The Asbestos/Carbon Nanotube Analogy: Will There be an Epidemic of Nanodiseases in 2030?

okay well thank you to everybody for coming to today’s earth sciences division distinguished scientist seminar today given by Agnes Brown Agnes Kane excuse me from Brown University so Agnes is an MD ph.d she’s both professor and chair of the department of pathology and laboratory medicine at Brown University and she’s also director of the training program in environmental pathology now in its 20th year of funding Agnes has received numerous awards research mentoring and teaching including i noticed that Dean’s Teaching Award at brown Medical School seven years in a row and I think you’ll see she’s a very clear communicator and talk today she’s done a lot of research on five the toxicology and more recently nano toxicology and participated in wh 0 working groups on the evaluation of carcinogenic risks to humans and Agnes has brought her experience of fiber toxicology and asbestos tops College in particular to the field of nanomaterial toxicology and is I think exactly the kind of person needed to address these really interesting and potentially hazardous new materials she’s co-author on numerous creative projects looking at the mechanisms of nanomaterials toxicology and we look forward to hearing about her research today Thank You Ben and I’m very happy to be here in California in my short visit so far I’ve learned there’s nothing nano about California in comparison to Rhode Island which as you know is a small estate so I’m going to be talking about the asbestos carbon nanotube analogy and asbestos most of us sort of consider is a historical problem although it is still used as a classical case study in occupational health so we’d really like to forget about that public health disaster that occurred as the widespread use of commercial use of asbestos fibers throughout the 20th century and put it behind us and we were about to do that until nanotechnology came on the scene and we see that nanotechnology is it is a new technology and the definition is rather new although nanomaterials are not new as we’ll see and one definition is that it’s working at the atomic molecular and super molecular levels and the scale is important at least for governmental definitions 1 to 100 nanometers in order to understand the create materials with fundamentally new properties because nano scale materials have frequently had very unique and unusual chemical and physical properties of the nano scale than their bulk counterparts and this is very fascinating and really this has opened up with some people call the third Industrial Revolution so today I’m going to talk about a comparison between asbestos fibers and carbon nanotubes from a physical and chemical viewpoint and then I will sort of compare the properties of these long thin fibrous materials that is related to toxicity and finally move from acute toxic effects that are commonly assessed in the laboratory to a question of more chronic diseases that take many years to develop particularly cancer which has been of course associated with exposure to asbestos fibers before I begin I dislike to acknowledge that my research funding is from the National stitute of Environmental Health Sciences in the past US EPA star grants and the National Science Foundation our trainees are supported by the National stitute of Environmental Health Sciences as well as a department education training grant I have served pro bono on numerous scientific committees and workshops for the World Health Organization the US EPA National stitute of Environmental Health Sciences and the Institute of Medicine I have not received any industrial funding I’m going to focus today on carbon nano and here you some examples of these they actually quite beautiful materials when you diagram these structures I need to focus on carbon nanotubes and it’s a rolled-up graphene sheet of hexagonal carbon atoms and the ends are usually capped with a fullerene which is a spherical molecule or c60 a nano soccer ball composed of hexagons and Pentagon’s if carbon nanotubes can be they’re single walled this is a high-resolution tem one ball very small in diameter for two maybe up to 100 nanometers in diameter but can be very long and of course thin carbon nanotubes also can be like Russian dolls concentric layers of these two to 10 or 20 layers and then a hollow core first i’ll talk about asbestos fibers the morphology the definitions and their commercial uses and then I’ll talk more specifically about carbon nanotubes when we think about asbestos fibres here’s an example of a rock and indie asbestos is the word used for minerals that have been exploited mind and exploited commercially since about the beginning of the 20th century although they were used in in previous times as well the characteristic feature of these fibrous minerals is the fact that they naturally cleave into long thin fibers longitudinally and they have various very very useful properties which are kind of unique in the mineral world again they’re fibrous in shape long and thin they have very high tensile strength but low thermal and electrical conductivity so they used widely for insulating devices chemicals was electricals was thoroughly insulation and very high stability millions of metric tons tons were used worldwide between about 1920 and 1970 in the 70s after it became widely people were widely aware of the consequences of the commercial use of asbestos fibers and realized the existence of asbestos related diseases that I’ll describe shortly asbestos was banned mostly in Western European countries and in Australia and it’s banned now in 52 countries worldwide interestingly enough it’s not banned in the United States and the you commercial use of asbestos fibers is increasing in rapidly developing countries such as Asia India South America and even parts of Africa asbestos fibers were used in a whole wide range of products of widely used in building materials and as such in our older buildings not your newer buildings here California but certainly on the East Coast most of our buildings are schools or universities even some of our homes have asbestos insulation in them or asbestos insulation around pipes for example I also use widely used industrial industrially friction products brake brake and clutch linings textiles gaskets cement pipes for water supply coatings of various sorts to coat the walls and the ceiling roofing tiles all sorts of applications now asbestos is actually a word that has been applied to regulate regulated minerals that is those that are regulated now by government regulatory agencies after it was realized they were associated with asbestos related diseases and this is the short list of those and their chemical composition there are two families the first is serpentine minerals and there’s only one example of asbestos fibers in this family chrysotile chrysotile is white and actually it’s the most widely used asbestos fiber commercially the rest are ansible minerals and these can occur not just only as fibrous minerals but also more prismatic or bulk minerals or even quite irregular cleavage fragrance crecido light is the name applied to the fibers form of Rebecca and amosite is the name of the fibrous form of coming tonight arite other minerals also can form long thin fibers that is they can be asbestos form similar to asbestos in shape and most notably is area night which is a member of the family of zeolites these are naturally occurring minerals and they’re found in turkey also Oregon North Dakota the United States actually quite right spread nature in nature but only when they are used as they have been in Turkey in just three villages for the construction of homes and in whitewash and caves where it was their significant human exposure area night fibers are very long and thin although their structures very different from the asbestos fibers but they are very potent and causing mesothelioma the other asbestos or mineral that’s of importance in the United States is a different type of amplifier called Richter I and winch ight these are found in Libby Montana a small town up here near the border with Canada and in Libby there’s a vermiculite mine that was operating throughout the 20th century up until about nineteen ninety and it produced about eighty percent of the world’s for Mick you like those of you know about the meteorites a clay-like mineral very light fluffy when it’s expanded heated used in kitty litter actually was used in home installations vary widely in the United States and other countries and also for spotting soils and things unfortunately that vermiculite mine was contaminated with anthropol asbestos fibers and the town of