor percentage of cases, about 10%, remains of unknown etiology and is indicated as GLUS (Granulomatous Lesions of Unknown Significance), (Brinker, H., 1990) From the histological point of view, such lesions are usually non caseating, with a few multinucleate giant cells and some surrounding chronic inflammatory infiltrate. Medical literature analyses local effects of wear from joint replacements, identifying the debris as the main result of in situ degradation of implants (Jacobs, J. J. et al., 1998). In addition to obvious local effects, particulate debris created by an implanted device has been reported as present in lymph nodes (Albores-Saavedra, J. et al., 1994), (Bos, I. et al., 1990) and in distant organs, but literature on the subject is rather scanty (Urban, R. M. et al., 2000). As a general rule, the larger particles thus generated cause a fibrous or giant-cell reaction, while the smaller ones prompt a macrophage phagocytosis reaction. Chapter 2 demonstrates that nanoparticles behave in a different way and do not induce “normal” inflammatory reactions. Our first case of liver granulomatosis is the one described in Chapter 1. In this later series, we analyzed a group of patients with phlogosis (13 cases) and “granulomatous phlogosis” (resulted from liver biopsies) (29 cases), since they are related to tissue inflammations also caused by foreign bodies . The relevant data are shown in Tab. 3.4.1. In order to have a reference, not shown in the table, five specimens of liver taken from presumably healthy young people dead in car accidents were observed in the same way under ESEM together with ten more samples from feti. Those specimens were in all cases free from any particulate contamination.

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Tab. 3.4.1 List of the pathologies investigated with the relevant number of cases. Pathology Phlogosis Fibrosis Granulomatosis Wegener’s granulomatosis Steatosis Hepatitis Siderosis Haemorrhage Wilson’s disease Cancer Metastasis Hodgkin’s Lymphoma non-Hodgkin’s Lymphoma Gulf War Syndrome Nephrocalcinosis Not diagnosed cases Reference samples Total

No. 13 2 29 1 7 6 3 3 1 28 17 3 1 1 1 23 15 154

All these cases were checked by a histopathologist. Just three of the cases chosen showed the presence of foreign material inside the granulomas, whose nature remained undetermined to him. In any case, he ruled out any infectious origin according to the results obtained with Pas, Grocott and Zihl-Nielsen reactions. Once again, the observations under ESEM proved essential in the identification of materials responsible for granulomatous reactions, as the presence of micro- and nano-debris in all samples except the references was consistently found. The particles detected were different in size (in general <20 µm) and in chemistry. As all the cases selected are meaningful in demonstrating the hypothesis mentioned above, some of them are discussed singularly because of their clinical history. Their anamnestic records confirmed the exposure we found in the tissue. Case No. 1: 61-year old female with a clinical history of rheumatoid arthritis; increase of GOT, GPT and gamma-GT. Use of corticosteroids and Gold salts injected directly in her joints. Histologically, the liver

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biopsy showed a mild chronic phlogosis and a small granuloma in the proximity of a portal tract, with black material inside. At ESEM analysis, particles trapped in a degenerated tissue containing Gold nanoparticles were identified. (Fig. 3.4.1). The oddity of such finding got us to analyse more in detail different areas of the same specimen. The EDS spectrum (Fig. 3.4.2) showed that Gold alone was not accompanied by any other element, the spherical, homogenous morphology of the particles as small as 50-nm excluded a dental origin. The study of the clinical record revealed that years before falling ill of liver granulomatosis, the patient had suffered from a knee arthrosis and had been subjected to a colloidal Gold therapy. We could not find any other reasonable explanation to the presence of that form of pure Gold in the liver, where it had behaved like a foreign body, but a migration from the joints, where it had been injected.

Fig. 3.4.1 Gold nanoparticles in a liver granulomatosis (marker 50 µm).

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Fig. 3.4.2 Cluster of Gold nanoparticles in a liver granuloma (marker 2 µm).

Case No. 2: A 25-year-old female was affected by a hepatic echinococcosis. At ESEM observation, small debris of Barium-sulphate could be detected in the section. The patient’s clinical file revealed that she suffered from digestive disorders, which suggested the possibility that she had undergone a gastroscopy with BaSO4 used as contrast medium. The fact was confirmed by the patient herself who also reported an allergic reaction from that chemical. Case No. 3: A 63-year-old male suffered from indefinite symptoms, and a constant feeling of fatigue. Liver biopsy showed the presence of a lympho-monocytic infiltrate, areas of macrosteatosis and an epithelioid granuloma lined with a lympho-monocitic halo. At ESEM, degenerated areas of the liver showed numerous particles of Calcium-sulphate (gypsum) and fillo-silicate (Fig. 3.4.3). Such chemistry suggested the possibility that the patient lived or worked in a particularly polluted environment. As a matter of fact, the clinical record reported that he was a mason.

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Fig. 3.4.3 Gypsum and fillo-silicate micro and nanoparticles in an epithelioid granuloma.3

Case No. 4: A 65-year-old African male was operated on for a spleen cancer. Surgery revealed that his liver was compromised and a biopsy of that organ was taken, which showed a large granuloma containing necrotic tissue. The ESEM observation in that tissue revealed the presence small spherical Calcium-phosphate particles with a narrow distribution of diameters (2-7µm) (Fig. 3.4.4). The hypothesis we advanced, a hypothesis never confirmed because of the patient’s death and the consequent stop of all investigations, was that those findings were of biological origin, perhaps a parasite.

3

This photo was published in Biomateriali, vol 23, issue 11, Gatti A.M., Rivasi F. “Biocompatibility of micro- and nanoparticles Part I in liver and kidney”, June 2002, p. 2381-2387.

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Fig. 3.4.4 400-µm granuloma with fibrous capsule (low-magnification). (marker 200 µm).

Fig. 3.4.5 Same sample as above. Higher magnification shows Calcium phosphate nanospherules (marker 5 µm).

The same morphology, size range and composition as those seen in this case was found in a number of other biological tissues (Fig. 3.4.5). Still today we do not understand if the reaction is a sort of unusual calcification (why spheres? Why Sodium and Magnesium?) or it is due to some other mechanism. Case No. 5: The only symptom of a 27-year-old male with a history of drug abuse was fever. Liver echography showed multiple nodules of a diameter never exceeding 2 cm. The liver samples we had the chance to

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check showed the presence of foreign bodies with different chemical composition (silicates). Such finding was probably due to the powder used to mix the drug. Case No. 6: The liver biopsy of a 62-year-old male with a clinical history of peritoneal dialysis showed granulomas in correspondence with the glissonian membrane and focal hepatic fibrosis. The spectrum of the debris seen in the pathological tissue reveals the presence of Carbon, Oxygen, Silicon, Sulphur, Calcium and Titanium. Such debris might be correlated with a possible pollution of the dialysis solutions or to the pharmaceutics taken in a chronic way. Diagnostics uses either light transmission microscopy with a maximum of up to 400 magnifications or molecular biology, that analyses the sequence of the aminoacidic bases, whose size is nanometric. Between this two terms there is a wide interval spanning about 2-3 orders of magnitude where information about particular pathologies, not otherwise explained, may be lost. And such information may be important or even essential to understand the problem. As mentioned above, no less than 10% of all cases of liver granulomatosis remains unexplained as to their origin, and that brought us to face this problem under the different point of view of our technique. The use of a novel technique, a technique typical of physicists, represented by the use of Environmental Scanning Electron Microscopy (ESEM) that allows the analysis of samples under different gradients of pressure, without any physical and chemical pre-treatment demonstrated that within this gap evidence can be found essential to issue a correct diagnosis. In fact, just as an example, the case of the patient with a fever and renal failure of unexplained aetiology described in Chapter 1, whose renal and liver biopsies were subjected to ESEM examination, showed a spectrum of elemental composition compatible with the porcelain his dental prosthesis was made of. This could explain the aetiology of his granulomatous hepatic lesions and was fundamental to set an efficacious therapy. In the same way, the X-ray microanalyses carried out on the particulate matter of case No. 3, gave information on the particles’

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chemistry and these data could be related, at least in part, to the clinical, environmental and employment history of that patients. A likely identification of the cause of the disease is certainly useful for the therapy, and, which is of extreme importance, to set prevention measures. On the basis of these observations, we can assume that defining “non toxic” what are just chemically inert (or hardly reactive) particles is erroneous. Below a critical size, still hard to establish, particles, whatever they are made of, are not biocompatible and may interact with biologic tissues just because of their presence, and they may be responsible of chronic inflammatory reactions that can lead to more severe forms of disease. Nano-scaled particles can induce other unknown phenomena not yet described in books or papers. This first part concerning the pathologies of the liver has been dedicated to cases of granulomatosis because such reaction by tissues against foreign bodies has been often described in literature. Local granulomatosis around the head of hip joint prostheses due to the wear debris of the matching surfaces (head against acetabulum) is a far from rare complication in orthopaedics. So, there is no problem in accepting the existence of a correlation between presence of foreign bodies and biological reaction. More difficult to accept are the following images. They show the presence of dust that induced more severe reactions, like cancer. For a better understanding of the subject, we are presenting a genetic disease involving the metabolism of endogenous Iron, in order to show the difference between a genetic and an acquired disease. The genetic disease we will deal with is siderosis. Siderosis is a pathological build up of Iron in the organism. The lungs are the most interested organs, but the liver and the urinary system can be also affected. An Iron overload is known to impair the immune response of the liver, and cause hepatic fibrosis and cirrhosis. There are different opinions about the relative risk of developing hepato-cellular carcinoma in patients with siderosis as compared with patients with hepatic fibrosis and cirrhosis and the mechanism of liver carcinogenesis in genetic hemochromatosis is still to be discovered. The main association with genetic hemochromatosis is with a defect in gene HFE which helps

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regulate the amount of Iron absorbed from food. In the case below (Fig. 3.4.6), precipitates of Iron in the form of numerous nanoparticles are clearly visible inside the cytoplasmatic area. The picture shows some nuclei surrounded by Iron precipitates (whiter).

Fig. 3.4.6 Image of a liver affected by siderosis. Globular Iron precipitates in the cytoplasm can be seen (marker 20 µm).

In two cases of liver cancer, we detected particles inside the nucleus of viable, “healthy” cells (Fig. 3.4.7). The mechanism of entry of those particles is still to be explained.

Fig. 3.4.7 ESEM image of a liver cancerous tissue with a viable cell containing nanoparticles in the nucleus (marker 10 µm).

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Fig. 3.4.8 Silicon-Aluminium-Phosphorus-Sulphur-Iron particles in a non clearly diagnosed liver pathology (marker 20 µm).

This sample of Fig. 3.4.8 was taken from a cylinder of hepatic tissue that showed a partially changed architecture. Portal spaces looked slightly dilated, with inflammatory infiltrate. No steatosis, no Iron or biliary accumulations were visible. Kupfer cells were not evident. Some hepatocytes looked glycogenated. Foreign-body giant cells with a marked lymphocyte halo were present at one of the edge of the cylinder. Silicon-Aluminium-Phosphorus-Sulphur-Iron particles were disseminated inside the area and could be detected in the tissue.

Fig. 3.4.9 Chlorine-Potassium-Sulphur-Nickel crystal in an apparently empty cavity (marker 20 µm).

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Fig. 3.4.9 is a cylinder of liver tissue with a preserved lobular architecture. A small quantity of inflammatory, lymphocyte infiltrate is visible in the portal spaces. No Iron or bile, but some small Iron accumulation were present. Kupfer cells were not evident. At an edge of the sample, some giant cells mingled with a thick lymphocyte infiltrate were visible. At ultrastructural observation, the sample revealed the presence of Chlorine-Potassium-Sulphur-Nickel particles. The edged morphology defines it as a crystal. The void space around it may mean that it is toxic.

Fig. 3.4.10 Two images of a neoplastic sample of the liver cancer where Sulphur-based debris are visible (marker 10 µm).

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The images of Fig. 3.4.10 show a wide dissemination of SulphurPhosphorus-Barium-Calcium-Potassium-Iron-Sodium-Copper micro and nanoparticles inside the hepatic tissue. The same chemistry was homogeneously found throughout all the sample. The patient suffered from a colon adeno-carcinoma, but his liver was already interested by metastases. The portal space was larger and infiltrated by lymphocytes and the metastatic tissue was interested by fibrosis, phlogosis and formation of ductal-like structures. The particles of the bottom image of Fig. 3.4.10 are attached to the surface of red cells. Another pathology of unknown origin is lung fibrosis. Among the cases examined, in one of them we found Lead-Iron nanoparticles in the lung and in the liver. Fig. 3.4.11 presents a preserved liver architecture with a lymphocyte infiltration, without neoplasm, and without Iron accumulation. No mycetes were present, but chemically toxic foreign bodies of Lead and Iron at nano-scale level.

Fig. 3.4.11 The image shows a hepatic tissue affected by a mild fibrosis. Lead-Iron-based nanoparticles can be easily seen (marker 20 µm).

After a cholecystectomy, the patient developed a diabetes and later a pancreas neoplasm. The liver presented a fibrosis, where we found the toxic Lead-Iron-based nanoparticles.

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Fig. 3.4.12 Zinc-Sulphur-Phosphorus particles in a liver. The same particles were found in the lung cancer of the patient (marker 20 µm).

Fig. 3.4.13 Liver cancer sample with two Zinc nanoparticles inside a cell nucleus (marker 5 µm).

Figs. 3.4.12 and 3.4.13 show a particular of the liver with small steatosic modifications. Zinc nanoparticles are clearly visible inside a cell nucleus. Fig. 3.4.13 shows that there is no efficient physiological barrier at cellular level for nano-scaled particles.

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Fig. 3.4.14 The image shows a particular of a neoplastic tissue of a cholangiocarcinoma full of Barium-Sulphate micro and nanodebris (marker 20 µm).

Fig. 3.4.15 Same hepatic tissue as that in Fig. 3.4.14, interested by cancer with roundshaped particles of a Magnesium-Aluminium-Barium compound (marker 20 µm).

Fig. 3.4.14 and Fig. 3.4.15 are about the same case of cholangiocarcinoma. A particle inside a cell nucleus is visible in Fig. 3.4.15. Particles gather and accumulate in preferential locations and finding those is essential to investigations. Another important point is the influence locations could have.

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Fig. 3.4.16 Liver with metastases from colon adenocarcinoma (marker 50 µm).

Fig. 3.4.16 shows a cancerous metastasis characterized by the formation of fibrosis and gland-like structures. The sample is full of nanoparticles composed of Barium-Sulphur-Iron. We did not have the possibility to check if the same particulate was present in what was called primary cancer located in the colon.

Fig. 3.4.17 Image of a small particle of a Aluminium-Phosphorus-Chlorine-SulphurCalcium-Iron-Lead-Strontium compound (marker 50 µm).

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Fig. 3.4.17 shows a small particle of a strange Aluminium-PhosphorusBarium-Chlorine-Sulphur-Calcium-Iron-Lead-Strontium compound found in the liver of a patient affected by the so-called Gulf-War syndrome. That alloy does not exist in any metallurgy handbook. Every time we find such strange chemical compositions, not having any application, we have reasons to believe they are created by uncontrolled combustions: Outside the chimney of an incineration plant or during the explosion of a weapon against a target or when an airplane crashes or explodes inside a building as happened in New York in 2001, a matter vaporization occurs with the creation of a new pollution whose composition changes from point to point. So, a coupling of elements is obtained that are not matched in any intentionally made alloy. 3.5 Kidney and adrenal gland From a certain point of view, the kidney tissue behaves like a mechanical filter, the way other organs, the liver, for example, do. The difference between kidney and liver seen as mechanical filters is that the former can trap tinier objects. Also for this organ we analyzed samples from subjects suffering from different pathologies, as reported in Tab. 3.5.1. Tab. 3.5.1 List of the renal diseases examined. Pathology Granulomatosis Wegener’s granulomatosis Glomerulo-nephritis Gulf War Syndrome Neu-Laxova Syndrome Cancer Nephrocalcinosis Kidney failure (IRA) Reference samples Total

No. 2 1 1 3 2 2 1 1 3 16

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The first case concerns a granulomatose reaction. Granuloma is a typical reaction of animal tissues to the presence of a foreign body that can be either organic (bacteria, parasites, etc) or inorganic (dust, etc). Sometimes, a fibrotic capsule is created in the attempt to isolate the exogenous inorganic presence from the biological context. Fig. 3.5.1 shows this type of reaction. When the foreign body is isolated that way, it can stay there for years without inducing particularly severe symptoms. An example may be the debris of a bullet that can remain sequestered in the tissues.

Fig. 3.5.1 Fibrous capsule in a kidney grown around a silicate particle (marker 20 µm).

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The image above shows the presence of a microsized debris of a silicate, i.e. a ceramic material, in the kidney. That debris can be “regular household” dust, but can as well be a material coming from sources like, for instance, ingested food. Chapter 1 showed the presence of dental prosthetic debris in a granuloma of the liver and the kidneys with the larger particles in the liver and the smaller in the kidneys. So, finding silicate debris present in the composition of chewing gums in those organs should not be surprising. As a matter of fact, chewing gums sold on the Italian market contain silicates under particular form, as the producer claims they can remove food remains from between the teeth when no water is available to brush them. During mastication, the gum matrix releases this particulate matter which gets swallowed and, thanks to its small size, can pass through the bowel barrier and be filtered by this tissue. Fig. 3.5.2 with two spectra is about a patient suffering from acute renal failure (ARF), i.e. a loss of kidney function occurring very rapidly. The immediate result is a retention of urea and creatinine and nonNitrogenous waste products that are normally excreted by the kidney. According to the severity and duration of the renal dysfunction, this accumulation is accompanied by a number of metabolic disturbances, such as metabolic acidosis, hyperkalaemia, changes in body fluid balance, and adverse effects on many other organ systems. The condition can be characterized by oliguria or anuria. The patient died of multiorgan failure after sessions of plasmapheresis and continuous arterovenous filtration. The histologic aspect was that of a tubular necrosis. Slight lymph-monocyte infiltrate in the interstitium

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Fig. 3.5.2 Silicate and Tin foreign body in a renal tissue (marker 50 µm).

Fig. 3.5.3 Right adrenal gland with adenoma containing Iodine-Copper-Sulphur particles (marker 20 µm).

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The patient to what Fig. 3.5.3 refers was a career soldier in the coastguard corps who had been on duty in Albania 2000-2001. In 2003 a Crohn’s syndrome was diagnosed, from adrenal-gland adenoma. In this case of Crohn’s syndrome, strange particles of Iodine-Copper-Sulphur were found. Copper is a toxic metal, but no literature could be found about the biocompatibility of this material in the adrenal gland.

Fig. 3.5.4 Chromium-Iron nanoparticles in a kidney, most probably a stainless steel (marker 5 µm).

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Fig. 3.5.5 Image of a particular of globular calcification in a kidney (marker 100 µm and 50 µm respectively).

Fig. 3.5.4 and Fig. 3.5.5 refer to a patient died of multi-organ failure. Fig. 3.5.4. shows the presence of what looks like stainless steel nanoparticles, while Fig. 3.5.5 shows globular entities, in fact CalciumPhosphate precipitates, formed inside the kidney. The calcification occurred modifies the physical properties of this tissue, like, for instance its elasticity. As a consequence of this irreversible phenomenon, the tissue grew harder and lost much of its functionality.

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Figs. 3.5.6a and 3.5.6b and spectrum. The images show a calcification (CalciumPhosphorus-Magnesium-Chlorine-Sodium) formed by single spherules (markers 200 µm and 5 µm respectively).

The patient the sample of Fig. 3.5.6 comes from suffered from a multi-organ failure with no clear diagnosis about its aetiology. He presented many different particles disseminated in all organs, but in the kidneys we found these Calcium-phosphate particles with a narrow range of size distribution (1–10 micron). The difference with the previous case is not only in the morphology but also in the composition. The spheres are also composed of Sodium and Magnesium. The case looks like that of the liver granuloma of Fig. 3.4.4, full of spherules very similar to these. Even after consulting one of the greatest experts of Calciumphosphates, Dr. Rachel LeGeros of the University of New York, we cannot tell if it has an organic or an inorganic origin, nor if it is endogenous or exogenous.

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The series of images shown demonstrates that environmental pollution can be trapped also in the kidneys. It is possible that the smallest nanoparticles can escape through the mesh of this filter, go through the renal tubules, the ureters and the bladder and are drained through the urethra. Our studies on urine could not confirm any possible hypothesis, since the salt crystallization masks any other presence. 3.6 Digestive system Our studies on the digestive system showed that every inorganic, non biodegradable, particulate matter can remain sequestered at different levels along the pathway between the mouth and the anus. This particulate pollution can be intentionally or unintentionally contained in the food, or in drugs as excipient, or can derive from the wear debris of dental materials. Part remains stuck in the oral mucosa and cases of “Burning Mouth Disease”, ameloblastoma, cancer examined confirmed the local entrapment. We present an example of a very small bioptic sample taken from the oral mucosa of a subject affected by a Burning Mouth Disease (Fig. 3.6.1). The patient was cured with tranquilizers and antidepressant drugs. In the one-by-one millimetre specimen we detected more than 600 particles, the debris from a dental prosthesis were found. During restoration works, the dentist drilled the old Gold-Platinum alloy prosthetic material without the protection of a dam and polluted the whole mouth cavity, but specially the mucosa, with the drilling debris. The mucosa responded with an inflammatory, highly disseminated, reaction.

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Fig. 3.6.1 Oral mucosa (marker 500 µm).

Our study of stomach cancer is just at an initial stage. The few cases, but particularly the too few samples we had available do not allow to put forward any hypothesis. Another important point as to the stomach cases is that we have never had the possibility to select in the explanted cancerous mass the interface between cancer and healthy tissue. From our point of view, that is the most significant position where inorganic pollution concentrates. The only meaningful oddity we came across was the finding of titania (Titanium dioxide) nanoparticles in a cancer tissue of a stomach of a 75-year old patient. Titania nanoparticles are something that is being employed more and more frequently in sunscreen creams and in paints as an application of nanotechnology, but whose use is rather recent. So, it is strange to find high-technology material in the stomach of an old person. A possible, though not demonstrated, explanation is that he may have eaten them mixed with some food. That and all the following images witness that those particles can remain entrapped inside the tissues and are not entirely eliminated through defecation. More than 50% of the 400 samples of food examined show a contamination coming either from environmental sources or from the wear of the machines used to work it (See Chapter 7).

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In the case of pathologies of the most distal tract of the digestive system, we analyzed different levels of seriousness of disease (Tab. 3.6.1): from mildly inflamed tissues to very severe illnesses like cancer. Tab. 3.6.1 List of the pathologies examined. Pathology Phlogosis Crohn’s disease Ulcerous rectocolitis Granulomatosis non-Hodgkin’s Lymphoma Cancer Mesothelioma Metastasis from hepatic carcinoma Reference samples Total

No. 2 3 2 2 1 30 1 1 3 45

One of the cases of Crohn’s disease we examined is particularly interesting. A 27-year-old patient had taken a antistaminic tablets since the age of 7. In his inflamed tissue we found silicate particles which, according to the drug’s producer, were part of the excipient used in those tablets. The next image (Fig. 3.6.2) gives an idea of what our colon is: a receptor of everything is introduced through the mouth. In this case, a large, curled debris of what is probably Teflon is adherent to the mucosa wall. The polymeric material is easily identified because of the presence of Fluorine in the Carbon-Oxygen spectrum. Observing the morphology of the debris, a suggestive hypothesis can be proposed: When food is cooked in an anti-adherent pan with a Teflon coating and is stirred or turned with a non appropriate tool like a knife or fork, the possibility exists of scratching the surface with the ensuing detachment of the Teflon layer with its typical folds. The deposition of such large debris on the mucosa surface can lead to the following consequences: They behave as mechanical barriers obstructing the adsorption of nutritive components. They are foreign bodies that the tissue tries to eliminate with spasms (probably), pain, inflammatory reaction, granuloma or fibrotic capsule formation, etc. But

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what may look strange is that also this macroscopic debris was not eliminated with defecation. The images below witness the presence of inorganic foreign bodies in the digestive tract wall. In a case we found 14 chemically different particles in a 2-mm square sample. In this situation it is particularly difficult to find a correlation between the particles, their origin and the disease. That is likely to be the result of multiple “insults”, all concurring to trigger a reaction.

Fig. 3.6.2 Bulk sample of a colon mucosa where a folded Teflon debris (arrow) is deposited.

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Fig. 3.6.3 Lead-Chlorine-Aluminium nanoparticles in a patient suffering from Crohn’s disease (marker 10 µm).

The sample illustrated in Fig. 3.6.3, containing Lead-ChlorineAluminium nanoparticles, comes from a patient affected by Crohn’s disease, a pathology characterized by granulomatous reactions that, in the case shown, were treated with Asacol (a non steroidal antiinflammatory drug). Inside the tissue we found also toxic particles containing Lead.

Fig. 3.6.4 Colon microvilli with Aluminium-Copper micro and nanoparticles (marker 100 µm).

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In the colon cancer shown in Fig. 3.6.4 we found, among other particles, Aluminium-Copper micro and nanoparticulate. In many pathologic instances, the colon contains particles presenting different shape, size and composition. It is likely that the reaction is mainly due to the presence of foreign bodies without a particular influence due to their chemistry.

Fig. 3.6.5 Probably zirconia nanoparticles in a cancerous tissue (marker 10 µm).

Fig. 3.6.6 Tin-Calcium-Silicon, Aluminium-Magnesium-Sodium-Phosphorus-Sulphur-Iron particles sequestered in a colon reaction tissue (marker 100 µm).

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The images of Figs. 3.6.5 and 3.6.6 present another case of colon cancer with particles of what is probably zirconia (Zirconium dioxide) and a strange compound whose complex composition can be seen in the spectrum attached. It is difficult to guess both the origin and the biological properties of these particles.

Fig. 3.6.7 A radioactive micro-sized particle in a peritoneal mesothelioma (marker 20 µm).

Fig. 3.6.8 Cluster of Lead-Silicon-Titanium-Chromium-Iron-Aluminium-Magnesium nanoparticles (marker 10 µm).

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Fig. 3.6.9 Round-shaped Silicon-Silver-based particle (marker 5 µm).

The images of Figs. 3.6.7, 3.6.8 and 3.6.9 show different samples coming from a case of peritoneal mesothelioma. We analyzed more than that 10 samples from different areas compromised by the disease and there we found numerous different micro and nanosized particles. What we found most frequently were stainless steel particles (non shown), but we could also identify toxic Lead particles (Fig. 3.6.8), Silver-Silicon particles and some ceramic radioactive material like that shown in Fig. 3.6.7. That material contained Titanium, Zirconium, Cerium but also Uranium, Thorium, Niobium and Yttrium. Industrial ceramic material could be the origin of such particulate.

Fig. 3.6.10 Particle aggregate of a salt in a sigma adenocarcinoma.

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Beside unusual chemical compositions detected in the intestine, we found also common products. In this case (Fig. 3.6.10) a SodiumChlorine particle is shown. The presence of a familiar composition like that is intriguing, because those are the elements composing soluble salts. So we wondered why such a compound could be present in the intestinal tissue in an evidently insoluble form. In an medium like urine, salts like Sodium chloride or Sodium chlorate solidify only during the sample preparation and at that time they crystallize forming dendrites. In this case we suppose they were already aggregated in an insoluble way before fixation of the sample. We are not in a position to tell if such a particle, with its composition and its being insoluble, therefore not a source of ions, is biocompatible.

Fig. 3.6.11 Particular of a colon mucosa affected by cancer with sub-micronic stainless steel particles (marker 10 µm).4

4

This photo was published in Biomateriali vol.25, A.M. Gatti “Biocompatibility of micro- and nano-particles in the colon (part II)”, 385-392, Feb 2004.

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Fig. 3.6.12 Higher magnification of Fig. 3.6.11.

The case Fig. 3.6.11 and Fig. 3.6.12 refers to is rather singular as the patient, suffering from a colon cancer, was a 19-year-old girl. Her pathologic specimens were full of stainless steel particulate matter.

Fig. 3.6.13 Cluster of Silver-Molybdenum nanoparticles in a cancerous colon mucosa.

The previous images are referred to colon cancer samples. In the same specimen we found metals like Silver-Molybdenum (Fig. 3.6.13) or Silver alone (Fig. 3.6.14). If we were somewhat surprised at finding those metals in the colon, the analysis of some food containing Silver (see Chapter 7) offered us a possible explanation for those presences.

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Fig. 3.6.14 Cluster of Silver nanoparticles deposited on the colon mucosa in a cancer case. Red cells are clearly visible at the right, lower corner.

A case of an amateur long-distance runner is shown below (Fig. 3.6.15). During his sports activity, a period of about 20 years, the subject had taken very large quantities of food integrators and stimulant drugs without any medical supervision. At the age of 50 a colon cancer was diagnosed. In this case, we were not in a position to blame either diet or drugs for his disease, since, because of the variety of substances introduced, many of which escaped from the subject’s memory, no traceability study was possible.

(a)

(b)

Figs. 3.6.15a and 3.6.15b5 Silver nanoparticles inside the colon mucosa (marker 20 and 2 µm). 5

This photo was published in Biomateriali vol.25, A.M. Gatti “Biocompatibility of micro- and nano-particles in the colon (part II)”, 385-392, Feb 2004.

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Fig. 3.6.16 Silicon–Magnesium particle, i.e. talc, trapped in a colon cancer.

The colon cancer of Fig. 3.6.16 contained talc particles. That mineral is generally ingested only as excipient in tablets. The samples above show particulate matter in a wide variety of morphologies, sizes and chemical compositions. Today’s world has grown particularly complex and taking into account all the variables that make it up and can interfere with living organisms is all but impossible. If we limit ourselves to food and consider it under the nanopathological point of view, we have to consider that: a- an overwhelming majority of primary products grow in polluted environments; b- cereals and flours are generally stored in silos, many of which are old and release debris that is inevitably added to food; c- manufacturing adds further contaminants like the wear debris of crushing and grinding tools; d- polluted ingredients can be added to clean ones, thus giving origin to a contaminated final product; e - animals fed with polluted food can retain contamination, and that contamination is transferred to man when he eats those animals which are never checked under this point of view.

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3.7 Vessels As demonstrated, environmental pollution can contaminate the blood, but also the vessel structure can be interested by those entities. Those pollutants are mainly metallic or ceramic and they are obviously harder than the red and white cells, the platelets, and the intima, the inner lining of the blood vessels. Because of that, when carried by the blood flow, they can graze those structures and damage them, for example scratch the vessel walls. A vessel damaged that way might be more prone to start thrombosis or host atheromasic deposits or calcifications. We saw that the particles carried by the blood can cross the vessel’s wall, perhaps pushed by the blood pressure or through other mechanisms we still do not know. During that passage, they can be trapped by the wall. It is only reasonable to accept the fact that they can interact with that tissue. Fig. 3.7.1 shows a silicatic particle that, probably because of its arrow-shaped morphology, scraped the endothelium and remained stuck in the vascular wall. Fig. 3.7.2 represents another example of entrapment of debris.

