21 May 2016

Most scientists believe that genes and environment each contribute to the onset of late-life neurodegenerative disease. However, solid evidence for environmental effects has remained elusive. Epidemiological studies can correlate exposure to chemicals with neurodegeneration, but fall short of demonstrating direct or causal relationships. A recent study estimated that more than 80 percent of the risk of chronic diseases comes from environmental exposures rather than genetics, and called on scientists to rigorously measure actual exposures in “exposome-wide” association studies by comparing biospecimens from cases and controls (see Rappaport, 2016). Two recent papers describe new approaches that strengthen the evidence for a harmful effect of chemicals, particularly pesticides, on the brain. In the May 9 JAMA Neurology, researchers led by Eva Feldman and Stuart Batterman at the University of Michigan, Ann Arbor, reported that people with amyotrophic lateral sclerosis harbored higher levels of several long-lasting toxicants, aka man-made toxins, in their blood than controls did, and were also more likely to have worked with pesticides. The authors estimated that pesticide exposure heightened the risk of developing ALS several fold.

Commentators called it an important paper and praised the inclusion of blood work as an objective measure of exposure. “It’s been difficult to establish clearly which environmental toxicants could be important for ALS. This adds weight to that effort,” Freya Kamel at the National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, told Alzforum. Robert Haley at the University of Texas Southwestern Medical Center, Dallas, agreed, adding, “Unfortunately, there are too few studies like this.” The next step will be to replicate the findings in larger groups, researchers stressed.

The other paper debuts a method for examining the risk posed by toxicants as well as shedding some light on possible mechanisms. Researchers led by Mark Zylka at the University of North Carolina, Chapel Hill, tested about 300 chemicals in mouse cortical cell cultures. As reported in the March 31 Nature Communications, they found a set of relatively new fungicides that harmed mitochondria and elicited nuclear gene expression changes similar to those seen in Alzheimer’s disease, Huntington’s, and autism. Ominously, use of these fungicides has mushroomed over the past decade, with some companies now planning to put them in building materials because of fears of toxic molds. Zylka hopes this study and others to come might prompt people to think twice about that. “The data support the idea that transcriptional readouts [from neuronal cultures] could be used to prospectively identify candidate risk factors before they become a problem,” Zylka told Alzforum.

Blood Evidence of Exposure
Epidemiological studies have long hinted at an association between ALS and exposure to pesticides, heavy metals, and industrial solvents (see Johnson and Atchison, 2009). Military service, especially during the 1991 Gulf War, has been found to correlate with an up to threefold increased risk for ALS in some studies (see Haley, 2003; Horner et al., 2003; Weisskopf et al., 2015). Feldman, Batterman, and colleagues previously reported a link between ALS and pesticide and/or fertilizer exposure in a small case-control study in Michigan (see Yu et al., 2014). Exactly what these chemicals were, however, remained murky.

To obtain better evidence, co-first authors Feng-Chiao Su and Stephen Goutman expanded the study to 156 people with ALS and 128 age-matched controls, and included the collection and analysis of blood samples. Participants completed detailed surveys on their occupational and residential histories. In keeping with previous studies, military service doubled the odds of ALS, while occupational exposure to pesticides pumped it up fivefold. Other occupations or behaviors did not affect risk. 

Surprisingly, jobs that involved working with lead correlated with lower risk. Although many previous studies report higher risk with lead exposure, there is conflicting data as well, Batterman noted (see Kamel et al., 2002; Callaghan et al., 2011). For example, one study associated lower blood lead levels with a shorter survival time in ALS patients (see Kamel et al., 2008). “I think the jury’s still out on lead,” Batterman told Alzforum. Lead has also been implicated in Alzheimer’s disease, with a recent paper reporting that mice exposed to the metal early in life had long-lasting epigenetic changes (see Wu et al., 2008; Bakulski et al., 2012; Eid and Zawia, 2016). 

