My name is Anne Summers. I’m from the University of Georgia. I have been working in the area of mercury biology for maybe 40 years and I’m trained in chemistry and microbiology and I work in the general area of metallobiology, and there has never been a better time to be working on the basic science of mercury than right now because of all the tools that are becoming available to understand this really fascinating element, which has been both a bane and a boon to human beings over their history, what it’s doing to our biology.
So I’m going to try very quickly to just update you on a couple of very recent observations from my laboratory that bear on this issue. One of them is just about to be published and the other is just about to be submitted. I do have handouts on these. They’re not here yet, but I do have handouts for all the Panel. It should be here within a half an hour.
So okay, the talk has an outline. I’ll tell you about some animal studies, some continuing studies on an animal model that we’ve worked on for many years. The monkeys that we work with that had the dental fillings implanted, we’ve done some new analysis on the fecal samples. I will also tell you, then, shifting gears very much down to the molecular level, talk about a very recent biochemical and cellular study at the level of what mercury exposure does to a model single cell organism, good old E. coli, which by virtue of evolution, I can say, very clearly does apply to our own biology.
So the animal studies basically show the microbial community of the large bowel methylates amalgam-derived mercury. That’s the bad news.
The other studies show that brief exposure to inorganic or organic materials results in modification of key proteins in every major metabolic pathway of the cell, and the proteins involved are especially the ones that are involved in redox metabolism. And I call that the good news. Okay. And the reason I call it the good news is because we are able to find these things now by virtue of the powerful new analytical tools that are available that we can bring to bear on looking at mercury in its interaction with proteins and other cellular molecules at a very refined and detailed level.
So here, for example, is the first piece and I’m only going to talk about the stuff that’s on the left side and I’m sorry that that’s not showing better. But on the left side of the screen there are two examples of monkeys. The upper one is monkey 78; lower one, monkey R-87, and those are timelines that go across there. Timelines early on the left side, you have monkeys that don’t have any amalgam fillings. And then about — a small bit in, about two weeks in one case and two months in the other case, 16 above, and in below, 12 amalgam fillings replaced in these animals.
The red and blue lines are the measurement of mercury and on the scale on the side there is — that’s a logarithmic scale and it’s a 4 order of magnitude increase in the amount of mercury coming out in the feces of these animals once the amalgams are placed.
And the yellow line is methylmercury. So we had previously analyzed these samples only for mercury and we’ve gone back now that the technology is available to analyze them for methylmercury. So the yellow line shows you the methylmercury.
Now, one point that needs to be made clear here is that the axis on the left side, which records mercuric mercury, is in the order of micrograms per gram. The axis on the left, which records methylmercury, is in nanograms per gram.
So typically the amount of methylmercury that’s found in biological systems, whether it’s in this case or in the environment, is about 500 to 1,000-fold lower than the amount of mercuric mercury that’s present. So only a small proportion is detectable as methylmercury in any setting that is actually metabolizing it.
And just to make the point about the scale here, just to put it in perspective, that red line on the left side that I just put in, that indicates 1 part per million. Okay, that’s the fish advisory level. We can’t sell fish that have 1 part per million of mercury in them or they advise you not to eat such fish.
And so that right there shows you that for the entire course of these experiments, these two animals — and there’s another pair of animals that I’m not showing that had the similar protocols — for the entire course of the experiment, except for in the top panel, when the mercury fillings were removed and replaced by glass ionomers towards the end of that time course, those animals had in their gut contents mercury levels that exceeded the level that you could have if you bought fish and ate it.
I mean, if they were eating fish continually during the course, which they weren’t, they were eating monkey chow, they would’ve had to have — that would’ve filled their gut contents with mercury below that red line. So the amount of mercury coming out of the fillings vastly boosted the amount of mercury that was in their gut contents over the course of this line.
Okay. One of the things we weren’t able to examine here, because we didn’t have the analytical skill for this, or techniques, is dimethylmercury. But in every environmental setting where it’s ever been examined for production of methylmercury, dimethylmercury was also being produced. It’s much more difficult to analyze, but every other environmental that’s been examined says, if there are bacteria there producing methylmercury, dimethylmercury is also being produced. Okay. So that’s all I say on that. I do think that’s bad news.
Then the next point that I want to make about this is that high concentrations affect the GI tract. And this is certainly physiology that all of you already know about. But the GI tract is a tremendously important and diverse and complicated part of our physiology and 90 to 95 percent of the mercury that does get out of our bodies goes out through the GI tract.
So it’s going down that pipe, through all those corners and loops and whirls and basically exposing all the cells that do all of that cool stuff that our digestive system does. It also has down there in the center, or the green thing at the bottom is the large bowel, which has a hugely diverse microbial community that medicine is now beginning to recognize as one of the most important parts of our cell biology and our cells.
There’s 10 times as many bacteria in our GI tract and on our skin and other places on our body, as there are cells — of eukaryotic cells in our own body. They are companions. They guide us. They help us, and a few of them can cause trouble. But those guys also metabolize mercury.
And we showed many years ago that the gut contents include — in people with amalgam fillings, the gut contents include bacteria that are resistant to mercury, take mercuric mercury and convert it to Hg0, which is highly volatile; it goes across the lung epithelium like a shot. And though we’ve never been funded to do the experiments, it certainly would likely go across the GI epithelium really quickly.
So we want to pay attention to the gut. We don’t want to just think of it as a sewer where stuff goes out and it’s gone and we don’t have to worry about it. Those cells are experiencing high levels of mercury.