Fig. 3.7.1 Silicatic particle in a vessel wall.

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Fig. 3.7.2 Silver-Chlorine particle trapped in an inflammatory aourtic aneurism case.

In Chapter 4, the case of a girl affected by vasculitis will be discussed extensively where Tungsten carbide nanoparticles were found. It is certain that they were present in the blood where they induced the disease, but also extravasated, i.e. they passed through the vessel wall and went into the muscles where they caused a different reaction. 3.8 Sperm Our first analysis of a sperm sample was made as a consequence of a telephone call from a mother, whose son had died after his return from a peace-keeping mission in the Balkans in 2001. Her son had decided to join the Italian mission after the end of the Balkan war, had left perfectly healthy and after a three-month mission had come home ill. His conditions were underestimated and misdiagnosed by the doctors who took care of him and he died of a Hodgkin’s lymphoma. Before undergoing chemotherapy, he donated his sperm in order to use it for a future fertilization in the hope he could recover from his disease. After his death, his parents were informed that the sperm should have been destroyed, but his mother asked to get it and gave it to us to be examined. That first analyses gave important results (see Tab. 3.8.1), since we found foreign bodies there. At the beginning, since it was our first match with sperm, finding foreign bodies in a fluid we believed to be protected

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enough surprised us, but, continuing our experience, we noticed that the fact is far from uncommon.

Tab. 3.8.1 List of the sperm samples analyzed with the relevant patient’s pathologies. Pathology Non-Hodgkin’s Lymphoma Hodgkin’s Lymphoma Lymphoma Testicle cancer Gulf War Syndrome Blood disorder Non diagnosed cases Reference samples Total

No. 3 1 1 1 3 2 2 5 18

The following images show some examples of the debris and their chemical compositions. The study involved mainly the sperm of veterans from Iraq and former Yugoslavia (see Chapter 5) given to us because of the fear of contaminations suspected be responsible for the children malformation sometimes observed among American veterans. At present, the epidemiological studies tend to deny any correlation among the “working site exposure” of the fathers and these pathologies, but it is true that the protocols of the US Army suggest to avoid procreation within a year after the mission or longer than that. Another case dealt with a soldier who had some health problems after having returned from the Balkans and asked to have his sperm analyzed and there we found metallic particles of different compositions, mainly of Tungsten-Cobalt-Copper-Titanium-Sodium-Aluminium-PhosphorusSulphur-Chlorine-Potassium-Calcium. The round-shape of most particles was the obvious consequence of fortuitous combustions, a fact confirmed by the great quantity of different elements present in the composition.

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Fig. 3.8.1 Tungsten-Cobalt-Copper-Titanium nanoparticle in a sperm sample. (marker 20 µm).

The soldier, to whom Fig. 3.8.1 refers, and his wife wished to have a child, but were afraid something wrong could happen as a consequence of the pollution we had detected and, they asked for our help. So, with the help of two young doctors, we tried a sort of decontamination, separating the spermatozoa from the plasma, and we saw that the particles remained in the plasma. Having seen that, we suggested to recur to artificial fertilization with the “clean” spermatozoa and the result was a couple of healthy twins.

Fig. 3.8.2 A viable spermatozoon is close to a Lead-Chromium particle (marker 20 µm).

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Fig. 3.8.2 shows a viable spermatozoon close to two Lead-Chromium (two toxic elements) particles. The obvious questions are: “What is the fate of this spermatozoon in contact with a toxic material? What is the fate of this particle when the sperm is donated during a sexual intercourse? What will the answer be of the vaginal mucosa and the uterus when a toxic particle remains in close contact with them? Is Burning Semen Disease (see Chapter 5) the response for this presence? All that is something really new, very little has been done in that field and we do not have the answers. We can formulate only hypotheses, and those hypotheses may show the way the research should follow. Another case we came across and illustrated in Fig. 3.8.3 is that of a soldier whose sperm was polluted by clusters of nanoparticles composed mainly of Lead and Iron.

Fig. 3.8.3 Cluster of Lead-Chromium-Iron-Titanium-Silicon-Aluminium-MagnesiumSodium-Chlorine-Calcium nanoparticles (marker 10 µm).

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Fig. 3.8.4 Image of an Iron particle together with a viable spermatozoon (marker 20 µm).

Both pictures (Fig. 3.8.4 and Fig. 3.8.5) show foreign bodies in a drop of sperm from two different subjects.

Fig. 3.8.5 Image of a spherical zirconia particle in the sperm (marker 20 µm).

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Fig. 3.8.6 Cluster of Lead-Chromium-Chlorine nanoparticles (marker 20 µm).

Fig. 3.8.7 Image of a viable spermatozoon with a spherical Antimony-Cobalt-SulphurSodium nanoparticle (marker 20 µm).

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Figs. 3.8.6 and Fig. 3.8.7 come from the same patient and show viable spermatozoa in contact with toxic foreign bodies. This finding interested us for two reasons: the first because the so-called physiological barriers prove to be inefficient against such form of assault, the second because it may represent a key to understand some medical mysteries of our age. For instance, the so-called “industrial sterility” that affects some workers in certain industries could be explained if we accept that the contamination in some working places, represented by a toxic pollution, reaches the testicles, pollutes the sperm and causes the death of spermatozoa or reduces their vitality. This kind of contamination could be verified also in the female partners suffering from Burning Semen Disease. In the case of contaminated sperm, the simplest prevention measure that can be adopted to avoid possible problems to the female partner is the use of a condom 3.9 A few considerations on reproduction We leave in an age whose sexual customs are somewhat looser than in the past, and meeting occasional partners without knowing anything about their health is common enough. Knowing that we do not know what we should and that ignorance may lead to grievous consequences should induce to adopt more prudent approaches. What we found in the sperm has other important consequences. What happens if an egg is fertilized by contaminated sperm? What happens to a fertilized egg that develops in a medium where particles of toxic material are present? All those questions remain without direct answers, but we had a chance to observe some of the possible consequences. When we analyzed malformed feti (both human and animal), we discovered something that had escaped consideration until that moment. We received two malformed lambs whose mothers had been grazing close to an interforce military firing ground in Sardinia, and samples of the internal organs of three feti affected by Neu-Laxova syndrome from Malta. The peculiarity of the latter was that they were born, along with

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three more, in a period of twenty moths, something really surprising, since that syndrome is extremely rare and Malta counts no more than 400,000 inhabitants. The animal cases presented two different macroscopic kinds of malformations: the first lamb had no developed brain nor eyes, in whose place sat its ears. The second had the extrusion of the intestine. Both survived just few hours. The three cases examined out of six feti affected by Neu-Laxova syndrome were sent to us by the University of Malta for the search of possible genotoxic material. Despite its extreme rarity, six cases were reported in Malta, a country with a population of 400,000 inhabitants, in the space of just 18 months. Neu-Laxova syndrome is a rare, incompatible with life, syndrome of unknown origin characterized by ichthyosis (scaly skin), intrauterine growth retardation, microcephaly, short neck, central nervous system abnormalities, hypoplastic or atelectasia of the lungs, deformities of the limbs, oedema, polyhydramnios (an abnormally high level of amniotic fluid), and short umbilical cord. As a matter of fact, inorganic particulate matter was clearly visible in all samples we examined. At the same time, we analyzed some internal organs of fetus from induced abortions and in all cases they appeared free from any particulate contamination.

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Fig. 3.9.1 Morphology of four different tissues: brain, thymus gland, spleen and kidney of a fetus whose delivery was artificially induced (markers: A = 20 µm, B = 50 µm, C = 100 µm, D = 100 µm).

Fig. 3.9.1 shows different tissues belonging to a healthy fetus from an induced abortion. The tissues appear free of any foreign body. They are composed of only Carbon and Oxygen. (Hydrogen and Nitrogen are part of the composition of the natural tissues, but it is impossible to see them with our equipment). All the images that follow regard samples taken from malformed feti. Surprisingly enough, since those beings had never lived in the environment we all share, particulate of exogenous origin could be detected in their tissues. The compositions of some of those particles were particularly strange and, in a few cases, certainly toxic.

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Fig. 3.9.2 Antimony particle inside the baby’s liver affected by Neu-Laxova syndrome (marker 20 µm).

Fig. 3.9.3 Strontium-Sulphur-Iron-Aluminium particle inside a baby’s liver affected by Neu-Laxova syndrome (marker 20 µm).

Fig. 3.9.2 and Fig. 3.9.3 regard the same case.

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Fig. 3.9.4 Lead-Bismuth particle surrounded by red cells in a liver sample of a case of Neu-Laxova syndrome (marker 10 µm).

Fig. 3.9.5 Antimony particle inside a baby’s kidney affected by Neu-Laxova syndrome. The subject is the same as the one of Fig. 3.9.4 (marker 10 µm).

Fig. 3.9.4 and Fig. 3.9.5 belong to the same subject. In the three cases we had the chance to examine, the particles found showed different compositions but Antimony was always present.

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Fig. 3.9.6 Zinc particle in a Neu-Laxova’s syndrome liver (marker 10 µm).

Fig. 3.9.7 Antimony particles inside a baby’s kidney affected by Neu-Laxova syndrome (marker 20 µm).

The pictures above (Figs. 3.9.6 and 3.9.7) belong to the same case. All the three cases of malformed feti showed the presence of micro and nanoparticles inside the liver and the kidneys. The spectral analyses of all the particles found in the small samples received revealed that they are contaminated with metallic foreign bodies, sometimes toxic, like the compounds of Antimony, Strontium or Lead. This presence is really peculiar, if we consider that feti are never directly exposed to the environment and its pollution. According to what we found, those specimens present different levels of pollution as far as concentration is concerned, but they all share the presence of Antimony compounds. A

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study aimed at searching the origin of this contaminant investigating parents’ life and environment they lived in is in progress.

Fig. 3.9.8 Iron-Zirconium-Silicon-Aluminium-Magnesium-Zinc-Copper-Chromium-CalciumAntimony particle in the liver of a malformed fetus (marker 10 µm).

Fig. 3.9.9 Lung tissue from a malformed fetus. Clustered Nickel-Chromium nanoparticulate is present (marker 20 µm).

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Among the oddities found in the lung pathologic tissues, we want to show also an indirect exposure. The direct exposure to pollution regarded a pregnant woman, but that pollution moved to the fetus through the placental blood circulation. The pulmonary tissue of a 20-week fetus is shown in Fig. 3.9.8 and Fig. 3.9.9. The artificial abortion was due to the discovery of a 2-cm vertebral fission. Clusters of micro and nanoparticles were visible in the lung tissue, composed of Nickel-Chromium. This debris is chemically toxic. The particle shown in Fig. 3.9.8 is rather large, about the same size of a red cell, and its composition is very complex. Also in the case of this fetus, Antimony was present. The next is not a human case, but a veterinary one (Fig. 3.9.10 and Fig. 3.9.11). It regards the malformed fetus of a lamb died immediately after delivery. Its mother grazed in the close vicinity of a firing ground in Sardinia where grass was contaminated by the dust coming from the explosions consequent to military experiments and drills.

Fig. 3.9.10 Antimony-Sulphur particle found in a malformed lamb’s brain (marker 10 µm).

Also in the cases of the malformed lambs we found a wide dissemination of nanoparticles in all its organs, including the brain and testicles. As in the human cases, Antimony was a constant presence.

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Fig. 3.9.11 Antimony-Cobalt-Sulphur nanoparticle in the liver tissue of the malformed lamb of Fig. 3.9.10 (marker 20 µm).

It was somewhat odd to find recurrent chemical compositions shared by either the human feti of Malta and the animal ones in Sardinia. But Antimony, the element in question, could also be found in combination with Cobalt in the sperm of a soldier who worked in a firing ground in Sardinia and in a Canadian soldier who served during the First Gulf War. This series of coincidences urged us to start a project on fetal malformation which is in progress.

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Nanopathology Tab. 3.9.1 Flow-chart of nanoparticle dissemination with the entrance points.

Note: The number 1 in the insets means a primary “door” of entrance of the particles inside the human body.

All the images shown in the previous pages suggest a particular dissemination of the nanoparticles once inhaled or ingested in the human body. Their persistence in the tissues can induce possible pathological reactions. The Tab. 3.9.1 is an attempt to summarize the achieved experience. Further studies are necessary to confirm the findings.

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3.10 Bibliography Albores-Saavedra, J., Vitch, F., Delgado, R., Wiley, E. and Hagler, H., (1994). Sinus histiocytosis of pelvic lymph nodes after hip replacement. A histogenic proliferation induced by cobalt-chromium and titanium. Am J Surg Path; 18: 83-90. Anderson, D. S., Nicholls, J., Rowland, R. and LaBrooy J. T. (1998) Hepatic granulomas: a 15-year experience in the Royal Adelaide Hospital. Med J Austr; 18: 148 (2): 71-74. Armaroli, N. and Po, C. (2003). Centrali termoelettriche a gas naturale, produzione di particolato primario e secondario, RICH-MAC Magazine – Novembre pagg. 45-51 Ballestri, M., Baraldi, A., Gatti, A. M., Furci, L., Bagni, A., Loria, P., Rapanà, R., Carulli, N. and Albertazzi, A. (2001). Liver and kidney foreign bodies granulomatosis in a patient with malocclusion, bruxism, and worn dental prostheses, Gastroenterology, 121, 1234-1238. Bos, I., Johaninsson, R., Lohrs, U., Lindner, B. and Seydel, U. (1990). Comparative investigations of regional lymphnodes and pseudocapsules after implantation of joint endoprosthesis. Pathol Res and Pract; 186: 707-716. Brincker, H. (1990). Granulomatous lesions of unknown significance: in biopsies from lymph-nodes and other tissues: the GLUS-Syndrome. Sarcoidosis; 7 (1): 28-30. Brown, L. M., Collings, N., Harrison, R.. M., Maynard, A. D. and Maynard, R.. L. (2000). Ultrafine particles in the atmosphere: introduction Phil. Trans. R. Soc. Lond. A 358, 2563-2565 Centeno, J. Selinus, O. (2005) Essentials in medical Geology, Academic Press New York Courter, L. A., Musafia-Jeknic, T., Fischer, K., Bildfell, R., Giovanini, J., Pereira, C., Baird, W. M.(2006). Urban dust particulate matter alters PAH-induced carcinogenesis by inhibition of CYP1A1 and CYP1B1, Toxicol Sci. 2007 Jan;95(1):63-73. Epub Oct 23 Denk, H., Scheuer, P. J., Baptista, A. et al. (1994). Guidelines for the diagnosis and interpretation of hepatic granulomas. Histopathology; 25: 209-218. Ebelt, S., Brauer, M., Cyrys, J., Tuch, T., Kreyling, W. G., Wichmann, H. E. and Heinrich, J. (2001). Air quality in postunification Erfurt, East Germany: associating changes in pollutant concentrations with changes in emissions, Environ Health Perspect. Apr;109(4):325-33. Fried, E. D. and Godwin, T.A. (1992). Extensive diffuse pulmonary ossification. Chest.;102(5):1614 Friedland, J. S., Weatherall, D. J. and Leedingham, J. G. (1990). A chronic granulomatous syndrome of unknown origine. Medicine (Baltimore); 69 (6): 325331. Gatti, A. M. and Montanari, S. (2006). Retrieval analysis of clinical explanted vena cava filters J. of Biomedical Materials Research: Part B. 77B, 307-314

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Gatti, A. M. and Monari, E. (1998). Bioactivity of the active glasses in New Biomedical Materials: Basic and Applied Studies, Ed. Haris, P.I., Chapman D., IOS Press Amsterdam, 73-80. Harrison, R. M., Shi, J. P., Xi, S., Khan, A., Mark, D., Kinnersley, R. and Yin, J. (2000). Measurement of number, mass and size distribution of particles in the atmosphere Phil. Trans. R. Soc. Lond. A 358, 2567-2580 Heffner, D. K. (2007). The cause of sarcoidosis: the Centurial enigma solved. Ann Diagn Pathol. Apr;11(2):142-52 Ikeda, Y., Yamashita, H. and Tamura, T. (1998). Diffuse pulmonary ossification and recurrent spontaneous pneumothorax in a patient with bronchial asma. Respir Med.;92(6):887-9. Jacobs, J. J., Gilbert, J. L. and Urban, R. M. (1998). Current concepts reviews. Corrosion of metal orthopaedic implants. J Bone and Joint Surgery 80A: 268-282. Kotani, J., Nanto, S., Mintz, G. S., Kitakaze, M., Ohara, T., Morozumi, T., Nagata, S. and Hori, M. (2002). Plaque Gruel of Atheromatous Coronary Lesion May Contribute to the No-Reflow Phenomenon in Patients With Acute Coronary Syndrome Circulation.;106:1672 Melato, M. and Rizzardi, C (2001). Malignant pleural mesothelioma following chemotherapy for breast cancer, Anticancer Research 21: 3093-3096 Menzies, R. and Fraser, R. (1987). Round atelectasis; pathologic and pathogenetic features. Am J Surg Pathol; 11:674-81 [8] Montanari S., 2000, Malattia tromboembolica e filtri cavali - Ed. C. Rabbia, G. Emanuelli – 90-140 Minerva Medica – Turin Naesens, M., Steels, P., Verberckmoes, R., Vanrenterghem, Y. and Kuypers, D. (2004). Bartter’s and Gitelman’s syndromes: from gene to clinic, Nephron Physiol. ;96(3):p65-78. Nemmar, A., Hoet, P. H., Vanquickenborne, B., Dinsdale, D., Thomeer, M., Hoylaerts, M. F., Vanbilloen, H., Mortelmans, L. and Nemery, B. (2002). Passage of inhaled particles into the blood circulation in humans, Circulation. 2002 Jan 29;105(4):411-415. Newby, J. A., Howard, V. 2006 Environmental influences in cancer aethiology, Journal of nutritional and environmental medicine, review 1-59 O’Shaughnessy, K. M. and Karet, F. E. (2004). Salt handling and Hypertension, J. Clin Invest.; 113(8):1075-1081. Peters, A., Doring, A., Wichmann, H. E. and Koenig, W. (1997). Increased plasma viscosity during an air pollution episode: a link to mortality?, Lancet. May 31;349(9065):1582-7. Ruuskanen, J., Tuch, T., Ten Brink, H., Peters, A., Khlystov, A., Mirme, A., Kos, G. P. A., Brunekreef, B., Wichmann, H. E., Buzorius, G., Vallius, M., Kreyling, W. G. and Pekkanen, J. (2001) Concentrations of ultrafine, fine and PM2.5 particles in three European cities. Atmos.Environ. 35 (21): 3729-3738.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 133 Samet, J. M., Dominici, F., Curriero, F. C., Coursac, I. and Zeger, S. L. (2000). Fine Particulate Air Pollution and Mortality in 20 U.S. Cities, 1987–1994, N Engl J Med 2000; 343:1742-1749, Dec 14. Stark, P. (1982). Round atelectasis: another pulmonary pseudotumor. Am Rev Respir Dis 125:248-50. Schwarze, P. E., Øvrevik, J, Lag, M., Refsnes, M., Nafstad, P., Hetland, R. B., Dybing, E. (2006). Particulate matter properties and health effects: consistency of epidemiological and toxicological studies Hum Exp Toxicol. Oct;25(10):559-79 Soini, Y., Kahlos, K., Puhakka, A., Lakari, E., Saily, M., Paakko, P. and Kinnula, V. (2000). Expression of inducible nitric oxide synthase in healthy pleura and in malignant mesothelioma. Brit. J. Cancer 83(7): 880-886. Tomonori, T., Masabumi, Y., Hiroaki, M. and Hitoshi, K. (2004). Roadside observation for chemical composition and size distribution of fine particles using Aerosol Mass Spectrometer in Tokyo. Japan Petroleum Energy Center presented at 18 Aug., 8th ETH-Conference of Zurich Trejo, O., Xaubet, A., Marin-Arguedas, A., Torres, A., Ramírez, J. and Luburich, P. (2002). Dendriform pulmonary ossification associated with idiopathic pulmonary fibrosis. Arch Bronconeumol. 38(8):399-400. Spanish. Urban, R. M., Jacobs, J. J., Tomlinson, M.J., Gavrilovic, J., Black, J. and Peoc’h, M. (2000). Dissemination of wear particles to the liver, spleen and abdominal lymphnodes of patients with hip or knee replacement. J Bone and Joint Surgery; 82-A (2): 457-477. Virchow R. Gesammelte Abhandlugen zur Wissenschftlichen Medizin. Arch Pathol Anat (Berlin) 10 (1856) 225 Wells, H. G. and Dunlap, C. E. (1943). Disseminated ossification of the lungs. Arch Pathol Lab Med.; 35: 420-46. Wilson, A. M., Ardehali, R., Brinton, T. J., Yeung, A. C. and Vagelos, R. (2006). Successful removal of a paradoxical coronary embolus using an aspiration catheter - Nature Clinical Practice Cardiovascular Medicine 3,633636doi:10.1038/ncpcardio0681] Schwarze, P. E., Øvrevik J., Läg M., Refsnes M., Nafstad P., Hetland R.B. Dybing E. “Particulate matter properties and health effects: consistency of epidemiological and toxicological studies” Human and experimental toxicology 2006 25, 599-578) Pope CA 3rd, Muhlestein JB, May HT, Renlund DG, Anderson JL, Horne BD. (2006), Ischemic heart disease events triggered by short-term exposure to fine particulate air pollution. Circulation. Dec 5;114(23)m:2443-8. Pope CA, Dockery DW (2006), Health effects of fine particulate air pollution: lines that connect. J Air Waste Manag Assoc. Jun;56(6):709-42. Review

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Chapter 4

Six “Detective Stories” ___________

7

he technique we developed allowed us to set-up a new investigative method to assess the patient’s actual exposure to micro and nanopollution. The identification of the particle chemistry found in the pathological tissues, along with their size and shape, induced us to investigate on the patient’s life and look for possible sources of that particular contamination, the real possibilities of his exposure and how that pollution contamination may have occurred. In some cases we saw that the exposure continued to be possible, so, some suggestions were given to the patient to avoid it. That kind of work had us discover unexpected ways of contamination and, quite often, unknown ways of dissemination of the particles inside the human body. Six of such cases are briefly described below. The 1st Case

The first case is related to a 25-year old non smoking girl. She was admitted to the hospital because of vascular problems affecting her hands and an altered electrical conduction of her peripheral nerves. The first diagnosis she received was sclerodermia, but later, after the neuro-electrical test results, a diagnosis of ischemic miopathy was advanced. The steroid therapy undertaken proved to be ineffective.

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A biopsy of the left-leg muscles (Fig. 4.1) was performed and specific tests on the fresh tissue revealed an endothelial vasculitis, diagnosis confirmed by Harvard University researchers. Another histological section was sent to us to be processed and analyzed with our technique. What we saw among other particulate were submicronic particles of Carbon-Tungsten outside a blood vessel.

Fig. 4.1 Image of Carbon-Tungsten nanoparticles inside the muscle biopsy (marker 20 µm).

The way we do in similar cases, because of the presence of such unusual foreign bodies which we took as a sort of marker, we started to investigate on the patient’s occupational life that had taken place all along in a small hairdresser’s shop. We checked a great variety of the products used there to see if Tungsten was present somewhere, but no trace of that metal was to be found. So, we analyzed the dust from the plaster of the shop premises, the indoor particulate pollution in the air, the dust inside the hairdryers (the electrical resistances of the hairdryers can be made of Tungsten) and the smoke of the cigarettes smoked by the only smoker who worked there. Again, no trace of Tungsten was to be found. Special attention was dedicated to parents’ story, also considering the fact that the patient’s father had died of a lung cancer. That diagnosis had been issued by observing bronchial mucus smeared on a glass slide, and we look for it in the hospital’s archive. Had we found Tungsten there, we could have identified a pollutant shared by both father and daughter and that would have put us on an easier track.

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It must be said that biological samples on glass are not ideal for our analyses, since glass issues signals that can interfere with those issued by the particles. In that particular specimen we found a few metallic particles, but no Tungsten. So, that hypothesis of domestic pollution had to be ruled out and we had to restart the investigation, which we did in the patient’s house. The indoor particulate contamination in the air was analyzed together with the wall plaster, the cigarettes smoked by her mother and the food more frequently eaten by the patient. But not even there any trace of Tungsten was found. We had to find a source of Tungsten if we wanted to solve the case. That metal or its carbide (Carbon-Tungsten particles were what we had found in the girl’s biopsies) are rather common in metallurgical industries, but the patient did not seem to be likely to be or to have been involved in such a contamination, as she had lived all her life in a flat downtown, far from industrial sites. In the course of our many interviews, the girl told us that two years ago she had had a bleeding from the endometrium which did not respond to any pharmaceutical treatment and that was solved surgically. Before the operation, she had also suffered from two bleeding episodes from the anus, and in both cases she was treated at the Emergency Room of the local Hospital. During the surgical operation, a small biological sample of endometrium (Fig. 4.2) was taken and processed for the usual histological tests. We analyzed the archived sample and found small particles of Iron-Chromium-Nickel (stainless steel) there. The presence of very small particles ranging from 0.5 to 8 µm in such an anatomical site seemed very strange. Their presence could have been the cause of the bleeding, since those are bodies foreign to the human organism and, in contact with the mucosa, can trigger an inflammatory reaction and, in some instances, a localized necrosis. The inflammation can also reach deeper into the tissues. That could explain the strange episodes of bleeding from the anus which could depend on an acute inflammation of the internal tissue in close vicinity of the vagina or the uterus.

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Fig. 4.2 Image of a sample of endometrium with stainless steel nanoparticles (marker 20 µm).

Such an event reminded us of the Burning Semen Disease suffered by the wives of the veterans after their coming back from the war missions in Iraq or Afghanistan during the 1st and 2nd Gulf War (see Cap. 5) This findings had us redirect the investigations toward the patient’s partner and we discovered that he had suffered from a testicle cancer, operated on four years ago. So, we thought that analyzing his pathological tissues could give us some important clues. At the time of the disease, the partner worked in a tile factory as a clerk, but his desk was located in a large room, close to a micronizing tool used to ground the clay before it was mixed with water, and to a special machine used to cut the edge of the tiles. The grinding and cutting tools were made respectively of stainless steel and Tungsten carbide. The partner told us that during the period he was employed there, the man who worked in direct contact with the latter machine had died of a lung cancer. The analyses on the four surgical samples we got revealed the massive presence of micron-sized particles of silicates (tiles) and debris of stainless steel and Tungsten. The finding of this material inside a testicle had us put forward a mechanism of dissemination and translocation: The partner had been exposed to the materials making up the tiles and to the wear debris coming from two different tool machines. Following a mechanism that

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we cannot yet fully explain but that we have observed in many instances, we think that those debris were entrapped in a testicle (we had no samples from the contralateral one, so, nothing about it may be said) after having been inhaled, and the metallic nanoscaled particles (stainless steel and Tungsten) extravasated and contaminated the sperm which was donated to our patient during sexual intercourse. The presence of non biocompatible, not biodegradable particles in the girl’s vaginal mucosa caused pain, inflammation and bleeding as occurs in the veterans’ wives. The surgery that followed the bleeding episodes exposed a compromised tissue, without any effective defense against sperm contamination. As a matter of fact, the symptoms referred to the sclerosis started a few months after the surgical operation. The probable reasons why Tungsten, and not stainless steel, particles were so widely disseminated were because of their size, much smaller than that of the stainless steel particulate, or because of a physicalchemical interaction withy the blood components, or both. Such dissemination in the blood circulation had been the cause of the vasculitis. The particles extravasated and came into contact with the muscle fibres, causing a fibrosis, which is the normal biological reaction to the presence of non biocompatible foreign bodies. Of course, we did not have the possibility to check but a few of the girl’s tissues, but, as far as we saw, the particle dissemination seemed to be ubiquitous. So, we suggested to have protected sex in order to avoid further exposure, but the physical elimination of every single particle from the organism looked impossible, at least to our experience and for the time being. In the case described, Tungsten acted as a marker and the search of its specific source of pollution allowed us to identify the kind of exposure and the gross mechanism of dissemination inside the organism. The following images show the Tungsten particles found in the spermatic vessel (Fig. 4.3), the stainless steel debris (Fig. 4.4) and the particles of silicatic material from the ceramic tiles (Fig. 4.5, 4.6 and its spectrum).

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Fig. 4.3 Image of a Tungsten particle found inside the spermatic vessel (marker 50 µm).

Fig. 4.4 Image of a stainless steel particle (Iron-Chromium and Nickel) found in the cancerous tissue of the testicle (marker 20 µm).

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Fig. 4.5 Image of the part of the resected cancerous tissue of the testicle with the spectrum of the normal biological tissue (marker 1.000 µm).

Fig. 4.6 shows the “dust” found in the resected tissue of the testicle cancer. The samples contained many different particles, mainly silicatic debris coming from the dust of the tiles (white particles indicated by arrows). They are composed of Silicon-Aluminium-Calcium. Normal biological tissue contains only Carbon-Oxygen-Sodium-PhosphorusSulphur-Chlorine. We do not know if the cancer was induced by the metallic particles or by the enormous pollution.

Fig. 4.6 Image of an agglomerate of silicatic nanoparticles trapped in a calcific tissue (marker 50 µm).