For the blood work, the authors tested for 122 toxicants that are known to persist in the body for decades. These fell into three categories: organochlorine pesticides, polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDEs). PCBs are commonly found in electrical insulation, while PBDEs are used as flame retardants. The list did not include heavy metals such as lead, nor short-lived toxicants. The authors analyzed toxicant levels singly and in combinations to find those that most consistently associated with ALS. They turned up three pesticides (pentachlorobenzene, β-hexachlorocyclohexane, and cis-chlordane), two PCBs, and one PBDE that were higher in cases than controls. Each associated with about twofold increased risk of ALS, except for cis-chlordane, which ballooned risk fivefold.

Curiously, a handful of chemicals associated with a lower risk of ALS. Some of the findings in this study might be a result of statistical chance in small subgroups, rather than a true effect, Batterman noted. “We need to look for consistency among different exposures, and confirm results using different datasets and larger samples,” he said.

Do the high blood levels of some chemicals mean that those particular toxicants predispose people to ALS? Possibly, but commenters noted that it is equally likely that some of these chemicals merely serve as markers for the ones that actually do the damage. For example, because people who work with pesticides are likely exposed to multiple different ones, the organochlorines measured in this study may simply flag those people with the highest rates of pesticide exposure. Quickly metabolized organophosphate pesticides such as malathion are believed to be more potent neurotoxins than the organochlorines, Haley said. Exposure to sarin nerve gas, a particularly deadly organophosphate, may explain the high rates of ALS in troops who served in the Gulf War, he added. Another possibility is that persistent toxicants, which are normally stored in body fat, are higher in ALS patient plasma because weight loss from the disease mobilizes fat stores and releases them into the bloodstream, pointed out Jacquelyn Cragg and Marc Weisskopf at the Harvard T.H. Chan School of Public Health, Boston, and Merit Cudkowicz at Massachusetts General Hospital, in an accompanying JAMA Neurology editorial.

Follow-up studies in larger cohorts are needed to confirm these associations, commenters said. “[The data] should be seen as a call for additional epidemiological and laboratory studies to identify mechanisms by which these chemicals and others may contribute to ALS risk,” Jason Richardson at Rutgers-Robert Wood Johnson Medical School, Piscataway, New Jersey, wrote to Alzforum (see full comment below).

In ongoing work, Batterman and Feldman are recruiting more volunteers into the study and taking additional blood samples to follow participants over time. They also plan to expand the control group to include a more diverse population. Initially, controls were recruited through the Internet and turned out to be better educated and more urban on average than the participants with ALS. Because urban populations might have less pesticide exposure than those who live near farmlands, that could have skewed the results, commenters noted. Batterman and colleagues will also examine how toxicant exposure interacts with other ALS risk factors, and how it affects survival.

What do the findings mean for the average citizen? Batterman noted that the blood exposure levels seen in this study are common in the population. Nearly everyone in the United States has been exposed to flame retardants, due to their use in furniture, plastics, and building materials, researchers said. Pesticide exposure comes through diet as well as yard work.

Many of these chemicals have now been banned, such as PCBs and most organochlorine pesticides, or are being phased out, like PBDEs. Perhaps the most infamous banned organochlorine pesticide was DDT, which some evidence has linked to higher risk of Alzheimer’s (see Jan 2014 news). Most exposures to these groups of toxicants occurred years or decades ago. Other recent regulations try to limit toxicant exposure. For example, Batterman noted that building codes in Michigan now require that garages attached to houses have tightly sealing doors or exhaust fans to prevent fumes from paints, solvents, pesticides, and cars from entering living areas.

Transcriptional Profiling Provides Another Method
New threats may hover on the horizon, however. More than 80,000 chemicals have been approved for use in the environment and, for most of them, data about their effects on the brain is scarce, Zylka told Alzforum. He wanted to find an efficient way to screen for potential toxicants that might increase the risk of autism, which he studies. To do this, joint first authors Brandon Pearson and Jeremy Simon developed a cellular assay using mouse cortical neurons. They tested 294 chemicals on these cultures, at concentrations that did not kill the cells, and measured gene expression changes by microarray.

