Listen: Chemist Bryant Nelson on NanoGenotoxicology
In Spring 2016, chemist Bryant Nelson visited Swarthmore to deliver a Sigma Xi lecture.
A bioanalytical chemist, Nelson is currently the nanogenotoxicology project leader in the DNA Science Group. His current research interests include investigating and characterizing the biological mechanisms of oxidatively induced DNA damage and its repair as they relate to the incidence and progression of age-related diseases such as cancer and metabolic syndrome. He is currently developing novel measurement platforms and mass spectrometry-based approaches for evaluating the impact of engineered nanomaterials on the induction of oxidative damage to DNA.
Sigma Xi is a national organization devoted to promoting scientific research. It has chapters at numerous college and university campuses, including Swarthmore College. Disciplines that are covered by Sigma Xi society are mathematics, statistics, engineering, biology, chemistry, physics, astronomy, and certain subfields of psychology and linguistics.
Bryant Nelson: I'm delighted to be here. Thank you very much Sigma Xi chapter. [inaudible 00:00:05] for inviting me. This is my first time with [inaudible 00:00:14]. Absolutely beautiful here. What I can't fathom ... I've been at NIST, National Institute of Standards and Technology, for 16 years now. The time has really flown by and I remember how I got this position. It was the senior, the final year of my graduate program and it was a lecturer, one of the scientists from NIST, actually came to the [inaudible 00:00:42] and was giving a lecture and I was asking lots of questions about the topics he was discussing, so many that after the seminar he came up to me, "You have to apply there for a position as a post-doc when you graduate at NIST."
Actually, I did that. It was a competitive process and 16 years have gone by, it's been fantastic. That scientist who invited me to come to NIST to do a post-doc is now the director of NIST and he's also the under Secretary for the Department of Commerce, so he's a great advocate back then and also now. I urge you to latch on to any advocates that you can find throughout your career and keep them close.
But today, I'd like to tell you a little bit about some of the work that we're excited about at NIST regarding the interactions of nano particles with DNA. I think it's a very important topic. We've been working in this area for about nine years now and we have some exciting data that I'd like to share with you, some early data and then some new projects that are moving forward.
So, what is NIST? NIST is actually part of the Commerce, we're an institute that is non-regulatory unlike the FDA. We don't have a lot of power to maybe do things, but we suggest that you do things in some way as far as measurements are concerned. We're the National Metrology Institute, what that means is that we set the standards and we measure things so that the United States can have a large economic impact on all sectors of the economy. We do measurements on objects such as proteins. We also do measurements on improving the technology of gene editing and we measure fuels, so we have a wide range of activities that we're involved in.
We were first thought of in 1788 in Article I Section 8 of the US Constitution where it was put out there that the United States needs to have a mechanism for setting the standards for our measurements and so the idea was there. It took another 120 years for our founders to actually create NIST. We were created in 1901 in D.C., established in actually downtown Washington D.C. as the National Bureau of Standards, you might have heard of that. We changed our name to the National Institute of Standards and Technology, not many people recognized that new name but we're still the same institute.
Just to give you a flavor of where we are located and how many employees work at the institute, we have two main locations: one in Gaithersburg, Maryland which is where I'm from. That campus is pretty much an academic type of environment, deer running around. It's quite beautiful, not as beautiful as [inaudible 00:04:18] with the sights. Our other main campus is in Boulder, Colorado. We have a total of about 5,000 employees including scientists and engineers and we have about 2,000 visiting guest researchers who visit all of our different campuses. Five different institutes around the United States going from D.C. all the way to Hawaii.
What do we actually do, day to day? Well we do measurement research, we measure things that are important for ensuring the economic well being of the United States. We put out standard records data, standard records materials. We perform calibrations and tests on airlines to make sure that, for example, the altimeters are working correctly, so our scientists actually go out and physically calibrate those instrumentations on those different types of airplanes. We do laboratory accreditation and we extend more than 400 standards committees. That's a general overview of the breadth of things that NIST is involved in.
Today I want to talk about, specifically, how we look at interactions of nano materials with DNA and this can be termed nano material DNA toxicology but that doesn't necessarily mean that there are things that are negative that come out of interactions with DNA. That's just the cover terminology for the field. We try to utilize the various types of models in our work, specifically we use three different models shown here on this graph. We use cellular models, we use cell models and we use also multicellular models to look at the effects of these nano particles and nano materials on DNA.