Libby is about 10,000 people it’s a very rural town but very beautiful nestled in between in a mountain valley and the whole town has been contaminated with asbestos fibers as a result of this vermiculite lining and there are documented adverse health effects of that lining both in the workers and also in some of the residents so they are at risk of developing asbestos related diseases in the last couple of years that whole town was placed on the national priority list it is a Superfund site so the consequences of exposure to naturally occurring fibrous minerals can be just as serious as the exposures occupational exposure to commercial use materials but only in certain circumstances remember in toxicology we sort of have a simple equation that risk that is the risk of developing disease is a function of hazzard the intrinsic toxicity of a material times exposure and if there’s no exposure if the mineral such as in the United States where they’re large areas shown in green where it is possible from geological considerations that asbestos I’ll exist if we keep it in the ground in those rock formations and don’t disturb it there’s not going to be any exposure no therefore no risk of human disease these yellow areas are where there have been identified positively asbestos fibers or what some people think are asbestos fibers one example is Fairfax County in Virginia where a housing development was being built on a rock formation that had tremolite asbestos in it and then of course here’s California big stay lots of yellow and in fact the state rock here is serpentine for chrysotile and there are deposits of asbestos in California but as yet there’s really no well established association between living or having recreational activities are going to school in these areas with asbestos related diseases and there’s some debate about what the minerals are here and whether they are indeed fibrous so with asbestos related diseases the association between exposure to asbestos fibers usually occupational exposure but also what we call para occupational exposure workers and minds or factories coming home and exposing the people in their household to asbestos fibers and developing certain types of diseases now one characteristic of these diseases is their chronic that is they don’t develop the day after you’re exposed to asbestos fibers they develop decades or many decades after the initial exposure the latent period between developing these asbestos related diseases exposure and asbestos related diseases is as short is 10 years for asbestosis or fibrous carring of the lungs but for these various types of cancers it can be 20 to 40 years after the initial exposure so we were using millions of metric tons of asbestos fibers throughout the early 20th century before clinically we recognize that these workers and their families were getting diseases related to that exposure and this is because of the long latent period between the exposure and the development of the disease that makes tokico logical studies a little bit complicated because of that long waiting period well what are these diseases the primary route of exposure that we are concerned about with asbestos fibers as well as carbon nanotubes that we’re going to talk about shortly it’s inhalation inhalation through the nose and the mouth going by passing here through the larynx down into the lungs the trachea the conducting Airways of the lungs the bronchi and even reaching the air spaces or alveoli these logs and fibers are able to penetrate deep into the lungs and also the alveolar spaces it is known that exposure to asbestos fibers is associated with can’t hilarious as well as cancer rising from these bronchial Airways and alveolar spaces lung cancer and asbestos fibers because they have a very high surface area can adsorb chemicals very easily for example the carcinogens the polycyclic aromatic hydrocarbons and cigarette smoke and exposure to cigarette smoke smokers who are occupationally exposed to asbestos fibers have a synergistic risk of increasing the incidence of lung cancer it’s like 80 fold greater than exposure to eat your aids agent alone asbestos fibers also frequently can migrate from the alveolar space is the air spaces outside of the lungs to the lining the space around the lungs called the pleura and even cross the diaphragm here into the abdominal or peritoneal cavity the pleura and the peritoneal cavity are lined by a thin layer of very delicate cells called mesothelial cells not very many things that we inhale get out here because we have very efficient defense mechanism for clearing particles that we inhale into our lungs however if asbestos fibres or other long fibrous materials get out to these spaces over a long waiting period to get 20 24 years in humans they can develop a tumor on the lignin tumor arising from the lining of the pleural cavity or peritoneal cavity called a malignant mesothelioma this is the tumor I’m particularly concerned about this is not synergistic with cigarette smoking not very many materials cause or exposures cause malignant mesothelioma but the risk of developing it into occupational exposure to asbestos fibers is very very high even worse this tumor usually grows very insidiously and a thin layer over the pleural and peritoneal surfaces and eventually will start compressing the lung so it’s not diagnosed until it’s quite big and extensive and it’s almost always deadly we don’t have a cure for it in most patients die within 6 to 18 months after diagnosis now when we looked at the burden of malignant mesothelioma over the years this is the cumulative 15-year mesothelioma mortality that’s what’s counted in these kinds of studies and it’s plotted as a function of the cumulative consumption they call it of asbestos by country and this is I think between 1920 and 1970 and then we’re looking at the development of mesothelioma beginning in 1994 these little circles are different countries and they’ve been adjusted for population size and up here the highest cumulative mortality are for those countries that consumed or used the most asbestos fibers in the 20th century right up here is good old US of A it’s Germany France Australia one of the highest rates of malignant mesothelioma in the world Great Britain young UK Japan Canada a major asbestos producer so all these countries that had the highest consumption of asbestos fibers have the highest mortality of due to malignant mesothelioma and these authors calculate a 15-year cumulative mortality from malignant mesothelioma 175,000 deaths that could have been avoided by preventing exposure to these fibers well I told you that since the 70s asbestos has been banned in most countries again except the United States however its use considered is continuing and accelerating in developing countries in Asia South America and the former Soviet Union Russia particularly and Russia’s one of the major suppliers of asbestos world well and I worked on a committee for the World Health Organization that predicts almost twice as many deaths from malignant mesothelioma due to this continuing use of asbestos through 2040 for so let’s turn now to carbon nanotubes the history of carbon nanomaterials is very very interesting carbon nanotubes are carbon nanomaterials aren’t really a new invention they occur in prehistoric times as the earth was being formed there’s a lot of volcanic activity of the earth and any combustion volcanoes or fires will generate suit or carbon nanoparticles and so is just an agglomerate or an Ag reduced aggregate of nanometer-sized carbon particles and obviously it was present in the atmosphere since the earth was formed the high pressures and temperatures in the Earth’s crust also produced another naturally occurring carbon based material diamonds which is carbon but in a very different chemical form than stood and in fact nano diamonds have been found in meteorites so nanomaterials even though we think we deliberately made them at the latter half of the 20th century actually they’ve been around for a long time well the history of carbon nanomaterials particularly carbon nanotubes and fullerene or c60 began in 1985 Richard Smalley at Rice University and his collaborators received the nobel prize in 1996 was synthesizing fullerene this nano soccer ball in 1991 iya Jima and in Japan received a county prize in nanoscience for synthesizing of single-walled carbon nanotubes shortly after the US as well as other countries Europe and Asia began heavily invested investing in nanotechnology not only in carbon nanomaterials but other types of of nanomaterials and in the United States this initiative is called the national nanotechnology initiative and since 2001 the United States has