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The 2nd Case A professor of Mathematics started to betray some problems in articulating the sound “r” during his lectures at the University. After different neurological examinations, a diagnosis of Lateral Amyotrophic Sclerosis was issued, a diagnosis he did not believe in and refused to accept. Thus, the professor started to search the Internet about that disease and learnt that the one of the first, more common symptoms is a slight claudication and this, among other symptoms he had read about, was not something he suffered from. In the enormous quantity of not always reliable information he found in the Internet, he read that dental amalgam, because of its content of Mercury, could be somehow related to the disease he had been diagnosed. So, the professor asked Dr Gatti her opinion as a biomaterial expert and a teacher of Dental Materials at the University of Modena and Reggio Emilia. Actually, he had either amalgam restorations and dental prostheses in his mouth and hardly in two teeth the amalgam did not look corroded. (It is only in the course of corrosion that amalgam releases Mercury, Silver, Tin and Copper ions which can be toxic, locally in the oral mucosa and in a systemic way after ingestion). But the observation of the mouth revealed an unusual situation: The surface of the tongue was not symmetrical and the right half was not as flat as the left one, but was corrugated and wavy. The surface of the mucosa seemed to have a larger extension than the substrate, and that created a sort of gibbosity, not adherent to the tongue base. In addition to that, we noticed a discoloration in the internal cheek mucosa at the level of the intersection of the teeth. We also observed that in the right part of the mouth some porcelain prostheses had been implanted. Having seen all that, we thought that if there is a wear phenomenon, debris must be released, and those debris can be entrapped by the nearby mucosa and induce a fibrosis. The tongue mucosa has a great aptitude to absorb, so a passage of some fine particles through it cannot be surprising and their entrapping occurs at the level of the underlying

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tissue called corium. That formation is a thick network of fibrous connective tissue strictly connected with numerous elastic fibres, forming the septa between the muscular bundles of the tongue. It contains the branches of many nerves and vessels that feed the papillae. The tissue can react to a foreign body giving origin to a fibrosis. A thickening and the following contraction of that layer could explain the humped morphology of the mucosa surface and the loss of agility of the tongue. It is also reasonable to think that all that exerts a compression on the underlying muscle and nerves. But, in any case, a collection of foreign bodies can give origin to a fibrosis, and a localized fibrosis could be responsible for the problem in articulating the sound “r”, because a fibrotic tissue is thick, less elastic than normal and does not allow fine movement. The first step we had to take was to demonstrate the presence of that fibrosis with the inclusion of prosthetic debris in a biopsy of the internal side of the cheek. As a matter of fact, the tissue presented a fibrosis and some porcelain debris were present there. The second step was to demonstrate a similar presence in a biopsy of the right, ipsilateral side of the tongue. The hope was that if a fibrotic reaction due to foreign bodies was present, probably the removal of this tissue or a cut made on the tongue could interrupt the phenomenon and its worsening. We thought that stopping the fibrosis’ progress could probably prevent further damage to nerves and blood vessels. In fact, the shrinkage of a fibrotic tissue can make the vessels occlude and stop of the supply of nutritious elements and Oxygen. The afferent and deferent nervous terminations can simultaneously stop the propagation of the electrical signal, thus causing the opening of the electrical circuit. While the histopathologist confirmed the presence of a fibrosis in the bioptic sample, we saw some strange precipitates of Zinc there. It is easy to tell the difference between exogenous particles and endogenous precipitates: The EDS signal is high in the case of “stones”, since the atomic structure is compact, while, in the case of precipitates, the inorganic element (Zinc in this case) is bound to proteins, forming insoluble metalloproteins. The density of these precipitates is very low and also the signal they issue is low (proteins contain Carbon, Oxygen,

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Hydrogen and Nitrogen, all very light elements). Also the morphology is characteristic. In our case, Zinc ions must have been absorbed by the tongue mucosa and in the sub-mucosa the interaction with proteins created insoluble precipitates. The sub-mucosa is reach of nerves and nervous terminations that can be compromised by the presence of toxic ions. Also the formations of metalloproteic precipitates can interfere with the cellular metabolism according to different mechanisms: protein depletion, protein immobilization or a change in the local pH. A further possibility is the entrance of ions within the cell where they can precipitate, forming aggregates that disturb the intracellular metabolism. The sequestration of proteins, namely the formation of insoluble precipitates, can interrupt cascade reactions that cause “alternative metabolic solutions”. It was not only Zinc what we detected, but a wide variety of particles ranging 0.8 – 2 µm. Gold particles were there, and this was compatible with the bridges the patient carried. 0.8 µm particles of Silicon, Aluminium, Calcium, Magnesium, Potassium and Iron were also present, and they could certainly be blamed for the fibrosis, the roughness of the tongue surface and the discolouration of the mucosa.

Fig. 4.7 Image of nanoparticles of Phosphorus-Zinc-Magnesium-Aluminium-SiliconCalcium-Iron found in the biopsy of the mouth mucosa (marker 20 µm).

But what puzzled us most was the presence of Zinc (Fig. 4.7). That is a metallic element commonly used as oxide because of its antibacterial activity, but its presence is unexpected inside the oral mucosa and an explanation for such a presence must be given.

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As usual, we questioned the patient about his diet, the use of chewing gum, drugs, etc., but no clue was to be found there. The truth emerged unexpectedly when we asked the patient about his visits to the dentist’s. The patient had been advised to use a mouthwash and he had used it in an obsessive way, many times a day, for more than 3 years. The label of the product reported the presence of a Zinc salt as a bactericide, but the solution contained also ethylic alcohol, a substance that increases the permeability of the tongue epithelium, thus making the entrance of the Zinc salt easier. That salt hydrolyzed, Zinc formed a metalloprotein and that metalloprotein precipitated. Its presence was the likely cause of the partial necrosis of the biological structures there present. Zinc is a well-known bactericide and is toxic to cells. Therefore, it could interact with the myelin sheath of the nerves and damage it. When we realized that, the patient had already quitted using the mouthwash and the damage to his nervous system had grown irreversible; so, all that was of no practical use to him and he died in six months. If our hypothesis is correct, this is a case where very simple precautionary measures could have avoided the gravest of consequences. The 3rd Case The manager of a very important company consulted our laboratory for a case of dermatitis afflicting his trunk, asking to check the biopsy of the derma interested by the affection. The observation of the very small sample showed the presence of numerous particles of stainless steel (Fe-Cr-Ni). Such a finding looked strange, because neither at home nor at the patient’s working place stainless steel particulate should have been present. He lived an apparent healthy life in a “normally” polluted big city. He had been a smoker in the past but in the last 10 years he had never lit a cigarette.

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The patient told us that in the course of a yearly check up he had been subjected to a chest X-ray examination that revealed light spots in his lungs and, because of that, some biopsies of those spots had been performed. The response was that the areas were calcified. We took those bioptic samples, observed them with our usual method, and found that stainless steel was present there in the form of small micrometric and submicronic particles. Having been found in the lungs, we supposed as a probable hypothesis that those pollutants had been inhaled; some of them had stayed in the lungs where they had triggered an inflammation with a consequent calcification, and others had been disseminated throughout the organism because of a size small enough to allow the passage through the lung barrier and enter the blood stream. Their presence in the skin (Fig. 4.8) could be explained with an attempt of the organism to get rid of them, but they were halted by the derma which is very compact and hard to cross, and there they caused the irritation the patients complained of. Such a hypothesis must be demonstrated, and to do that we had to find out the origin of that particular pollution and the way our patient had been exposed to it. Questioning him, we got to know that he had suffered from nasal congestion for more than twenty years and for that reason, to try and breath better, he had been operated on his nasal septum one year ago. But for the previous twenty years, he had put drops in his nose to decrease the congestion. During all that period, he had used only two types of herbal drugs, one coming from Italy and one from Switzerland, and we checked both products from bottles the patient still conserved, even if he did not use them any more after the successful surgery he had undergone. What we saw was that only one of the two oily liquids contained particles, and they were the same size and composition as those we had detected in the pathological tissue. That could be explained by guessing that the herbs used by that particular laboratory were ground with a stainless steel tool that, because of the obvious wearing of the machine, released particles to the product. We wondered why the presence of metal particles did not induce a more serious disease like, for example, a cancer?

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A possible explanation is that the quantity of particles was relatively small and the oil used as a vehicle protected them from corrosion, thus preventing a release of Iron, Chromium and Nickel ions. Besides, that oil was organic and the tissues had an interaction with something they recognized and did not see the inorganic bodies below. So, all that occurred was a comparatively mild inflammation with a consequent calcification. In that case, the exposure had been interrupted and nothing else could be done as a preventive or therapeutic measure.

Fig. 4.8 Image of three particles of stainless steel embedded in a biopsy of derma (marker 20 µm).

Fig. 4.9 Image of a small stainless steel particle found in a lung biopsy (marker 20 µm).

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Fig. 4.10 Image of a stainless steel particle found inside the nose drops used by the patient (marker 20 µm).

In Fig. 4.9, a single stainless-steel particles found in the patient’s lung is visible. Fig. 4.10 and Fig. 4.11 show the contamination of Iron-based particles detected after filtration in two different nasal drops used by the patient.

Fig. 4.11 Image of the metallic contamination of the nose drops used by the patient, and deposited on a paper filter (marker 200 µm).

The 4th Case In this case, the patient was a 45-year-old lady suffering from a peritoneal mesothelioma.

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She had been operated on and some bioptic samples of her peritoneum were sent to us to be analyzed. There we found many debris singularly disseminated in the specimen: metals like, for example, stainless steel were present, but one particular chemical composition attracted our attention. The EDS spectrum indicated that a number of submicronic particles were a silicate containing, among other elements, Uranium and Thorium. In that very period we were busy with specimens coming from Italian and French veterans back from former Yugoslavia, and Uranium was what we were looking for, but without any result: No trace of Uranium was to be found in those pathological tissues. Now we were confronted with the case of a lady who had never been in a war theatre nor, to her knowledge, in the vicinity of a source of radioactivity, but had spent her whole life in a quiet Italian town working as a clerk in an office, yet she had Uranium and Thorium in her body. So, we started a hunt to identify the origin of such an unusual and unexpected particulate. Being a silicate, we immediately suspected that the origin of the pollution found could be an area about 20 kilometres south of the place where the patient lived, where numerous ceramic factories are located. As to Uranium, it is used to give a yellow colour to enamels and Thorium is used as well. The study of the winds, though, seemed to rule out such a hypothesis or, at least, to make it very unlikely.

Fig. 4.12 Image of a particular debris containing radioactive elements found in the peritoneal mesothelioma (marker 20 µm).

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Fig. 4.12 shows a particle that contains Titanium-ZirconiumCalcium-Iron-Aluminum-Yttrium-Niobium-Uranium-Thorium-CeriumManganese-Copper. That can be originated by tiles industries that use a silicatic or Zirconium-based earth for the tiles.

Fig. 4.13 Image of a particle found on a leaf surface. Its composition includes radioactive elements (marker 20 µm).

Fig. 4.13 shows a particle composed of Silicon-PhosphorusAluminium-Uranium-Thorium-Lanthanum-Cerium-Neodymium-IronPotassium-Calcium. That composition is typical of a type of earth used for ceramic tiles. Uranium and Thorium are likely to have been added as components of some glazes. Since those particles had been found in the peritoneum, i.e. anatomically close to the digestive system, we assumed as a likely, though not exclusive, hypothesis that ingestion could have been the way they were introduced into the organism. According to that assumption, we began to search the food the patient ate more frequently, despite the fact that what was in the peritoneum could have entered long ago with a food, if food was indeed the Trojan horse, that had been used in the past and then forgotten. Nevertheless, we tried. All the family composed by a husband and a girl, both healthy, was involved in that sort of treasure hunting. The patient told us that both her parents had died of liver cancer, without any doubt something worth investigating, but we could not find

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their biopsies and, of course, the histopathological records did not contain anything that could help us. Questioning the family about their preferences in the matter of food, we got to know that the lady preferred vegetables to meat, unlike her husband and daughter, and she was particularly fond of a kind of wild chicory that her parents had been bringing to her at least once a week from a village in the hills, about 30 kilometres away from town, where she had moved at the age of 22 when she married. That chicory had been a usual dish to her since her childhood and had never ceased to be. Chicory was what we had to check and we went to the small valley were it had always been picked. It was winter time and no chicory could be found in that place nor other edible vegetables, but, looking for possible clues of pollution, we saw a plume of smoke in the distance and we drove there, four or five kilometres away as the crow flies. There we saw a group of small ceramic factories whose existence was unknown to us. Then, we went back to the valley and picked some oak leaves still hanging from a few trees that we brought to the laboratory and observed under ESEM. On those leaves we detected particles containing silicatic compounds with Uranium and Thorium. According to what we discovered, the lady and her parents had been eating something they believed to be very safe but which, in fact, contained toxic (and slightly radioactive) particulate. Now, a final piece was missing to the solution of the puzzle: why had our patient contracted a peritoneal mesothelioma, while her parents had died of liver cancer (granting the fact that the aetiology was the same)? Twenty three years ago, our patient had spent her honeymoon in the North African desert and, when a guest at some Bedouins’, she had drunk tea which gave her a typhoid fever. Intestinal problems lasted a long time and, probably, that inflammatory condition altered the permeability of her intestinal mucosa which became more negotiable to particles. If we accept that hypothesis, had she had a healthy gut, she would have contracted not a mesothelioma but a liver cancer, the way her parents did.

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The 5th Case A couple of somewhat unusual patients, two lady doctors working in the neonatal department of a big hospital, came to our laboratory, asking us if we could help solve their case. Four doctors, including the chief of the department, and two nurses suffered from different diseases: cancer, sarcoidosis, and two missing diagnoses, as the operators developed symptoms that could not be referred to any known disease. One of the two ladies suffered from a lung sarcoidosis and inside the granulomatous formations typical of that disease we found micro and nanoparticles of stainless steel (Fig. 4.14) and silicates (Fig. 4.15).

Fig. 4.14 Image of a small stainless-steel particle (indicated by arrow) found in a lung biopsy performed to confirm the diagnosis of sarcoidosis (marker 20 µm).

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Fig. 4.15 Biopsy of a nodule containing Silicon-Aluminium particles (marker 10 µm).

Fig. 4.16 Bacterium (inside the circle) containing Aluminium particles collected by gravity on a Carbon sensor (marker 10 µm).

The second case was different. The doctor-patient contracted a bacterial infection in the ring finger of her left hand, an infection that proved resistant to the specific antibiotic administered. Those bacteria had been detected within the Intensive Care Unit of the department, where the patient was usually busy (Fig. 4.16). In a short time, the patient had started to suffer from fatigue, than from acute episodes of sometimes monolateral and sometimes bilateral pain at the lower limbs followed by the formation of nodules that were surgically removed as many as 18 times without any advantage to the patient, since the pathology continued its progress. Our investigations in

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those nodules discovered the presence of stainless steel and Aluminium particulate. A section of those nodules was sent to the Harvard School of Medicine in Boston and the diagnosis issued was of a vasculitis. Our hypothesis was that phenomena of extravasation of those particles, running in the circulatory system, had been occurring, and that could explain the acute pain. Then, the presence of foreign bodies in the muscular tissue could have reasonably induced a biological reaction that drove to the formation of the nodules. We had to look for the source of pollution that could have caused the pathologies in doctors who had been living what for doctors is a normal life. So, we decided to sample the air in two adjacent rooms where the doctors work more frequently and we did so by means of an air pump that worked for one hour. The dust was collected on cellulose filters where particles smaller than 2.5 µm were detected. We were told that just outside the building where the department was located, another building had been standing and that building had been very slowly pulled down raising great clouds of dust. The still standing broken stainless-steel reinforcement had been left on place for a long time. The pollution created was aspirated by the air conditioning system set on the roof of the department. Every room was equipped with a filtration system that can only stop particles larger than 2.5 micron, while smaller particles can pass easily and their presence inside the rooms was concentrated because of a recirculation system which created an accumulation of fine pollutants. It is only natural that everybody who works in such environmental conditions is exposed to the effects of that sort of pollution. The main difference between the two cases was the much more regular presence of the second doctor in the Intensive Care Unit, where a bacterial contamination had been found, and those bacteria were, in turn, contaminated with Aluminium and had served as a carrier. Aluminium particulate was inside the bacteria we found and that bulky presence changed their morphology. It was probably for that change in shape that the antibiotics administered did not have any effect. In the first case, the inhalation of foreign bodies had induced a granulomatosic reaction,

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while in the a second one the infective carrier of the pollution was predominant. To the same way of entrance, two different pathologies had ensued. The cases proposed and the solution found should have us reflect. The solution of the twin cases had been possible because we started from looking for and finding the real exposure the patient had undergone. Every patient has a personal history and epidemiological studies are not always helpful or are not meaningful at all if the number of cases is very restricted. So, a single-patient oriented, “customized” investigation should be carried out in such circumstances. That is certainly far from easy, expensive and time consuming, all things our society does not look willing to accept, but we do not see any other way to deal with patients like the ones we were confronted with. Far from easy, expensive and time consuming but not useless. The knowledge gained from such cases is of the utmost usefulness to prepare prevention guide lines and, in a very pragmatic way, prevention means saving money. In the case in point, we got a further proof that any uncontrolled release of nanoparticles in the working places must be avoided. Masks, specific filtration means and effective systems to abate acute dust pollution, especially when it is the nanometric size, must be studied and readily adopted. When dust is released in the environment, everybody must take proper care. How, it is the scientist’s task to say. The 6th Case The case of a dentist who had developed a lung sarcoidosis showed the existence of a close correlation between his disease and the powder he had used to bleach his patients’ teeth. In 20 years he had used about 300 kilograms of that fine powder, a part of which he had certainly inhaled and whose concentration in his organism had probably exceeded his tolerable threshold concentration. That dentist, male, 52 years old, never been a smoker, gradually developed shortness of breath during exercise, cough and retrosternal pain, which prompted for investigation of lung and heart function.

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Plain X-ray chest film showed signs of pulmonary fibrosis. A subsequent CT scan confirmed that fibrosis and showed the presence of several micro-nodules. All other diagnostic tests were normal. Sarcoidosis was suspected and a bronchoscopy was performed, during which a biopsy of the bronchial mucosa and bronchoalveolar lavage were carried out. Optical microscope observation of the biopsy revealed chronic inflammation of the bronchial mucosa, with edema and eosinophilic granulocytes. Bronchoalveolar lavage showed an increase in total cell number and CD4/CD8 lymphocytes ratio. A diagnosis of sarcoidosis was established and a steroid therapy was started. An investigation was performed in order to determine whether the disease could be traced to an occupational environment agent. The activity of dental surgeons is associated with several possible hazardous exposures (such as X-ray, dental material components, bloodborne pathogens and so on). Among these, aerosols of biological material and inorganic substances are specifically responsible for airborne exposure. The instruments used during dental surgical procedures may produce intense heat (electrocautery, laser) and such procedures usually generate fumes containing biological material (even partially unburnt). High-speed, air-driven dental handpieces, ultrasonic scalers, the polishing of composite and ceramic restorations and the use of the milling cutter on metallic prostheses disperse a large amount of aerosol and spatter including fine particulate matter. During the polishing and whitening of natural teeth, chemical compounds (composed by particles of Sodium di-carbonate and tricalcium phosphate) are sprayed on the tooth surface detaching part of its enamel and dispersing particles in the environment, thus exposing both patient and operator to risk of inhalation. For example, a standard hygiene procedure involves the use of prophylactic material and ultrasonic and/or manual instrumentation. The prophylaxis phase can be substituted by an artificial bicarbonate aerosol and that was what our subject had been doing. Artificial cleaning aerosols are formed by a Mini-Clean device (Castellini, Bologna, Italy)

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with air pressure set to 6 to 7 atmospheres and water flow to 1 atmosphere. Each individual run lasts 20-30 minutes for each patient. It has been reported that 95% of the particles measure less than 5 micrometers and are mainly concentrated within 2 meters of the patient where they can be easily inhaled by dental clinicians. For this reason, the dentist always protected himself with a face mask [3M ESPE 1942 F.B. (1800 NL) resistant molded face] whose level of efficiency was 90-92%. To test our hypothesis that sarcoidosis could be in this case related to a possible environmental agent, a further examination of biopsy specimens was performed. So, sections of the lung biopsy were sent to us to be analyzed. The label on the product used by our subject declared that the powder contained particles of Sodium carbonate (Fig. 4.17) and tri-Calcium phosphate (Fig. 4.18).

Fig. 4.17 Sodium-carbonate particle of and its EDS spectrum (marker 20 µm).

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Fig. 4.18 Tri-Calcium phosphate particles and their spectrum (marker 20 µm).

Fig. 4.19 Silica particles: a component non declared by the Manufacturer (marker 50 µm).

Fig. 4.19 shows other silicatic particles found in the polishing material. Those particles were not declared in the product label. Considering the patient’s long exposure to dust, a hypothesis was advanced that the particulate matter could originate from the wear of materials used during the abrasion and polishing operations. It was hypothesized that the material used for dental cleaning procedures could have a chemical composition similar to that of the

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debris found. A sample was observed under ESEM and EDS analyzed. One of the spectra obtained proved similar to that of the debris found in the lung. (Fig. 4.19). In the case we described, the diagnosis of sarcoidosis was issued according to standard criteria (ATS 1999). Additional information about the possible origin of the disease was provided by ESEM with EDS of biopsy specimens (Fig. 4.20 and Fig. 4.21), which demonstrated that the material found inside the sarcoidotic granulomas was identical to that found in the powder the dental surgeon had used in large quantities for several years. More Calcium-phopshatic particles were found in the bioptic specimens. Fig. 4.22 shows spherical particles composed by Calcium-phosphate with Sodium and Magnesium ranging from 0.5 to 2.5 µm. The prolonged inhalation of soluble triCalcium-phosphate and Sodium-carbonate pollution provided a large supply of Calcium, Phosphorus and Sodium ions. So, a logical correlation can be established between the inhaled pollution and the formation of those Calcium-phosphate entities.

Fig. 4.20 Particular of the lung biopsy (marker 200 µm).

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Fig. 4.21 Silica particle found in the lung biopsy (marker 20 µm).

Fig. 4.22 Spherules of Calcium-phosphate (marker 10 µm).

Such result cannot be considered a conclusive proof, but it does however induce a strong suspicion that the disease could have been initiated by the material used by the surgeon.

Chapter 5

War and Nanoparticles ___________ fter the 1st Gulf War many soldiers (mainly American, British and French) became ill. After the Balkan War also Italian and in our direct knowledge also Spanish and French soldiers, in the course or soon after their peace-keeping missions, developed strange collections of diseases, called later with the name of the site where that condition seemed to have had its origin. Then, after the 2nd Gulf War, other soldiers became ill. Medicine can neither explain the collective, simultaneous presence of all those symptoms nor cure them. Also the epidemiological studies carried out on that problem are not conclusive, also because they involve a relatively small number of cases, in a limited time. A new vision is now presented with some specific pieces of evidence that clarify the impact of the new war technologies with human life. It is a well-known fact that every kind of combustion generates particles, and explosions can rightfully be classified as combustions. Since the invention of gunpowder, probably in the middle of the thirteenth century, explosions have been increasingly used in warfare and have produced as a consequence clouds of dust, but it is only relatively recently that blasting bombs have started to be widely used. To our knowledge, the first connection between explosion and micro and nanoparticles dates back to the late Seventies of the last century, when the Air Force Laboratory, Armament Development and Test Center, of Eglin Air Base, Florida, issued a report about the results of a few experiments carried out on depleted-Uranium weapons which included the Air Force GAU-8, the Army XM 774 and M735E1 and the PHALANX gun systems (Technical Report, 1978).

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Depleted Uranium (DU) is a by-product of the enriching process of natural Uranium meant to be used in nuclear reactors, when the fissile and more radioactive isotopes are removed. Another, though less common, source of DU is reprocessed spent reactor fuel. DU is not so radioactive as natural Uranium and definitely less than enriched Uranium (the specific activity [the activity in becquerels (Bq) per unit mass i.e. one radioactive decay takes place per second] of DU is about 15 Bq per mg compared with 24.5 Bq per mg for natural uranium), but some not negligible radioactivity remains, and, in any case, DU is toxic. Once natural Uranium has been deprived of much of its precious 235 isotope or enriched Uranium is not usable any more, DU must be disposed of, and doing that is rather expensive. So, whoever faces that problem is more than willing to rid himself of that metal in a quite inexpensive way. In the late 1940s, the US and the then existing USSR started their respective nuclear programs and, in a short time, began to store huge quantities of DU in stockpiles, in the hope that future technologies would allow to further extract the 235 isotope, but the expectations of both armies were disappointed. In the 1970s, the USSR developed a particular armor plating for their tanks that US ammunitions could not pierce, so the Americans began to test materials dense and penetrating enough, and found that the best compromise between efficiency and cost was DU, of which there was an enormous availability. Uranium, be it natural, enriched or depleted, is an excellent penetrator and, in addition to that, is pyrophoric, i.e. gets oxidized and ignites spontaneously upon impact with a solid target in the presence of Oxygen, and the temperature that combustion reaches exceeds 3,000°C. The study mentioned above and completed between October 1977 and October 1978 at the Ford Farm Firing Range, Aberdeen Proving Ground, MD, placed a particular emphasis on size (just out of curiosity, they describe the formation of ultrafine particles less than 0.1 µm in diameter as an “unexpected phenomenon”), structure and stability of the particles resulting from the impact against multiple-armor-plate targets of penetrators consisting of 3.5 kg DU containing 0.75% Titanium by weight.

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The airborne particles were collected on a double-stick cellophane tape which was cut in small portions, and those portions were placed on Carbon-coated Aluminum stubs, coated with 100%-Gold and observed under SEM. The soil samples were sieved and treated in order to select and concentrate particles in the respirable range and observed as described above. The elemental composition of the particles observed down to the size of 0.5 µm in diameter was identified with an X-Ray energy spectrometer. As could be expected, a large number of particles in a broad range of sizes (< 50 µm - > 1 µm) resulted from the explosions, and most of them were either spherical or ellipsoidal in shape, thus showing that the heat generated was enough to melt Uranium and its oxides. On page 6, the document reads: “Fragments produced are ignited spontaneously by a combination of shock and friction heating at impact. Combustion of fragments in air is exothermic and self-sustaining. Flash temperature reached during impact of depleted Uranium penetrators with armor plate have been shown to fall in the range of 5500 to 5600°F (…) Test results show further a nearly constant (3037 to 3093°C) impact flash temperature over the entire range of impact velocities from 4010 to 5560 feet per second. Such temperatures (…) are sufficient to initiate combustion of the numerous particles produced.” And further on: “ Examination of numerous particles revealed several distinct morphologies. The great majority of airborne particles exhibited a rugose or convoluted structure which frequently appeared, at surface level, to consist of large, interconnecting concave plates (…). Each plate, formed independently from the solidification of molten material, was observed to radiate from a common origin or focal point upon the surface. At or near the junction of adjacent plates considerable overlapping and fusion occurred, resulting in distinct but irregularly delineated boundary edges. Higher magnification revealed numerous imperfections in the overall crystalline structure along the surface, thereby serving to further subdivide each major plate. (…) some rough particles exhibited a more uniform surface morphology, devoid of major plate divisions. The convoluted surface, although similar to that previously described, consisted of deeper, more numerous folds and extensive dimpling.

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Fissures and pore-like invaginations were frequently observed and progressively developed into deep fractures, presumably as a consequence of thermal expansion or through collisions with other objects. These fractures, which traversed irregular courses along the convoluted folds, were the eventual cause of extensive particle breakup (…). Internal morphology was frequently revealed by examination of fractured particles, particularly those in which large portions had become detached. Both solid and hollow particles were observed, the latter clearly demonstrated by the presence of hemispherical fragments (…). Although wall thickness varied greatly, the inner surface morphology of hollow particles consistently resembled that of the outer surface. (…) Prior to weathering, the surfaces of most airborne particles were covered to a varying extent by immense numbers of nearly spherical, ultrafine particles less than 0.1 µm in diameter (…) Identity of these particulates as pure or alloyed Uranium was confirmed by X-ray analysis. “An example of the uniform dispersal of ultrafine particulates on the surface of a depleted Uranium/iron particle is demonstrated (…) Any further accumulation, however, generally resulted in extensive coagulation and hence the formation of billowing aggregates (…). “Although generally found in association with larger particles, the ultrafine particulates were also detected in the free state directly upon the surface of the collective tape. At low magnification these particulates appeared as large concentric masses often reaching several hundred micrometers in diameter (…). At greatly increased magnification (…), these masses were revealed as consisting of vast numbers of small aggregates and long angular chains. The ultrafine spherical particles comprising these aggregates measured approximately 0.01 to 0.1 µm in diameter. (…) “Morphology of particles removed from soil samples was quite unlike that of their airborne counterparts. Far greater numbers of irregularly shaped fragments were present, presumably the result of interaction and fusion with sand and other materials within the soil (…). Spherical particles, although quite numerous, generally lacked the convoluted surface morphology so apparent in airborne samples. Their surfaces were consistently smoother and frequently speckled with knobby blebs (…).

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Some of these blebs were clearly continuous with the surrounding surface whereas others appeared nearly detached. “The relative fragility of these Uranium particles was clearly evident following brief exposure at the laboratory to ultrasound (…). Although sonification lasted no longer than 15 seconds, particles showed extensive fracturing and in many instances compete disintegration. “(…) “The elemental composition of individual particles was qualitatively determined by energy dispersive X-ray spectroscopy. Depleted Uranium particles frequently contained iron, aluminum, silicon, magnesium, potassium, titanium, and tungsten as a result of contamination during impaction and settling.” Unfortunately, a document of such importance was never published to the scientific community and is still very hard, if not impossible at all, to come by, in spite of what is declared in the preface: “This report has been reviewed by the Information Office (OI) and is releasable to the National Technical Information Service (NTIS). At NTIS, it will be available to the general public, including foreign nations.” It was only in the 1990s, starting from the first Gulf War, that DU weapons were largely and regularly employed in warfare, and it was soon apparent that both soldiers and civilians involved in that conflict showed unexpected health problems whose nature was unclear. The collection of symptoms they suffered from was called “Gulf Syndrome” since they did not fit in any already classified pathology and its origin was attributed to various causes, from the radioactivity and toxicity of Uranium to the improper use of vaccines and other drugs, but that was done without any convincing scientific evidence. Much of it was due to an emotional wave caused by the fact that some people, especially journalists, had discovered that the Allied Forces had used bombs containing DU, and Uranium, with all that that name recalls, looked an excellent scapegoat and also the spectre of the diseases induced by the radioactivity of the A-bomb resurfaced. Uranium is dangerous if it is inhaled, ingested or its debris are incorporated in biological tissue. It is chemically toxic and its radioactivity can induce lethal effects in biological tissues. The effects of the radiations emitted by Uranium can be deterministic or stochastic.