I’m not going to stop with this. This gets us into the business of the molecular biology of cellular damage. I won’t go through this in great detail. This is what we call the mercury damage-ome (ph.), using E. coli as a model system.
The field with proteomics allows you to look at all of the proteins in the cell at once, okay, and see every protein that the cell is making at that time and enumerate it. And you can also look for things that have modified it. So you can look for what are called adducts. And we had devised a means to use proteomics to look at, specifically, mercury modified proteins inside the cell. And we started off doing this, of course, with a simply model system, good old E. coli. It has a manageable number of proteins. But this technique, proteomics, is widely applied in all kinds of diagnostic regimes and research regimes, cancer biology and even function and so forth.
So this project had two parts. One part at the top, in the blue box, we send the treated cells out to our colleagues at the Pacific Northwest Laboratory of the DOE in Richland, Washington, and they complete the proteomics, which involves a lot of very advanced machinery and highly skilled operators, and we get back the data on the peptides.
And then in the bottom part, the brown box, my own laboratory does very detailed biochemical studies of these cells to be sure that everything that we get from out there, we know what the story was with the cells whose proteins we were looking at. And we call that benchmarking.
So I’ll show you one of the benchmarks.
Mercury exposure increases the intercellular free iron. You may know that free iron is a major player in initiating REOX damage to the cell. Iron is supposed to be sequestered very tightly when it’s working inside the cell. And nature has evolved many very sophisticated proteins, including heme proteins, but also still has a lot of primitive proteins called iron sulfur proteins, and these iron sulfur proteins are the ones that are most vulnerable to mercury damage.
And I will only point out a typical oxidizing agent that is in the second position there. The first position is no treatment of the cells. The second position is treatment with 4 millimolar hydrogen peroxide. And hydrogen peroxide is a standard oxidizing agent and the cell is well enough buffered that it just boosts it a little bit; even 4 millimolar, it goes up a little bit in free iron. All the way over to the far side, 80 micromolar mercury can take the cells up as high as releasing 80 percent of the iron in the cell is now free.
And so we have other measures of oxidative damage that I will not look at, but this is a very strong indication that mercury essentially catalyzes the release of things that are going to be doing serious damage oxidatively inside the cell.
The other point that I want to go to and then finish up is the most vulnerable proteins, the proteomic data, the stuff that we get from the Pacific Northwest Laboratory after they have enumerated the proteins and been able to — a computational filter that we devised, been able to tell which ones have mercury on them. We can do a profile of all the proteins in the cell that we see that are modified by mercury.
So on the blue lines up there are the percentage of a given protein functional class. The functional classes are across the bottom and those include things like regulation of gene expression. And the three boxes that I’ve highlighted particularly: red is energy metabolism; the green one is protein synthesis; and the blue one is amino acid synthesis. And I’m probably just going to stop with talking about the energy because that will take me to my almost last slide.
And so that big red bar there above the energy metabolism, which is the first box on the left, indicates that as many as 16 percent of the proteins that we find that are modified by mercury are in the category of energy metabolism. That’s basically talking about the mitochondrion, as far as our cells are concerned, because all of these proteins that bacteria use for their energy metabolism eventually got synthesized, became part of what evolved into the eukaryotic cell’s mitochondrion.
So energy metabolism, for example, in this category we have the ATP synthase, which is the major agent of making ATP. We also have glucose glyceraldehyde-3-phosphate, which is a major player in glycolysis. And there are a couple of other enzymes, including succinate dehydrogenase, which is right there at the bottom. And that’s a major interface between the crib cycle and restoration. So those are all involved.
And what I’ve listed is taken from our data to list human mitochondrial homologs that we find to be mercury vulnerable in E. coli. Okay. So we’ve taken our dataset and we’ve looked at the proteins that we find to be mercury vulnerable by the proteomic method and we’ve said, okay, where do these fall in human biology?
Okay. So this is a subset, only the list that has to do with mitochondrial proteins. And right at the top you can see succinate oxidase with its wave and sub-unit, a high peptide homology, and it has multiple disease associations. In other words, succinate dehydrogenase has been implicated in many diseases that have already been described.
And then the other one that I want to highlight particularly is the ATP synthase. I mean, that’s the major player in the mitochondrion. That’s the thing that makes ATP. And that also has a very high level of homology. It’s involved in respiration and it’s been implicated in many diseases.
The last point I want to make is over a paper that was published just a couple of weeks ago and this is from Dr. Giulivi’s group at
Davis. And it shows “Mitochondrial Dysfunction in Autism” as the title of the paper. The reference is right down at the bottom of the screen. This was published in JAMA in — December 1st, in JAMA.
And the two columns that I have highlighted, in fact, on the left is succinate dehydrogenase, or oxidase it is also called, and then the one to the right is adenosine triphosphatase or ATPase. And so those two proteins, which I’ve just pointed out, were heavily damaged.
In fact, the proteins that I showed in that slide before, all of them had 80 to 100 percent of a given important cystine residue was mercurated in our data. Okay. And this was from a brief 15-minute exposure of E. coli to mercury at 80 micromolar.
So just wrapping up, it’s already been mentioned, chronic diseases from Donna Lack’s (ph.) paper and on a survey on the NHANES data, shows that over a lifetime the chance of having mercury in your body increases. That’s already been mentioned and I think that it’s important for us to keep in mind that mercury may be involved in many, many diseases and certainly part of what I’ve shown you is the reason why. There’s almost no important system in the cell that is not hit by mercury.