invested sixteen billion dollars into this technology this is a quite amazing investment we been through over this time in two thousand for a new nano material was discovered graphene a single atomic a single atomic layer thick of hexagonal carbon atoms and dime and novo received a nobel prize for this discovery 2010 so the bottom line here is if you want to win a Nobel Prize discover a new nano material and as of 2008 there are about a hundred commercial products on the market using carbon nanotubes and they’re in all sorts of things usually consumer products right now a lot of sports equipment any sport you like mass for yachts tennis racquets keys baseball bats all have been reinforced with carbon nanotubes there in cars to make them stronger and more lightweight until they get better gas mileage and tremendous potential for electronics batteries various types of chemical biological sensors and defence and even biomedical applications so that now we have about a hundred and twenty eleven hundred companies worldwide making carbon nanotubes commercially another thousand companies are involved in R&D activities and the market is down about here however there’s tremendous growth predicted over the next several years up to about twelve thousand metric tons worldwide in 2016 so given what we know about the similarities at least some of the similarities between carbon nanotubes and asbestos fibers is growing commercialization this is why scientists are concerned whether there’ll be an epidemic of nano diseases in 20 22 24 T okay so nanotechnology is very exciting discipline those of you who are working in it it’s a very interdisciplinary field it’s new you don’t have a whole pile literature you have to worry about because it’s so such such a new field but concern about the possible threats of nanotechnology we’re already being raised as early as two thousand by Robert service in an article in science Vicki Colvin one of the earliest nanotechnologies working at Rice University with Richard Smalley raised concern about the potential environmental impact of these materials in 2003 and this continues to be under investigation the ethical and the scientific issues of occupational exposure started to be recognized shortly thereafter and then finally in 2008 in the New York Times it was reported that researchers find that nanotubes may pose a health risk similar to asbestos fibers and this is where the asbestos carbon nanotube analogy began most carbon nanomaterials carbon nanotubes are manufactured in high temperature furnaces and they produce initially in after their production very very dusty materials you can see the black foot on the fingers of this worker he is wearing gloves and a dust mask but neither of these are really sufficient to protect against inhalation of these materials people have measured levels of carbon nanotubes and various manufacturing and research labs and found dust levels in the micro gram per meter cube range now let’s compare that to what what is the recommended permissible exposure limit or threshold limit value for carbon black which are carbon nanoparticles it’s 3.5 milligrams per meter cube actually carbon black particles unless you have very very high exposures are not very toxic however NIOSH the National Institutes of Occupational Safety and Health then one of the laboratories involved in the investigation health risks of carbon nanotubes has just proposed last year a PEO a permissible exposure limit to carbon nanotubes of just seven miles per meter cubed so much less than exposure to carbon block and that that proposal is now open for public comment so there has been some activity being more proactive limiting exposure again in the Occupational worksite in the workplace to prevent any adverse effects following inhalation in the lungs alright so why are we concerned why do we have this this asbestos carbon nanotube analogy why was this term coined anyway and the initial analogy really reflects mostly two properties that are similar between these materials they’re both long and thin and mostly their bio persistence and so I have some examples here of asbestos whoops it’s pretty sly we gotta go through this asbestos fibers versus carbon nanotubes and this is the appearance under usually the transmission electron microscope in this case scanning electron microscopy serpentine your chrysotile asbestos is very curly inflexible and in fact it is is composed of a silicate sheet that’s actually rolled up here like a jelly roll it’s really pretty the other types of asbestos are empty bowls and they are much more rigid they’re more broad like and they usually are not as flexible as chrysotile asbestos notice that there’s tremendous variability in the diameter and the length of these naturally occurring materials these are images of carbon nanotubes and you can see also that there’s quite different differences in the morphology and the sizes of carbon nanotubes these are very thin and curly somewhat like chrysotile asbestos these are somewhat curl and these are very rod-like and stiff and even look like they have sort of a dimensional chicken wire type network so while they’re both long and thin they have some even physical differences well we know from extensive toxicologic study since it was realized beginning in the 60s that workers exposed to asbestos fibers are developing asbestos related diseases what the attributes what the chemical and physical properties of asbestos fibers are that are relevant for their toxicity and they include surface reactivity durability or bio persistence in the body after their inhale and fibrous shapes and dimensions and we’re going to be talking about each of these three properties head-to-head comparison with asbestos and carbon nanotubes now there are some differences there’s a very important differences at the chemical level and also the surface properties between carbon nanotubes and asbestos fibers although their shape and durability or file persistence are quite similar so we have to understand that the structures as we said with carbon nanotubes depending on whether they’re single layer or multiple layers can be either rigid or very flexible chrysotile asbestos is quite flexible curled fibers and football is rigid fibers because at the chemical level they’re composed of rod-like structures and then they’re cations between these layers much like a sandwich whereas remember chrysotile asbestos has rolled up like a jelly roll carbon nanotubes are graphene sheet rolled up with a single or multiple concentric layers the chemical composition carbon nanotubes is carbon for the most part but there can be both grow Phoenix or amorphous carbon as well as because of the the growth of these carbon nanotubes at high temperatures in order to make these long thin structures usually you require a metal catalyst and the the product may not be pure and it could be to have 20 to 22 twenty percent transition metal catalyst iron nickel cobalt yttrium the cations invo in the sandwich layers of anchor bolts bestest can also be iron and the fibers cleave along the cab layers exposing iron and other cations at the surface a crocodile asbestos is a magnesium silicate however it’s not uncommon in mineralogy in many of you know this more than I do for iron Fe 2 plus to substitute for magnesium two plus in the crystal structure the surface properties also are quite different the chrysotile asbestos is a high positive charged in magnesium the silica Tampa balls have a high negative charge and carbon atoms for existence for the most part have a low negative charge one very important property of the nanotubes unless they specifically functionalized are very hydrophobic whereas these crystal and minerals are hydrophilic both of these materials have defects which I’ll talk about shortly and the presence of iron can determine whether they are able to undergo redox cycling iron catalyzed redox activity which is characteristic of asbestos fibers however depending on the purity carbon nanotubes can either generate or catalyze redox activity or scavenge them the durability of carbon nanotubes and anthem polis bestest is very very high they’re expected to be bio persistent after inhalation the lungs whereas chrysotile asbestos is susceptible to acid leaching the magnesium news that of progressively removed and then they fragment into shorter and thinner fibers where they can be cleared more easily so we consider the first property relevant for biological activity with asbestos fibers the surface reactivity associated with iron catalyzed generation of the hydroxyl radical from hydrogen peroxide is a very very important property and these radicals that have an unpaired electron their outer orbital