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That means that in the former case, if a threshold dose is reached, the probability to contract a medullar aplasia or cutaneous lesions or sterility is equal to 1; in the latter case, a malignant disease originates in a fortuitous way without a critical dose having been administered. Not much later, another undeclared war broke out, that time in what remained of Yugoslavia, and DU weapons were used there as well. Like in Iraq, also there soldiers and civilians showed symptoms of disease, mainly, but far from exclusively, of oncologic nature, and those symptoms were classified under the common expression of “Balkan Syndrome”. A few mistakes were made in the epidemiologic studies that were implemented in order to try and quantify the actual extent of what someone described as a disaster and others as something of no importance when non existing at all, and that delayed and then slackened the scientific investigations. In any case, it was a matter of fact that the collection of symptoms was not consistent with any already known pathology and that alone was enough to stimulate our curiosity. Stress may be blamed for disorders like fatigue, shortness of breath, headache, sleep disturbance, forgetfulness and impaired concentration, but cancer, diseases of the genitourinary system (GC Gray et al., 1996), diseases of the blood and the haematopoietic organs, and some forms of mental disorders can probably be ascribed to other causes. But also now, long after the end of the war, final reports are delivered from the Departments of the Veterans Affairs and new suspicions of associations with known diseases, (but of unknown origin) are rising; among others, for instance, with the Lou Gehrig’s disease (web ref. 1, 2001), and unexpected deaths (web ref. 2, 2002) Studies on cohorts of soldiers who had served in different wars had already shown the existence of previously unknown collections of symptoms related to the particular conflict they had taken part in. Already in the XIX century, after the US civil war, the so-called Da Costa Syndrome was described (JM Da Costa, 1871). Then, after the first World War, the so-called effort Syndrome was observed, while psyconeurosis was reported as a consequence of the participation in the second World War. Anxiety, neurosis, panic disorders, mitral-valve prolapse and chronic fatigue Syndrome were seen in soldiers employed in the Korean conflict, and a post-Vietnam Syndrome was described in

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veterans from the long war fought in Vietnam, but in that conflict an important contribution was most probably given by chemically toxic agent as Agent Orange, i.e. dioxin. The Syndrome suffered by the soldiers who were engaged in the first Gulf War included fatigue, headache, muscle and joint pain, diarrhoea, frequent fevers, skin rashes, shortness of breath, chest pain, sleep disturbance, irritability and depression, but also, in some cases, increased birth defects among the veterans’ children. In the UK that condition was called SSIDC, by that acronym meaning Symptoms and Signs of IllDefined Conditions. The symptoms suffered by the veterans from the Balkans were somewhat different from those of the ones who took part in the Gulf War, but, in that latter case, some veterans had died in a very short time. Then, between the end of the 1990s and the beginning of the new century, the claims that radioactivity was to be blamed grew louder and louder. To verify the truthfulness of that claim a specific Committee from the U.N. Environment Protection Agency checked the radioactivity in ex-Yugoslavia on January 2001. After 340 samplings in 11 sites, only a few of them resulted to have a high level of radioactivity apparently linked to DU ammunitions (web ref. 3). Though not overwhelming, evidence that DU ammunitions was not only used (something NATO had then already admitted) but a certain radioactivity was still present was there. Circular red lines are still present in the ground in many areas, delimiting an area around a DU bullet non exploded entrapped in the soil. Nevertheless, a few Italian soldiers who were not deployed in those zones reported some of the symptoms listed above, so the probability that radioactivity could be the cause of the diseases grew much weaker. Another group of people identified the cause of the Syndrome in the toxicity of multiple vaccinations that part of the soldiers were subjected to in a short period of time, but also for this claim there was no scientific evidence strong enough, though that factor may not be ruled out. The multifarious and often unexplained symptoms shown by a comparatively high number of veterans of the Desert Storm Conflict induced many researchers to call it a Syndrome, commonly referred to as the Gulf War’s. As a matter of fact, the collection of symptoms was not

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consistent with any already known pathology. Also the delay in showing up and the duration of the symptoms was something hard to explain still today. The studies on soldiers who had served in different wars previously mentioned had already shown symptoms related to that particular conflict: 1 - after the US civil war: DaCosta Syndrome 2 - I World War: so-called Effort Syndrome 3 - II World War: Psyconeurosis 4 - Korean conflict: anxiety, neurosis, panic disorders, mitral valve prolapse and chronic fatigue Syndrome 5 - Vietnam war: post-Vietnam Syndrome (In the course of that war, an important contribution was given by chemically toxic agent as Agent Orange [dioxin]) 6 - Gulf War Syndrome: fatigue, headache, muscle and joint pain, diarrhoea, frequent fevers, skin rashes, shortness of breath, chest pain, sleep disturbance, irritability and depression, but also (in some cases) increased birth defects among the veterans’ children. In the UK that condition was called SSIDC, i.e. Symptoms and Signs of Ill-Defined Conditions. There is a similarity of symptoms as fatigue, headache, shortness of breath, forgetfulness, sleep disturbance, impaired concentration, but they can found also in adult population under psychological stress. It is singular the symptom of diarrhoea. The variety of the pathologies and their intensity, that in some cases led to death, seem to have different aetiologies. That is the conclusion of many researchers even if many veterans have expressed concern that their unexplained illnesses may result from their experience in the war. Also after the end of the war in what was Yugoslavia, the soldiers engaged there denounced a collection of symptoms that has been called the Balkans Syndrome. The symptoms were different from those of the Gulf War, but, in this case, some veterans died in a very short period. After the first casualties, somebody tried to explain why young and healthy (before the war) soldiers died and to find a possible correlation among pathologies like the various forms of cancer and their presence in the war theatre.

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Some people, especially journalists, discovered that the allied forces used bombs containing Depleted Uranium, and Uranium looked an excellent scapegoat. Uranium is a high density, metallic element that, in its natural form, consists of three radioisotopes: 238U, 235U, 234U.(2). It decays spontaneously emitting alpha, beta and gamma radiations. This peculiarity was sufficient to suspect that some soldiers were highly exposed to its radioactivity due to the enormous quantity of DU bombs used in the conflict and developed cancers and others diseases. The correlation radiation = cancer was already been demonstrated in people who lived in Nagasaki and Hiroshima during the second World war. Uranium is dangerous principally (Grandolfo et al., 2003) if it is inhaled, ingested or debris are incorporated in biological tissue. It is chemically toxic and its radioactivity can induce lethal effect in the biological tissues. The rumours of weapons containing radioactive materials dropped in the Balkans were verified by a specific Committee from the United Nations for the Environmental Protection Agency on January 2001. After 340 samplings in 11 sites, only three sites resulted to have a high level of radioactivity apparently linked to the DU ammunition, for instance Hadzici, a small village, 60 km far from Sarajevo. (web ref. 3). So there is evidence that DU ammunition were used and a certain radioactivity is still present, but some ill Italian soldiers were not deployed in those zones, so the radioactivity cannot be the cause of the diseases.

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Another cause was identified in the toxicity of multiple vaccinations in a short period of time, but also for this cause there is no scientific evidence. The Italian Commission (web ref. 4) considered 44 cases reported in Tab. 5.1 and the first conclusion was that there was no relationship with their permanence in the war theatre. One year later the Commission elaborated again the data and reached a different conclusion. They found that there was a slightly higher incidence of Hodgkin’s lymphoma and Acute Lymphatic Leukaemia among the military people deployed in exYugoslavia or in close contact with DU ammunition in fire grounds. Specific observations were carried out in soldiers exposed to hightemperature combustion processes involving the blast of Depleted Uranium weapons. Our original aim was to find Uranium nanoparticles in the bioptic samples we received, but what we found instead of Uranium was metal dust coming from target and bomb volatilized by the heat generated by Uranium and re-condensed under nanoparticle form. Inhaled and ingested nanoparticles can negotiate either the alveolar and the digestive system walls, migrate to the blood and be carried virtually to any organ. Sperm samples belonging to dead soldiers who were active in war territories were referred to our laboratory and we developed a novel technique to be able to test them. In all cases, nanoparticles were present, while nothing was found in similar samples coming from healthy subjects we used as reference. [See chapters 3 and 5] Having found dust dispersed in the sperm, we checked if malformed human and animal feti contained that particular form of pollution.

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War and Nanoparticles Tab. 5.1 Cases examined by the Mandelli Commission in Italy. Pathology Hodgkin’s lymphoma non-Hodgkin’s lymphoma Acute lymphatic leukaemia Thyroid Carcinoma Colon or rectum cancer Larynx and Pharynx cancer Lung and Bronchus cancer Kidney cancer Stomach cancer

N. of Cases 12 8 2 3 4 1+1 1+1 1 1

In her capacity of coordinator of the European project on nanopathology and consultant of the 2nd Italian Governmental Commission, Dr Gatti had the opportunity to check a small cohort of 52 soldiers (for a total of 59 samples, because from autopsy we had more samples for 1 subject) with our technique. The aim was to look for Depleted Uranium inside the pathological tissues of the sick veterans. Some of the Italian soldiers had already died before getting a clear diagnosis, while others had developed symptoms typical of well-known diseases (for instance, Parkinson’s or Alzheimer’s). All declared to suffer from a common symptom: chronic fatigue, a symptom usually complained for also by the New York fire fighters who took part in the rescue operations of 9/11. So we analyzed autoptic specimens, biopsies, surgical samples or “fresh” specimens as blood or sperm. (See Tab. 5.2) In eleven of them, colon cancer had been diagnosed and the rest suffered from non-Hodgkin’s lymphoma. In all the small specimens inorganic particulate could be detected and its chemistry verified. The samples were very small, and there is the possibility that further debris included Uranium could be entrapped in other organs and tissues, but what we found is meaningful of a certain contamination and it responds to a logic behavior of the bombing: it creates a new “special” environmental pollution.

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No. of Cases 12 6 8 3 3 2 5 3 3 2 1 6 2 1 2 59

In none of the cases we checked could we find trace of DU debris, either micro- or nano-sized. In many of them, instead, we detected the presence of nanoparticles, often showing very strange elemental chemical compositions, different from case to case. The explanation to that phenomenon is relatively easy: As described above, when a bomb goes off, the explosion induces a great quantity of heat and involves a large number and variety of objects and materials. Those objects and materials volatilize and most of the molecules they are made of break into chemical elements that recombine, often under the form of an alloy, as soon as the atmosphere cools down. Since all that matter is cast far away from the spot where the explosion had taken place, it finds an environment cold enough to allow it to recombine very quickly, in a matter of seconds. Because of the variety of the materials that take part in the explosion and the fact that the soldiers we checked had been deployed in a comparatively vast territory, the alloys we found were rather different from each other. That finding agrees with the deployment of the soldiers. They did not go in the same bombed places and at the same time.

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Like in many other, not war-related, cases we could study, nanoparticles were scattered in many different tissues, including red cells (an excellent carrier), brain and gonads.

Fig. 5.1 Image of red cells spread on a plastic sheet, of a contaminated spouse, observed under ESEM, with a white particle of Antimony –Cobalt attached (marker 5 µm).

The first of the cases we checked was particularly interesting and was related to the 1st Gulf War (web ref. 5). A Canadian officer with a history of amateur marathon runner and, also because of that, enjoying an excellent physical condition, was repatriated after a six-month stint on a wheel-chair, despite the fact that he had never been injured, but for three days he had been highly exposed to bombing pollution. The symptoms since February 1991 were loss of motor control, chronic fatigue, respiratory difficulties, chest pain, insomnia, short-term memory loss, testicle pain, aching bones, diarrhea, and depression. After his return he survived for 8 years. During that time he developed symptoms specific of different pathologies, included Parkinson’s and Alzheimer’s diseases.

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Among the unusual aspects of this Syndrome there was the change of his eye colour, originally brown, but after 2 years turned to greyish, then to intense blue. Then, just before his death, they turned to grey again. His widow did not find any doctor who could give an explanation to this strange phenomenon. Besides his difficulty in walking, he had problems with his sexual life, as, after sexual intercourse, his wife felt a smart burning sensation in her vagina, which started to bleed and, in a short time, developed sores which did not respond to any therapy. That particular condition, later on reported by other partners of veterans when we interviewed them, was called “Burning Semen Disease” (see also Chapter 3). Blaming a particular chemical composition for that disease does not seem possible, but the cases we had the chance to study so far are still few and deeper investigations on a sufficient number of cases are necessary to be able to say something conclusive. The analyses were carried out on 4 different types of samples: the lung (Fig. 5.2), the spleen (Fig. 5.3), the liver (Fig. 5.4) and the kidney (Fig. 5.5). In the liver we found Cobalt and Antimony nanoparticles. The presence of Cobalt debris attracted our attention and pushed out our fantasy, beyond the border of the present knowledge. Cobalt is blue (Fig. 5.4). The patient’s eye became blue and all his body contained Cobalt nanoparticles. All his body contained metallic, toxic nanoparticles, finely disseminated. To explain the colour eye mystery we can remind the symptoms of a genetic disease called Wilson’s Syndrome. In this case the lack of a protein does not allow to metabolize Copper that precipitates, mainly in the liver. One of the symptoms considered for the diagnosis is a yellow circle around the iris. Copper is brown-yellow in colour. Can this comparison be sustainable? For now there is no possibility of epidemiological studies, since that was a unique case. The particles we found in the liver were present in the blood circulation and reached also the eye microcirculation. We found also nanoparticles of Mercury-Selenium in the kidney. A Canadian doctor performed analyses on his urine and he found traces of Uranium, but in the samples we analyzed the amount of toxic particles was sufficient to explain the symptoms.

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This case was deeply analyzed since we had sections from autoptic samples of different organs, and we could understand that the dissemination is total, and some of the particles accordingly to their chemistry can be trapped selectively in some organs. The disease and the symptoms are related to this wide contamination.

Fig. 5.2 ESEM image of a solitary spherical nanoparticle of Bismuth-Chlorine-Sodium found in the lung (marker 20 µm).

Fig. 5.3 ESEM image of a post mortem section with spherical nanoparticles of Antimony-Chlorine found in the spleen (marker 20 µm).

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Fig. 5.4 ESEM image of a post-mortem sample of the liver. Nanoparticles of Cobalt were found (marker 10 µm).

Fig. 5.5 ESEM image of a post-mortem sample of the kidneys. Nanoparticles of toxic Mercury-Selenium were found (marker 20 µm).

Especially in the kidney the particles were found as singlets (Fig. 5.5). This fine dissemination is peculiar. Until now it is impossible to reproduce that phenomenon in an in-vitro simulation test for the capacity of clustering of engineered nanoparticles. We analyzed also soldiers not directly involved in the war, but who served in peace-keeping missions in bombed sites. The Italian soldiers’ samples we analyzed were provided by the patients themselves or by their families. We present a selection of some results. It is possible that if we had the opportunity to analyze other tissues we could have found more or less debris with further

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compositions, since a selective capture from an organ of specific chemistry is possible. The soldiers we analyzed served in the Balkans in different periods and in different areas, so the difference in compositions of the pollution is a logic consequence. Many slept and/or worked in destroyed buildings, other visited polluted areas or were busy destroying weapons. It is a common procedure, after the destruction of enemy ammunition collected inside a hole dug in the ground, to verify directly on the explosion site, on the smoking ashes, if everything had been actually destroyed. That is a dangerous procedure: the risk exists of inhaling the pollution generated by the explosion. That habit was kept also during the 2nd Gulf War. That means that soldiers slept close to primitive open-air incinerators, no more than holes in the ground where they dispose of daily wastes of food, but also many other kinds of unwanted materials, including captured weapons. The first 3 cases (Fig. 5.6, Fig. 5.7, and Fig. 5.8) show images of pollution inside biopsies of lungs and bone marrow of Italian soldiers.

Fig. 5.6 Image of micro and nanoparticles of compounds containing Iron-SiliconPhosphorus-Aluminum-Sulphur-Chlorine-Sodium-Potassium (marker 20 µm).

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Fig. 5.7 Image of nanoparticles of stainless steel inside the bone marrow (marker 50 µm).

Fig. 5.8 Image of spherical particles of Iron-Manganese-Chromium-Silicon found in a bone marrow biopsy (marker 5 µm).

Figs. 5.7 and 5.8 show different particles found in a bone marrow sample. They are metallic debris both irregularly and, in most cases, round-shaped; their origin was from combustive processes at a temperature higher than 900°C.

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Fig. 5.9 Image of a cluster of nanoparticles of Iron found inside the liver (marker 10 µm).

Fig. 5.10 Image of particles of a compound of Zirconium-Sulphur-Chlorine-Calcium found in the liver (marker 10 µm).

Figs. 5.9 and 5.10 are related to the same soldier who died after having contracted a lymphoma. His organs were contaminated mainly by Iron-based particles, found mainly embedded in the bone marrow. Also Zirconium-based particles where found in the spleen biopsies after the organ was removed.

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Fig. 5.11 Image of a debris of Antimony found in the lung (marker 20 µm).

Fig. 5.12 Image of a particle of Tungsten found in the lung (marker 20 µm).

Figs. 5.11 and 5.12 are related to the case of a soldier who had a lung neoformation removed. Solitary particles of Antimony and Tungsten were found inside. Those are recognized as chemically toxic materials.

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Fig. 5.13 Image of a cluster of Cobalt particles inside a bladder cancerous tissue (marker 10 µm).

Fig. 5.14 Image of round-shaped particles inside a bladder cancer (marker 2 µm).

Figs. 5.13 and 5.14 show particles found in the same sample of bladder cancer. They belong to a mine clearer who had served for 25 years in the Army and had been present at very numerous explosions for the elimination of ammunition. For that reason, he was exposed to the pollution created by that kind of combustion. Inside his pathological tissue we found nanoscaled particles of Cobalt, Tungsten, stainless steel, etc.

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Fig. 5.15 Image of a solitary particle of stainless steel surrounded by red cells (marker 50 µm).

The picture above (Fig. 5.15) is relative to a rhino-laryngeal neoformation in a soldier affected by non-Hodgkin’s lymphoma.

Fig. 5.16 Image of a wide dissemination of debris of stainless steel inside the bone marrow in an (marker 50 µm).

The sample of Fig. 5.16 is related to an acute mieloid leukaemia a veteran from the Balkan War suffered from.

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Fig. 5.17 Iron-Zinc micro and nanodebris inside a calcific tissue found in the liver (marker 20 µm).

The soldier whose liver biopsy was observed in Fig. 5.17 suffered from pancytopenia. The tissue contained a wide variety of particles with different compositions. Calcium was detected together with Iron, Zinc, Magnesium, Silicon, Phosphorus and Sulphur.

Fig. 5.18 Image of an Antimony-Cobalt debris found inside the sperm of a soldier who served in a fire ground (marker 10 µm).

Hodgkin’s lymphoma was the disease diagnosed to the soldier, now died, whose sperm we examined. It contained 800-micron sized Antimony-Cobalt particles (Fig. 5.18). The patient was never deployed in the Balkans but served in a firing ground in Sardinia.

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Fig. 5.19 Image of Iron micro and nanoparticles disseminated in a brain tissue affected by glioblastoma (marker 20 µm).

Fig. 5.19 shows Iron-Silicon-based nanoparticles finely disseminated in a soldier’s brain affected by glioblastoma. The presence of Silicon witnesses that the particles are of exogenous origin.

Fig. 5.20 Image of a solitary debris of Lead-Chlorine-Antimony found in a ganglioneuroma (marker 20 µm).

Fig. 5.20 and Fig. 5.21 present two solitary particles disseminated in a ganglioneuroma (Antimony-Lead-Chlorine) and in an adrenal gland (Mercury-Silver). The elements composing those particles are recognized as toxic.

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Fig. 5.21 Image of a Mercury-Silver-Calcium-Phosphorus singlet particle found in an adrenal gland. (marker 20 µm).

Fig. 5.22 Image of two Strontium-Sulphur-Calcium-Sodium debris found in an adrenal gland (marker 20 µm).

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Fig. 5.23 Image of three Copper-Iodine-Sulphur particles found in an adrenal gland. (marker 20 µm).

Figs. 5.21, 5.22 and 5.23 show three different types of particles found in a removed adrenal gland. They contain chemically very oddly composed toxic materials. The pathology that affected the patient who served in a peace-keeping mission in the Balkans was adrenal adenoma.

Fig. 5.24 Image of a 5-micron sized particle of a silicate containing also radioactive materials like Uranium-Thorium-Yttrium-Cerium-Neodimium (marker 10 µm).

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Fig. 5.25 Image of micron- and nano-sized (white arrow) debris of a silicatic composition. The debris on the left is composed of nanoparticles found in a bone marrow sample (marker 2 µm).

Figs. 5.24 and 5.25 show mostly nanometric debris found in the same bone marrow biopsy that contains mainly ceramic materials, some of them radioactive (Fig. 5.24). The patient had served in Bosnia and developed a thyroid carcinoma with lymph-nodal metastases, and acute leukemia. In that biopsy, we found ceramic debris containing, among other materials, radioactive elements like Uranium, Thorium and Yttrium. We do not know when and where the soldier was exposed, but these materials may come from the pollution of buildings, perhaps containing tile with radioactive glazes. After the buildings were destroyed, a part of them was aerosolized. Among the samples we received, there were some coming from a few French soldiers who had taken part in the peace-keeping missions in the Balkans. One of those soldiers had served for six months as a clerk in an office based in Kosovo and had developed a Hodgkin’s lymphoma because of which he eventually died. In the biopsies we got, we found nanoparticles composed of alloys containing Silver and Gold, among other elements like Iron, Chromium, etc. (Fig. 5.26). No such alloy is to be found in any metallurgy manual or, at least to our knowledge, in any commercially available product. As a matter of fact, Gold is very often used for its anti-corrosive properties and is never

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mixed with stainless steel, which is what the rest of the alloy looked like. So, as happens in many cases, we guessed that that strange composition could be the result of a combustion and the consequent interaction of the elements occasionally present. Explosions are obviously very common in war theatres, and explosion is a form of combustion. But the subject we were dealing with had never been involved in battlefield actions or was close to actual war theatres.

Fig. 5.26 Image of the cluster of nano-debris composed of Silver-Gold-Chlorine-IodineChromium,-Iron- Gold,-Magnesium-Silicon-Sulphur (marker 5 µm).

In the course of our investigations, though, we were informed of a kind of exposure our subject might have undergone. He had slept and eaten for two months in a truck, and that truck had been previously used in Iraq, from where it had been moved to Kosovo without having been cleaned. Of course, we did not have any chance to get samples of the dust contained inside the truck, but it is only natural that such a condition can rouse suspicion about the possibility that that dust may have polluted the people who had stayed there for a relatively long time. Long enough, in any case, to get contaminated. The sleeping bags could have been particularly interesting to check, as dust trapped there would have kept the soldiers closely exposed to particles for many hours a day. The small size of the particles found and their composition made their origin clear: combustion at high temperature. But we have no answer to the question about their origin, Balkans or Iraq.

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One of the questions we wondered about was: Can only Depleted Uranium and Tungsten bombs create nanoparticles, or is the temperature developed by collections of more conventional weapons enough to make them? Thus, we carried out a simple experiment with the Italian Governmental Commission Dr Gatti worked with. Since the Italian soldiers stationed in Baghdad confiscated all kinds of small armaments and bombs, put them in large holes and had them blow, we had gravimetric passive sensors set at distances of 100, 200 and 300 meters from the hole and exposed their surface just a few seconds before the explosion. Then let them exposed to the environment for 30 minutes and analyzed them to check for the presence of nanopollution. In all samples we found a great quantity of nanosized dust and, in addition to that, we detected there particles with the same, unusual composition we had already found in the bioptic specimens of soldiers we had had the possibility to observe. That was a simple demonstration that very fine pollution can be generated also by “low-technology” weapons. The next images show the pollution created during the firing of a pool of ammunitions. Fig. 5.27 and Fig. 5.28 show particles with their chemical composition found 100 m away from the explosion, while Fig. 5.29 and Fig. 5.30 represent the dust collected 200 m away.

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Fig. 5.27 Image of dust created 100 m away from the firing of a pool of weapons in Baghdad. The big 20 micron-sized particle is a cluster of nanoparticles (marker 5 µm).

Fig. 5.28 Image of a 700 nm-sized round-shaped particle of Iron-Aluminium-Silicon found at 100 m from the explosion (marker 10 µm).

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Fig. 5.29 Low-magnification image of the dust collected at 200 m from the explosion (marker 200 µm).

Fig. 5.30 Image of a spherical Zirconium-Sulphur-Sodium particle found 200 m away from the site of explosion (marker 20 µm).

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Fig. 5.31 Image of another group of particles found 200 m away from the explosion site. (marker 20 µm).

Fig. 5.31 shows debris collected on the passive sensor 200 m away from the explosion site. Micro and nanoparticles were found in all the three points of measure (100, 200, 300 m). Some showed strange chemical compositions like that of Fig. 5.31 and Fig. 5.32. The first spectrum shows the presence of Lead together with Chromium, Iron, Silicon, Aluminium, Calcium. That composition is the result of an occasional melting. This alloy is not be found in any metallurgy handbook and is the occasional result of elements present when high-temperature combustions occur. The particle of Fig. 5.32 shows a round-shaped morphology formed by other smaller “balls” composed of Iron, Silicon, Sulphur, Aluminium, Sodium and Calcium.

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Fig. 5.32 Image of a composite round-shaped particle composed of Iron, Silicon, Sulphur, Aluminium, Sodium and Calcium from the explosion occurred 200 m away (marker 10 µm).

We had the opportunity to analyze the oil used in the engine of trucks that had worked for 2 years in Iraq. That oil contained dust of different compositions (Fig. 5.33 and Fig. 5.34). Some of the particles detected presented the same morphologies and chemical compositions as we found in some pathological tissues. Zirconium, one of the rather unusual elements detected in the oil, was also present in the specimens of two soldiers.

Fig. 5.33 Image of a round-shaped, hollow particle of Iron found inside the engine oil used in the truck in Baghdad (marker 5 µm).

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Fig. 5.34 Image of a Zirconium bar found inside the engine oil used in the trucks in Baghdad (marker 10 µm).

So, we observed something that probably did not need any particular demonstration, but just common sense, i.e. that explosions cause a new pollution contaminating the environment. For how long? Are those particles somehow degradable? In our opinion, seen their composition, we can hardly believe that they, or, at least, the vast majority of them, can “disappear”, even after a long time. And that persistence applies both to the environment and to the organism. Once they have been released in the atmosphere, they float in the air and can cover long distances, carried by the wind. When they eventually fall to the ground - and rain and snow can be the occasional carriers of particles - a gust of wind is enough to raise them again and have the circle restart. As to the organism, we have demonstrated that particles, no matter how they are introduced, can escape most physiological barriers and settle virtually everywhere. Their presence in the lymph nodes, where some physiological waste collects, looks almost natural, but their being in the bone marrow, in the brain and in the sperm is somewhat less expected. Now, having observed all that evidence, one may ask whether their presence in the pathological tissues is actually related to the pathology? Is their chemistry a crucial, or even just an important, factor? And do all

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subjects polluted in the same way develop identical symptoms and disease? We try and answer those questions checking places where explosions occur on a regular basis, i.e. in firing grounds. Italy has a few of them, but one in particular attracted our attention because of the rumors and the actual complaints we heard from the people living in its vicinity. The firing ground of Salto di Quirra is located in the middle of the east coast of Sardinia, close to a village called Quirra, along a beach of “uncontaminated” beauty. There are no industries worth mentioning in a radius of many tens of kilometers and traffic is scarce. The village has no more than 150 inhabitants. The firing ground specializes in testing missile aiming systems (web ref. 6) and is used not only by different Italian services but also by private companies producing weapons and foreign armies. A few soldiers serving there had died in the same short period of time of Hodgkin’s disease. Among those soldiers we could analyze the case of a 22-year old boy whose Hodgkin’s lymphoma had not been diagnosed in time and he had died. In a supra-clavicular neoformation we found particles of Antimony and Iron-Copper, while in his sperm we found round-shaped particles, about 1-micron-large particles of Antimony-Cobalt and Iron-ChromiumSulphur. Their spherical shape and chemical composition witnessed a high-temperature combustive origin. Some of the inhabitants of the village had developed forms of cancer and the activities of the firing ground had been blamed by them as the responsible of the diseases, and in another village of the inland called Escalaplano, twelve malformed babies were born, but that was limited to the period between 1988 and 1990. By request of the authority responsible for public health of that district, we analyzed pathological samples (see Tab.5.3) from ten of the inhabitants of the village of Quirra, located closer to the firing ground than Escalaplano.

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Examined sample

Melanoma Non-Hodking’s lymphoma Chronic Myeloid leukemia Breast cancer Adenocarcinoma Thyroid carcinoma Lobular carcinoma

Skin Lymph node Aspiration biopsy Breast Uterus Lymph node Breast

In order to verify if this pollution was related to the activities of the firing ground, a specific investigation was carried out inside the area with the permission of the military authority and under their supervision. At the time of the investigations, no activities were planned, so, we collected on adhesive disks the dust already present in that environment, touching the areas behind the missile launching pads on the ground and on the special metallic devices that adsorb the fire. We collected samples also from a pool under a platform where special, big engines for rockets were tested (Fig. 5.35). The water of the pool served to absorb the heat of the exhausts.

Fig. 5.35 Particle found attached to the wall of the pool. It is composed of LeadPhosphorus-Iron-Copper-Silicon-Aluminium-Sodium-Calcium (marker 10 µm).

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The samples collected there contained dust with different composition, but one attracted our attention since it contained Lead. A specific research verified that the engine used a solid red propellant that contains Lead. The sample was not analyzed in our laboratory, but by RIS (Crime Scene Investigative Institute) of Parma (Italy) which gave us the result shown in Fig. 5.36.

: Fig. 5.36 Image of a particle of solid fuel with its spectrum. It is composed of CarbonOxygen-Lead and Aluminium (marker 20 µm).

Lead was important for our study, since some patients showed its presence in their pathological tissues.

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5.1 Civilians living around a firing ground In some cases the patients living around the firing ground and who developed a disease presented metallic particles, some of them Leadbased. That presence is puzzling for three reasons: Firstly, the village where those people live is very small, with little traffic car and without any industry. Secondly, this contaminant is nanosized and its production needs a very high temperature. Finally, Lead is bound to elements like Titanium, Chromium, Iron, Silicon, Aluminium and such an alloy does not exist in any metallurgical handbook and can only be the result of an occasional meeting of all those elements, which is typical of a combustion where some presences are accidental. For these reasons, we think that there is a certain correlation among some activities of the firing ground (rocket engines or weapon elimination), the chemistry of the pollution generated and some cases of the patients examined. The activities of launching missiles are controlled and the population is alerted when a launch is planned, but the bench tests of the rocket engines might be considered a non risky activity, so, no precaution is taken. There are also other specific activities we had no opportunity to investigate, that probably were responsible for the creation of the Antimony–Cobalt pollution we found in the samples of the soldiers.

Fig. 5.1.1 Image of a cluster of nanoparticles found in a lymph node affected from nonHodgkin’s lymphoma (marker 10 µm).