Just to make it clear, nano particles or nano material are these really small materials that have at least one dimension that is on the order of 100 nanometers or less. You can have other dimensions but that's the main definition. There are a lot of controversies about the definition of nano materials, both in China and in Europe, but for today's talk any material that has one dimension that is at least 100 nanometers or less is what we're going to be talking about.
The organism model shown at the bottom is the newest type of model system that we've been working with to understand these interactions. There's controversy still about whether or not it's best to use in vitro models or in vivo models for doing nano material toxicology. The best case, of course which we would never do, is use human models. That would never happen, so we need something between the cells and the human model to actually understand physiological ramifications for nano materials and DNA. We're using in our laboratory now plants and organisms, specifically we're using some Arabidopsis and Rye grass and we're also doing a lot of work with [inaudible 00:08:06] so I'll get into that. That's the exciting area that we're heading into.
How do nano materials actually induce or modify DNA, if they can? The main mechanistic rationale is that nano materials may be able to produce free radicals such as hydroxy radical and free electrons or hydrogen atoms. Those free radicals can attack at any of the four bases of DNA or any of the phosphates on the backbone. When those free radicals, if they're formed from nano materials, they can form different types of DNA damaged products, such as DNA base damage or DNA sugar damage and a variety of different types of these.
We want to be able to measure the formation of those lesions at very low amounts so we have to come up with a technique to do this and we have approached the measurement of these different lesions using mass spectrometry techniques. Some of the lesions that are formed from free radical attack are mutagenic and some are cytotoxic and some are just markers of oxidative stress. Some of this gene graph are 26 of the lesions that are formed that we can measure using our mass spectrometry techniques. How do we do this?
We use hyphenated [inaudible 00:09:46] mass spectrometry to actually measure these lesions, either in the cellular DNA or DNA extracted from cells or DNA extracted from organisms. The process is simple, it's a five step process. You take the DNA, you clean it up with ethanol, you determine the amount of DNA, whether you extract this DNA or it's raw DNA, then you add isotope labeled entero-standards, you chop up the DNA using basic repair enzymes. I'm showing FPG and Endo-3 here. Then you actually incubate that and then these enzymes actually excise, or remove, the damaged bases or the lesions that I showed you on the previous slide. Then you just simply measure the levels of them and identify them using liquid chromatography, mass spectrometry or gas chromatography mass spectrometry.
Using those techniques you can measure as low as one lesion in 100 million bases. That's what we've been able to go down to. One of the first studies that we did, way back in the day, back in 2012, was we looked at the effects of carbon monoxide nano particles on some terrestrial plants. We used this model to look at radish, the effects on radish, annual rye grass and perennial rye grass. The work was picked up by the New York Times because it had a lot of implications on could nano particles actually enter plant systems and cause DNA damage. So, just to show you the results from the radish incubation and exposures, when you expose radish to the nano particles and bulk particles, bulk particles have a diameter of 200 nanometers, they are outside that 100 nanometer range I was talking about and the nano particles had a diameter of 69 nanometers.
Right off the bat you can see that compared to a control, unexposed radish the bulk particles inhibited the growth of the radish at two different concentrations. But more so, the nano particles caused a tremendous decrease in growth and stunted the formation of the radish plants. As far as the DNA damage that may or may not have occurred we basically focused on this study and looking at it there's three different reasons, two guanine oxidative stress products, 8-hydroxy guanine everyone has heard of that lesion. It's very mutagenic, causes transversion mutations. Then a reduction product from guanine, affecting guanine, and that is a form of mito prohibidines that are formed from free radical attack.
Just looking at those three lesions only, and the effects of formation in plants, radish, you see that there's a dose dependent increase in the number of lesions. On the y-axis is shown the lesions per 1 million DNA bases and so as you increase the concentration of the exposure to copper oxide you do get a dose dependent increase in the number of types of lesions that are formed. Now I'm not showing the perennial rye grass or the annual rye grass because what happens is the profile is completely different from the radish. It's not dose dependent but it does have a pattern that's independent of the nano particle or the bulk particle. So there's a species dependence on what happens in terms of DNA damage in plants.
