PADRECC NATIONAL VANTS AUDIO CONFERENCE Agent Orange, Pesticides and Parkinson’s Disease Jeff Bronstein, MD, PhD January 13, 2011 Thanks, Virginia. In the next hour, I am going to speak a little bit about the evidence that pesticides may be involved in contributing to the causes of Parkinson’s Disease and we will end with the evidence that Agent Orange might be associated as well, which is particular relevance to our Veterans who served in Vietnam. When we try to understand the causes of Parkinson’s Disease, they think about two big areas. One is the environment and the other is genetics. So, today we are going to talk about the environmental studies. The way we approach environmental studies are two different models. One is taking clusters or small groups of patients with very high incidence and try and figure out what caused it in them. There are larger studies called case-control studies, a population based study, where we will identify a much larger group, say in the 100’s of patients with Parkinson’s and then you try and find people who live in the same area that have many of the same characteristic agents that match and then try and find out what is different about them, different exposures, different lifestyle variations. Then finally, the prospective studies. These are much harder to do, but offer several advantages as well. So, in the next several slides, I am go through some examples of each one of these that have supported the idea that pesticides may be involved in contributing to Parkinson’s Disease, at least in some people. Next slide. So, one of the first clusters that was noticed in modern times, at least, was in the 1970’s and then in the early 1980’s. In 1976, there were a few people that we exposed to a compound called MPPP Plus and they were given Demerol and they developed an acute onset of Parkinsonism, but the main discovery of MPTP was made by Dr. William Langston in Santa Clara back in the early 80’s. He first described four patients, but later followed as many as 30 or so that developed acute onset Parkinson’s after shooting some drugs that were later found and had been manufactured by some students at Berkley. They were trying to make Demerol. Obviously, MPTP is not something that any of us have been exposed to, but it did provide some very important insight into some underlying mechanisms that may be in play in regular old Parkinson’s Disease. Next side. This slide demonstrates what we know about how MPTP works. Basically, MPTP gets converted quickly by an enzyme called MAO-B and this generates the compound called MPP+. Now MPP+ can only get into a cell using a transporter and more specifically, dopamine transporter, which is shown as DAT in this picture. Once it binds to the transporter, it gets transported into the dopamine cells and then binds the mitochondrial, which are the power generators for all of us. That’s where we convert oxygen in ATV. MPP+ blocks what we call Complex One or the first enzyme complex, which is critical for making ATV. Not only does this decrease the function of mitochondrial, the decrease is pretty radical. Next slide. Another interesting aspect of MPP is its remarkable similarities of some other compounds that we use in pesticides. So, for paraquat, as you can see in this picture, is very, very similar MPTP and MPP+ actually is a pesticide, called cyperquat. So, these are some of the compounds that are actually out there in the environment already. This lead to a number of studies seeing if some of these pesticides in general could be associated with Parkinson’s Disease. Next slide. So, the first studies we looked at, these are the larger case control and population studies, looking at large groups of people and you will notice in rural areas have a higher incidence for Parkinson’s then in cities and specifically, farming seem to have a higher incidence. Now, on a number of slides, you are going to see OR, which stands for odds ratio and anything above one (1) means that it is a higher risk and anything below one (1) means it has a lower risk. To the right of that you will see competence intervals or CI. So, if it is above one (1) as it is in farming 1.1 – 1.9, that means that it is highly statistically significant. You can see that drinking well water, farming and professional pesticides are associated with a higher incidence of Parkinson’s from anywhere from 30% - 90% increased risk. Next slide. Now, there have been several studies recently reviewed by Brown and Colleagues approximately five (5) years ago and there have been additional studies since that have looked at pesticide use in PD. Almost all of these are case controlled studies, but you can see in this analysis that almost every one has been associated with a higher incidence for Parkinson’s Disease. Now, we are well aware of publication ??, but even when taking into account that the vast majority of studies have found the association with exposure