are highly reactive with biological molecules proteins lipids and even DNA and are associated with much of the acute toxicity of these men a carbon nanotube can donate electrons in the presence of oxygen although this is usually not a particularly efficient reaction and this will generate high superoxide anion which also has an unpaired electron carbon nanotubes have some other properties relevant for their service reactivity I talked about the catalyst residues that are necessary to grow these tubes and the catalyst residues are very electron-dense materials they can be at the surface isn’t this commercial candle carbon nanotubes or they can be encapsulated in the lumen that all this catalyst residue is bioavailable to participate in redox cycling and that should be assessed specifically in assays to assess the viability io availability under physiological conditions by high-resolution tem these carbon nanotubes are not always as nice as we draw them in their idealized structures there can be defects in the layers there can be topological defects not all the rings can be six membered there can be five or seven membered rings it also can be vacancies or holes in here and these holes will then leave dangling unpaired bonds which are sites of reactivity there also can be other types of defects these are sort of manufactured right now you’re very crude conditions there can be folding and even holes in the in the in the carbon nanotubes and even on the surface it’s very common to have a layer of amorphous carbon and all of these different features contribute to different levels of surface reactivity and carbon nanotubes so there’s no real good structure-activity relationship that has emerged yet for these diverse types of materials second property is bio persistence and it’s well known that asbestos fibers are bio persistent particularly the amp of all types of fibers like chrysolite asbestos shown here and the relationship between bio persistence and carcinogenicity was realized in the 70s and there were lots of experiments using animal exposure lifetime animal exposures usually rodents to asbestos fibers and new materials that were being developed as asbestos fiber substitutes in an attempt to develop a safer asbestos fiber substitute and for example in these studies in a rat we look at the lifetime of the t one half of chrysolite asbestos in the lungs is virtually longer than the lifetime of the rat and even in humans when we inhale amplivox bestest fibers they persist in our lungs essentially for our lifetime and in the rodent asses these are carcinogenic in contrast will last tonight is a fibrous mineral but if they’re calcium silicate it’s highly soluble in physiologic conditions it only lasts about 20 days and it’s not carcinogenic so industry developed substitutes for asbestos fibers mostly fiber glasses that are more readily soluble in the lung fluid and these were found not to be carcinogenic and so we can cleverly design fibrous minerals to substitute for asbestos fibers and conserve many of your useful properties now what about carbon nanotubes well the breakdown of minerals in the lungs or anything that’s inhaled into the lungs we mostly evolve this kind of elaborate system to deal with inhaled microorganisms we don’t live in a sterile environment we’re breathing in fungal spores and bacteria all the time but we have cells our lungs called macrophages and they’re highly phagocytic cells that like to eat particles and they not only like to eat them they have evolved a very specific system in their phagocytic vacuole here’s a chain of bacteria for example that generates reactive oxygen species that are designed to kill that bacterial organism and this phagocytic vacuole is a very low pH it’s only 4.5 and it generates endogenous oxygen superoxide and I hydrogen peroxide iron is also present in here and forms hydroxyl radical very potent killing so my collaborator at Brown Robert hurt in the School of Engineering and I decided that we’re going to try to mimic this a lysosomal environment to try to attack or degrade a single-walled carbon nanotubes and we use sort of a flow through fluid flow through systemno cells and we delivered hydrogen peroxide and iron in the presence of a score bait to cause this read outside the reaction in the hopes of breaking down their carbon nanotubes at the same time valerie kay ganda the University of Pittsburgh I was conducting very very elegant experiments to show that carbon nanotubes actually can fit into the active site of a plant peroxidase horseradish peroxidase which in the presence of hydrogen peroxide will also slowly degrade these materials so our experiments we took single-walled carbon nanotubes either pristine or unfunctional eyes or functionalized them various ways and then looked at their degradation in fatal lysosomal simulant fluid up to three months and we found that only those materials that had surface carboxylation CO 02 on them which actually is breaking that hexagonal crystal lattice and of creating active sites that can be attacked by the hydrogen peroxide in this flow through system so that made us comfortable and happy actually that engineers can design or post-process carbon nanotubes so that they can be biodegradable after inhalation so the third property that we’re going to be concerned about is fibrous shape and dimensions long thin fibers long thin materials regardless of what they’re composed of induce a phenomenon in macrophages those cells that like to eat things that we inhale into our lungs called frustrated phagocytosis the macrophages are only about 10 to 20 microns in diameter this kind of smallest cells go most of our cells are 20 to 50 microns in diameter but they like to eat they rapidly eat fibers microorganisms dust particles whatever is around indiscriminately if they’re small if they’re shorter than the diameter of the macrophage us in about 10 microns they are closed and that phagocytic vacuole in the lysosome and they’re either degraded or they stay there however if they’re long they’re too long to be completely engulfed by the macrophage the macrophage still thinks is dealing with a bacteria it’s going to try to kill it generate a lot of reactive oxygen species and produce additional mediators that will stimulate inflammation that is recruitment of other macrophages and inflammatory cells to deal with this bio persistent of noxious stimulus and when we look at macrophage is in culture in cell culture disk we did this in my laboratory many years ago this is a crystallite or amber ball asbestos fibers and this is a macrophage undergoing frustrated a phagocytosis it cannot completely engulf this material and Joseph brain at the Harvard School of Public Health instilled these sample fibers into the lungs of gratitude confocal microscopy the asbestos fibers are pseudo coloured in red here and we see this frustrated by phagocytosis in the air space the alveoli of a rap and also penetration of these fibers into the alveolar walls and the interstitial that is the tissue surrounding the alveolar walls and in experiments that we did a couple of years ago in collaboration with Allison elder and Gunther Obrador stir at the University of Rochester who are really skilled in doing animal installation and installation experiments they instilled carbon nanotubes into the lungs of rats and we were looking seven days after installation to see whether the carbon nanotubes were still in the lungs and we know that the clearance mechanisms following phagocytosis of particles or fibers by macrophage is in the lungs usually they will penetrate through the alveolar walls and go to the lymph nodes or they will be on the what’s called the mucosal iary escalator that lines the branca the bronchi or the conducting Airways of the lungs and those silly i Priscilla are beating upwards and remove the macrophages and the particles from the lung that mucociliary clearance mechanism is usually very very efficient and inhaled particles or fibers are cleared usually within 24 hours however this is seven days after exposure this is a scanning electron micrograph this is a section of a lung and this is an alveolar wall this is the alveolar space these are the epithelial cells lining the alveoli of the air sacs this is an alveolar macrophage and there’s a carbon nanotube still there sticking out these long materials long fibrous materials impair the movement and the clearance of these materials from the lungs and here we have opening longitudinally of the bronchus the conducting airways and Afghanistan electron micrograph the cilia you can see here and then these little alveolar macrophages each of about 10 microns in diameter and a long carbon nanotube here seven