This study showed, for the first time, that multiple lesions can form in common plants. We dug a little in the study and it showed that there's a really interesting effect on the uptake of these copper nano particles compared to copper bulk particles. As a control we used copper ions so shown in A are the uptake in radish of copper ions, nano particles and bulk particles. You can see that for the nano particles there's a tremendous increase over the bulk particles in the uptake. It's an amazing increase and the scale here is going from 0 to 1500 micrograms of copper per gram of plant, completely different from what happens in the perennial rye grass. The scale is going only up to 100 micrograms per gram of plant, so there's a species dependence on the uptake and also on the DNA damage in plants. It's a remarkable finding.
So we went a little deeper to find out where are these nano particles going in these plants? Well it turns out they're going into the root cells. We did a scanning transmission electron microscopy on the root cells and we located nano particles in the root cells and identified them using elemental analysis, so that's what is going on with the plant systems.
Things are not all bad with nano particles. Not to say that uptake in plants in bad, but we have started a new program where we're looking at how can we use certain types of nano particles to potentially inhibit DNA repair proteins. Why would we want to do this? Well cancer is a type of system where the DNA repair proteins are highly up regulated so compared to normal cells, cancer cells have inefficient DNA repair protein system that's highly redundant. There may be a way that we can use nano particles in combination with other types of therapies, such as radiation or chemotherapy, to inhibit and selectively inhibit their DNA repair proteins.
Now this is not a new idea in terms of using chemicals. There have been a number of trials done by major pharmaceutical companies, such as Astra-Zeneca and Pfizer, that have used different types of chemical based DNA repair inhibitors to actually selectively look at and inhibit different types of DNA repair proteins or pathways, such as PAR or basic excision repair protein pathways. The idea is there, but can you do it with nano particles, the same type of thing that actually could be safe.
As an example, there is one human base excision repair protein called NEIL1. This is just a graph showing the excision specificity of different repair proteins and how they excise and remove certain types of oxidatively modified lesions. For NEIL1, here, it shows that it specifically only removes b-adenine and b-guanine so in a DNA strand that has a number of b-adenines and b-guanines, NEIL1 will go in and specifically chop those lesions out and repair the site. That's why NEIL1 is so important. It's also important because NEIL1 is based in the nucleus and the mitochondria and it's involved in two different DNA repair pathways, single strand break repair and also nuclear [inaudible 00:18:35] repair. It only has specificity for those two lesions, b-adenine and b-guanine, and no specificity for 8-hydroxy guanine. What that means is when it encounters 8-hydroxy guanine it cannot excise and remove that.
Luckily, we have this nice gold nano particle called Au 55, means that it only has 55 atoms, it's composed of 55 atoms and it's 1.4 nanometers in diameter. It turns out that this Au 55 cluster has very unique properties like most nano materials. It can have catalytic properties that you can tune to do certain things. We took this Au 55, 1.4 nanometer gold nano particle and we incubated it with the NEIL1 and it turns out that when you do this and perform a concentration study of the gold nano particle going from zero to 100 micro molar exposure conditions, that it can specifically inhibit of b-adenine and b-guanine.
The first row is showing the background level in DNA that has been incubated, that is exactly 40 grade DNA, so it's been exposed to 40 grade ionizing radiation so we have a high level of DNA lesions. The background level of lesions in the sample is very low without any NEIL1 repair. Heat activated NEIL1 has no activity as expected. Then if you don't add any gold nano particle in, you get this amount of lesions excised. This is normalized data here, but if you increase the concentration of the gold nano particle, the lesion excision starts to be inhibited. We've shown this in two different model systems now. This is all excision work. We've used the NEIL1, we've also used a bacterial derived protein called FPG and it works the same. We get inhibition of the activity of the repair protein. I show here that has an IC 50 of about 30 micromoles.
This is only the first step in this direction. The next step will have to focus on actually putting targeting ligands on to this Au 55 and using cells, actual cells, cancer cells, and doing the same sort of thing. It's a much harder thing to do but I think we're on the right track.
Alright, I'm going to switch gears for a little bit and go to that new area I was talking about at the beginning where we're actually trying to bridge the study and understanding of nano particles with living organisms. I'd like to talk about this excellent organism called C. elegans. It's a beautiful beautiful creature that has been said to be the most numerous nematode on the planet Earth. It's a transparent worm that has been completely sequenced, back in 1998. It was actually the first multicellular organism to have its genome sequenced. It has approximately 20,000 genes with many of those genes are homologous with human genes. It has a great lifespan, you can do all your experiments in less than about two weeks so from birth to full adult you can expose the worms and extract their DNA and look at what the effect of those nano particles and nano materials on the induction of DNA modifications.