and development of Parkinson’s Disease. Next slide. These associations have been found in four (4) different continents and in all of the continents where it has been studied and appears to be independent of farming. But, there are still some very important methodological concerns, which quickly gave us false/positive results. The most important one is called recall bias. This is a real concern. If you ask a Parkinson patient if they have been exposed to pesticides and toxins, they are much more likely to remember every time they have been exposed to something than in somebody who doesn’t have this disorder or normal controls. In almost all these studies, this self-identified exposure. This is a major weakness, which has really concerned many people before they were convinced that this association is real. There are issues with the control population, while some of them are not properly matched, so there may be other co-variables that may associate with it falsely. Then, our study in pesticides has really been hindered by the fact that we hadn’t identified any individual agents. There are thousands of different pesticides out there with many different mechanisms of action and it would be unreasonable to believe that all of them or many of them are associated with Parkinson’s Disease. There are going to be some very important selected toxins that are more likely involved and to be able to study it to be able to identify them. So, these are some of the problems that many groups have tried to address and we are going to review a few of those now. Next slide. The one way to address the Recall Bias is to do a prospective study. So, you take a large group of people, thousands of people that are well and then you ask them every question you can think of, follow them for ten (10), fifteen (15), twenty (20) years, some of them are going to get Parkinson’s and then you can go and review those questionnaires and see what people said different in their lives when they were well. So, what we call Recall Bias because they answered questions before they had developed Parkinson’s Disease. So, the first study is the Honolulu – Asia Study, which some of these slides were generously provided by ?? who is the driving horses of Parkinson’s for part of this study and they followed over 1,000 Japanese – American men and asked a few questions early on in this study about Asia and pesticides, etc. When this was reported in 2002, 116 cases of Parkinson’s among these people had concurred. Next slide. You can see that the longer these people working in the plantation, the higher risk they had of developing Parkinson’s Disease. It was about a two-fold increase in risk and there’s kind of dose response, the longer, the higher the risk. Next slide. When asked cumulative years of pesticide exposure and again, a crude measure but the Recall Bias, that was also showing the same trend, the more pesticide, the higher risk. Next slide. The second study was a much larger one by Alberto Ascherio and Alberto looked at questionnaires in the cancer prevention study, which followed 143,000 individuals over approximately a nine (9) year period. The exposures were very crude and it was assessed whether you were exposed or not and the length of exposure and the cases, in other words, whether who had Parkinson’s also by a questionnaire throughout this. So, there’s no Recall Bias, but there may be some problems with identifying the cases accurately, as well as the degree of exposure is somewhat crude. Next slide. But, even with those weaknesses, they found a 70% increase of risk of Parkinson’s in those people that self-identified as having PD and were exposed to pesticides. What makes this even more believable is that there were several toxins asked about in this study and you can see in this. The only one that showed a positive association with pesticides. That adds a pretty strong evidence that there is an association. Next slide. Now, these studies are strong because they are large, they control well for many confounds that we are not going to talk about, other risk factors with the kind of things like smoking, which is ?? related and these are controlled for in these studies. They are prospective, which really helped with the Recall Bias. So, those are very important strengths. There are some important weaknesses or limitations as well. We have very little data on the duration and intensity of the exposures and really no information on specific pesticides and in most of these studies there are a fairly high rate of misdiagnosis, somewhere maybe in the neighborhood of 25%. Now, generally these sorts of weaknesses are the ones that we lose significance instead of having false/positives, so it very well may mean that we are underestimating the effect, but we still need to identify pesticides as individual agents if we want to go further with our studies. Next slide. So this is addressed by a study by Carly Kamel