days after halation and robert mercer the national institute of occupational safety and health in west virginia looked at the pleural lining around the lungs of rats instilled with the same type of carbon nanotubes as we used and C’s penetration from the alveolar space is under here through the pleura into the pleural space and here are these thin layers of metal filial cells so indeed if there’s frustrated phagocytosis impaired clearance of carbon nanotubes they can reach the pleural space where they may induce some of the same diseases produced by asbestos fibers in this compartment so this was a sign for serious concern now how do these fibrous nanostructures enter cells and I’ll go over this fairly quickly because we have already published this work in a paper in nature nanotechnology and basically I was collaborating again with Robert hurt and wash and Gao in engineering at Brown why Jim does molecular dynamics modeling of how various types of structures react with phagocytic cells and it uses models of membranes and looks at the biophysics and energy dependent processes of this of this internalization process and we were using a variety of target cells in a very simple model in vitro let me show that there’s an energy-dependent uptake of carbon nanotubes it can be there occur by phagocytosis as we see in with macrophages or endocytosis they both require ATP and energy and they end up in same compartment their license of low pH we looked at the initial stages of entry of these long fibrous materials carbon nanotubes here or even gold nanowires bestest fibers into these cells and they’re usually going in here at a 90 degree or so angle we asked why was this the case and he did the modeling and he showed that the elastic strain generated in the membrane following the clustering of receptors that recognizes this tip of the carbon nanotubes or a bundle of carbon nanotubes provides a driving force to flip the carbon nanotubes or the asbestos fiber the gold nanowire to approximately 90 degrees to allow internalization whereas it’s the carbon nanotubes on capped you take off that ball there isn’t a sufficient density of receptors at the membrane to recognize it and if it’s flat on the surface receptor density is too low and it won’t be reading internalized so that sort of it provided the explanation at least with a biophysical level for frustrated endocytosis or uptake of these long fibers nanomaterials so in this work we wanted to know then well what happens then in cells that after they’ve tried to engulf these these long thin materials not just the process of entry but what happens when they get inside the cytoplasm of the cell and how do they interact with the organelles this is really kind of interesting work we’re looking at here there we are we’re looking at confocal microscopy and transmission electron microscopy of cells and culture and this top panel here is uptake endocytosis or phagocytosis of carbon black nanoparticles and we see that there’s aggregates of carbon black nanoparticles large aggregates they’re enclosed in membrane bound vacuoles or lysosomes in the cytoplasm of these we then used to die that accumulates in the lysosomes and flores’s orange at low pH and here we see these red or orange fluorescent vesicles in the cells are usually surrounding the nucleus and when these lysosomes contain a whole wide range of hydrolytic enzymes that are usually very active at acid pH to break down lipids proteins DNA anything that’s engulfed into the lysosome one of the components of the lysosome a group of protease is called cathepsins these are unusual because cathepsins function at neutral pH and they can be released from lysosomes when they are damaged and they can be released into the cytoplasm so we did an immunofluorescence assay for cathepsin V and these are cells that have been exposed to these carbon nanoparticles and the conception bees and red fluorescence it colocalizes with the lysosomes and very distinct granular kind of fluorescence we look at uptake of carbon nanotubes on the other hand we see this very large vacuole in the cytoplasm a carbon nanotube here but it’s sort of swollen and here they’re even carbon nanotubes free in the cytoplasm and we evaluate the integrity of the lysosomes following uptake of carbon nanotubes and we see that there aren’t as many of these discrete red equity an orange enclosed acidic vacuoles but they’re there the inequity and orange is released from that acidic environment there’s increased green fluorescence and cytoplasm and similarly we looked at cassettes and a localization and we see that there’s diffuse red fluorescence indicating that this lysosomal permeability or leaking this in some of these cells the consequences of release of this neutral protease into the cytoplasm in cells is is is a very specific kind of mechanism that will activate additional protease it’s called caspases in a cascade reaction that kills the cell in a very specific pathway called apoptosis and indeed we demonstrated activation or caste based in these cells exposed to carbon nanotubes and apoptosis by various morphologic endpoints and here we quantified this these are two types of carbon black nanoparticles these are two types of carbon nanotubes these induce apoptosis the carbon black spherical nanoparticles do not so the consequences of frustrated into psychosis also extend to inability to compartmentalize long fibrous structures in the cytoplasm of the target cells and setting off a mechanism whereby there’s release of proteases from the lysosomes that trigger cascade reaction that kills that cell okay so we then go from these acute effects to the chronic effects that may be more related to potential carcinogen icity we’re still going to be doing head-to-head comparison now spherical carbon black nano particles that are not carcinogenic and not toxic remember they a much higher permissible exposure level asbestos fibers and Phobos bestest fibers and carbon nanotubes and basically we talked about the chronic events that may link these acute toxicity to carcinogenesis II there are two properties were concerned about that have been shown with asbestos fibers first is chronic inflammation inflammation that persists over weeks two decades where the macrophage is are still trying to deal with these bio persistent fibrous materials try to kill it generate continually reactive oxygen species and second in the target cells not the macrophage is but in other cells where the long thin fibers may translocate to the epithelial cells lining the out air sacs are alveoli or out into the pleural space in contact with mesothelial cells so specific effects that cause chromosomal and DNA damage that may be associated with cancer so first we’ll look at this chronic inflammation and it’s not easy to assess the chronic effects of these types of fibrous materials especially when you consider the wide range of carbon nanotubes that are being produced commercially today and probably many many more in the future in a short term asset using cells for 24 hours we wanted to try to prolong that situation and still use in vitro systems because we didn’t want to kill thousands and thousands of rats and mice and see if we can mimic those chronic effects so Vanessa Sanchez have just finished her graduate degree in my laboratory developed a 3d culture system for macrophages it could prolong their viability for 21 days and we expose them in this kind of culture system to carbon black nanoparticles Amphipolis bestest fibers or carbon nanotubes the same carbon nanotubes incidentally that we used in the experiments of the University of Rochester and in these confocal microscope fluorescent micrographs the nuclei are stained blue and most important here is red red indicates the increased expression of a protein or a cytokine called tumor necrosis factor-alpha tnf-alpha and this is a very potent inflammatory mediator it activates macrophages and we see the morphology of activated macrophages in this 3d culture system the cells become very large even larger than 20 microns they have very prominent interdigitating surface microvilli or finger-like projections their nuclei are enlarged they are prominent and clear light they’re very active in making RNA protein cytokines like tnf-alpha growth factors all very important in stimulating a persistent inflammatory reaction that it recruits additional inflammatory cells in the blood and really trying an all-out defense against these bio persistent materials so the chronic inflammatory reaction is thought to be very very important in response to bio persistent fibers but also at the same time that’s more of an indirect mechanism at the same time this is a diagram of a