Why do they use them in neurobiology and [inaudible 00:23:01] biology over the years and a number of great discoveries have emanated from the use of C. elegans. We're heavily going in this direction now in our laboratory. The work is on that now. It is our model system.
What do we need to do to really take advantage of this? Well, for mass spectrometry, which are the techniques that we're using in our work, we need to be able to get the DNA. There are currently not very many easily usable DNA extraction methods for removing DNA from worms. The kits that are out there are very expensive and all of the procedures that we've tried that are published for extracting DNA have not been reproducible. We're developing a new method for extracting worm DNA based upon using high salt enzyme procedure that is we're trying to benchmark that against the usual phenyl chloroform extraction procedure that is common for worm cells.
Being NIST, what we like to do is when we develop a new procedure we like to look at all the different types of attributes for that procedure to make sure it's of very high quality. In this case we're going to look at the amount of DNA extracted, the extent of DNA fragmentations, level of RA protein impurities, contamination of DNA from other sources and also finding the DNA lesions. We've got to do all of this first, and then we'll have a method that we can use reliably.
I just wanted to give a preview of the work that we already initiated. The worms, they have this tough cuticle on the outside that allows them to pretty much be impervious to all kinds of extraction procedures. I've shown here three different extraction procedures, the results from those procedures under light microscopy. Control worms are in A versus free thawing worms, high salt buffer, lysis or grinding the worms in liquid nitrogen. Okay, so we took 500,000 worms to start with and we performed all of these procedures. Free thawing the worms compared to control didn't do very much, so this is where you take liquid nitrogen and then thawing the worms, liquid nitrogen and thawing again. Not much happened. However, when you took our new high salt buffer, and I won't say anymore than that about it, you see all of this fragmentation of worms. Clear fragments, worms we have been able to cross the worms and possibly get to their DNA, right?
You can grind the worms in liquid nitrogen, in D, and nothing happens to them. You can physically do this for 30 minutes and you can still see worms swimming around right here in this one. It's crazy. It takes a lot of work just to get the DNA out of the worms. But fortunately we were able to do that. I'm showing here the amount of DNA we could get from 250,000 worms versus 100,000 worms. We only need 50 micrograms for our mass spec procedures so using just 100,000 worms was sufficient, pretty much.
Finally, we'd done some background measurements on the lesions that are formed and shown here we're comparing the high salt procedure, the new procedure that has yet to be published, compared to a phenol extraction and as expected the phenol extraction, in blue, you see a larger level of scars disappeared, but there's a larger level of oxidation products over these different lesions that we measured. We did get a nice signal for all the other lesions, the background lesions, so we're confident that using this new high salt extraction procedure that we can actually start to look at the effect of nano particles on C. elegans.
Whenever anyone wants to do any type of exposure study, no matter what the organism is, whether it's fish or C. Elegans or plants, you have to be able to expose what you're interested in and then remove the nano particle because you want to know how much, you have to know how much nano particle is in the dose and how much the organism actually is exposed to. We have to come up with a procedure that reliably separates the nano particle from the organism. That's part one, the organism work. Part two is then you need a reliable and robust procedure to measure how much of the nano particle is actually [inaudible 00:28:41] into the worm. I'll speak to both of those at this moment.
It turns out that if you take a look at the literature, you will find that most people just, most scientists just perform exposure using whatever model that they want to and then wash the organism off with water. That is not suitable, it doesn't work, and I'll show you why. Imagine that you were doing an exposure in a flask of nano particles and your organism and then you transfer it back to a fountain tube and you want to remove the nano particles because you need to know that the organism is only exposed to a certain amount of nano particles and they're not sticking on the outside of the organism.
You can centrifuge, in our case, this fountain tube at a very low speed and what that does is it forces the C. elegans to the bottom of the fountain tube and nano particles stay in suspension and so what you can do is you can remove the super native containing the nano particles and do that three times. Go ahead and do your experiment, do your DNA then the experiment and you will be in the finding of lots of artifacts. Why? A simple wash with either water or buffer is not sufficient in the organism studies. We've done some scanning electron microscopy on samples that were clean using those procedures and this is a C. elegans mote and this is an adult. You get an SEM and scanning the outside of the worm and you see these nice little bright spots. It turns out that you do this with gold nano particles that are 60 nanometers in diameter, you can find 60 nanometer in diameter particle on the outside sticking to the tube. We confirmed that using elemental analysis, x-ray dispersion spectroscopy, showing nice gold signals.