at the Parkinson’s Institute, along with several researchers and they did the prospective study in a very highly exposed group of people. These were pesticide applicators in Iowa in North Carolina. So, they took applicators and asked them who had Parkinson’s and who didn’t. The ones that said they have it already are prevalent cases and the ones that developed it over the five (5) years or so that they followed these people are called incident patient cases. They asked questions about exposure and what they were exposed to prospectively, just like in the previous prospective studies and what they have found so far is that a few of the pesticides have had a higher incidence of Parkinson’s. So, they identified 2, 4, 5 – T, which interestingly is a component of Agent Argent, which we will talk about in a few minutes and trifualin is another one. Now one of the problems with this study is that so far it has only been five (5) years and they have only identified 90-something incident cases, which really limits statistical analysis, in other words, dividing them up into many different pesticides. It should develop more patients as we go on and strengthen the statistic. But, again, it gives us more support for the association pesticides and now we are starting to see some specific agents being identified. Next slide. The problem with this study, as I mentioned is that so far ?? and the exposures were self-reported. So, what the agents were and duration is clearly going to be less accurate because people are still reporting and they have motivations to over report, regulatory agencies, etc., and the preciseness of those reports are by memory. The diagnosis is also self-reported. These are some weaknesses. But it did control a variety of other potential compounds and it did address a couple of the specific agents that may be involved. Next slide. So with those limitations, Dr. Beate Ritz, who’s on the faculty at UCLA and somebody that I have been fortunate enough to work with, came up with a strategy to try and address the weaknesses in previous studies. He called the study PEG or Parkinson’s Environment Gene Study, which has been funded by the NIEHS. Basically, the idea here is to identify 400 incidence cases of new diagnosed cases within a few years of diagnosis that we did in the early 2000’s and 400 population controls that live in the same areas and are age and sex matched. We can determine the lifetime exposure of at least 35-year exposure to pesticide based on a state mandated pesticide use registry. So, these patients were all identified in Central Valley, which is a very large farming community and 20% of the nation’s pesticides are used there. Now, all the pesticide use commercially needs to be registered, the amounts, what acre of land, what parcel of land, when it was applied, all put in this database. Most of the time, it’s being sprayed. So, something that we learned through some early elevating studies is that if you were in the neighborhood, say 500 meters of where it is being sprayed, that is a good marker for the amount of exposure. Next slide. This slide you are seeing here is actually from the Central Valley of somebody spraying a pesticide. I confirmed the diagnosis of most of these patients. The ones that weren’t confirmed by me, were evaluated by one of my colleagues. These aren’t self-reported, these are actually confirmed by a movement disorder specialist. Next slide. Dr. Ritz and her colleagues developed a very sophisticated program using Google Earth and merging it with her database in the pesticide use registry database can now estimate exposure by where people lived and worked during those 35 years. Next slide. The number of papers that were published, I want to show you one because it presents data and a few aspects. One is that the context that people are exposed is important. In other words, not everybody who is exposed to pesticides is going to get Parkinson’s and it is almost certainly going to be their genetic background will have some influence. In this study, it looked at the dopamine transporter, polymorphisms, using either common variance, the application for common variance that are in the community. If these have the common variance shown here by risk alleles, they have very modest and nothing statistically significant effect on you developing Parkinson’s Disease that shown with zero or low exposure on the left part of that graph. If you look at the right part of the graph, the people that we found that were highly exposed to, in this case, maneb and paraquat, that had a very little effect unless you had a more common risk alleles. So, if you happen to have two of the risk alleles and for exposed maneb and paraquat, you had a 4.5 increase, 450% increased risk of getting Parkinson’s Disease. A very significant finding. We have been able to do this for a number of genes now and for a number of exposures and this is just one example of