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In the technical report of 1978, Dr. Joe A. Framer declares that “Depleted Uranium as the most desirable candidate is based primarily upon on its 1-high density 2- pyrophoricity 3-metallurgcalproperty, 4availability and 5- relatively low cost.” But after the test, he says that “additional research is necessary to better define the physical and chemical nature of the fragmentary depleted Uranium generated as a consequence of its military use as weaponry. Foremost attention should be focused on its potential for dissemination within the environment and entry into biological systems, particularly that of man” In addition to that, “Such specific knowledge is required for determination of potential safety hazards associated with the respiration and deposition of Depleted Uranium aerosol within the lungs. Particles in the 0.1 – to 0.5 micron size range are of great concern because of high efficiency for deposition in the lungs. This range has been defined appropriately as the respirable size range.” Back in 1978, nobody talked about nanoparticles and nanopathologies, but the concept is clear. The concern for the environment and humans caused by the effects of explosions was already expressed almost three decades ago, but nothing was made to control or to avoid the problem. Now we extend the concept: All the particulate matter generated by high-temperature combustive processes is greatly aggressive to environment and human and animal health. 5.2 A few reflections We are all aware that the new technologies we invented or what we call, without much insight, progress have always a more or less important negative side. In this case we see that bombing creates a new environmental pollution, sometimes nanoscaled, but always with a chemical composition that can be very dangerous for human and animal life. The new wars create very small bullets, impossible to see and for now undetectable by soldiers since they have no sensors for them. But nanotechnologies can help find the right solution to develop equipment

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sensitive enough. Through them we can develop sensors for nanoparticles, filters to capture them, masks to avoid their inhalation, personal protections for contamination. But for now no technology is available or even thinkable for an environmental remediation. When the environment (ground, vegetables, water) is contaminated, it is practically impossible to clean it. The effects of such contamination will be paid not only by the populations living there, where that contamination had actually occurred, but by the neighbouring countries as well, perhaps not directly involved in the war but, unfortunately, pollution does not stop at frontier stations. Wind carries particles far from their origin, but so does water through torrents and rivers that flow into the sea. Fish eat particles along with food and are contaminated, thus becoming a contamination carrier to animals they are food for. Our planet is too small to stand all that. So, before starting another war, governments ought to be aware of the hardly soluble problems they are triggering, last but not least among them that of the money that will be spent in what is going to be the unsuccessful attempt to reclaim a so polluted territory. 5.3 Bibliography Da Costa, J. M. (1871) On irritable heart: a Clinical study of a form of functional cardiac disorders and its consequences Am. J. Med. Sc., 61, 17-52 Grandolfo, M., Mele, A., Ferrigno, L., Nuccetelli, C., Risica, S and Tosti, M. E. (2003). Uranio impoverito e linfomi di Hodgkin nei soldati italiani in Bosnia e Kossovo: una possibile associazione?, Not Ist Super Sanità; 16 (7/8) Gray, G. C., Coate, B. D., Anderson, C. M., Kang, H. K., Berg, S. W., Wignall, F. S., Knoke, J. D. and Barrett-Connor E. (1996). The postwar hospitalization experience of U.S. veterans of the Persian Gulf War, N Engl J Med. 1996 Nov 14;335(20):1505-1513. Technical report of the Air Force Armament Laboratory – Armament development and test Center, Eglin Air Force Base, Florida, USA, From October 1977 to October 1978, Project n° 06CD0101 Web Ref. 1: www.gulfwarvets.com/gehrig.htm, Web Ref. 2: www.gulfwarvets.com/study.htm, The Washington Post Company 4 March 2002, by Suzanne Gamboa

War and Nanoparticles Web Ref. 3: http://balkans.unep.ch Web Ref. 4: www.uranioimpoverito.it/mandelli.htm Web Ref. 5 (http://www.umrc.ney/riordon.aspx) Web Ref. 6: www.perdasdefogu.it/MILITARE.htm

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Chapter 6

Nanoparticles in the Environment and Working Places ___________ 6.1 Introduction

3

ollution taken in a broad sense depends both on the source and the environment. Restricting the subject to particles generated at high temperature, their composition depends mostly on what is being burnt and, in part, on the physical conditions at which that combustion takes place. The environment where particulate matter is issued affects in a determinant way its concentration and scattering. It is only natural that, in general, a windy place will suffer less from concentrated dust pollution than a place where the calm of the wind is a predominant condition and, even more so, an indoor environment will be very critical in comparison with the open air, because of stagnation. Also atmospheric pressure and inversion phenomena influence diffusion in the environment of that dust. The traceability method we use takes all those factors into consideration. A few examples are described in this chapter where traceability has been of the utmost importance to understand some phenomena otherwise difficult to make out. In some of those examples, a chemical element has been taken as the marker and the guiding thread that led us to the solution of some cases. There are instances where the technical attempt of solving a problem linked to pollution leads involuntarily to the creation of different, unexpected problems that, in some circumstances, can be more serious than the original ones. Filters used in Diesel cars (FAP, see Chap. 6.5) to capture the particulate matter produced by the combustion of gasoil are

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just one possible example and are briefly taken into consideration as the producers of a novel form of pollution. Another example is that of incinerators, meant to solve the problem of getting rid of wastes but causing the formation of pollutants far more dangerous than what was to be eliminated. Distribution of particulate pollutant is of the utmost importance and may be expressed in terms of mass, volume, surface area or number. Already back in 1998, toxicological studies on rat models showed that nanoparticles are much more toxic than coarser ones made of the same components (K. Donaldson et al., 1998) and even earlier, in 1995, ultrafine particles were demonstrated to be able to penetrate in the pulmonary interstitium, thus causing cardiovascular diseases (A. Seaton et al., 1995), something that larger particles are unable to do. As already explained in Chapter 1.1, nanoparticles are dominant in the particle number count and are significant in contributing to surface area, but mean little in terms of mass. Tab. 6.1 shows this concept. Tab. 6.1 List of the ratio size-number and surface of nanoparticles.

Diameter (in µm)

Relative number

Relative surface

10 1 0.1 0.01

1 103 106 109

1 102 104 106

Nevertheless, it is a well-known fact that size is a critical parameter when the point of view is that of human health, as the smaller the particles, the easier they can penetrate the organism and the deeper they can reach. It is another fact that the larger the surface area of those particles, the more chemically reactive they become. And, finally, count is also determinant, since it represents the number of impacts the organism may undergo. So, evaluating particulate pollution in terms of weight alone has very little significance, at least when health is the issue. Condensation of non volatile materials from supersaturated hot combustion gases is a process responsible for the formation of airborne nanoparticles, the size of about 1-2 nm. Those particles can grow very

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rapidly in size by condensation of low-volatility materials. It must be remembered that inorganic, microsized particles formed at high temperature are often fragile and can break into much smaller, often nanosized, particles. In that case, the number of actual particles is increased and so is their surface area, while their mass stays constant. The following examples are presenting places and situation were nanopollution is already unintentionally released in the environment. This knowledge can be very useful since, being aware of the problem, we can study precautionary measures, preventive procedures and countermeasures. 6.2 Chimneysweeps: a historical case “Ramazini [sic, correct spelling is Ramazzini] has written a book De Morbis Artificium. The colic of Poictou is a well known distemper; and every body is acquainted with the disorders to which painters, plumbers, glaziers, and the workers in white lead, are liable: but there is a disease as peculiar to a certain set of people, which has not, at least to my knowledge, been publicly noticed; I mean the chimney-sweepers’ cancer. It is a disease which always makes its first attack on, and its first appearance in, the inferior part of the scrotum; where it produces a superficial, painful, ragged, ill-looking sore, with hard and rising edges: the trade call it the soot-wart. I never saw it under the age of puberty, which is, I suppose, one reason why it is generally taken, both by patient and surgeon, for venereal; and being treated with mercurials, is thereby soon and much exasperated. In no great length of time, it pervades the skin, dartos, and membranes of the scrotum, nod seizes the testicle, which it enlarges, hardens, and renders truly and thoroughly distempered; from whence it makes its way up the spermatic process into the abdomen, most frequently indurating and spoiling the inguinal glands: when arrived within the abdomen, it affects some of the viscera, and then very soon becomes painfully destructive.” That is how Dr Percivall Pott (1819) [The Chirurgical Works of Percivall Pott, F. R. S. Surgeon to ST. Bartholomew’s Hospital with his last corrections. To which are added, a short account of the life of the

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Author, a Method of curing the Hydrocele by injection, and occasional notes and observations by Sir James Earle, F. R. S. Surgeon Extraordinary to the King, &C. First American, from the last London edition. In two volumes. Vol. I. Philadelphia: Published by James Webster, No. 24, South Eight Street. William Brown, Printer. 1819] in one of his numerous texts on this subject, starts to describe a sort of cancer that had affected chimneysweeps for centuries and for which no therapy had been found. The chimneys built in North Europe especially in the XVIII century had a very small diameter and followed long and tortuous courses. So, people with a very slight build and a nimble body, and scantily dressed to reduce their bulk, were needed to clean them and, in fact, children were usually employed to do that job. And, already in their early adolescence, a non negligible number of those young workers contracted scrotum cancer. What Dr Pott did was to perform a surgical ablation of the tissues involved, but, unfortunately, to no avail, thus inflicting further sufferings and pain to his patients. Already in 1775 Pott had realized that soot was responsible to the disease, in this way admitting that some “poison” could induce a cancer. The practical problem was solved in a very pragmatic way by the Danish government that obliged all chimneysweeps to take daily baths, thus avoiding a too long exposure of the skin (children worked all but naked) to the soot that smeared their body. Great Britain followed later with a law that forbade the employment of children under eight years of age and imposed weekly baths (G. Paganetto. 2001) The same type of disease was observed at the first half of the 20th century in mule spinners in cotton textile factories, where they were exposed to shale oil, which was used as a lubricant for cotton spindles. Between 1920 and 1943, 824 out of the 1,303 skin carcinomas identified in textile-industry workers affected the scrotum (S.A. Henry 1943). In both cases, for the onset of the pathology polycyclic aromatic hydrocarbons (PAHs), particularly some 3-, 4- and 5-ring PAHs such as benz(a)pyrene and dibenz(a,h)anthracene were blamed. Two things are particularly noteworthy: An environmental agent was recognized as cancerogenic, and effective laws were passed on the sole

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basis of medical observations without waiting for a complete theoretical explanation of the physical, chemical and biological mechanisms involved, the three of them then completely missing. 6.3 Welding metals Welding is used to join different metal parts by having them coalesce, and that is done by melting them. Many different energy sources can be used to carry out that process, and, among them are gas flame, electric arc, laser light, electron beam, friction, and ultrasound. All those methods generate particles that scatter in the environment. Their shape is generally spherical and their size depends mostly on the temperature at which the procedure has occurred (Fig. 6.3.1). Most of those particles are composed by a thin, crystalline skin, are hollow inside and are very fragile.

Fig. 6.3.1 Image of a hollow particle obtained by laser welding (marker 20 µm).

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Even a sight collision can break them up and the fragments thus originated become particles in their turn. Those particles are obviously irregular in shape and smaller then the objects they come from, growing that way more numerous and more aggressive. Many of them are likely to be nanometric in size, so a single microparticle may give birth to a high number of nanoparticles (Fig. 6.3.2 and Fig. 6.3.3). It is only natural that welding is more critical from the point of view of the health impact it may have, when it happens in a closed space, and among the effects the dust produced can cause is its being trapped in the welder’s clothes. Besides rather uncommon circumstances, those clothes are eventually handled, often to be washed, by the welder’s wife or, in any case, by somebody who is not the welder himself and it is not rare the case when that person, never directly involved in the workman’s job, falls ill with the same occupational diseases that typically affect welders.

Fig. 6.3.2 Image of an agglomeration of spherical particle of Iron-Copper-Zinc-SiliconSulphur-Calcium found on the ground around a welding site (marker 5 µm).

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Fig. 6.3.3 Image of spherical Zinc debris collected by air filtration during a flame welding (marker 10 µm).

Some of the particle have a submicronic size and, if inhaled, can pass through the lung barrier and enter the blood circulation, causing a chemico-physical pollution. Also the microsized particles that have been blocked in the alveoli because of their large size and the weakness of the aggregation bounds can degrade along the time. Or, better, the material that induced their aggregation degrades, releasing all the nanosized debris, that, being so small, have a high probability to pass through the lung barrier. That means that pollution can have a delayed effect, practically impossible to predict. In the case of these workers, it is mandatory that they use an effectively protective mask when at work; that they wear overalls that are never brought back home but are washed at the working place; that they do not eat in the working place; that they change their cloth when they eat. Many working places are potential sources of pollution. So, a dedicated risk analysis is necessary, along with the proper education of every single worker. In most cases, the protective measures necessary are simple enough and even inexpensive.

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6.4 Toner Nowadays printer toner is widely used in offices and even in household environments. No particular attention is usually reserved to it, but when there are many printers gathered together in the same room, black dust is often visible on the furniture and on the machines themselves. We had the chance of checking a few toners made by different producers and saw that the Carbon granules contain metal micro and nanoparticulate whose composition depends on the effect the manufacturer wants to obtain. Fig. 6.4.1, Fig. 6.4.2 and Fig. 6.4.3 show Carbon-based spherules containing respectively Iron, Titanium-Tin-Antimony-Silicon and Titanium-Strontium-Sulphur nanoparticles.

Fig. 6.4.1 Image of dust of toner (Olivetti) composed of tiny Iron particles contained in larger Carbon-Oxygen particles (marker 5 µm).

Toner can be dangerous particularly when the cartridge is being changed, and especially so if that operation is made in room without any proper air aspiration. Repeated exposure, like the one undergone by technicians assigned to service of photocopiers, may be particularly risky because of accumulation of this kind of dust.

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Fig. 6.4.2 Small Carbon-Oxygen particles contaminated by Titanium-Tin-AntimonySilicon (marker 5 µm).

Fig. 6.4.3 Coloured toner containing Titanium-Strontium-Sulphur nanoparticles (marker 10 µm).

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6.5 The particulate active filter or FAP Because of a more favourable taxation as to gasoline engines come into force decades ago, Diesel cars have grown very popular in Europe. One of their drawbacks, though, is the great quantity of particles they give out, a major problem in European cities where pollution has already gone beyond any sustainable limit, and PM10 may not exceed by law a certain mass per cubic metre (now, in 2007, the limit is 40 µg/m3), a mass limit that will become smaller and smaller along with the approval of new rules. In the year 2000, a filter invented the year before, meant to capture that dust, started to equip some French cars. Owing to its capability of having particles coalesce, Cerium oxide contained in a small tank is let in the filter, so that the dust becomes coarse enough to be detained. In order to avoid obstruction and keep the filter clean, every 300-400 kilometres the system breaks up what has been captured and emits gases and ultrafine, mostly Carbon-based, particles from the tailpipe. Those particles contain inorganic nanodust and are much finer than the relatively big ones produced originally by the combustion in the engine. That “cleaning” operation occurs when the car is running at high enough speed for a comparatively long time, so, presumably, not in urban areas, but nevertheless, from the nanopathological point of view, the inorganic dust let free is far more dangerous than the one that comes out of cars that are not equipped with that device and, because of its size, it can stay suspended in the atmosphere a long time and travel very long distances. Another problem is Cerium, an element belonging to lanthanides whose toxicity has not been exhaustively investigated but which is not negligible, which is excreted from the organism rather slowly when in ionic form (but, as explained all over this book, particles behave in a different way) and which we start finding under particulate form in urban surroundings. Fig. 6.5.1 shows a Cerium particle detected in the environmental pollution of Mantua, an Italian 48,000-inhabitants town. In the case of the Cerium particles of Fig. 6.5.2, they contain also smaller Platinum particulate that may be used as a marker indicating a particular source of pollution.

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Fig. 6.5.1 Image of a Cerium particle found on a trafficked street in Mantua (Italy) (marker 2 µm).

Fig. 6.5.2 Platinum nanoparticles contained in a larger Cerium particle (marker 500 nm).

Such pollution was unknown until a few years ago. In fact, we performed a similar analysis on a trafficked street of two North-Italy streets, but the dust we found, in spite of its complexity, did not contain any Cerium.

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One of the elements we found was Silver in particle form, maybe coming from the coating of low-cost catalytic mufflers (Fig. 6.5.3). During the periodic ignition and killing procedures, the muffler is subjected to heating and cooling cycles that cause the detachment of the Silver coating and the expulsion of the debris in the environment. Environmental pollution can depend on current technology and, therefore, can change along with progress. An example of that can be that of Sapporo (Japan) where snow tyres with steel spikes where largely used. The wear debris of those spikes was related to an increase of lung cancer incidence and, out of prudence, those particular tyres were forbidden. Epidemiologic studies will tell if that measure was indeed effective.

Fig. 6.5.3 Micro and nanoparticles of a Silver compound released by cars in a trafficked street (marker 20 µm).

From Lavoisier’s principle’s point of view (the total mass of an isolated system is unchanged by interaction of its parts), it is obvious that no inorganic pollutants is disposed of, particles are made smaller and, as a consequence, more penetrating and, in addition to that, a new element, in this case Cerium, is introduced into the mass of pollutants. What is surprising is that this aspect of the problem has never been taken into consideration and cars equipped with those devices are allowed to circulate when car traffic is forbidden by municipal authorities because pollution limits have been exceeded.

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If more sensible, more adherent to science, regulations were enforced, devices like that would have never been proposed by industry. 6.6 The environment around a foundry In January 2006 we had the chance to check the territory around a huge steelworks at the outskirts of Udine, a medium-sized town in North Italy (95,000 inhabitants). As it often happens in similar circumstances, the people leaving in a radius of a few kilometres from the plant had complained for years about the poor quality of the air, the ubiquitous presence of dust that made it impossible to hang out the washing and forced them to keep their windows constantly closed, and the fact that fruit trees and greens withered and died. An unusually high rate of cancer was also unofficially reported, but, not unlikely what happens in most similar cases, no epidemiologic evidence (or, to be sure, serious research) existed to support that widespread persuasion.

Fig. 6.6.1 Image of micro and nanoparticles of an Iron-based compound found in the vicinity of a foundry (marker 5 µm).

Fig. 6.6.1 and Fig. 6.6.2 show some examples of round-shaped pollutants found in the environment.

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Fig. 6.6.2 Image of an Iron-Manganese particle (marker 10 µm).

The environment around the factory was full of the material worked inside and dispersed in the environment through the chimneys. Dross was also heaped outside, that contributed to disperse particles in the environment (Fig. 6.6.3).

Fig. 6.6.3 Images of the two morphologies of the Iron-based particles released in the environment. The first is hollow but filled with smaller spherical debris, the second one is an aggregation of very small debris on an Iron sphere (marker 5 µm).

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So, the municipality of Udine together with those of three neighbouring small towns interested by the same problem asked us to carry out an investigation limited to ten specimens sampled inside and in the vicinity of the plant. Unfortunately, the person in charge of the factory did not allow us to enter the premises where metals were actually worked nor to take samples from the dross that were heaped outside the buildings. So, we had to be content with what we could gather in the air, on some vegetables and a few standing objects within about 800 meters from the area of the plant. What we found was a huge quantity of micro- and nanoparticles ranging from 0.1 to 20 µm, in many cases spherical in shape and with an elemental chemical composition mainly based on Iron, but containing also other elements. Failing a way prescribed by law to characterize particles and in presence of an undeniable pollution, car traffic is generally the source blamed. In this particular case of Udine, the spherical shape prevalent in the particles, the fact that those spheres were hollow, the nanometric size of most of them and the unusually high content of Iron, in that case considered as a marker, made it evident that the origin could not be traffic exhausts. Non spherical, nanometric particulate matter was also present, and also in that case, due to its size and composition where Iron was again predominant, blaming cars did not look reasonable. Besides, in the case in point, traffic was not particularly heavy and no other industry, big enough to justify such a degree of pollution, stood in the territory. It must also be considered how fragile hollow particles are and how the breaking fragments inevitably released in the environment are nothing else but irregularly shaped nanoparticles. That the particles detected were formed at high temperature is further proved by their composition. Just as an example, in one sample we saw the presence of 12 elements (C, Fe, O, Zn, Si, Ca, Mn, S, Al, Mg, Cl, Cr), while in another one, the elements were as many as 14 (Fe, O, Ca, Si, C, Al, Mn, Mg, Cr, P, K, S, Ti, Zn). In both cases- but similar conditions are present in the majority of the particles examined - those elements are alloyed and the alloy is formed in a fortuitous way,

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according to the elements present in the crucible that vaporize and subsequently combine as soon as they go up and find a cooler environment, a fact, this, shared by all similar cases like, for example, those that pass in the course of explosions. A comparatively small number of particles were also detected, whose origin was most probably different from the former. Calcium, Potassium and Magnesium are typically found in the soil and it is only natural that some particles thus composed are airborne and are detected among the others. And it is as likely that traffic, however scarce, is responsible for a fraction of the pollution present in that territory, though with a particulate which is not the same as the one generated by the steelworks. All samples were taken in places where dust had the time to accumulate, with the exception of a sample, which was obtained sucking air for 45 minutes in a place 200 meters away from the factory. Also in that case a great quantity of Iron-based micro and nanoparticles were filtered, most of which were spherical and clustered like those harvested elsewhere. Fig. 6.6.4 shows a fully contaminated acarus found on a leaf picked about one kilometer away from a foundry. Also its mouth (Fig. 6.6.5), a sort of open door to its organism, is surrounded by the metallic debris. Unfortunately, only the first part of the investigation could take place. What should have followed to come to a meaningful and much more useful conclusion, i.e. a research on biopsies of people suffering from diseases likely to be due to particulate pollution, could not be carried out because of pressures that convinced the municipalities not to pursue the matter further.

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Fig. 6.6.4 Image of an acarus found on a leaf. It is widely contaminated by the Iron-based particles (marker 500 µm).

Fig. 6.6.5 Image of the mouth of the acarus at higher magnification with the chemical composition of the homogenous pollution (marker 100 µm).

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6.7 The environment around a power plant The Italian river Po flows into the Adriatic Sea forming a delta, and that delta was transformed into a regional park. Between 1970 and 1984, an electric power station was built on a small island called Polesine Camerini situated in those wetlands, and the combustible it used was heavy oil at a yearly rate of 3 million tons. The not many people living in that territory started soon to complain about occasional oil fallouts and, in a relatively short time, about some health problems that they ascribed to the pollution generated by the power station. Their suspicion was impossible to prove, as no epidemiological study had ever been carried out and the population was too small to justify such an undertaking. In addition to that, epidemiology is often tainted by mistakes in what should be the population taken as a reference, as in many cases that population is living in conditions that are not the ones where contamination is absent. In 2005, together with other experts in fields different from ours, we started to investigate the problem on behalf of the cognizant criminal court. Our idea was to cover the whole course the possible particulate pollutants, if any were present, had followed. We had, then, the possibility to check a certain number of samples that were suspected to contain micro and nanoparticles with the obvious aim of discovering if the power plant could be blamed for the pollution of the park it was accused of. The samples had been collected years before, when the plant was fully operational, since at the time of the trial it was closed. The first specimen we took into consideration was a cotton singlet which had been washed and then hung to dry in the open. After a short time, yellowish oily drops had fallen on that garment, leaving easily visible spots. Different varieties of oil had been used in the station, according to their content of Sulphur, and we analyzed all of them according to our method. Then, we checked the different ashes deriving from combustion. Lichens are considered excellent biological indicators and we check numerous samples of those vegetables. We also received dried salad

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leaves picked in the territory, some of them grown outside and some inside greenhouses. Those grown indoor did not carry particulate pollutants, unlike those grown outside. Another kind of specimens we checked were the particles captured by the filters of the devices used by the local agency for the protection of the environment. The following images (Fig. 6.7.1) show the content of the oil polluted by Lead, Iron-Sulphur, Barium-Sulphur-Strontium and SiliconAluminium-Sulphur-Iron and some Vanadium-Sulphur compounds. Part of them remains in the ashes, but part is released through the chimneys to the environment, with a chemical composition that a combination of these elements.

Fig. 6.7.1 Image of a drop of oily fuel. The oil contains Carbon-based material and also different particulate matter, for each of which the relative spectrum is shown (marker 100 µm).

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After having been burnt, the elements contained in the oil may recombine in particulate form. In addition to the spectra shown, small quantities of elements such as Vanadium, Calcium and Bismuth were also present and, as a consequence, were found in the ash (Fig. 6.7.2 and Fig. 6.7.3).

Fig. 6.7.2 Image of the ash with its chemical composition. It contains mainly Barium sulphates, Vanadium-Calcium-Iron-Nickel-Strontium-Magnesium-Silicon (marker 400 µm).

Fig. 6.7.3 Image of a particular of the ash. The particle is round-shaped and is composed of Iron-Sulphur-Vanadium-Magnesium-Aluminium-Silicon (marker 20 µm).

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The flying ash is particularly light and is easily carried by the wind. For that reason, it can be found in the environment, including vegetables like, for example, lichens (Fig. 6.7.4).

Fig. 6.7.4 Particulate pollution in dry lichens (marker 10 µm).

In the dry lichens we analyzed, we found a great number of particles. Most of them were spherical, as is typical of a combustive origin. The spectra of Fig. 6.7.4 shown two different types of particles: the former contains mainly Sulphur and Iron with a ratio similar to that found in the oil, along with other elements in a much smaller quantity. The latter contains a mixture of many elements like, for instance, Silicon, Lead, Titanium, Iron, Zinc, etc.

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Fig. 6.7.5 Round-shaped hollow debris made of Iron, Aluminium, etc. found in a lichen (marker 5 µm).

Fig. 6.7.6 Sulphur-Iron-based nanoparticle cluster found in a lichen (marker 2 µm).

Fig. 6.7.5, Fig. 6.7.6 and Fig. 6.7.7 show single and composite roundshaped particles found over the lichen surface. Though they have different chemical compositions, they contain a combination of the same elements present in the oil.

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Fig. 6.7.7 Round-shape particle containing, among other elements, Vanadium (marker 10 µm).

The lichens examined contained particulate whose composition recalls that of the elements found in the oil. The same elements have been combined in a different way as a consequence of combustion. It is important to remember that the power plant was placed in a regional park, close to the sea and rather far from any other pollution source. Besides lichens, we analyzed also some lettuce on which we found Sulphur and Iron in the same ratio as in the oil and in the particles detected on the lichens. It is only natural that the lettuce grown in greenhouses, which we examined as well, was free from particulate contamination (Fig. 6.7.8).

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Fig. 6.7.8 Spherical particles on a lettuce leaf, containing Sulphur-Iron and SulphurBarium (marker 20 µm).

On the basis on the evidence described, along with that of other experts, the criminal court condemned to damages the property of the plant and sentenced its president and its manager to imprisonment (suspended sentences). On behalf of the criminal court, we checked about thirty cases of cancers suspected to be related to the power-plant’s pollution. Not all the specimens we received were particularly useful for our kind of investigation, since they were not taken from the interface between cancerous and healthy tissue. In the case of pathological lymph nodes, particles are inside their structure and, for that reason, we did not meet any problem. In ten of those samples, we found pollution compatible with the combustion of the oil previously checked. In Fig. 6.7.9 particles are shown with the same Iron-Sulphur ratio detected in oil, lichens and lettuce.

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Fig. 6.7.9 Debris detected in a supra-clavicular lymph-node in a patient living in the power-plant’s area, suffering from Hodgkin’s lymphoma (marker 20 µm).

The same kind of Iron-Sulphur particles were found in dust fallen on car bodies. That dust induced corrosion of the metal parts. The obvious question is: If those particles are aggressive enough to corrode a car, can they be accepted by the organism? 6.8 The case of a ship A ship is a sort of maze made up of an unbelievable number of rooms. Some of them are enormous, some are very small. Some received an abundant supply of air directly from the atmosphere, some, and those are the majority, are ventilated only through air which is forced there through a complex network of pipes. One of our investigations concerned a ship under construction and we had thus the possibility to observe men at work, the particulate pollution they produced and how those particles behaved in such an environment. Samples were taken close to welders busy in a huge shed placed in the dock yard. As could be expected, the vast majority of the particles were made of Iron and were spherical. A very similar type of particles we found in the air inside the ship, where workmen were doing the same job as their colleagues in the shed, and those particles had been captured by the bulk insulators and the varnish used to paint the walls of the

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rooms. Obviously, those particles may be slowly released in the scarcely ventilated room and be a continuous source of pollution. 6.9 Incinerators We would have much liked to study the pollution generated by incinerators, but that proved all but impossible. The reason is not a technical one, but is due to the huge economical interests involved in the building and running of those systems used to dispose of urban and industrial wastes, interests that involve powerful lobbies of builders, businessmen and politicians that oppose any form of serious control. Those lobbies describe incineration as a way to have waste disappear, but such a claim is in jarring contrast with the law of conservation of matter, that states that the mass of a closed system of substances will remain constant, regardless of the processes acting inside the system, provided no nuclear reactions take place. That means that matter may change form, but cannot be created or destroyed, a notion that was already familiar to Greek philosophers of the fifth century B.C. and was experimentally demonstrated by Antoine Lavoisier in 1786. It is hard to think that burning tons and tons of matter every day, whatever that matter is made of, produces just steam, Carbon monoxide, Nitrogen oxides and little more. Besides organic pollutants as dioxins, furans, polychlorinated biphenyls (PCB), polycyclic organic matter (POM) and a very long list of further pollutants, many of which probably unknown, non existent in the original load but the result of chemical transformations, mainly oxidations, and depending on what is being burnt, incinerators process virtually all sorts of materials and many components of those materials are of inorganic origin. Since chemical elements cannot be transformed, at least in the working conditions of an incinerator, into other elements and experience proves that combustion, when occurring in the presence of inorganic materials, generates inorganic particles, it is evident that those particles must be taken into consideration when dealing with the fumes coming from waste incineration. Those particles can either form directly where combustion occurs or farther than there, in the latter circumstance beyond the

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filtration system any incinerator is equipped with. In the former case, it must be kept in mind that the majority (not in mass but in number) of the particles generated are most probably nanometric in size and escape any possibility of capture by the filters used, hardly mattering what filters are made of. In the latter case, what passes through the filter has not yet condensed into particulate and, for that reason, the filter is hardly effective. In both cases, even in the event the filter has trapped all the nanodust generated upstream, something that waits to be proved in an indisputable way, what the fate of that material is remains dubious and the subject is always dealt with in an ambiguous and far from exhaustive way. As a matter of fact, that dust is somehow, sooner or later, introduced into the environment. As an example, activated charcoal may be cited. Because of the high surface area of its granules, activated charcoal is sometimes used as a filter, but as soon as it grows no more usable because it has already captured all it could, it is thrown into the incinerator the way any waste is, thus introducing the dust it had trapped into the cycle again. In a way, incineration as a means to dispose of waste is a sort of naïve conjuring trick. It is a fact that most of what is burnt is not visible any more, but Lavoisier teaches us that nothing has actually disappeared, but must have been transformed into something else. And that something else is heavier than the rubbish we did not want to see. The reason for that is that burning means oxidizing, and, in the case in point, oxidizing means involving atmospheric Oxygen in the reaction, i.e. adding mass. But more mass is being added, as, depending on the different techniques used, chemicals like, for example, bicarbonate, lime and ammonia, are employed for various reasons. Water is also generously used to cool parts of the plant and that water is poured in streams at a higher than natural temperature, thus influencing negatively the habitat of plants and animals. It is only natural that also those substances contribute to increase the mass that results from the combustion the original waste undergoes. So, if we consider all the addenda, what comes out of the process weights more or less twice as much as what we wanted to get rid of. Setting aside organic pollutants, in most cases much more aggressive than the original rubbish, and setting also aside what are called secondary

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particles, i.e. those that form in the atmosphere from gaseous pollutants, particularly Sulphur dioxide, Nitrogen oxides, ammonia, and volatile organic compounds and that can be the carriers of other particulate, incineration produces a wide variety of particles, presumably often in the form of alloys whose composition is actually impossible to prognosticate because of the almost infinite assortment of materials entered into combustion. So, guessing what their toxicity will be is impossible. All we can say is that thinking that dust is not formed by incineration is a preposterous notion and one of the many dangers is that particles, even if microsized, are very likely to break up into a higher quantity of nanoparticulate. We had the chance of analyzing some of the ash produced by an incinerator and scattered on the ground for a radius of many hundred metres and we also collected dust from vegetables grown in the vicinity of one of such plants, but we were never allowed to carry out more systematic investigations. Biomass like dead trees, tree branches, yard clippings, left-over crops, wood chips, bark and sawdust from lumber mills is used in some circumstances as a fuel to produce energy. Apart from the generation of pollutants like, for instance, dioxins because of the presence of Chlorine in the wood, such process is responsible for the release in the atmosphere of inorganic particulate, since inorganic molecules and metallic elements are contained in vegetables. In spite of the fact that vegetable biomass used for that purpose should not contain chemical additives, in fact, in the vast majority of cases, those vegetables contain pesticides and evident traces of chemical fertilizers. In other, almost as numerous cases, the timber used is the industrial production reject of furniture, frames or other wooden objects, containing all the chemicals used to enhance the characteristics of those materials, chemicals that end up, in chemically and physically changed forms, often as more toxic substances, in the fumes given off by through the chimneys. In many circumstances, because actual biomass coming from comparatively short distances is not enough to fuel this kind of plants, they are gradually transformed into waste incinerators. Also in this case, we have never been allowed to investigate.