This will defeat all of the answers that you will get from your DNA damage studies so we had to come up with a new and better procedure to actually confirm and remove particles from the surface of C. elegans. The procedure that we came up with is actually quite simple and elegant. You take the same exposure scenario where you have the particles in a flask, transfer them to a fountain tube, the particles and C. elegans, spin them three times, let them settle, capture your C. elegans down here. You can see the particles still embedded. Then simply you can transfer this whole sample to this tube that contains a density gradient of sucrose.
What happens is you place the C. elegans and nano particles on the top of this gradient, this increasing gradient of sucrose going low to high concentration, and you can perform one simple spin on this at low G and what this causes is the separation of the nano particles from the C. elegans. The nano particles move down from this initial point into this initial gradient concentration, but the C. Elegans actually migrate down even further and stop where their buoyant densities matches the density of the sucrose. At this point you have a nice, clean separation when you can take a [inaudible 00:33:01] that is purely C. Elegans, it's still alive at this point, and you can perform further studies. We confirmed this using ICPMS for total gold and those super flakes. As you go down each layer, go down each layer and we can quantify the amount of gold that's present. As you can see, between zero and two at the C. Elegans layer, there's hardly any gold left.
We really like this new procedure, but we wanted to make sure that it really works, so we did further testing and looked at the bioaccumulations of the gold nano particles in the C. Elegans that I showed previously. This time we used single particle ICPMS to determine the size of the particles inside the worms. We expected that the worms would eat 60 nanometer gold nano particles and what did they do? The outcome shows that compared to a control of 60 nanometer gold nano particles that for three samples, the worms actually ingested 60 nanometer gold nano particles.
I have a little movie here to show you, if I can figure out how to get this to start. Paul do you know how the clicker does without the ...
Speaker 2: [inaudible 00:34:43]
Bryant Nelson: That should be the icon right here.
Speaker 2: [inaudible 00:34:48]
Bryant Nelson: Oh there it is. I saw it.
Speaker 2: Yep, let me [inaudible 00:34:53]
Bryant Nelson: There it is. What I'd like to show you, this is a focused ion beam scanning electron microscopy image of slices of the worms, 50 nanometer slices of the worm longitudinally, where you can see the lumen, or the gut tract, of the worm. Pay close attention, there are bright spots that appear right here. Those bright spots are what we believe to be gold nano particles. The particles are entering the lumen and then staying in the lumen. You can see the microvillae right here, that might be some particles on the microvillae. To actually prove that those are gold nano particles, we then did SEM on those slices and we looked at all of the bright spots that were available. The bright spots have higher contrast than lipids and so performing elemental analysis on those bright spots, let's focus in on the spot that I just highlighted right here. We're able to show, if you zoom in, that we think those are three different nano particles, but elemental analysis shows without a doubt that those are gold nano particles.
Now we know that our method for cleaning up the nano particles from the outside of the organism works well. We have a method for looking at the bioaccumulation of the uptake of the gold nano particles into the worm and I'd like to end with some uptake data.
So I'm going to show the size dependent uptake of the gold, this is total gold using ICPMS into the three different exposure sizes. We used 80, 100, and 150 nanometer size gold nano particles and the total gold that is uptaken is actually quite ... there's a size dependence. These two, my post-doc actually switched these two, so you can see that for this should be 80 nanometers. The uptake in terms of total gold is decreasing by size but it's shown more clearly here. When you use single particle ICPMS you can see 80, 100, and 150 nanometer gold nano particles. Non-organic particles per nematode, as you increase the size, the number of particles decreases. You have here approximately 30 particles and then ten particles then two particles as you go up to 150 nanometers.
With that, I think I'd like to acknowledge many of the collaborators on this project. A number of these are students, undergraduate students and post-docs, collaborators from outside of NIST. The undergraduate students are really excellent, the ones that come to NIST, we have a Summer Undergraduate Research Fellowship Program that is a full three months during the summer. It's a competitive program where the students apply, I think now, and through the months of December and January and February. It's fully paid, housing and you get to work with one of the outstanding researchers at NIST. It's a very very solid experience and a lot of fun. If you're interested in finding out any details about this program please feel free to ask me after the lecture, but with that I'll end my lecture and thank you for your attention.