that. So, these are large increases in risk. The other aspect of this is that the risk was evaluated not for the whole 35 years. WE broke it up into two. The first 25 years of exposure and then the most recent 10 years. The reason why we did this was that we don’t think that the recent 10 years was probably as important in developing Parkinson’s as 25 years based on various studies suggesting the disease begins many years before it’s clinically diagnosed. Sure enough, the exposure from that previous 25 years was much more highly associated with developing Parkinson’s Disease than in the people were only recently exposed. Next slide. So, at this point, we know that pesticides appear to be associated in developing Parkinson’s, but we still are not sure if it actually causes it. Associations don’t prove plausibility. So, I thought long and hard about how can we prove this or at least figure out whether it is positive or not. So, I listed a few of what I think are important criteria that are going to support a causality. One is do we figure out what they are doing biochemically to the cell and are those processes consistent with the processes that we think are involved in the pathogenesis of Parkinson’s Disease. So, a plausible mechanism of action. The second one is one we already talked extensively about, which is there association with people that are exposed more likely to get Parkinson’s in epidemiological studies. I think the answer is yes right now for that. Then, another important criteria is can we give these pesticides to animals and cells and recapitulate the pathological and behavioral features of Parkinson’s Disease. So, let’s review some examples of what I think support this, but we are not 100% there yet. Next slide. So, one of the plausible mechanisms that have been implicated in Parkinson’s is mitochondrial dysfunction and that was first implicated by the MPTP story that I talked about before and that is a Complex I inhibitor and it turns out that Complex I activities have been found in Parkinson’s patients in platelets and the brain, not knowing what their exposure status is or their genetic status. Some of the rare genetic that have been associated with Parkinson’s, actually are involved in normal functioning of mitochondria, suggesting that if you injure the mitochondria, either genetically or with the toxins such as MPTP, it can lead to Parkinson’s Disease or something that looks like Parkinson’s Disease. Next slide. So, it turns out that Rotenone is the classic Complex I inhibitor. It’s also a pesticide. We don’t have good exposure data with as we do for many of the commercially used pesticides because Rotenone is often used in residential areas, such as rose gardens, it is used a lot in organic farming in the 70’s and 80’s because it is considered an organic pesticide, despite its serenely potent toxicities, it is considered organic. What we do know is there are some case control studies, not perfect studies, but they have linked Rotenone to Parkinson’s Disease and a few of the references are listed here. I think importantly, the Agriculture Health Study has found this association as well, although much like our study in the Central Valley, they are very few people exposed to Rotenone. In our study, there were essentially none or less than 1% were exposed to Rotenone, so we really couldn’t evaluate it. In the Agriculture Health Study, they also have very few low exposures, but it is in the direction that we would expect. Then, there are many anecdotal reports, especially from the organic farmers of young onset Parkinson’s patients who use lots and lots of Rotenone in their farming. Next slide. The ?? is still not clear with Rotenone, but maybe there may be a suggestion. Now, we do know that Complex I inhibition is probably a plausible mechanism. Rotenone we know leads to oxidative stress and energy failure, oxidative stress is few radicals. We also know it leads to inhibition with proteasome. The proteasome is one of the key mechanisms for us getting rid of proteins that are either damaged or ?? in ourselves. We know that alpha synuclein is probably central somehow in pathogenesis Parkinson’s Disease because we see accumulations of alpha synuclein in the brains of people with Parkinson’s and it turns out that the proteasome is one of the ways that we get rid of alfsanuclean. So, if you block it, you are going to be increasing the amount of alpha synuclein is not good. We know if you double the amount you make, a gene duplication, you get Parkinson’s Disease. So, it’s reasonable to assume that blocking its derogation will increase the concentrations and also be toxic in a similar matter. Rotenone, also we know, inhibits microtubule function and that has been a mechanism that has been implicated in the development of Parkinson’s Disease as well. Next slide. A landmark study was performed by Dr. Greenamyre, who is out in Pittsburgh, who wanted to know