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6.10 Tobacco smoke About 15 billion cigarettes are smoked every day in the world, but, though the most popular, cigarettes are not the only system to inhale burnt tobacco, and pipes, cigars and less common systems are currently used. Smoking tobacco is a concentrate of particulate atmospheric pollution because its leaves are relatively large and dust falls on their surface. In order to be suited for consumption, those leaves must be desiccated and that procedure makes them loose an enormous fraction of their weight. So, dust, being not volatile, concentrates on the final product. Smokers inhale smoke on a voluntary basis, and that smoke, produced at a temperature of about 800°C, contains more then 4,000 known pollutants, among which are Carbon monoxide, Nitrogen oxides, Hydrogen cyanide, ammonia, and other toxic irritants such as acrolein and formaldehyde. Inorganic particulate is more difficult to define chemically as it depends on fortuitous, variable circumstances like the place where the plant has grown and dried and what was actually present in the atmosphere during those periods of time. Everybody, particularly a non-smoker, is aware of the fact that tobacco smoke does not interfere exclusively with smokers but reaches also, though in a more diluted way, the respiratory system of whom is unwilling to breath burnt tobacco. That way, cigarettes, cigars and pipes become the producers of environmental pollution which is particularly perceptible and aggressive in confined rooms, and that pollution contains suspended particles. The following pictures show the particles of what we found in some of the cigarette tobacco we observed and their relative elemental spectrum.

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Fig. 6.10.1 Biological morphology of a tobacco leaf with its spectrum (marker 1,000 µm).

The elemental composition shown in Fig. 6.10.1 is the one typical of tobacco leaves. The elements shown (Potassium, Carbon, Oxygen, Calcium, Chlorine, Magnesium, Phosphorus and Sulphur) in the spectrum are homogeneous throughout the whole leaf and are to be subtracted from the following spectra. A few pictures of particulate pollution found in cigarettes from Sarajevo, Baghdad and the US are shown below. Some of the particles are nanosized, while others are microsized but equally interesting as they contain typical war pollutants, some of which are radioactive. Those cigarettes had been produced in 2003, but we do not know when that tobacco had been grown. Fig. 6.10.2, Fig. 6.10.3 and Fig. 6.10.4 show the pollution on the tobacco leaf strips in cigarettes (Drina King Size and Aura Light) from Sarajevo after the war, in 2000. Fig. 6.10.5 represents the war pollution in a cigarette from Baghdad, (Sumer Star King Size) in 2004.

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Fig. 6.10.2 Different debris found in a cigarette from Sarajevo (marker 100 µm).

The heavy metals present in the particles are notoriously toxic and their penetration may be enhanced by the hot surface of burning tobacco.

Fig. 6.10.3 Drina King Size Cigarette from Sarajevo (after the Balkan war). The particles found contain Uranium and Thorium (marker 20 µm).

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Fig. 6.10.4 Aura Light Cigarette from Sarajevo with very unusual particulate: Lanthanum, Praseodymium, Neodymium, Cerium and Samarium are present in its composition (marker 20 µm).

Fig. 6.10.5 Sumer Star King Size Cigarette from Baghdad with particles containing Uranium and Thorium (marker 50 µm).

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Fig. 6.10.6 Cigarette from the US (Marlboro) polluted by particles containing rare earths like Lanthanum-Cerium-Neodymium (marker 10 µm).

Fig. 6.10.7 Cigarette from the US (Marlboro) polluted by particles containing rare earths and Lead (marker 10 µm).

The Fig. 6.10.6, Fig. 6.10.7 and Fig. 6.10.8 show the environmental pollution on an American tobacco leaf. During smoking this pollution is burnt and inhaled.

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Fig. 6.10.8 Cigarette from the US (Marlboro) In this case, the particles polluting the leaves contained Gold, among other inorganic pollutants.

Nowadays world is vastly polluted by anthropic activities and sooner or later we will realize that we have no other choice left but face the problem instead of clumsily try to hide it the way most politicians are doing. A political action, common to all countries, is inescapable and the sooner the better. But, beyond politics, the problem is also a technological one, as, in many circumstances, no available technology can clean where we have made a mess and something must be contrived. Our work is meant to contribute to solve this problem, showing the presence of a very particular, infinitesimally small but highly dangerous pollution inside Man and animals in the deepest parts of their body, and that presence is mainly caused by Man himself. As soon as this contamination exceeds a certain threshold now impossible to tell, and do it in a continuous and stable way, our species is doomed to extinction. Let us call all that catastrophism, if we like it better, but let us get up and do something.

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6.11 Bibliography Donaldson, K. and McNee, W. (1998). The mechanism of lung injury caused by PM10 In Issues of environmental science and technology, Ed. R. E. Hester and R. M. Harrison, no. 10, pp- 21-32. The Royal Society of Chemistry Henry, S. A. (1943). Cancer of the Scrotum in Relation of Occupation”, 16, Oxford University Press, Paganetto, G. (2001). Percorsi storici di cancerogenesi chimica. Basell Poliolefine Italia. Ferrara Seaton, A., McNee, W., Donaldson, K. and Godden, D. (1995). Particulate air pollution and acute health effects. Lancet 345, 176-178 The Chirurgical Works of Percivall Pott, F. R. S. Surgeon to ST. Bartholomew’s Hospital with his last corrections. To which are added, a short account of the life of the Author, a Method of curing the Hydrocele by injection, and occasional notes and observations by Sir James Earle, F. R. S. Surgeon Extraordinary to the King, &C. First American, from the last London edition. In two volumes. Vol. I. Philadelphia: Published by James Webster, No. 24, South Eight Street. William Brown, Printer. 1819. (http://www.mindfully.org/Health/Chirurgical-Works-Percivall-Potts.htm.)

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Chapter 7

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anotechnological items are already on the market and we interact with them everyday even if we are not informed and we are not aware. Label on the product rarely contains the definition “nanotechnological” or the notice “contains nanoparticles” or something of the kind, because industries are afraid of a negative perception people may have and the a-priori rejection of a nanoproduct. Singular is the case occurred in Germany involving “Magic Nano”, a protective glass and bathroom sealant in the form of a spray, which claimed to have antibacterial activity (web ref. 1). Within a few days, almost 100 people were hospitalized for pulmonary problems, some of them edema. The producer Kleinmann GmbH, a multinational industry, assured that it did not contain nanoparticles or other nano-things, as confirmed also by Rene Zimmer of the Federal Institute for Risk Assessment (BfR) in Berlin (web ref. 2), (web ref. 3), (web ref. 4), (web ref. 5) and that the toxicity was due to the propellant. No matter what the origin of the problem was, the product had to be withdrawn in a hurry. We do not know what the truth is, but propellants are contained in many sprays and there is no information about other similar cases. In any case, people rightly or wrongly, perceived that “nano” is suspicious. And that event made also the scientists ill at ease. L’Oreal, the French cosmetics company, invented liposomes, nanostructures capable of crossing the derma and penetrate deeply. They are investing a lot in these technologies and the hope is that they are putting money also in safety, because beauty is certainly something 239

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pleasant that may be rightfully pursued, but safety is far more important. So, biocompatibility of the nanostructures they add (web ref. 6) and their long-term reactions must be ascertained beyond any possibility of doubt. Matter at nanoscale level can hide surprises. For instance, Aluminium is stable in bulk shape, but explosive at nano-levels. Carbon nanostructures are not soluble and not biodegradable. So, taking advantage of that characteristics, they are used for the construction of golf balls, tennis rackets and skis. From our point of view, these items can be considered safe, since the nanomaterials used are trapped in the solid structure and cannot be released. “Nanotea” by a Chinese company will increase tenfold the amount of Selenium adsorbed with green tea through nanocapsule engineered to bypass the acid environment of stomach and dissolve in the bowel. Canova Activa oil contains nanocapsule-delivered chemicals in rapeseed cooking oil that will stop cholesterol entering the bloody stream. Slim Shake chocolate is a powdered drink that uses nanotechnology to cluster cocoa cells. Among their all but infinite applications, nanotechnologies can be applied also to create sensors for food. In fact, there is a project using that technology to detect bacteria inside meat, doping 60-nm sized silica nanoparticles with molecules of fluorescent Ru(bpy) dye. The scientists of the University of Florida attached antibodies for antigens that are present on the surface of the bacteria to nanoparticles. That can work for Escherichia Coli bacteria present in mincemeat and the test takes only 20 min. Nanotechnological devices have started to be used in washingmachines, air conditioners and freezers made by two industries: Samsung and Daewoo. (web ref. 7), (web ref. 8), (web ref. 9) Samsung inserted a device that releases “Silver Nano” ions (Ag+) that bind themselves with the fabric at a molecular level. “This draws out impurities and bacteria from the clothes, leaving them completely sterilized and totally fresh”. “Silver Nano employs the safe and sanitizing power of silver to eradicate airborne bacteria and germs”.

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Obviously, something like nano-sized silver ions (Ag+) is a nonsense. Does the device release nanoparticles or does it release ions? The difference is enormous. When the advertisement of this product line appeared for the first time, it publicized Silver nanoparticles and nanotechnology. The toxic activity of Silver nanoparticles is well-known (see Chapter 9) and is being taken advantage of by Monsanto that use it in pesticides to control the Colorado potato beetle. FDA say they do not have the authority to regulate pesticides and that is an EPA’s job. EPA say that FDA has responsibility for requiring a label, because potato is a food (web ref. 10), (web ref. 11). What is occurring is that nanotechnologies, creating new products, create at the same time new situations that are not regulated. So, when Samsung changed their advertisement from just Silver nano into Silver nano ions, it is possible that they did so as soon as they knew of the possibility that the material used in the device is a pesticide. Daewoo uses Silver particles mixed with a plastic resin. “Using Nano Poly Technology in “Pulsator” & “Tub U” in Washing Machines many hurtful bacteria in clothes shall be sterilized perfectly”. That is what they claim (web ref. 9). All that is very exciting, but we cannot exclude that there may be a risk that we inhale Silver nanoparticles directly from our clothes and/or dispersed in our house from air conditioners. And no studies have been undertaken about a potential skin contamination. But further hypotheses can be put forward. The waste water from the washing machine is drained to the water treatment plant through the sewer system, but at present there are no filters that can capture nanoparticles. So, there is a real possibility that they reach the sea, through the rivers, and are ingested by fish. The end of the story is that we can eat a material released by our own washing machine. That waste water can also pollute the environment, contaminating through irrigation the grass eaten by animals as well as the vegetables that are our food. So, we should not be too surprised by what we found in a hamburger bought in a shop: a cluster of Silver nanoparticles (Fig. 7.1).

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Fig. 7.1 Image of a cluster of Silver nanoparticles contained in the meat of a hamburger (marker 20 µm).

We do not the actual origin of that Silver, but our hypothesis is not unreasonable. After some time from this finding we had the possibility to analyze dried hay in a small Italian farm where a case of mad cow had been reported. The hay contained the environmental pollution we usually find in that area, but we detected also numerous Silver particles, though in a composition containing also Magnesium, Aluminium and Silicon (Fig. 7.2). We supposed that they could come from a pesticide, but we had no possibility to check.

Fig. 7.2 Image of small Silver particles on the surface of a hay blade (marker 50 µm).

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Silver or Silver oxide nanoparticles are already present in socks and T-shirts, as the aversion for body smell induces industries to make yarn with bactericidal properties. Fig. 7.3 shows a thread coated with an already scratched Silver layer. What is the appearance of this layer going to be after many washings? If the metal coating is no more there, we will have good reasons to think that probably some fish, clams and other beings have been contaminated.

Fig. 7.3 Image of a thread of a T-shirt with Silver-oxide nanoparticles (top marker 20 µm; bottom marker 2 µm).

There are famous examples of “translocation” of environmental pollution. The people of Minamata City in Japan suffered from the toxicity of Mercury eaten along with contaminated fish (web ref. 12). That Mercury was ingested in particulate form and not as ion, but the corrosion the particles underwent in the body had Mercury ions released and their toxicity induced the pathology.

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Another example not yet entirely investigated is related to the Balkan war. A refinery in Pančevo was bombed for three days and a 800-m column of soot was created that moved also toward the American headquarters (web ref. 13). Airplanes took off and bombed the cloud with Silver iodide in the attempt to make it precipitate with the rain. Unfortunately, that operation caused the river Danube to be contaminated by this tiny, not biodegradable pollution as well as the Black Sea, where the Danube flows. Having fish polluted by Silver-iodide particles is a logic probability, but nobody seems to show any particular interest in that and no proper prevention measures are taken, except a control for Uranium and a few other heavy metals on game and mushrooms imported to Italy from former Yugoslavia. Heavy-metal pollution is something we detected in 140 samples of bread or biscuits coming from 10 different countries in three continents. 40% of them were contaminated by environmental or industrial particulate pollution.

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Fig. 7.4 Image of Tungsten-Cobalt micro and nanoparticles detected in non-industrial bread (marker 1000 µm, image above and 20 µm, image below).

Fig. 7.4 shows a metallic contamination in a bread sample. Because of the particle shape, we may say that that is probably due to the wear of some crushing or milling tools, probably from the anti-wear metal used as coating of those machines. In “nanotechnological” food, non biodegradable nanoparticles can be added intentionally, for commercial, technological or aesthetic reasons. One clear example is represented by a chocolate bar called Mars where Titanium dioxide nanoparticles are added to avoid the separation of cocoa and cocoa butter due to aging and changes of temperature. Inside our body, there is no way to degrade titania nanoparticles. They are insoluble, non biodegradable and can remain as foreign bodies inside the digestive system or migrate elsewhere. There is no evidence that they are eliminated with defecation and, to our knowledge, no studies have been undertaken on the subject.

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Fig. 7.5 Image of Iron-Chromium nanoparticles in a chocolate (Mars) (marker 10 µm).

Fig. 7.5 shows nanoparticles of a type of stainless steel that were probably unintentionally released in the chocolate during a specific industrial manufacturing process. Fig. 7.6 shows titania nanoparticles contained in a chewing gum. It contains also silicates and silica micro particles added to remove food debris from the teeth.

Fig. 7.6 Image of a cluster of titania nanoparticles in a chewing gum (marker 2 µm).

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But nanoparticles can be present also in toothpastes and even as coatings of toothbrushes (head and bristles). Nano-UP, a Japanese company, has a product line composed of toothbrushes (Fig. 7.7) and toothpastes where Gold and Silver as coating and nanoparticles respectively are used. Their toothpaste contains Silver and few coarse Gold debris.

Fig. 7.7 Image of a toothbrush bristle with Silver nanoparticles (marker 100 µm).

It is only obvious that the presence of unwanted, toxic, not biodegradable contaminants (pesticides, dioxin, etc.) in food decreases its nutritive value and has a negative impact on human health. Among the numerous contaminants that can be present, heavy metals make up a well-known group. Recently, with the diffusion of nanotechnology, the problem of toxicity related to nanoparticle size (and not just to its chemical composition) has started to be investigated (Donaldson, K. et al., 2004), (Kipen, H. M. et al., 2005). Indeed, we can say that some in-vivo studies suggest that nanoparticles are not inherently benign (Hillyer, J. F. et al., 2001), and that they affect the biological behaviour at the cellular, subcellular and protein levels (Donaldson, K. et al., 2001), (Zhao, Z. et al., 2006) The possible presence of metals as salt has been investigated in food (Charbonneau, J. E. 2001), (Szefer, P. et al., 2003).

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So far, inorganic pollutants not as soluble salts (i.e. ions) but under particulate form has not been investigated in depth. The previous chapters proved that exogenous, inorganic, micro- and nanoparticles can be found in biological and, more specifically, human pathological tissues, in tissues affected by pathologies of unknown origin like Crohn’s disease (a granulomatous disease of the colon) or various forms of cancer (e.g. colon, stomach cancer). Food could be an important carrier for “dust” since, in many cases, row materials grow in polluted environments, more and more contaminated by industrial wastes. Nanotechnological food can be very useful, but also very dangerous for human and animal life. The information gathered will be used to set up prevention program. Then we should study food “from the fork to the farm” and detect what the sources polluting it are. The study should also include animal feed. As our method allows to work on archived biological samples, “mad cow” disease could and should be part of the study, in order to verify if cattle were actually fed with exhausted lubricants mixed with animal flour as is sometimes reported. A focused study could be carried out on the poultry food in order to verify if there is a nano-contamination. If so, the infected animals must be verified for this contamination and the nanoparticle-virus interaction should be evaluated. It is desirable to analyze as basic research ice carrots in the Antarctica to check if environmental nanopollution was already present in the past and to what extent. Hiroshima and Nagasaki destruction and Chernobyl accident with its Cesium dust could be particularly interesting. Then, again as basic research, it would be desirable to know how bacteria and parasites behave and develop after having interacted with nanodust. It would be of great interest to study how viruses adhere to nanoparticles and how (and if) they change, for example in pathogenicity, after having interacted. Nanodust seems to be teratogenic and this is an issue that certainly deserves to be deeply investigated.

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We should also develop traps and filters to capture nanodust and sensors to detect and measure it. Without any doubt, this will prove very useful to nanotechnology industries to protect workers. Then, it would be very useful to develop a network of infrastructures to monitor nanodust in the environment, capable of alerting people in case of danger (see, for example, what was not available at Chernobyl). Setting up courses and schools to educate scientists and technicians in this new, particular field of environmental science is of the greatest importance and an urgent necessity to nanotechnology companies producing or working with engineered nanoparticles. Special attention must be paid to security, since nanoparticles can be inexpensive and silent bullets to the cell nucleus and could be used as such. Further research directions can be the study of the so-called industrial sterility, malformed feti in specific industrial areas, rare diseases and new pathologies investigated, but not yet understood. Epidemiological studies with homogenous cohorts of patients should be carried out, where, starting from the analysis of the embedded particulate matter, exposure is verified and the source of pollution is identified. The results of our research have been readily applied by the Italian senatorial commission dealing with the problem of pathologies contracted by peacekeeping troops in former Yugoslavia. A non negligible number of them were in the vicinity of the explosion of hightemperature rounds (e.g. depleted Uranium or Tungsten weaponry) or were involved in the destruction of weapons which was carried out by heaping them up in large holes dug in the ground and having them burn. Some of those soldiers were also close to burning oil wells. In all those cases, they inhaled particulates produced be the combustion of large quantities of matter and may also have ingested them. Part of those soldiers developed diseases that can be classified as nanopathologies. 7.1 Bibliography Charbonneau, J. E. (2001), Investigation of corrosion and container integrity in metal food containers using scanning electron microscopy-X-ray microanalysis, Scanning, May-Jun;23(3):198-203.

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Charbonneau, J. E. (2001), Investigation of foreign substances in food, Scanning, JanFeb;23(1):51-7.Donaldson, K., Stone, V., Tran, C. L., Kreyling, W., and Borm, P. J. (2004b), Nanotoxicology. Occup.Environ.Med. 61, 727-728. Donaldson, K., Stone, V., Clouter, A., Renwick, L., and MacNee, W. (2001a). Ultrafine particles. Occup.Environ.Med. 58, 211-6, 199 Hillyer, J. F. and Albrecht, R. M. (2001), Gastrointestinal persorption and tissue distribution of differently sized colloidal gold nanoparticles, Journal of Pharmaceutical Sciences 90(12):1927-1936 Kipen, H. M. and Laskin, D. L. (2005), Smaller is not always better: nanotechnology yields nanotoxicology. Am J Physiol Lung Cell Mol Physiol, Nov;289(5):L696-7. Szefer, P., Domagała-Wieloszewska, M., Warzocha, J., Garbacik-Wesołowska, A. and Ciesielski, T. (2003), Distribution and relationships of mercury, lead, cadmium, copper and zinc in perch (Perca fluviatilis) from the Pomeranian Bay and Szczecin Lagoon, southern Baltic, Food Chemistry 81, 73-83. Zhao, Z., Hyun, J. S., Satsu, H., Kakuta, S. and Shimizu, M. (2006), Oral exposure to cadmium chloride triggers an acute inflammatory response in the intestines of mice, initiated by the over-expression of tissue macrophage inflammatory protein-2 mRNA, Toxicol Lett. Jul 1;164(2):144-54. Epub 2006 Jan 18. Web Ref. 1: www.washingtonpost.com/wp-dyn/content/article/2006/04/05/AR2006040502149.html Web Ref. 2: http://www.bfr.bund.de/cms5w/sixcms/detail.php/7750 Web Ref. 3: http://www.quackwatch.org/01QuackeryRelatedTopics/PhonyAds/silverad.html Web Ref. 4: http://www.fda.gov/bbs/topics/ANSWERS/ANS00971.html Web Ref. 5: www.smalltimes.com/Articles/Article_Display.cfm?ARTICLE_ID=270664&p=109 Web Ref. 6: www.wired.com/medtech/health/news/2005/09/68683) Web Ref. 7: www.samsung.com/in/products/washingmachine/thesamsungwashingmachinea dvantage/index.htm) Web Ref. 8: www.samsung.com/ph/presscenter/samsunginthephilippines/productnews_20040608_00 00058341.asp) Web Ref. 9: www.daewoo-electronics.de/eu/products/living_washing_glos.asp Web Ref. 10: (www.biotech-info.net/seeds.html, Web Ref. 11: (www.lawbc.com/fda.html). Web Ref. 12: (www1.umn.edu/ships/ethics/minamata.htm) Web Ref. 13: (www.ieer.org/reports/bombing/index.html)

Chapter 8

New York 9/11 ___________ n 9 September 2002, Dr Gatti sent the following message to the Herald Tribune, as an answer to a specific request by the editor to remind the anniversary of the Towers’ collapse with a 100-word message.

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“I was in London, at Harrod’s, when I watched the Twin Towers collapse. The immediate slaughter was something everybody could understand, but there was something more sadly sophisticated I thought of. All people who survived the disaster inhaled and ingested particles of dust which got stored in the tissues of their respiratory and digestive tracts. There they will induce more or less slow, but often deadly, reactions, what a European research started relatively recently calls a ‘nanopathology’. (An innovative technique makes it possible to detect and diagnose them.) As a scientist involved in the research, I know that New York is bound to suffer more mourning that will pass unrecognized.”

Probably somebody remembered that letter, since a little less than one year later we received a phone call from New York, and the person who called was a lady who had been heavily involved in the event of 9/11. She was one of those people who had their working place inside one of the Twin Towers and survived the attack; but she inhaled a huge quantity of dust and, because of that and the respiratory problems that followed, she spent two months inside an Oxygen tent. She called to know something about the theory of Nanopathology mentioned in the letter and about the possible existence of remedies. So, we started to exchange messages. 251

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After two years, she developed an angioma in her brain, she was operated on and eventually recovered after a long convalescence, during which she followed what looked a strange detoxification program (Web. Ref. 1) The Council of Down Manhattan had accepted the proposal made by the Foundation for the Advancement of Science and Education located in Los Angeles to apply a particular procedure aimed at detoxifying a certain number of workers employed by the Council and exposed to dust: firemen, sanity workers etc. That detoxification consisted of a 3week treatment based on physical exercise, sauna and a sort of vitamin cocktail. Dr Gatti was invited to take part in meetings where medical doctors and patients presented their data and discussed their experiences and, in case, their progress. At the beginning, she was sceptical, but, before rejecting any theories or hypotheses, knowing them well enough is necessary, in order to be able to find out their possible weak points, if any. So, every judgement was left suspended. From what is a traditional scientific point of view, such a treatment looked indeed much of a nonsense and a very poor remedy. Moreover, we are accustomed to believing that the higher the cost, the more effective the result is, and, in that case, the cost of the treatment proposed was in fact very low. The particulate pollution generated by the collapse was extremely fine, in our opinion especially the one formed in the part of the buildings crossed by the aircrafts that had “disappeared” there. In that particular point, the temperature had been very high, high enough to have the metallic structure and the airplanes vaporize and it was particularly so in the vicinity of the blast, and that blast involved a great variety of different things, from glass to plastics, from steel to computers. Everything was inside disappeared from sight. But, in fact, as every student knows, nothing had disappeared: everything had been sublimed and transformed into something else: an aerosol whose composition depended on the elements present in the reaction matter. That new pollution thus generated had a huge variety of chemical compositions that did not exist before. The Environmental Protection Agency (EPA) workers looked for asbestos in the pipelines of the air conditioning systems three years after

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the collapse to demonstrate a possible correlation between the diseases become manifest and the presence of such pollution. But blaming just asbestos is a limited way of facing the problem which is much bigger and with more culprits than just that. As a consequence of the collapse of the buildings, another form of pollution was created and that was the one originated from the pulverization of concrete, whose particles were larger in size than the ones generated by the parts of the buildings where the planes had entered. The constant, persistent cough of the firemen is a demonstration of the inhalation and entrapment of those micro-sized particles that, because of their larger size, had not penetrated deep enough to reach the alveoli but had remained in touch of the bronchial walls where the ciliary activity tried, not so successfully, to get rid of them. One of the subjects belonging to the group had started his work September 11, 2001, but grew seriously ill next May. After such a comparatively long time, that person developed chronic fatigue, that did not allow him to stand up from his bed without exhausting all his energies. A number of other, apparently not homogeneous, symptoms like blurred vision, nightmares, depression, etc. affected him and, for all those reasons he was unable to work. For him, like for all the other patients, no current drug was effective against those symptoms and some of the subjects even suffered from the side effects those drugs produce. So, this person entered the group and underwent the “detoxification“ treatment after which he recovered and we lost his track. During the meetings held at the place where doctors and patients gathered and carried out their program, and thanks to long discussions with the patients, Dr Gatti got to know interesting details about the onset of those people’s diseases, exposure, latency period, symptoms, their getting better and their personal feelings. In particular, a sentence of a survivor attracted her attention: while he talked about his impressions during the detoxification treatment, he told of his surprise when he observed the dramatic change in colour of his own sweat which had turned to a rich brown. None of us had ever heard of such a phenomenon. Sweat’s main functions are regulation of body temperature and elimination of some waste products and it was probably those products

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that had caused that reaction. As we work on inorganic debris, we thought it natural to check if that liquid contained them and, in case, what could their size and composition be. Had we found what we wanted to look for in those people’s sweat, the next question was: are those things, expelled from the organism, responsible for the partial recovery experienced by the majority of those subjects participating in the programme? If the particulate matter inevitably inhaled, dispersed in the blood circulation and in the internal organs, can induce a body reaction with those unusual collections of symptoms, its complete, or even only partial, elimination can eliminate or even just decrease the biological reaction. So, we proposed to analyze the sweat of those patients and some of them collected it during their saunas following a protocol simple enough not to be a bother, but strict enough not to yield polluted samples. We asked the patients to clean their skin scrubbing it with alcohol, not to use soap, creams or other potential contaminants. They were provided with a sterile vial where they collected the sweat drops during the sauna, mainly from the breast and the arms, and not from the armpits. The analyses on the sweat we got were very interesting. They revealed that Nature is not unkind and a possibility exists to get rid of so unusual a pollutant from the pores of the skin. When the body temperature rises and sweat is produced, the pores open and a certain amount of liquid is thus eliminated. In that liquid we actually found the debris we were looking for. The following images show the particles we found in the fluids examined. We present below a series of unusual chemical compositions found in the particles detected in the sweat, that could be related to the collapse.

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Fig. 8.1 Particles of n. 1 rescue-worker’s sweat containing a particle composed of Copper-Zinc-Gold-Chlorine-Potassium (marker 20 µm).

Fig. 8.2 Particle from n. 2 worker’s sweat containing Zinc-Lead-Iron-Chlorine-SiliconCalcium (marker 5 µm).

Fig. 8.1 and Fig. 8.2 show small particles found in two different workers that present two different peculiarities. The first chemical compound (Copper-Gold-Zinc) contains Gold and Copper, typical of a commercial golden alloy, but in commercial alloys Zinc is never present. Such a composition is not used in any known material. The second one contains again Zinc but now that element is bound to Phosphorus-LeadIron-Chlorine-Calcium-Silicon. The oddity is represented by the hard-toexplain presence of Phosphorus. This can be the result of an occasional combustion: a residual of the melting of pipelines of water-gas, window structures, etc.

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Fig. 8.3 Particle from n. 3 rescue-worker’s sweat containing Copper-Silver-Gold-Zinc (marker 20 µm).

Fig. 8.4 Particle from n. 4 worker’s sweat containing Iron (marker 10 µm).

Fig. 8.3 shows an 8-micron sized particle of Copper-Gold-SilverZinc. It is strange to find golden compounds in the sweat of two different workers who shared only one comparable exposure. The presence of Gold and Silver as parts of computers, furnishings, jewels kept inside safes as well as Copper in electrical wires is understandable. Zinc is another element whose presence can be explained as it may derive from a coating (zincing), but is never used to form golden alloy. That means that this type of particles can be originated by an accidental, local, uncontrolled combustion. Fig. 8.4 presents a particle containing a mixture of elements coming from two different origins. Chlorine-Sodium is the elemental composition of a salt commonly and predominantly present in the sweat,

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so of biological origin. Iron-Sulphur, instead, with the specific morphology observed (a sharp-cornered debris) is of exogenous origin. The particle appears immersed in the sweat and surrounded by other nanosized debris (Fig. 8.5).