target cell could be an epithelial cell lining the bronchus of the alveolus or a measle filial sell these rigid long thin materials may interfere with mitosis and we’ve done some studies to look at this mitosis this is in the process of a cell it wants to duplicate it to DNA and divide and produce daughter cells if this happens at a low rate in the epithelium of our lungs and also in the mesothelium and its characteristic of cancer or during the development of cancer that mitosis and proliferation in and here we see the diagram of the late stages of mitosis in these diagrams as well as in these confocal immunofluorescence images the nucleus and the chromosomes are indicated in blue and the microtubules that are very important in attaching to the chromosomes dividing them in half between the daughter cells making sure that each of the daughter cells as the normal number of chromosomes is in green and at this point at the late stages of mitosis we have two daughter cells the nucleus has been reformed around these chromosomes and then there’s a contractile ring structure here called the mid-body and we also visualize this green fluorescence sustain that mid body region and then this is confocal microscopy we dimmed the fluorescent image and we could visualize a long carbon nanotube in the same plane as that mid body so the consequence of this physical steric hindrance of long thin structures nanostructures with the midbody will result in failure of separation the daughter cells and formation of multinucleated daughter cells here’s one that has three nuclei and that’s kind of an unstable chromosomal situation in addition we look at the mitotic spindle in earlier stages of the chromosomes here in blue and then the spindle for each of the daughter cells in green and we look here they’re actually carbon nanotubes sort of again in the same plane as these chromosomes and the result of this steric hindrance with separation of the chromosomes during mitosis can result in multipolar mitosis for poles here so that the chromosomes will not be divided equally among the daughter cells producing cells with abnormal numbers of chromosomes or a chromosome that sort of loses its way and becomes detached and its forms what’s called a micro nucleus which is a very standard say for geno toxic materials formation of micronuclei and we score this under the microscope and we see that car mod particles do not really increase the numbers of micronuclei in these dividing cells in culture whereas these carbon nanotubes do and we look at the consequences of this and this is a really beautiful head-to-head comparison of what happens in these target cells in mesothelial cells or in lung epithelial cells exposed to Amphipolis bestest fibers chrysolite asbestos or carbon nanotubes in response to this chromosomal and DNA damage cells will respond by activating a DNA damage pathway and key in this pathway is a protein called p53 the initial response in the cell exposed to these fibrous materials and undergoing this type of DNA and chromosomal damage is a phosphorylation and we can extract all the proteins from the cell into a Western blot and we can detect and these cells exposed to Chris it alight asbestos dose-dependent time and the phosphorylation of p53 this stabilizes the protein increasing the level of protein the protein moves to the nucleus acts as a transcription factor to turn on genes that tell the cells to stop proliferating we’ve got to repair your DNA or if we have lots of DNA damage you’re going to die by apoptosis you’re going to die by suicide and we see here up regulated expression of one of these p53 regulated gene products p21 so this DNA damage response pathways induced in target cells by asbestos fibers and we look at this up regulated gene expression in a more global way looking at genes associated other p53 target genes in response to DNA bcl-2 dad 45 sestra all of these genes are upregulated and quite high levels of upregulation to fold is usually the limit of statistical significance but if we go up to nine or ten fold that’s a tremendous of regulation of gene expression also because it seems that these cells are undergoing oxidative stress they’re up regulating their antioxidant defenses many fold and also this is interesting releasing inflammatory mediators that will sort of cooperate with the macrophages in triggering inflammation and chronic inflammation and head to head comparison similar magnitudes of up regulated expression of these genes following exposure to these materials but not to carbon black nanoparticles so we’re getting close to the end okay go to this one now the way we would have to test if we were given the mandate by EPA or not to test carcinogenicity of carbon nanotubes is to do a lifetime SI in rodents now the best thing you do would be to do an inhalation asset but that’s really technically very challenging and I don’t know if we’d all want to work in the same building where this inhalation exposure was going on for two years which is the lifespan of these animals and even so when we take when investigators took types of asbestos fibers that could produce mesothelioma if you directly inject it into perineal cavity which is lined by mesothelial cells it when these rats inhaled asbestos fibers there was a very low incidence of mesothelioma so that’s a very technologically difficult thing to do it’s very expensive so the direct injection intraperitoneal injection has been widely used to assess whether various materials are carcinogen its carcinogenic or not and what caused all of the concern about the asbestos carbon nanotube analogy in 12 2008 was an experiment that Japanese investigator that the same carbon nanotubes that we have been using in our studies in mice that are genetically deficient in that p53 gene that’s very important in response to DNA damage they developed a higher incidence of mesothelioma in response to injection of chrysolite asbestos i actually just published that model initially and also multi-walled carbon nanotubes induced mesothelioma was in these months now at the same time investigators in Belgium worried studying whether another type of carbon nanotube that is produced commercially in Belgium nano steel is carcinogenic by this assay and they also use crystallite asbestos as a positive control in rats and they found a much lower potency of these carbon nanotubes to induce mesothelioma than this particular type and you look at the structure of these carbon nanotubes are quite different the Belgian investigators used a very short more flexible form of carbon nanotubes that actually form these little balls or aggregates and proudly the cells think i’m looking at a spherical material whereas the carbon nanotubes used in japan were the same ones that we were using they’re very long and rigid and also almost have a 3d kind of a structure and here you see the crystallite asbestos materials that were used in these studies and they also are very long and rigid most both of the materials have widely different lengths but i think it’s the high proportion of materials that are long and rigid that is very important in this kind of reaction so here we are we have all of these new carbon nanotubes being developed and many many more are going to be developed because they’re going to be very very useful not just for tennis rackets and and skis but because they can conduct electrons they are probably going to replace lithium in many batteries situations they’re going to be used flatscreen video display terminals we are learning how to modify them and to purify them and perhaps use them for chemical and biological sensors to be implanted into our bodies if they are very pure and maybe biodegradable after some time to guide regeneration of nerves or tendons to diagnose cancer at an early stage to deliver drugs selectively to cancer cells tremendous potential for these materials but we cannot do these chronic lifetime assays and rodents to assess them so we have to develop more clever ways of looking at it so here’s where we are we’ve talked about the properties related to carcinogenicity of asbestos fibers the geometry long thin rigid maybe redox activity at the surface bio persistence a very important property the long thin fibers at higher so frustrated phagocytosis our clearance they can translocate then to the lungs and to the pleura where the mesothelial cells are at the same time there’s a chronic inflammatory reaction set up additional recruitment of macrophage activation producing oxidants stimulating self-liberation growth factors and in this kind of setting in together with this chronic inflammatory environment and DNA damage that can occur if these cells are taking up those long rigid