how Complex I inhibition was involved in Parkinson’s Disease. So, he used Rotenone because that was the Complex I inhibitor and it just happens to be a pesticide, so it is very pertinent to this conversation. What this paper demonstrates is that you infuse into rats over the course of a month, many of them will develop lesions on the brain, such as loss of dopaminergic neuron and an aggregate shown on the right that look much like the alpha synuclein aggregates that we see in Parkinson in the brain. So, by using, not in the brain, but in the subcutaneously, you infuse Rotenone to develop many of the behavioral and pathological features of Parkinson’s Disease. Next slide. More recently, people have been able to actually feed small amounts into the gut of mice and slowly, progressively they develop the Parkinson’s Disease syndrome, much like the one that Dr. Greenamyre showed. What’s interesting about this is that it shows a progression that has been proposed for humans in Parkinson’s by a well known pathologist, Dr. Brock and it fits in with the Brock theory of Parkinson’s progression. Next slide. Another plausible mechanism is something I mentioned earlier, which is proteasome dysfunction. This first became ?? pathway when Parkin and another gene called UCH L1 was identified as a cause of Parkinson’s Disease. These are key components of the system in patients cause proteasome dysfunction. Again, we also know that alpha synuclein degradation is partially dependent on proteasome and we also have found that proteasome activities decrease in the brains and blood of Parkinson’s patients. So, another pathway that implicated Parkinson’s Disease. Next slide. So, we question several years ago whether pesticides may be increasing the risk of Parkinson’s Disease by inhibiting proteasome. So, we did our screen and found four major classes of pesticides that actually may inhibit the proteasome that we call relevant concentration. Interestingly, Rotenone, which is an inhibit Complex I, actually inhibits the proteasome in a very potent way and is through the Complex I inhibition, which turns out to cause inhibition of the proteasome. A large group of fungicides called dithiocarbamates. Ziram being one that seems to be the most potent, at least in our studies, was a major class that we found inhibited the proteasome and then there’s benomyl, which is another fungicide that is widely used and then the organocholorines, dieldrin and endosufan, also inhibited, but dieldrin has been taken off the market and the organochlorines are actually used at much lower levels, so they may not be quite as relevant. So, we have been focusing our studies on the first categories that are listed here. Next slide. The first question that we had when we found this problem with the diethyldithiocarbamates, ziram in particular and the proteasome inhibition was whether exposure to ziram associated with a higher rate of Parkinson’s. So, we probed our database from our PEG study and found sure enough that ziram was associated with an almost three fold increased risk of Parkinson’s Disease. You can see that this is a study that’s in press right now and it’s associated with paraquat exposure because there are very few people that were exposed to ziram alone. So, ziram and paraquat are often used or these people that use ziram and paraquat are often exposed to both. We had to put them together. Paraquat by itself, actually does not have any increased risk of Parkinson’s. This is by the way, a very potent effect. You can see it’s very highly significant. Next slide. We wanted to see giving these animals systemically the ziram or diethyldithiocarbamates we would recapitulate some of the factors. Now, others have done ?? which is in this category and it wasn’t quite as potent as ziram, along with paraquat, it may have demonstrated very nicely that that recapitulates with many of the aspects, pathologically and behaviorally. In this paper that published years ago, shows that ziram alone injected subcontaneously with infusion pumps over the course of a month ?? develop long lasting behaviors that are shown in the graph on top. The black bars are showing behaviors are getting worse, these are motor behaviors that assess Parkinson like behavioral features. You can see that eight weeks after the last exposure, they continue to progress with motor deficits. The gray bars are the controls. Below in the graph, you can see that the fibers dopamine, the density of those fibers was decreased. It’s interesting that there were decreases in the pattern that we see in the Parkinson’s patients, the ventral lateral aspects, moving the most severely and most significantly affected. Next slide. When we count 58 positive neurons, we did not see a significant decrease, but there was a trend or approximately 5% - 10% decrease in the number of neurons, dopamine neurons. So, the trend was in that direction, but we did not see significant