Fig. 8.5 Image of a Sodium chloride crystal trapping other nanometric particles containing Iron-Barium-Sulphur-Magnesium-Aluminium-Silicon-Calcium-Potassium (marker 20 µm).

Thanks to a lucky coincidence, we could get the dust deposited on an object on display in a shop that was located inside one of the Towers that its owner found completely covered with dust just after the collapse, and on a fireman’s helmet. Unfortunately, we did not have the possibility to analyze the flying ashes that hovered over what is today called Ground Zero and flew toward the other boroughs of New York. Those are likely to preserve the memory of the two airplanes and the parts of the skyscrapers where they stroke. The following images show the dust collected on the firemen’s helmet used for 3 months and put inside a bag together with his overalls. Neither helmet nor overalls were cleaned. After 3 years from the collapse, when we formulated the hypothesis that the dust on the helmet represented the exposure the subject had suffered and caused the disease he suffered from, he asked us to check and try to explain why his weight had increased and why his breath had grown short. The helmet was very dusty, and the main composition was concrete (Fig. 8.8). In that dust we found some particles with compositions similar to those we had found in

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his sweat samples. Much of our attention was focused on golden alloys, not common in the normal pollution, and on debris composed of numerous elements, since this is one of the clues left by a random combustion.

Fig. 8.6 Image of micro and nanosized debris containing Gold-Silver-Copper-CalciumSilicon (marker 50 µm).

Fig. 8.7 Image of particles of the dust collected on the helmet. They contain SulphurCopper-Silicon-Calcium-Chlorine (marker 20 µm).

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Fig. 8.6 and Fig. 8.7 show particles detected in the dust found on the rescue worker’s helmet. They have different morphology and size. The bigger and sharp-edged ones are concrete, while the smaller have different origins. Some of them present a matrix where very small particles are embedded. The first contains noble metals that were also present in the sweat. That should not look strange, but what is just obvious to find in such circumstance. When the disaster occurred, the atmospheric temperature was still hot and many air conditioners were working. (In New York, air conditioners are kept on even if there is no actual need of them.) For that reason, the dust produced, much of which was very thin and behaved more or less like a gas, entered the pipes. That transferred and probably even concentrated that kind of pollution indoors. A problem that is somehow similar to that is due to the rubble and remains of the collapse moved elsewhere and disposed of in sorts of landfills. Those too were the obvious origin of dust. Since some of those particles are still nested somewhere, are actually impossible to get rid of and can have only been diluted in the atmosphere, it is only natural that they can still have an impact on the people who live in the territory.

Fig. 8.8 Debris of the concrete found at Ground Zero with the spectrum (marker 100 µm).

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Fig. 8.9 Image of round-shaped Iron-Sodium-Silicon-Sulphur-Chlorine-Calcium debris. (marker 20 µm).

Fig. 8.10 Image of composite debris found on the overalls of the fireman. It includes nanosized, round-shaped Lead-Iron-Sodium-Aluminium-Silicon-Phosphorus-ChlorineCalcium particles (marker 5 µm).

Fig. 8.9 and Fig. 8.10 show particles of compounds of Iron and Lead: similar debris were found also inside the sweat specimens. There is a correlation between the chemical compositions of the particles found in the sweat and those found in the pollution.

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Fig. 8.11 Image of a round, submicronic Chlorine-Cerium-Sodium-MagnesiumAluminium-Silicon-Sulphur-Calcium-Iron particle (marker 20 µm).

Fig. 8.11 shows a 500-nanosized spherical particle with a strange composition: Chlorine-Cerium-Sodium-Magnesium-Aluminium-SiliconSulphur-Calcium-Iron, found on a firefighter’s fatigue dress.

Fig. 8.12 Image of New York debris. The whiter particle is composed of Gold-CopperNickel-Zinc-Sodium-Aluminium-Silicon-Chlorine-Potassium-Calcium-Iron (marker 10 µm).

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Fig. 8.13 Image of a round-shaped particle containing Calcium-Lanthanum-CeriumNeodymium-Sodium-Magnesium-Aluminium-Silicon-Sulphur-Potassium-Iron. (marker 10 µm).

Fig. 8.12 and Fig. 8.13 show particles detected on the object found intact below the ruins, inside a shop of sanitary fixtures by the owner. He collected that as a memento of his destroyed activity and life. Also the chemistry of this dust containing Gold-Copper, but also Zinc, Iron, Nickel was related to that found in the sweat samples. Once again, it is a random combination of elements that are not related to the melting of a single material or object, but to the presence of many other objects in the same crucible. In many of these spectra there is the presence of Sodium-Magnesium-Aluminium-Silicon-Calcium-Iron, i.e. is the basic composition of a ceramic glass material. It has been observed that during the melting in some parts of the Towers, there was the formation of glass debris entrapping other materials. The composition of the image of Fig. 8.13, a spherical debris, is rather peculiar. Its spectrum contains Sodium-Magnesium-Aluminium-Silicon-Calcium-Iron, but that spectrum could be due to the part that surrounds it. Certainly, it contains earth elements like Cerium-Lanthanum-Neodymium, and Sulphur. In our experience, though not exclusively, the earth elements’ triplet signal may be related to an environmental contamination of tobacco smoke. A more accurate investigation about the history of the object, of the shop and of the owner (was he a smoker?) could explain much about those presences. In any case, nothing can be completely destroyed.

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Most of the dust we analyzed was related to concrete. In this chapter we presented the more meaningful or unusual debris to try and demonstrate if a passage from inside the organism to outside is possible. We considered those strange compositions as traceability markers and the conclusion we came to was that a passage looks possible. Can this finding help to devise an efficient detoxification method? There is a possibility, but a limited one, since, probably, the passage is allowed only to the dust trapped in the fat. For the one remained in the blood or in other tissues there seems to be no way to be exteriorized. Let us hope that scientists and technologists are smart enough to find a solution. 8.1 Bibliography Web. Ref. 1 http://www.nyc.gov/html/doh/html/wtc/index.html

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Chapter 9

The Future and Prevention Criteria ___________ 9.1 The state of the art an has always lived with nanoparticles: proteins and colloids, when observed according to their size, may be so classified, but proteins and colloids are organic compounds and, at least in most cases, can be tolerated by man or even used for his metabolism. Inorganic micro and nanoparticles are constantly generated by nature: belching volcanoes throw out huge quantities of minerals in the form of tiny specks of dust, fires produce particulate, sand is blown by the wind in the atmosphere and part of the products of rock erosion caused by the weather ends up in the air. So, man has been confronted all along with that kind of pollution, but very little can be said of its interactions with his organism, since, with rare exceptions, that has always been considered as an inescapable condition, almost a sort of background noise, wherein man is bound to live. Therefore, little or no attention at all has been paid by medicine to that phenomenon that in most cases was not seen as a problem. One of the characteristics that distinguishes man from all other animals is his unique way of using the planet where he lives. Man is the only animal who can lit a fire, and this now apparently very simple and harmless action has been the first source of non natural pollution, as all combustions produce particulate matter whose composition is different from the one of the materials it derives from. For millenniums, though, fire was very sparingly used, and in the vast majority of applications just to warm oneself or cook food, and the pollution generated represented a comparatively trifling problem in a planet inhabited by a scanty population. Energy has not been in great demand for the largest part of

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human presence on the Earth: Muscles, be they offered by animals or by man, have been the primary form of energy for a very long time, only much later boosted by natural sources like wind and flowing water, and it was only in Europe, in the second half of the eighteenth century, with the so-called First Industrial Revolution, that combustion started to be used on a relatively large scale, and that was done to heat water meant to produce steam exploited to drive machinery. A few decades later, with the Second Industrial Revolution, coal became quantitatively the most important source of energy, thus supplanting wood and peat as a fuel and transforming water-driven mills into plants whose output was strictly dependent on man’s decisions. Thus, man started to impinge noticeably upon nature by producing through combustion particulate matter that, when inorganic, could not be degraded. Nowadays the sources of anthropic micro and nanoparticulate are all but endless. Heat has become very easy to come by and very cheap, and higher and higher temperatures can be reached with no technical difficulties. This allows us to produce energy and materials we commonly and often absently use in our every-day life without considering that one of the by-products is inevitably dust, and the higher the temperature, the tinier the particles. And what we produce nowadays is particles much smaller than and very different from those produced by nature. One of the most obvious and visible responsible for this kind of pollution is certainly car traffic. And not only with its exhaust fumes, but also with the wear of the tyres and the brakes. Heating systems, when using fossil fuels, are also polluters, and so are all plants that have recourse to combustion: incinerators, cement works (often used, more or less legally, as incinerators as well), power plants, foundries and many factories in general. But great quantities of heat can also be generated by the explosion of weapons, especially when they are high-technology ones like those based on depleted uranium or tungsten, and much heat and dust was generated in New York when the World Trade Center collapsed. Besides unintentionally produced, noxious nanodust, there is dust of the same size we manufacture and whose properties, for which we keep finding new applications, we exploit more and more frequently.

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Engineered nanoparticles are already used in a number of commercially available products, while scientists and technicians are busy experimenting new possibilities for their use. Huge amounts of money and human resources are being invested in nanotechnologies and great expectations, both technical and economical, are placed in the indeed extraordinary properties of nanosized matter. If, on one hand, one can share this enthusiasm, some prudence is necessary if we don’t want to run the risk of being sorely disappointed. There are a few applications, mainly in food and in medicine, that require the introduction of nanoparticles in the organism, and that is done without having carried out a sufficient experimentation and without having allowed enough time to elapse to observe and evaluate the consequences of such use. In too many cases it is taken for granted that our organism can get rid of those particles, but no scientific evidence does really exist to confirm such an assumption. As far as we have been the possibility to see, we could not detect any biological mechanism of elimination for inorganic particles once they have been trapped in a tissue, and evidence is being collected that shows that those foreign bodies are far from harmless. In our opinion, this should be regarded as an invitation not to act in a hurry in the hope of outrunning one’s competitors. 9.2 What is next? Nanotechnologies, creating new materials and structures not far from an atomic level, at least from the dimensional point of view, will represent a societal revolution. But the other side of the coin inherent in every progress may be not so rosy and it is impellent and mandatory to find it out before the side effects are the cause of phenomena that may be hard to tackle or even be or become irreversible. The synthesis of new matter and the discovery of its incredible properties excite not just the researchers involved but also businessmen and a market always searching for new possibilities of gain. The previous chapters showed pieces of evidence that are hard, when not impossible at all, to deny.

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Everybody remembers how GMOs (Genetically Modified Organisms) flopped partly because of a generally bad perception but, above all, because research organizations and media were not wise enough and lied on the possible effects those organisms could induce on human bodies. So, now, great care is taken on the news released by scientists, and this, in some cases awkward, prudence may cause a bad perception of nanotechnologies. Booklets are prepared for the instruction of scientists in order to alert them of the danger and teach them how to present the subject to the public opinion. Specific European projects (web ref. 1) of communication in the specific field are being financed and a sort of censorship has been set up by the European Community in order to prevent the release of news that may be unfavourably interpreted by the public. But if, from one side, there is a need to control information in order to protect the investments and avoid that huge amounts of money go wasted, from another, certainly more important, point of view, we need to set up all the possible preventive measures we are capable of in order to avoid risks for the workers, the end-users and the environment. It is a matter of fact that a number of nanotechnological products are distributed on the market without informing the consumer of that particular origin and something like that cannot be accepted. The fear of a flop surrounding these new technologies due to a possible bad, emotional, reaction of the public, induces the producers perhaps not to lie, but to issue only partial information. When, within 3-4 years, no adverse side effects are reported, it is very likely that they will not only reveal but even advertise the nanotechnological origin of their product. But not to run risks in the event of something having gone wrong and their products having caused some sort of damage to their users, producers have no interest in furthering the research on nanotoxicity so to be able to maintain that there is no evidence to blame their goods. One of the dangers inherent in that kind of attitude is that the full understanding of what is a scientific problem involving health be delayed and a possible wide dissemination of the diseases originated by a misuse of nanotechnology, deriving from the globalization of the market and the dissemination of the products worldwide, go unrecognized even by the cleverest epidemiologist. In such a scenario, what will be registered will

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be nothing more than a “normal” increase of different types of cancer, leukaemia, lymphoma, etc. of “hard-to-explain” origin. Another problem is the attitude of some scientists, hopefully a minority, but in some instances an important one, at least from the acquired prestige point of view, to yield to the allurements of money or power. The way it happened some decades ago with tobacco, those scientists agree to write papers that are then published even by serious journals, and in those papers the essence of science gets twisted and, in the most favourable of hypotheses, truth is concealed and facts are passed over in silence. People who obtained money for their research or people who invest their money to develop nanotechnological products, will hardly look for side effects. So, all that is used as an alibi based on a mistaken feeling of authority to escape from responsibilities and, in our case, the risk is that the use of nanoproducts and its relevant exposure do not enter among the parameters examined and are not considered in the statistical evaluations. What is written above must not be seen as a rejection of nanotechnology but just a sort of warning intended to make it stronger, more effective and much less open to attack. A discipline that comes often into the picture is epidemiology. Research in that field requires huge numbers of cases and a long time, and, for that reason, a research whose conclusions are presented today was actually started ten or fifteen years ago when the conditions, due to the rapid evolution of pollution, were not comparable with the present ones. If, as an example, we take into consideration a population exposed to the pollution originating from an incinerator built according to the most modern technology, we must know that the particulate matter it eventually produces as a final result of its processes is probably much finer than that produced by the older plants. So, the modern particulate’s behaviour in the environment is much more similar to a gas than the old one’s and limiting the epidemiologic evaluation to areas were gross particles are likely to fall to the ground becomes meaningless and deceptive. And equally meaningless and deceptive is considering just respiratory, oncologic and cardiovascular diseases, neglecting all those belonging to nanopathologies, including, for example and among others,

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Parkinson’s and Alzheimer’s diseases, diabetes, fetal malformations, etc. On top of that, many studies are published only if they are “reassuring” and some of them draw conclusions that should be rejected by any serious epidemiologist or, in any case, looked at with suspicion. It is not exceptional to find articles maintaining that increasing pollution has no effect on the morbidity of diseases notoriously due to pollution, and even taking for granted that that had been written in good faith, something like that, in contrast with science and common sense, should be considered as a false negative, waiting for more and stronger evidence. We must keep in mind that nanoparticles behave in a very peculiar way, unsuspected until not long ago, and probably much is still to be learnt and psychologically accepted. Nanoparticles are sets of atoms, but, because of their size, they have properties that are not ruled by the atomic-physics laws. And, in more than a way, their adverse effects are different from those of the chemicals containing the same elements but in a form that is not that of nanoparticulate. At certain threshold concentrations, nanoparticles have been found to be toxic (Brayner et al.,2006), and capable of inducing oxidative stress (RDS) to cells (Nell, A. et al., 2006), but under that critical concentration they can present new, maybe long-term, toxic aspects never investigated or suspected before. (Hansen, T. et al., 2006) This reveals the particular physico-chemical aspect of inorganic nanoparticles. In fact, in addition to the particle chemistry, their inorganic nature and particulate structure makes them respond in a special way in a biological environment. They have greater densities (and greater inertia, in a world small enough to be dominated by Brownian motion), high electron densities (the response to light is very different and this is the basis of the remote photoablation), they have strong redox potentials, they have a marked thrombogenic activity, etc. Some of them have another peculiarity: they are not soluble and not biodegradable, so, when they penetrate the organism, since it is likely that we have no elimination mechanisms made available by nature, they stay there forever. An example of such a behaviour can be found in silicosis. Inhaled silica microparticles are attacked and incorporated by macrophages that

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try to digest them but, because of their absolute non biodegradability, they do not succeed. So, when those macrophages die, they are “digested” by the organism the way all macrophages are. Not so their content which is indestructible and is released again in the biological environment to be, in case, attacked by another macrophage. This continuous, repetitive behaviour causes a state of disease. Single nanoparticles represent a very small stimulus to cell, too small to be recognized by the membrane sensors and activate the normal cell defences (the activation of the macrophagic reaction), but they disturb the cellular metabolism. So, the presence of inorganic nanoparticles in biological media, even without being directly toxic to the cell, may cause environmental stress. Cells are very sensitive to signals from the environment. For example, cellular reproduction is regulated by complex exterior signals and synchronized with the neighbouring cells. In the presence of a stress (i.e. a non expected modification of the environment), the cell stops temporarily its reproduction cycle until it re-adjusts to the new situation. The responsible for that are the Stress-Activated Proteins (SAP), kinases which transmit the alarm signal to the cell. If this process does not function properly, erroneous cellular duplication may be induced, which might result into tumour cells. In addition to that, nanoparticles can be a reservoir of perturbing or toxic cations which are slowly released and induce noxious effects in tissues. Furthermore, nanoparticles may modify ternary structure of proteins, changing their functionality. In addition to that, they can be not recognized as self, and give an immunological disease. Prion-related pathologies as Creutzfeldt-Jacob’s and amyloidosis as Alzheimer’s are classified as conformational diseases where misfolded proteins induce nearby proteins to change their conformation. Moreover, the biodistribution of the molecule attached to a nanoparticle can be very different from the monomeric form. For example, compared to free chemotherapeutic molecules, delivery to cancerous tissues may be favourably biased by several mechanisms, including particle size (Tang Z. et al., 2001) and by attaching targeting ligands to the surface of the particles.

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All this is enough to understand that conventional tests to assess the cyto- and eco-toxicity are not designed nor suited to study nanoparticles– cell interaction and that the study of acute toxicity may not be as relevant as other slower processes, where inorganic contamination progressively induces stress and damages tissues. There are no homogenous and certain results about the in-vitro and in-vivo toxicity of nanoparticles, so floods of words are being written to debate the problem. Failing data, writing has the effect to remove the fear of the unknown. Many articles, reports, strategic documents, risk assessments, contain recommendations. An article by Dr. Maynard of the Woodrow Wilson International Center for scholars in Washington (Maynard, A. D. et al., 2006) is a series of good intentions mixed with fundamental, theoretical rules of risk assessment and management. The most used words are “we must develop”, and, in particular, what we must develop is instruments, tests, validations of tests, and models to predict toxicity. That sounds rather premature since, without knowing the laws ruling the nanoworld, it is impossible to predict anything meaningful. The incredible properties we verified in some nanoparticles witness that nanoworld presents rules that can be either wonderful or terrifying. All the reports of different associations and organizations underline the “must” task (Research Needs on Nanoparticles, 2005), (web ref. 2), (web ref. 3) A far from negligible quantity of money is dedicated to research, much of it by the European Community and the US. But now there is a risk: nanotechnologists who had new institutes built, money and honours as pioneers may not want to kill “the goose that lays the golden egg”, so it may be not so likely that they declare the existence of a possible risk. The death of Richard Smalley, co-discoverer of the so-called “buckyballs” and Nobel Prize laureate, occurred October 31, 2005, could be a sign of danger not to be ignored, as he died of lung cancer and leukaemia, simultaneously. There is a possibility that such an unusual combination of diseases be the expression of the exposure to the nanoparticles he himself had created and that possibility should be considered among the risks related to handling nanoparticles.

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A hypothesis that can be put forward is that part of the inhaled particulate matter forming clusters may have remained trapped in the lungs where, because of its non biodegradability and non biocompatibility, it may have induced a cancer. The nanoparticles that did not form clusters or with a size small enough, for example, below 100nm, may have passed rapidly, before having the time to cluster, through the lung barrier and gone into the bloody stream. There, they may have created bounds with some proteins or cells specific of this environment, so they were not filtered by organs but went to the bone marrow. We do not know for certain if the cell reaction is due to the particle’s chemistry or its physical structure or only because nanoparticles are foreign bodies interfering with the normal cell metabolism, but a reaction is possible at the deeper levels. What is sure is that this approach leads to a “customized medicine” to cure diseased people rather than diseases as a sort of abstract entities, since each individual has necessarily undergone his own exposure to pollutants. This implies more time and more money dedicated to patients and this, in its turn, means that only affluent people will be able to afford it. If we want to avoid such a situation, the only possible solution is primary prevention, that set of measures aimed at removing the causes of a disease, rather than diagnose it precociously or cure it when it has already grown manifest. At present, no pharmaceuticals show any real activity against the effects the interaction between non biodegradable and non biocompatible nanoparticles and organism has, and the most they can do is a simple palliation, and that in case they are correctly prescribed, something that means a correct diagnosis has been issued. From our point of view, we have already a basic knowledge to understand what the possible risks are and how and where they can be expressed. Identifying risks does not mean at all rejecting nanotechnologies on principle and the business hoped for as some scientists and entrepreneurs claim and fear, but means the intention to drive their development safely in order to exploit their great potential in the best and most lasting way. Nanotechnology-based industries must have taken precautions and probably check their staff from the health point of view, but the question is: How? And how much do they really

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want to know? If, for example, one of their employees develops a cancer, how willing are they to investigate on a possible correlation between working place exposure and disease? Cancer morbidity is growing more and more common in industrialized areas, and children are not spared by that trend, too often hastily dismissed as an inescapable fatality caused by wrong parental genes. One of the likely causes, almost certainly not the sole but nevertheless not negligible, is particulate pollution and there is no reason to believe that engineered particles differ under this special aspect from unintentionally produced ones. But there are also diseases other than cancer that must be kept under control, and among them troubles like chronic fatigue, insomnia, loss of memory and irritability that are often attributed to stress and treated accordingly but that may have nanoparticles as cause. For all these reasons, referable to the prudence of common sense, we suggest to apply the precautionary principle before an intentional, uncontrolled, systematic, massive release of engineered nanoparticles in the environment occurs. Something we will be bound to face soon is the nanoscaled pollution unintentionally released in some specific working places and, later, in the environment. If it can be easy or, at least, feasible to check the release of engineered nanoparticles in the place where they are produced or worked, it is extremely complicated and expensive, if not sometimes impossible, to control them when they are already disseminated in the environment. The need to be competitive in the market has already induced industries to develop a fair number of nanotechnological products. It is the case of Samsung and Daewoo that constructed washing machines with the NanoSilver technology. Inside the machines there is a tablet releasing Silver nanoparticles (Silver oxide is well-known for its bactericide activity). So, their presence in the fabric guarantees no odours since the bacteria responsible for the problem are killed. Those manufacturers claim also a lesser use of detergents. The use of molecule of Silver oxide as a bactericide is regulated in “Private area and public health area disinfectants and other biocidal products” under the Directive 98/8/EC (web ref. 4) but in the case of Silver oxide in nanoparticulate

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form, its properties are due mainly to their being nanoscaled, so, the reference to the disinfectants regulation is improper and should not be considered valid. It may be curious to observe that after the news was spread that the US EPA (Registration Review Schedule: Antimicrobial Pesticides of October, 4, 2006) had declared that Silver Oxide (AgO) is a pesticide, the Italian advertisements for those products changed and they claim a release only of Silver ions and not nanoparticles. A direct measure of this presence in the washing water could solve the mystery. With a similar technology, Samsung make also air conditioners and refrigerators (web ref. 5), (web ref. 6), (web ref. 7). From our point of view, the clothes, having come out of washing full of “free” nanoparticles, are very close to people who wear them and, as a consequence of such a proximity, those people can inhale them. But those particles can also be disseminated in the house and outside and the waste water, not filtered efficiently at nanolevel, can disperse the nanoparticles in the rivers and in the sea, providing unwanted “food” to clams, fish, etc. It is somehow absurd that the scientists that discovered the nano phenomena be still busy assessing their potential toxicity, that the funding agencies be just starting broadly funding the assessments of risks and funding research, while industry is already prepared to release massively and in an uncontrolled way biocide nanoparticles. Such nanoparticles, despite being used as a substitute of bleach to disinfect water for a long time, should be banned from therapeutic use because their mechanism of action is still unknown, because, in contrast with common antibiotic agents, nanoparticles may persist indefinitely active since they are not degraded during their bacteria killing function and one has to bear in mind that attacking the microbe world means to attack the life substrate itself, and, for example, it has been found that biocide Al2O3 nanoparticles reduce root growth in different crops, due to the perturbation of the soil composition (Yang, L. et al., 2005). Of course, releasing nanoparticles through air conditioners inside a close ambient is a nonsense. We fear to inhale dust from incinerators, from cement plants, from diesel engines, etc. and allow industries to install a source of indoor pollution with nanotechnological particles in the house. This pollution can be inhaled by children or pregnant women,

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which is something that must be taken into very serious consideration and not dealt with so superficially as we are doing now. The case of refrigerators releasing bactericide nanoparticles is somewhat different. Only when its door is open the Silver pollution is released in the domestic environment, but that pollution is likely to contaminate food. Thus, the same device can potentially give two different pathologies, the former starting from inhalation, the latter from ingestion. Special attention must be paid to what happened with asbestos. It was used in brakes, buildings and innumerable further applications, from where it was expected not to be released, but actually it was. The next observation was that it accumulates in the lung, but as it did not produce acute toxicity, it was happily overlooked until many years later with a burst of lung and brain (where they may have entered through the olfactory nerves) diseases related to cancer. The results obtained through our research should put scientists, technicians and politicians on the alert and, maybe, induce them to revise their way of understanding the impact of environmental pollution upon our health. We should start by considering a simple natural law, stating that nothing can be created and nothing destroyed, but everything can just be transformed. It is a matter of fact that all forms of combustion change matter and produce micro- and nanoparticulate whose size depends mainly on the temperature reached in the crucible, and that most of that particulate, which, in many instances, is neither biodegradable nor biocompatible, gets dispersed in the environment. So, the air, the soil, water and vegetables are polluted and both humans and animals are the victims of that condition through the inhalation of air and the ingestion of food. The situation may grow worse to humans who eat animals whose flesh is polluted, and nowadays that pollution may be imported from places that are very far from where those humans live. In our century, it is particularly hard to set boundaries to everything and pollution is no exception. What the majority of the systems aimed at getting rid of wastes do is reduce them to a very small size, sometimes incomparably smaller than the original, and disperse them. As already pointed out, the problem is that the smaller the size of that dust, the more penetrating and aggressive

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to the organism it is, and making it smaller and smaller does not seem particularly wise. One of the most promising results of our research is the possibility it offers to trace back the source of pollution and the kind of exposure the subject underwent, by comparing what we detect in his pathological specimens with the environment where that subject lives or work, or the food he eats. So, it will not be too difficult to understand whether the strategy of having wastes “disappear” by making them small enough to become hard to detect or we had better resort to different solutions. To summarize, there are some pathologies strictly related to the environmental pollution; so, in order to understand them it is necessary to know and characterize that. The following Tab. 9.1 summarizes the parameters we need to know to explain these pathologies.

Tab. 9.1 Scheme of the factors that can trigger a disease.

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As a result of our research, but much is still ignored, being a foreign body seems to be the most important among the factors inducing pathologies. No matter what particles are made of, they are not recognized as compatible with the environment they have entered and that lack of compatibility is mainly due to their being a physical entity. This is something that could be somewhat hard to classical toxicologists, accustomed to reasoning rather in terms of ions or molecules and their chemical noxiousness. Size is also of the utmost importance, since, as often mentioned, the smaller particles are, the worse their effect on organisms is. Clustering in a tissue is a common phenomenon and, under certain aspects, for instance in inducing the formation of granulation tissue, clustered particles may behave like larger entities, but the sum of their surface areas is in any case much larger than the one of bigger particles and the result is an enhanced reactivity. As just mentioned, surface area also influences pathogenicity and, in particular, its ratio with volume, as the higher it is, the higher reactivity particles have. Another factor worth considering is particle shape. According to our experience, still in need of being confirmed by more observations, a needle-like shape like that of asbestos fibres is more capable of penetrating tissues than a bulkier one. Concentration is also a critical factor: unless particle penetrate cell nuclei, in which case this consideration may lose much of its meaning, it seems that a comparatively low concentration of particulate matter can pass unnoticed and be tolerated by the host tissue that, on the contrary, reacts when a threshold concentration is exceeded. That such concentration exists is a fact hard to doubt of, but how to measure it is now beyond our possibilities. We strongly suspect that that may depend on a number of factors including size, shape, surface-area/volume, chemical composition, tissue involved and its health condition, along with the subject’s health and habits. If and how other chemicals or pollutants in general interfere with that hypothetical threshold we do not know, though it is reasonable to think so, and even to think that in some cases a sort of synergy may occur.

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A phenomenon we have often noted is how intake velocity can influence the onset of a disease. In some cases, that can be rather easily explained: Let us take a non-smoker and a smoker and have them inhale quickly the same amount of dust. A non-smoker is likely to be able to get rid of it because of the better efficiency of his respiratory system and, in particular, of his muco-ciliary cells. In other instances, a quick intake may happen when a certain organ is especially prone to capture those particular particles and the consequence is they are not more evenly distributed throughout the organism. As to radioactivity, we have no direct experience, since we never found any trace of it in the cases of soldiers we had a chance to observe and in the case of peritoneal mesothelioma described in Chapter 4 we did not measure it. So, radioactivity has been listed as a likely factor but, as a matter of fact, we have no direct experience to support its influence. Though probably not so important as being a foreign body, chemistry is certainly a weighty factor of pathogenicity. It is only obvious that a particle made of Arsenic is more toxic than an Iron particle sharing the same shape and size. Most of the particles we observed, though, are alloys and their behaviour is notoriously not the same as the sum of each of their elemental components. If a particle gets corroded in a tissue, the substances resulting from that chemical phenomenon are something different from the material composing the original entity and, in that case, the resulting toxicity can be the one well-known by toxicologists. It is reasonable to think that chemistry influences the possibility of a nanoparticle to be bound, for example, by a protein and thus acquire a different dynamics. It should not be surprising that every individual reacts to nanodust in a way that can be different from his neighbour’s. The same individual variability occurs with exposure to bacteria, viruses, parasites and most other aggressions.

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9.3 The future As described in Chapter 5, we know from war experience that as soon as the hot, volatilized materials generated by explosions cool down, nanosized particles are created and scattered in the environment. The inhalation or ingestion of those, mainly metallic, particles by humans and animals can bring about pathological effects. But for the formation of nanoparticles as pollutants, there is not only warfare to blame. Car engines, industry, incineration and, in general, all high-temperature procedures are just a few examples of particulate pollutants producers. So, it is easy to guess that in more than one case, the environment is already contaminated. As far as we know now, it is not possible to reclaim the environment from nanoparticles and even in the future, with better technologies than those available today, that may be extremely difficult. In many cases, the proposed environmental remediation obtained with techniques making use of nanoparticles does not solve the problem but simply shifts it from a variety of pollution to another, this time induced by the nanoparticles employed, nanoparticles that in most cases are not biodegradable and stay in the environment, perhaps not the same as the one that had been “cleaned”, but still somewhere on this planet, forever. But, being aware of their possible adverse effects on the human health, even now it is not so hard to confine them in a laboratory and study the safest procedures to handle them. At present, we have sufficient knowledge to suggest some simple criteria to try and avoid toxicological problems in a “not alerted” population, criteria that should be adopted as a precautionary measure. The following ten “golden rules” can be adopted immediately and at trifling costs: 1 - Nanoparticles contained in any product should be declared in the product’s label, so that consumers are free to make a conscious selection. 2 - A notice should be clearly visible in any rooms or sites where nanoparticles are released (e.g. some new air conditioners). 3 - Do not disperse intentionally, non biodegradable nanoparticles in the environment, specially indoors, and take all necessary measures not to disperse them unintentionally.