fibers I ain’t afraid we might ptosis my physical mechanisms are also secondarily from the oxidants generated release from those macrophages the cycle of persistent inflammation cell proliferation DNA damage leads to lung cancer or mesothelioma well we have more work to do I guess researchers always say that because we’re always looking for grants we have to really know whether asbestos carbon nanotubes are going to translocate to the pleura filing inhalation it’s been shown following intratracheal installation but not ventilation their surface reactivity it’s very likely to be modified in vivo so we don’t know whether asbestos fibers or carbon nanotubes particularly because of their hydrophobicity may be different once they reach their target sites and may not show so much surface redox activity and most importantly do these acute toxicity assays that we commonly do in the laboratory for 24 hours or 48 hours can we be more clever and design assays that would be more sensitive more specific in predicting more chronic disease endpoints like malignant mesothelioma so I’ve reached the end you just summarize that there is certainly significant potential for nanotechnology I am NOT someone who advocates banning nanotechnology at this point I think the benefits much out rate the way the potential risk but we do have to be aware that there have been reports of cell toxicity and in animals adverse health effects we know what the properties are and perhaps we can more intelligently modify them one at a time to minimize some of the toxicity and that is why I collaborate very actively with engineers and hopes that they will be able to design less toxic and biodegradable biocompatible materials that will minimize both the adverse human health impacts occupationally but also environmental impacts and I’d like to thank all the people who actually do the work it is a very close and productive collaboration between Bob hurts lab at Brown and engineering in my own lab is the people working in his lab as well as washing gal in engineering and in my lab there are a whole wide range of students postdocs and technicians involved in this work most important is Paula Weston who has done a lot of the beautiful electron microscopy that you’ve seen and the my collaborators at Rochester Alison Oh bird or stir who do the in vivo exposures and work I didn’t have time to talk about where we’ve done actually Mouse assays to produce malignant mesothelioma was following intraperitoneal injection asbestos fibers in and identify some of the tumor suppressor genes that are lost as a result of that chromosomal damage that are linked the development of those cancer this network was done at fox chase cancer center in collaboration with debbie al tamari and Joe testa thank you and I’ll be happy to answer any questions right right so how are the exposures of carbon nanotubes are they comparable in magnitude to occupational exposure to asbestos fibers now this is one of the problems in the field and we assess most occupational exposures to particulate materials like carbon black are measured in micrograms or milligrams per meter cube of air and asbestos fibers because they were originally developed the exposure limits were developed using phase contrast microscopy they were defining a fiber is anything longer than five microns that’s a number of fibers per cubic centimeter of air there’s no direct head-to-head comparison available so this is a real problem that the permissible exposure limit for asbestos fibers in the United States is point one fiber per cc or middle ear that’s really low but and you know carbon nanotubes very impurity some of them have a whole lot of amorphous carbon with them and not very many tubular structures and others are very pure so that that head head comparison is not really very useful certainly it’s less dusty than the original mining operations with asbestos less dusty than many of the earliest bestest manufacturing facilities where you see a cloud of white dusts in the photographs but what was disturbing was what Andrew Maynard from NIOSH really went into one of the factories and showed that there’s the dust on the workers gloves and I’ve used carbon nanotubes in my lab one of the graduate students opened up a new container she was trained to do this under either fume hood or biohazard laminar flow hood with HEPA filters which will catch those nanomaterials but she opened it up because the hoods drawing up up there as soon as she did the whole container just flew out coated the whole walls and everything in the hood with black dust it was a mess so it can they can be very dusty but i don’t think the dust is anywhere as high as it was early days of asbestos fibers that said niosh still wants to limit the exposure and in a my laboratory I at Brown in general I’m very very careful to limit exposure we only handle these materials under class to be dual HEPA filtered hoods with external exhaust I don’t want any black dust and the asbestos fibers also handle the same way roundabout way of answering a question yes yes yes certainly even when we inhale it and and the macrophage is get on that mucociliary escalator and we cough it out or swallowing ingestion news is a route of exposure and of course if it gets into the air from a factory on stack and you get into the water as well and ingestion is a serious concern and interestingly enough very few studies of the toxicity of nanomaterials via ingestion have been done well you know asbestos that that exposure route has been studied in asbestos yeah but it really didn’t do much with asbestos fibers I’m not so worried about exposing rats by ingested what I am more worried about environmental exposures to carbon nanomaterials is not mammals it’s it’s the smaller organisms in there in the water up there important for the food chain invertebrates fish amphibians those are the organisms that are actually probably more sensitive to nanomaterials than most rodents are and that’s really what’s being studied I would not consider an ingestion chronic ingestion exposure to carbon nanotubes is a very high priority thing to worry about for other organisms model organisms in the environment there’s tremendous concern and there’s quite a bit of work going on about that right now yes yes absolutely alright so when cells die in the body they are always ingested by other cells macrophages and macro fibers will ingest them and try to break them down to their very simple components if there’s a lot of necrosis and it happens very rapidly the response of the body will be to encapsulate it as you described but that usually only occurs in diseases like tuberculosis where we have a lot of death of the macrophages by those bacteria which are very very pathogenic very toxic and and even with something like the response to tuberculosis the bacteria that causes tuberculosis there’s a whole area of necrosis then it becomes surrounded by activated macrophages and they elicit fibroblasts and try to wall off this whole thing called a granuloma with fibrosis but even so with that walling off with fibrous scar tissue collagen scar tissue there are always some viable organisms that remain inside and in people whose immune response responses are weakened they can come out and be reactivated and cause an infection so we’re never very good at walling off those kinds of bio persistent toxic insults and in fact one of the lesions that is induced in animals in the lungs following installation of carbon nanotubes are greedy granulomas have frequently progressed to fibrosis and that fibrosis is what we worry about because fibrosis in the lungs that deposition of thick collagen in the alveolar walls of the air spaces can interfere with gas exchange and that’s a very serious kind of complication particularly if it’s progressive with this chronic inflammation that’s triggering it so we might be able to do some da bat walling off necrosis but at that there’s an expense we always pay a price we’re going to cost fiber scarring and impaired function now right I think on carbon nanotubes and even the ones that we are using those that are carcinogenic the ones from Japan electron spin resonance assets which are pretty good and sensitive for detecting direct generation of oxidants on the surfaces of minerals or materials and they’re not very potent in that regard whereas asbestos fibers are very cold so the direct generation of oxes and oxidants at the surface of these carbon nanotubes probably is not very important secondary generation of oxidants by the cell during phagocytosis particularly macrophages if they’re frustrated phagocytosis it is much more important the other thing is that these these long rigid fibers we’ve shown