decreases. Although, it must be remembered that we did a short two week exposure and only had one concentration. I think we are pretty fortunate to see the changes that we did, without having done several different dose finding experiments. Next slide. ??, ziram and other studies, but I just wanted to give you a few others that other people have done as well. The paraquat has been associated with an increased risk of Parkinson’s Disease, but mostly in combination with maneb and ziram, although there are a few studies that have suggested that they have been by itself in the risk factors. It’s not clear, but does seem to be linked somehow. The mechanism was surprising. We all thought it would be the Complex I inhibitor based on the structure, much like MPTP, but it turns out ?? and its structural similarities, it is not a sub?? for the dopamine transporters, because ?? that fit in, nor does it ?? Complex I. It does, people who call redox-cycling, which is a way of propagating oxidated stress and radical and that may be the mechanism. What’s interesting is if you inject systemically paraquat, using approximately 20% loss in dopamine or cells in animals, some very mild behavioral deficits, suggesting that it can recapitulate both behaviorally and pathologically some of the features. Dino Dalante has done a lot of this important work. Benomyl is actually a very interesting fungicide that we have been studying more and more. The Ag Health Study as well as PEG has shown that it is found in association with increased risk of Parkinson’s Disease with benomyl. We have been studying the mechanisms that, as I mentioned before, it’s a proteasome inhibitor, but it also inhibits the ALDH-2, we would ?? very important activity of the dopamine neurons because you need ALDH-2 to help get rid of very toxic dopamine adux and some other toxic aldahydes that seem to accumulate in Parkinson’s patients in some of these models. So, inhibitors of ALDH-2 may in fact be important contributors and benomyl is a very potent ALDH-2 inhibitor. It also microtubual, much like rotenone and some other pesticides just another possible mechanism. The recapitulation of some of the characteristics of Parkinson’s are still underway. We have looked at primary neuronal cultures, where these are big sculptures of dopamine cells and non-dopaminergic neurons, we exposed them to benomyl or its ?? selected loss of dopaminergic neurons and in fact ALDH seems to be the key mechanism in the ?? is the toxicity of the culture, but the animal models have not been done yet. Next slide. What about Agent Orange? Well, its been over a year now since it was announced that people in Vietnam that were exposed to Agent Orange will now be able to get service connection for Parkinson’s Disease among a couple of other disorders listed here. Next slide. Agent is a defoliant herbicide, so it is a type of pesticide. It was used to clear the jungles so there wouldn’t be good hiding places for snipers. The name was derived because the drums were colored orange. It’s really composed as two herbicides, 2, 4 – D and 2, 4, 5 – T and there is small amount of dioxin or TCDD, which is also in the Agent Orange. Next slide. You can see it was heavily used over approximately a ten year period and many of the people were exposed to that large scale spraying and my understanding is they were very poorly protected against exposure and were told that it was quite safe. These ?? Next slide. There is very limited epidimiologic data. There’s essentially none that I am aware of associating Agent Orange specifically with Parkinson’s Disease, the Vietnam Vets or anybody else, although in the Ag Health Study, they looked at this and you can see for 2, 4, 5 – T, that in incident cases, which were the ones we are most interested in, there was an increased risk of odds ratio of 1.8. This is some fairly good data showing that one of the components of Agent Orange, at least in this one study was associated with Parkinson’s Disease. We haven’t looked into the PEG study yet. Next slide. We don’t know really how these chemicals are working, what the mechanisms actually are. They may inhibit microtubule assembly and there was one small study in cerebellar granule cells. We know there is some acute toxicity, although it’s not basically limited or specifically associated with dopaminergic cell loss. We really have very limited information on what’s causing the neurotoxicity of the Agent Orange. Next slide. I just want to summarize the key point, which is we now do believe that, or many of us do, that there’s a clear association with pesticides and Parkinson’s Disease and that there are clearly some very plausible mechanisms that several lines of evidence suggest that these mechanisms may be involved in Parkinson’s Disease, both genetic and non-genetic forms and that some of these toxins and pesticides interfere with these processes. Finally, many of them will recapitulate