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4 - Do not add non biodegradable nanoparticles in food. 5 - Do not introduce in any way (inhalation, ingestion, injection, instillation, etc.) non biodegradable, non biocompatible nanoparticles in living organisms. 6 - Do not put non biodegradable, non biocompatible nanoparticles in contact with external or internal parts of living organisms. 7 - Do not have sexual intercourse with persons who were exposed to micro- and nano-pollution. In case, the use of condoms is mandatory. 8 - Do not procreate if the male partner was exposed to pollution. Assisted fertilization can help in such a situation, if only decreasing risk. 9 - Do not eat vegetables grown in a polluted area. Water can be polluted as well. 10 - Do not smoke tobacco leaves grown in a polluted environment, especially the one created by war. Weapon explosion can create a new, mainly inorganic, form of pollution. The criteria above are simple enough and should be applied immediately for the sake of human and animal health, but more will certainly be added as our knowledge advances. Those criteria give also clear indications to the laboratories and companies busy in the fabrication of new nanoproducts. That does not mean that nanotecnologies must be sacked, it means that certain products are safe, other that release nanoparticles not. We need nanotechnologies to face the nanosized world and contrast that for our safety and of our society. 9.4 A few reflections “Memento, homo, quia pulvis es et in pulverem reverteris.” (Remember, man, that you are dust and unto dust you shall return.) That is what priests say when they put a pinch of ash on the head of the members of their flock on Ash Wednesday. Is that sentence adapted from the Genesis a prophecy? Like a parasite, man has already destroyed a part of his habitat and any being doing that is doomed to extinction. If he has done so in an irreversible way is hard to say.

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Every day we can create forms of pollution that did not existed before and whose impact on this planet where vegetables, animals and men belong on an equal basis, has never been experimented. Can living beings adapt themselves to the attack of those unexpected pollutants? In many cases the question has no answer. What we can say is that Nature rests on a delicate equilibrium and her times are long, much longer than today’s Homo sapiens sapiens. Nanopollution is a stimulus living beings had never been confronted with before; at least, not to the present extent. We should not be mistaken and think it does not have an effect or has a minor one just because those particles are so small. What they do in comparison with the larger dust generated by Nature or by less technological human activities, is change their target. As an example, let us take 10-micron particles and 0.1-ones. When inhaled, the former are likely to penetrate no farther than the alveoli, and that just in case they can get that deep; the latter, instead, reach the alveoli very easily, stay there no longer than few seconds and enter quite easily the blood circulation to invade virtually all the organism and be sequestered inside it by some tissue impossible to guess. What we did in the last few years is create new forms of pollution as side effects of novel, very sophisticated technologies. At the beginning, we did not pay attention to it. We did not want to. And now that pollution has already shown some of its possible interferences upon human and animal organisms, but we are not entitled to say that we have already seen everything. Much is likely to be still unknown and we shall have to wait many years before being able to say something conclusive. Let us take as an example asbestos. We can inhale it again and again and in most cases nothing visible happens for a long time. But we know that mesothelioma may take up to forty years after exposure before growing manifest and we have not the least evidence allowing us to say that something similar is not happening with nanodust. Nature does not come to terms with what we do: she just lives according to her rules and is blind and deaf to any protest. Once again, the way we have been doing for millennia using our brain to develop more and more deadly weapons, challenging Nature instead of living in harmony with her as all living beings do, we are showing all our naïve arrogance and lack of wisdom.

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As it always happens, those who will suffer the severest consequences of nanopollution belong to the weaker part of society: children and old people. Children can be affected even before they are born as may be seen reading what is reported in Chapter 3 on malformed feti, while old people are obviously more prone to be affected because of their generally poorer health condition and less efficient defence systems. But, if we look at the problem from a social standpoint, we must admit that less affluent people will suffer more or, better, will be the first to suffer. In many cases, large factories and incineration plants are built in the poorest quarters of cities and in already environmentally compromised areas where the rich do not live in a sort of more or less conscious attempt to escape disaster, a disaster, however, that in the long run cannot be dodged. As already described, nanoparticles, the most aggressive particulate, behave like gases and their distribution is homogeneous over large areas, knowing no boundaries. In addition to that, as far as we know, physiological known barriers are hardly effective. Anthropic pollution will probably cause an increase in sterility and miscarriages may become more and more common. This is not psychological terrorism: more than one medical association has already alerted governments and international agencies about that danger, while symposia, congresses, meetings on nanotoxicity are being held all over the world, articles are being written, TV and radio programs are being broadcast, but the truth is that no meaningful political decision has ever been taken while we keep approaching a no-return point at increasing speed. Politicians are not only short-sighted but are often in bad faith, as what they keep trying to do is hiding the problem. And they do that by hiring scientists available to produce literature fit for that aim, and by calling terrorists all the others. So, an honest, effective political action on a global scale is mandatory, as we cannot afford to live the way we are doing, with a small - small from the population size point of view - country like the United States consuming a disproportionate amount of energy resources and being responsible for not much less than half the production of greenhouse gases. All that, while emergent countries like China and India dream to adopt as behavioural models our most reprehensible

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habits in terms of respect for planet we live on. We are spoilt children and are the model which billions of people aim at. Nature does not care for money, political colours, philosophical ideas, sociology or any other superstructure we created. Nature does not even care too much for Man. Man is just another animal and if the new equilibrium is not compatible with Man’s life, let Man be extinct. Dinosaurs reigned on this planet for a very long time, far longer than we Men have done. Yet, something happened that had the scale tip just a little and those enormous animals simply disappeared. We are not sure, but perhaps a meteorite hit the Earth creating a huge, long-lasting cloud of very fine dust, and that dust became an impassable screen to some solar radiations, causing the extinction of organisms that were at the base of the food chain. And the same dust has certainly been inhaled by animals and ingested along with vegetables so that only the more resistant to that kind of unusual aggression survived. That pollution may cause disasters is something even some historians maintain. One of the factors they add to those which led to the fall of the Roman empire is heavy-metal pollution in their aqueduct pipelines and in their wine. And if we look at the tragedies of Hiroshima and Nagasaki in the light of what we know now, we must accept that the two very high-temperature explosions which destroyed the cities and had all sorts of materials sublime must have created millions of tons of nanodust whose existence is impossible to deny and is proved by the black rain that fell about half an hour after the bombing. And if nanodust was present, it must have behaved the way all nanodust does. So, it is reasonable to think that part of the pathologies that affected the inhabitants of those cities and the malformations so common in their offspring is due to particulate matter entered in their tissues. One of our present themes of research is aimed at finding out if that hypothesis is correct and will be one of the subjects of our next book. 9.5 Bibliography Brayner, R., Ferrari-Iliou, R., Brivois, N., Djediat, S., Benedetti, M. F. and Fievet, F. (2006), Toxicological impact studies based on Escherichia coli bacteria in ultrafine ZnO nanoparticles colloidal medium NANO LETTERS 6 (4): 866-870 Apr.

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Escote, X., Zapater, M., Clotet, J. and Posas, F. (2004). Hog1 mediates cell-cycle arrest in G1 phase by the dual targeting of Sic1. Nat Cell Biol. 2004 Oct;6(10):997-1002. Epub 2004 Sep 19. Gatti, A. M. (2005). Risk assessment of micro and nanoaprticles and the human health, Chapter of Handbook of Nanostructured biomaterials and their applications ed American Scientific Publisher USA, cap. 12, 347-369. Gatti, A. M., Montanari, S., Gambarelli, A., Capitani, F. and Salvatori, R. (2005). In-vivo short- and long-term evaluation of the interaction material-blood” Journal of Materials Science Materials in Medicine, 16, 1213-19 Hansen, T., Clermont, G., Alves, A., Eloy, R., Brochhausen, C., Boutrand, J. P. Gatti, A. M. and Kirkpatrick, J. (2006). Biological tolerance of different materials in bulk and nanoparticulate form in a rat model: Sarcoma development by nanoparticles, Interface.2006 J.R.Soc.Interface., 3, p.767-775. Lucarelli, M., Gatti, A. M. Savarino, G., Quattroni, P., Martinelli, L., Monari, E. and Boraschi D. (2004). Innate defence function of macrophages can be biased by nano-sized ceramic and metallic particles, Cytokin Network, Vol 15 No. 4 Decembre, pag 339-346 Maeda H. (2001). The enhanced permeability and retention (EPR)effect in tumour vasculature: the key role of tumour-selective macromolecular drug targeting. Adv Enzyme Regul 41:189–207. Matsumura, Y., Oda, T. and Maeda, H. (1987). General mechanism of intratumour accumulation of macromolecules: advantage of macromolecular therapeutics. Gan To Kagaku Ryoho 14:821–829. Maynard, A. D., Aitken, R. J., Butz, T., Colvin, V., Donaldson, K., Oberdorster, G., Philbert, M. A., Ryan, J., Seaton, A., Stone, V., Tinkle, S. S., Tran, L., Walker, N. J. and Warheit, D. B., (2006), Safe handling of nanotechnology, Nature. Nov 16; 444(7117):267-9. Nel, A., Xia, T., Madler, L. and Li, N. (2006), Toxic potential of materials at the nanolevel, Science, Feb 3;311(5761):622-7. Peters, K., Unger, R., Gatti, A. M., Sabbioni, E., Gambarelli, A. and Kirkpatrick, J. (2006). Research Needs on Nanoparticles, (2005), Proceedings of the workshop held in Brussels, on 25-26 January Ed. R. Tomellini, C. de Villepin Tang, Z., Mori, T., Takarada, T. and Maeda, M. (2001), Single nucleotide polymorphisms (SNPs) assay using reversible association and dispersion of DNAlinked colloidal nanoparticles, Nucleic Acids Res Suppl (1) 165-6 Impact of ceramic and metallic nanoscaled particles on endothelial cell functtions in vitro. - Nanotechnologies for the life Sciences Vol.5 Nanomaterials- Toxicity, Health and Environmental Issues Ed. By Challa S.S. R.Kumar Wiley –VCH Verlag GmbH &Co. KGaA 108-129. ISBN: 3-527-31385-0, vol 5, 108-125. Yang, L. and Watts, D. J. (2005).Particle surface characteristics may play an important role in phytotoxicity of alumina nanoparticles, Toxicology Letters 158 122–132. Web Ref. 1: www.nanodialogue.org Web Ref. 2: www.innovationsgesellschaft.ch/images/publikationen/Schlussbericht5.pdf

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Web Ref. 3: nano.foe.org.au/filestore2/download/125/FoEA%20nano%20cosmetics%20report%20 web.pdf Web Ref. 4: eur-lex.europa.eu/LexUriServ/site/en/oj/1998/l_123/l_12319980424en00010063.pdf Web Ref. 5: www.samsung.com/in/products/washingmachine/thesamsungwashingmachineadvantage/ index.htm Web Ref. 6: www.samsung.com/au/products/airconditioners/split/sh30za2.asp#silver_nano Web Ref. 7: www.samsung.com/au/products/refrigerators/topmount/sr518sd.asp#silver_nano

Appendix

Our analyses are mainly made with an Environmental Scanning Electron Microscope (ESEM-Quanta, FEI-Company, The Netherlands) The reason why that instrument is called “Environmental” is because, besides being able to work in high and medium vacuum, it can analyze samples at room, i.e. environmental, conditions. Thanks to that feature, it can easily accept biological specimens. (Report, 1996) In addition to that, there are 3 major characteristics that make ESEM excellent for our use: Pure secondary-electron detection; Compatibility with water; High chamber pressure. 1- Secondary electrons come from the atoms of the sample that derive from interactions with the primary electrons of the beam. Those electrons are responsible for the best sample resolution at low energies. ESEM can detect pure secondary electrons in a gaseous environment. In conventional scanning electron microscopes (SEM), the electronic gun must work at high-vacuum conditions because of the high voltage it needs. Because of that, the equipment accepts only dried and clean samples, since humidity and dirt are incompatible with high vacuum. In addition to that, the samples must be electron-conductive and so they must be coated inside a vacuum chamber through a sputtering process with Carbon or with a Gold/Palladium alloy. In order to remove the electrostatic charge created by the electron beam, their morphology must be flat and simple enough, so as to allow the coating to be homogeneous. 2- The imaging process is directly affected by the ionization characteristics of the gas inside the chamber. Different gases can be employed. There is no difficulty in having water vapour ionise, and that yields excellent imaging performance, so biological samples can be kept fully hydrated virtually indefinitely. 287

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3- The lowest pressure necessary to keep water in a liquid state is about 4.6 Torr. In a conventional low-vacuum scanning electron microscope, the highest chamber pressure does not exceed 2 Torr. High chamber pressure permits to study samples with high outgassing rate as well as to perform dynamic experiments like re-crystallization, or to observe living cells in the medium. Our ESEM is equipped with a Tungsten filament, an ionisation secondary-electron sensor, a back-scattered sensor, SW controlled Peltier cooled specimen stage and an Energy Dispersive System (EDS by EDAX, USA). The electron beam issued by the ESEM hits the sample surface and produces X-ray fluorescence from the atoms in its path. The energy of each X-ray photon is typical of the element which generated it. The EDS microanalysis system collects the X-rays, sorts and plots them by energy, and allows to identify the elements that produced the peaks in this energy distribution. The system can identify the elemental composition of the materials imaged for all elements with an atomic number greater than Boron. Most elements can be detected at concentrations of order 0.1%. In general, when analyzed by the EDS, biological samples from healthy tissues show just the peaks of Carbon and Oxygen, but some specialized tissues reveal also a content of other elements like Calcium and Phosphorus in the bone, Iron in the liver and Phosphorus in the brain. When their ion size is below the sensitivity threshold of the system, some elements undoubtedly present in a tissue are not detectable with this method. Iron bound to haemoglobin in the red cells, for example, cannot be seen, while when it precipitates in the liver as it does in patients affected by siderosis, detecting it becomes quite easy. If the tissue to be observed has been fixed chemically, dehydrated and then embedded in paraffin, it can show small peaks of elements like Sulphur or Chlorine not belonging to the specimen, and that depends on the chemicals present in the liquids used for the treatments. In this case, the spectrum of the biological tissue is necessary as a reference and must be subtracted from that of the foreign body contained in it.

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Sample Preparation The biological sample must be properly prepared in order to detect inorganic particulate it contains. Biological samples can be divided into two groups: fresh tissues (not chemically fixed), bulk or sections; and chemically fixed and paraffined tissues. a – Fresh tissues are: 1- the bulk samples from surgery, including bioptic and autoptic specimens, 2 – the blood , 3 – the sperm, and 4 – the living cells from in-vitro simulation. As briefly described above, they can be observed in wet conditions, keeping the sample at a temperature of 5°C degree and at a relative air humidity of 90-95%. Since these samples did not undergo any additional chemical treatment, no pollution is possible. According to the kind of sample, different preparation methods have been developed. The samples obtained from surgery are usually bulk and, to compare them with the histo-pathological sections on which diagnoses are performed, they are frozen at –20°C and then cut into 20-micron slices. Then, they are deposited on an acetate sheet and observed under ESEM. Acetate was selected since, under Energy Dispersive Spectroscopy, it emits only minor signals for Carbon and Oxygen, ever-present elements in any biological specimen. When the section is very thin, the contribute from the acetate substrate is just added to that from the biological specimen and does not affect the measure carried out on the inorganic debris. Since every fresh sample represents a potential a risk of infections (HIV, hepatitis) for the operators, we decided to subject them to a halfhour’s immersion in formalin before handling it. Bulk samples may be subjected to a critical-point dehydrating process through the exchange of water with Nitrogen. The blood and the sperm are smeared on the acetate sheet and then covered with another sheet of the same material. A few seconds later, they are delicately turned and separated in order to obtain a cell

290

Nanopathology

monolayer. The sheet is glued with a Carbon disc on a stub and observed immediately. The living cells, not treated in any way, are put on a support and observed under ESEM directly in the medium. As, because of the medium surface, observing the cell morphology is impossible, humidity is slightly and smoothly decreased, thus allowing a mild dehydration without prejudice to the morphology. Sometimes, fixation is performed (a few drops of 2,5% glutaraldehyde) so the morphology is preserved and, what is most important, the interaction between cells and nanoparticles is preserved as well. b – The following method allows the observation of old samples preserved in archives. Those samples had already been fixed, dehydrated and embedded in paraffin and, from those blocks, we cut 20-micronthick sections. The slices are then suspended in warm water and deposited on the acetate sheet. To remove the paraffin, the samples are covered with a few drops of xylol and 98%-alcohol and, a few seconds after, the excess of liquid is slid along the sheet edge and absorbed in blotting paper. The samples can be observed in different modalities: in high and low vacuum; in secondary and in backscattered electron mode. Bibliography Report from Environmental Scanning Electron Microscopy: An Introduction to ESEM by Philips Electron Optics, Eindhoven, The Netherlands 1996, Robert Johnson Associates California.

Index

1st Gulf War, 161 2nd Gulf War, 161 9/11, 251

antibodies, 21 Antimony, 125 anti-wear, 245 arteries, 45 arthrosis, 5 Asacol, 106 asbestosis, 22 ash, 230 atelectasis, 49

A-bomb, 165 acarus, 218 acrolein, 231 Acute Lymphatic Leukaemia, 170 acute renal failure, 97 adenocarcinoma, 65 adenoma, 99 adrenal adenoma, 186 adrenal-gland, 99 Adriatic Sea, 220 Agent Orange, 167 air conditioners, 259 Air Force Laboratory, Armament Development and Test Center, of Eglin Air Base, Florida, 161 Allied Forces, 165 alloy, 9, 95 Aluminium, 2 alveoli, 43 alveolitis, 55 Alzheimer’s disease, 13 amalgam, 13 ameloblastoma, 102 amiotrophic lateral sclerosis, 13 ammonia, 229 amyloidosis, 271 angiogenic, 26 animal feed, 248 Antarctica, 248

Baghdad, 189, 232 Balkans War, x Barium, 3 beauty and sun-screen creams, 24 bicarbonate, 229 biocompatibility, 16 biocompatible, 16 biomass, 230 biscuits, 244 Bismuth, 222 Black Sea, 244 Blebs, 62 blood, 1 blood-brain barrier, 18 blood-placenta barrier, 23 blurred vision, 253 bombing, 54 bone, 47 bone marrow, 177 Bonfield, William, vii Boraschi, D, 24 Boron, 288 Bosnia, 187

291

292

bowels, 4 brain, 18 brakes, 266 bread, 244 bronchial mucus, 136 bronchoalveolar lavage, 156 Brownian motion, 270 bucky-balls, 272 Burning Mouth Disease, 102 Burning Semen Disease, 121 Cadmium, 21 calcification, 100 Calcium, 2 cancerogenic, 28 cancerogenicity, 40 Canova Activa, 240 Capitani, Federico, vii car bodies, 227 Carbon, 1 Carbon monoxide, 42 cardiogenic mortality, 79 cardiovascular, 23 cardiovascular diseases, 77 caval filtration, 5 cell, 16 cellular duplication, 271 Cerium, 55 Cesium, 248 Chernobyl, 248 chewing gum, 24 children malformation, 116 chimney-sweepers’ cancer, 205 China, 283 Chlorine, 1, 76 chocolate, 245 cholangiocarcinoma, 93 cholestasis, 4

Nanopathology

Chromium, 1 chronic fatigue, 40, 253 cigarette, 22, 231 cirrhosis, 87 clams, 275 clothes, 208 coal, 266 Cobalt, 2, 24 colloids, 265 colon, 5 combustion, 7 composites, 13 concrete, 259 coronary vessel, 78 cough, 155, 253 Council of Down Manhattan, 252 COx, 46 Creutzfeldt-Jacob, 271 criminal court, 220 Crohn’s disease, 5, 40 cryptogenic diseases, 39 customized medicine, 273 cystic fibrosis, 5 cytokine, 15 Da Costa Syndrome, 166 Daewoo, 240 Danube, 244 De Morbis Artificium, 205 defecation, 245 dendrimers, 17 dental filling resins, 13 dental prosthesis, 5 dental restorations, 13 Departments of the Veterans Affairs, 166 depleted Uranium, x, 54 depression, 167, 253

293

Index

derma, 145 dermatitis, 145 Desert Storm Conflict, 167 detoxification, 252 diabetes, 91 digestive system, 5 dioxin, 8, 42, 167 DNA, 12 Drexler, Kim Eric, 6 drug, 85 EDAX, 288 edema, 239 Effort Syndrome, 168 enamel, 156 endometrium, 137 endothelial cells, 23 endothelium, 45 Energy Dispersive Spectroscope (EDS), 1 energy, 266 engineered particles, 274 environment, 22 Environmental Scanning Electron Microscopy, 86 enzymes, 18 EPA, 241 epidemiologic studies, 166 epidemiological studies, 249 epidemiology, 269 Escalaplano, 195 Escherichia Coli, 240 ESEM, 28 Europäische Akademie, 11 Europe, 266 European Commission, vii European Community, 5, 43 excipient, 102

explosions, 161 exposure, 39, 253 eye, 174 faeces, 14 fat, 263 fatigue, 83 FDA, 241 Federal Institute for Risk Assessment (BfR), 239 FEI-Company, 287 fertilizers, 230 feti, 41 fever, 85 fibrosis, 3 fibrotic capsule, 29 filters used in Diesel cars (FAP), 203 firemen, 252 firing ground, 121 First Industrial Revolution, 266 fish, 200 Fluorine, 104 food, 88, 248 Ford Farm Firing Range, Aberdeen Proving Ground, MD, 162 foreign-body, 3, 20 forgetfulness, 168 formaldehyde, 231 fossil fuels, 266 Foundation for the Advancement of Science and Education, 252 foundries, 266 Framer, Joe A, 199 Free Royal Hospital of London, 4 fullerene, 18 furan, 8, 42, 228

294

Gadolinium, 20 Gambarelli, Andrea, vii ganglioneuroma, 184 gastrointestinal system, 4 Gatti, 171 Germany, 239 giant cells, 58 Gitelman’s syndrome, 54 glass, 8 glioblastoma, 184 globalization, 268 GMOs (Genetically Modified Organisms), 268 Gold, 5 gonads, 173 granulation tissue, 278 granuloma, 31 granulomatosic reaction, 28 granulomatosis, 3 granulomatous tissue, 29 Ground Zero, 257 Gulf War, 54 Gulf War Syndrome, 43, 95 Hadzici, 169 haemolysis, 4 haemolytic anaemia, 2 hamburger, 241 Harrod’s, 251 Harvard School of Medicine in Boston, 154 headache, 167 heart, 19 heavy oil, 220 hepatic echinococcosis, 83 hepatitis, 289 hepato-splenomegaly, 2 Herald Tribune, 251

Nanopathology

HIV, 289 Hodgkin’s lymphoma, 115 hydrocarbons, 42 Hydrogen, 2 hydroxyapatite, 14, 15 immune system, 50 immunological disease, 20 incinerators, 8 India, 283 industrial sterility, 121 infarction, 45 inflammation, 4 inflammatory pathologies, 39 Information Office (OI), 165 inhalable, 19 inhalation, 22 insomnia, 173 internalization, 23 interstitial pneumonia, 55 intestinal mucosa, 4, 151 Iodine, 99 Iraq, 54 Iron, 1 irritability, 167 Istituto Tumori (Cancer Institute) of Milan, 5 Italian Commission, 170 Italian senatorial commission, 249 Kleinmann GmbH, 239 knee, 5 Korean conflict, 166 Kosovo, 188 landfills, 259 Lanthanum, 55 Lateral Amyotrophic Sclerosis, 142

295

Index

Lavoisier, 214 law of conservation of matter, 228 Lead, 21, 91 LeGeros, Rachel, 101 leiomyomatosis, 64 lettuce, 225 lichens, 223 lime, 229 liposomes, 239 London, 251 Los Angeles, 252 Lou Gehrig’s disease, 166 lung, 5 lung barrier, 209 lymph-nodal metastases, 187 lymph-nodes, 19 lymphocytes, 30 lymphomas, 40 macrophage, 15 mad cow disease, 248 Magic Nano, 239 Magnesium, 1, 2 malformed lambs, 121 Malta, 121 Manganese, 150 Mantua, 212 Mars, 245 masks, 155 mast cells, 30 Maynard, 272 medical devices, 16 Medical Geology, 43 medullar aplasia, 166, 169 Mercury, 13 mesenchymal tumors, 31 mesothelioma, 46 mesoworld, 6

metalloproteic precipitates, 144 metalloprotein, 145 metastasis, 19 mieloid leukaemia, 182 Minamata City, 243 miopathy, 135 Molybdenum, 1, 111 monocytes, 30 Monsanto, 241 mouth, 44 mouthwash, 145 muco-ciliary cells, 279 mule spinners, 206 multi-organ failure, 100 multi-organ-diseases, 43 multiple sclerosis, 13 muscles, 266 Nagasaki and Hiroshima, 169 Nano Poly Technology, 241 nanocomposites, 13 nanopollution, 135, 205 nanoproduct, 239 nanospheres, 19 Nanotea, 240 nanotechnologies, ix nanotube, 18 nasal congestion, 146 National Technical Information Service (NTIS), 165 NATO, 167 neck, 20 Nemery, Ben, 69 Neodymium, 55 nephrocalcinosis, 43 Neu-Laxova syndrome, 121 neurological, 19 New York, 171, 251

296

Nickel, 1 nightmares, 253 Niobium, 66, 150 Nitu, Lavinia, vii non-Hodgkin’s lymphoma, 43 NOX, 46 nucleating agents, 71 oil wells, 249 olfactory nerves, 276 osteoblast, 15 oxidative stress, 27, 270 Oxygen, 3 paints, 103 Palladium, 287 Pančevo, 244 pancreas, 91 pancytopenia, 183 Parkinson’s disease, 13 peace-keeping missions, 176 Percivall Pott, 205 peritoneum, 150 pesticides, 230 phagocytosis, 80 Phosphorus, 1 photoablation, 270 Phynox, 2 physiological barriers, 121 platelets, 114 Platinum, 102, 212 PM10, 22 PM2.5, 43 pneumoconioses, 22 pneumothorax, 5 Po, 220 Polesine Camerini, 220 pollutants, 8

Nanopathology

polychlorinated biphenyls, 228 polycyclic aromatic hydrocarbons, 8, 206 polycyclic organic matter, 228 post-Vietnam Syndrome, 166 Potassium, 2 power station, 220 Praseodymium, 234 precautionary principle, 274 pre-neoplasia, 32 prevention, 87 primary electrons, 287 pro-inflammatory, 27 prostate, 19 protein, 16, 265 pseudotumour, 49 psyconeurosis, 166 pulmonary, 19 pulmonary intestitium, 204 pulmonary thromboembolism, 70 Pulsator, 241 PVC, 24 pyrophoric, 162 quantum dots, 21 quartz, 22 Racemose ossification, 48 Ramazzini, 205 rats, 28 red cells, 65 redox potentials, 270 renal failure, 2 respiratory, 23 respiratory system, 5 retrosternal pain, 155 Revell, Peter, 4 rhabdomyosarcoma, 32

297

Index

rheumatoid arthritis, 81 RIS (Crime Scene Investigative Institute) of Parma (Italy), 197 risk factors, 22 Ru(bpy), 240 Salto di Quirra, 195 Salvatori, Roberta, vii Samarium, 234 Samsung, 240 Sapporo, 214 Sarajevo, 169 sarcoidosis, 5 Sardinia, 121 sclerodermia, 135 sclerosis, 139 Second Industrial Revolution, 266 second World War, 166 secondary electrons, 287 secondary particles, 229 Selenium, 240 September 11, 253 Shake, Slim, 240 shale oil, 206 ship, 227 shortness of breath, 155 siderosis, 87 Sighinolfi, Gianluca, vii Silicon, 1 silicosis, 22 Silver, 14 Silver Nano, 240 skin, 205 skin carcinomas, 206 skin pesudolymphoma, 43 sleep disturbance, 167 Smalley, Richard, 272 Sodium, 2

soldier, 99 SOx, 46 sperm, 42 spleen, 84 squamocellular carcinoma, 52 stainless steel, 72 steatosis, 89 steelworks, 215 stomach, 44 Stress-Activated Proteins (SAP), 271 stroke, 19 Strontium, 95 Sulphur, 2 sunscreen creams, 103 surface/size-ratio, 22 sweat, 254 Symptoms and Signs of Ill-Defined Conditions, 167, 168 talc, 113 Taniguchi, Norio, 11 Tantalum, 66 tea, 240 teeth, 13 Teflon, 104 teratogenic, 248 testicle, 121, 205 Thorium, 109, 150 threshold, 10 thrombectomy, 70, 78 thrombi, 5 thrombus, 23 thyroid, 187 tiredness, 2 Titanium, 2 tobacco, 55, 231 Tokyo Science University, 11

298

toner, 210 tongue, 142 toothbrushes, 247 toothpaste, 14, 247 Tossini, Daniela, vii toxicity, 212 traceability, 203 Tub U, 241 tuberculous pleuritis, 46 Tungsten, 66 Twin Towers, 251 typhoid fever, 151 tyres, 266 U.N. Environment Agency, 169 U.N. Environment Protection Agency, 167 Udine, 215 ultrafine, 23 umbilical circulation, 41 University of New York, 101 University of Siena (Italy), 5 Uranium, 54, 150 urine, 2 US, 272 USSR, 162 uterus, 118, 137 vaccines, 165 vagina, 137 vaginal mucosa, 118

Nanopathology

Vanadium, 221 vein thrombosis, 40 vena cava filter, 1 veterans, 116 Vietnam, 167 Virchow’s Triad, 77 volcanoes, 265 wall paints, 24 Washing Machines, 241 wastes, 204 water, 266 wear, 14 Wegener’s granulomatosis, 43 welding, 207 white cells, 114 wind, 266 Woodrow Wilson International Center, 272 World Trade Center, 266 X-ray, 288 Yttrium, 109, 150 Yugoslavia, x, 116 Zimmer, Rene, 239 Zinc, 21 Zirconia, 13 Zirconium, 13