that they’re not very well encapsulated or enclosed or sequestered and lysosomes they’re causing leakage of the lysosomal memory lysosomal membrane permeability they’re out there in the cytoplasm I’m not sure they may even be able to interact with the mitochondrial membrane remember the mitochondrial membrane is rather always involved in electron transport it’s generating a lot of radicals all the time because it uses oxygen to generate ATP and maybe in that scenario carbon nanotubes and other nanomaterials have been shown to interfere with a mitochondrial membrane polarization and that also can trigger cell death by apoptosis so I think the secondary generation of oxidants by these cells and also the perturbation of their normal and doggedness oxidant generation is more likely to contribute to toxicity those are subtle things and what we really have to work hard to entangle him yes well I think we can incorporate them relatively easily that carboxylation reaction of all the types of oxidative reactions was very specific it’s it you can control the extent of it so you can still retain the properties that you want but allow or maybe even program in the rate of biodegradation either in the lungs or even in the environment by horseradish peroxidase for example I think we can do this use the desirable properties for what we want it for is a sensor drug delivery device or whatever let it degrade and everything will be fine it’s interesting you know both we and Valerie kegging to publish those papers and Valerie Kagan’s papers were very elegant and published in very high-profile journals and I don’t know to what extent the manufacturing are trying to do that kind of design I think certainly for people who are involved in drug delivery there’s thinking along those lines but they just so tell you about it because of pen yes great yes there was a while yes yes well I’m Porsha I was involved in the review sup sup not the original but subsequent ones and they never really did well not only I suggested but other reviewers suggested to prove that they are mesothelioma is I mean I’ve been working for many many years in producing mesotheliomas and mice with direct intraperitoneal injection of asbestos fibers and I went to great lengths to show because i don’t have ologist that they were mutual Philly illness and even that they had the same type of molecular alterations as human mesothelioma and those techniques are not that difficult I mean but of course these people are not molecular biologists or pathologist at toxicologists but they have not done that and there is a concern about that diagnosis malignant mesothelioma is a difficult diagnosis pathologically unless you really used to seeing a lot of it and so I think it is somewhat in question there that we have to resolve these things some way or another it’s just that people do not want to do these studies because it’s not as expensive as an invasion study which is a one or two million dollars for one material one species but it’s it’s quite an undertaking to do these things alright those weren’t asbestos there’s bestest fiber substitutes hmm if you believe those kinds of studies yeah yeah but actually we’ve used now those fiber glasses they are in commercial use they’re not classified as human carcinogens and they’re widely used and so far everything seems to be fine so that’s good oh no that was enough self oh oh yes that wasn’t an animal I haven’t done more chronic studies myself but the investigators at NIOSH have on their persistent for six months anyway they they have yes and there’s no degradation yes and you would expect them to be filed persistent unless you carboxylate them yeah yeah those those if they were if they were pristine they’d be expected to be well we tried that even with single-walled carbon nanotubes that were pristine and very clean and we did the 90-day flow through system with one milli molar hydrogen peroxide one milli molar iron and ascorbate no degradation so they’re pretty good unless you carboxylate them that seems to be what you have to do yes so some some investigators are very much concerned about that and the person who’s most most active in that investigation is Jamie Bonner at North Carolina State University and there have been studies where they have mouse models of allergic asthma which are pretty good models for human asthma and they definitely show that these carbon nanomaterials exacerbate those kinds of allergic reactions as well as the inflammatory responses another experiment that was done by investigators at NIOSH National Institutes of Occupational Safety and Health use mice that were genetically engineers to develop atherosclerosis they inhaled single-walled no interest that was into tracheal insulation single-walled carbon nanotubes and they had exacerbation of their appt atherosclerosis which is an inflammation based mechanism so it seems that even introduction of carbon nanotubes in the lungs no matter how you do it will exacerbate local allergic reactions in the lung but also generate inflammatory systemic inflammatory responses which they measured at NIOSH that will exacerbate atherosclerosis that’s those are the more systemic effects outside of the lungs are a serious issue in nanotechnology we’re concerned about it with carbon nanotubes certainly but most mostly with other types of nanoparticles they’ve been shown to translocate into the brain they reach the liver the kidney and even some investigators have shown in small numbers they cross the placenta and cause the blood test is barrier so those all the systemic translocations is a very serious issue we’re concerned about oh yes they have put out a draft limit seven micrograms per meter cubed it’s an eight-hour workday set up five days a week it’s out for comment they proposed that limit based on the animal studies that I’ve been describing but they’re a lot more and that the draft the whole draft recommendation all the detailed data there the rationale it’s all freely available on the web you just go to the NIOSH website it’s all there yes we did that yeah yeah one of the first studies i did with robert hurt we met we were having these little talks at brown trying to put an interdisciplinary grant together and we each got it for 10 minutes explain to our research and i talked about asbestos and he came up and said i have some carbon nanofibers you want some I said you bet and I also want you to dope him with iron and those were the first paper is among the first papers we studied and we did it with a very simple system with DNA plasmid DNA and a test tube and we did had dead confessions carbon nanoparticles sweetie carbon nanofibers commercial carbon nanotubes with different levels of iron and dispenses fibers and we showed DNA damage certainly when you doped iron dope the carbon nanofibers tremendous amount DNA damage was produced depending on the level of doping and also the time they were exposed tear so their bids several types of studies along those lines definitely if you have high levels of bioavailable redox-active iron in any nano material it’s a cause for concern it’s not only iron I’m worried about we also did some work with nickel nickel is another common catalyst nickel is a human carcinogen on metal as a carcinogen I think it room has some other problems cobalt also redox-active again it’s whether it’s vile available or not and that seems to be the consensus now manufacturers are taking great pains to purify their carbon nanotubes after synthesis get rid of the bioavailable metals they actually heated to high temperature vaporize them all off that also burns off that amorphous carbon coat and they’re making some pre pure products now which was required as you said for those electrical applications it’s interesting because the ones that we’re using from Japan they did wash them quite a bit these are the ones that that are not very active in generating radicals by ESR electron spin resonance but yet they’re quite toxic and they’re carcinogenic apparently so it must be oh yes but it’s in low levels is less than point one percent at the end of the product so i think it’s not the whole explanation everyone would like to look for what’s the one physical property or chemical property that to deal with i think it’s a combination bio persistence I think this rigidity is really really important length is important and redox activity intrinsic as importantly it’s there but if it can change tell the cells to screw up their reactive oxygen species generation or scavenging or normal aerobic respiration in the mitochondria it’s just as much of an issue thank you all I appreciate your attention and your interest

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