both pathologic and behavioral features of Parkinson’s Disease in animal models. I think that accumulation of these pieces of evidence and many other studies, really strongly supports causality. The final piece of causality would be removing these from the environment and watching the incidents go down, but unfortunately, we have terrible incident data, both in the U.S. and in most countries. It takes so long to develop them, I don’t think this is going to be possible in our lifetime. The Ag Health Study has published and they have looked at some data suggesting that the people that were exposed to pesticides and this is a prospective study, the ones that took very good precaution to exposure, they were exposed, but they wore gloves and masks and all of their protective equipment, their risk was no greater than people that weren’t exposed. That’s not a bad surrogate marker from removing it from the environment and watch as the risk goes down. Again, the work is ongoing, but it’s been pointing that pesticides may be one of the contributors. I do want to stress, I am not saying the pesticides are the cause of Parkinson’s. I think there are going to be many causes and it may be one cause that may be a very small part of the population. We are all exposed to pesticides and some people are going to be more susceptible than others, obviously based on their genetic background and other exposures, but looking at the mechanisms may help us find other toxins and other genetic causes that may ?? other mechanisms that are involved. I think at this point, we have a few minutes to take some questions and thank you for your time. A few of the questions that were asked in the last session were, are there any forms of subtypes of Parkinson’s patients that have been associated with pesticides? So far, it seems like it is run of the mill Parkinson’s, so we can’t distinguish them at least typically, their characteristics, whether they are trend or dominate or a kinetic rigid, it does not seem to be a specific subtype. So, I don’t think right now we can say there’s a pesticide induced Parkinson’s Disease like a MPTP induced Parkinson’s Disease, but it looks like it increases the risk of the bread and butter Parkinson’s Disease that we all see. One other question that was asked, was there evidence that Vietnam Vets have a higher incidence of Parkinson’s degeneral and I think it’s a very important study to do, but as far as I know, has not been done. There are apparently databases of deployments and the use of Agent Orange, so it is doable, even though there are a number of obstacles to overcome, but it is something that would be very important and interesting to do. What is the age of onset of patients with PD in the Central Valley? The age of onset is about the same as the age of onset for most that we see. The average was in the early 60’s if I recall. I do want to say you have to be careful with this. There’s a large migrant population in the Central Valley. There are a lot of farm workers that are not documented and we were initially surprised that we had very few Latinos in our cohort. When we realized that most of those people when we reached out into these small health clinics that these people that were doing a lot of the direct farm work, were actually seeking medical care in Mexico. They are also quite younger. So, the ones that were exposed 20 years ago are almost certainly back in Mexico based on these trends. So, even though our cohort with pesticide exposure with the average age of onset, there are some limitations in our study, not being able to capture everybody that was exposed and that makes me hesitate and say that’s it is not definitely the same age of onset. Do pesticides really cause Parkinson’s Disease, wouldn’t you expect the age of onset to be earlier? To answer the question correctly, we don’t know if it is really early or not. Like I said, in our study, we don’t capture everybody. So, that’s one problem. We have broken down our cohorts, which is under 60 and over 60. In support of the fact that you expect it to be earlier, the odds ratios are much higher in the younger exposed. We have odds ratios for ziram if I remember of 7 or 8 that were under 60 where what I showed there was an average odds ratio of everybody. In every exposure I can think of, at least for our cohorts, the under 60 group of age of onset at the age of onset had a much higher odds ratio. That would be supporting it earlier on. On the other hand, I could argue that it’s not important because we don’t know what causes the average with Parkinson’s Disease, so maybe what all those exposures our genetic background would have the same influence. I don’t think it’s a must that it should be younger, but actually the evidence does support that. END OF CONFERENCE. PADRECC National Vants Audio Conference Agent Orange, Pesticides and Parkinson’s Disease Jeff Bronstein, MD, PhD January 13, 2011 Page 1