The assay turned up a group of eight chemicals that all produced similar gene-expression changes, suppressing synaptic and ion channel genes while promoting inflammatory ones. When the authors compared this profile to published expression data for various brain disorders, they found that the changes mimicked those seen in autism, Alzheimer’s, and Huntington’s diseases, as well as in normal aging (see Feb 2004 news; Durrenberger et al., 2015; Voineagu et al., 2011). The chemicals in this group included rotenone, a pesticide that poisons mitochondrial complex I and induces Parkinson’s-like symptoms in animal models (see Nov 2000 news). Other chemicals belong to a recently developed class of fungicides that act on mitochondrial complex III.

Because mitochondrial complexes I and III are involved in superoxide production, the authors measured levels of this reactive oxygen species in neuronal cultures treated with these toxicants. As expected, the fungicides hiked up superoxide levels (see image above). The chemicals also destabilized microtubules in the cytosol and caused neurons to swell. Treatment with a microtubule stabilizer largely prevented these phenotypes, as did treatment with sulforaphane, an antioxidant found in broccoli that has been used to treat autism.

The findings suggest these fungicides could pose a health risk. They are present on foods, particularly leafy greens such as spinach, in concentrations as high as 20 parts per million. This equates to a concentration of roughly 5 μM, Zylka said. The most abundant fungicide, pyraclostrobin, damaged neuronal cultures at concentrations of 1 μM. While it is still unknown how much fungicide would enter a person’s blood or brain through diet, the numbers indicate a plausible risk of toxicity, he suggested. Moreover, use of these fungicides has been climbing since the early 2000s. “The more I learn about these fungicides, the more worried I get,” Zylka told Alzforum.

In the big picture, Zylka believes his cellular model might be an efficient way to identify potential toxicants before they harm people. He plans to profile thousands of additional chemicals, and will test leading candidates in animal models to see if they affect behavior.

In addition, by administering toxicants to animals with susceptibility genes for autism, Zylka hopes to uncover gene-environment interactions. William Atchison at Michigan State University, East Lansing, noted that such interactions are difficult to detect in epidemiological studies because many genetic risk factors are so rare. However, these interactions could explain why some people develop ALS or other disorders after exposure to pesticides, and others do not, he noted. For example, polymorphisms in paraoxonase genes have been associated with ALS in several studies (see Saeed et al., 2006; Slowik et al., 2006; Cronin et al., 2007). These genes encode proteins that detoxify pesticides, but the polymorphisms have so far not shown up as risk factors in ALSGene, perhaps due to their rarity in the population.—Madolyn Bowman Rogers

Comments

1. • Jason Richardson
     Northeast Ohio Medical University
   • Posted: 25 May 2016

   I think this research is significant for a couple of reasons. As with our previous work, this study actually measures the levels of persistent toxicants in the body so that specific chemicals that may contribute are identified. This is particularly important so that follow-up studies in the laboratory can be performed to determine whether or not specific chemicals may be part of an established or novel pathogenic pathway. Secondly, it highlights the need to also consider genetic susceptibility. Although the odds ratios established are not what one would consider consistent with causality, it does provide information that may shed light on genetic pathways that may work in concert with exposures to increase risk of ALS. On the other hand, some of the chemicals identified may really only be a measure of exposure rather than being the specific chemicals that may contribute to the etiology of ALS.

   There are a couple of weaknesses that we also dealt with. As with our study, Su and colleagues could only reliably measure "legacy" chemicals, such as the persistent organic pollutants. Because of this, we are still limited with self-reports on current use pesticides that may also contribute to ALS susceptibility. Secondly, some of the blood concentrations are a bit surprising and appear to be present in greater frequency than in the general population, as reported by the CDC’s National Health and Nutrition Examination Survey. This may indicate that there was something a bit unusual about this population.

   Overall, I found this to be a very strong preliminary study that should be seen as a call for additional epidemiological and laboratory studies to identify mechanisms by which these chemicals and others may contribute to ALS risk. Finally, it further reinforces the need to include environmental factors into the thought process about complex disease, even in those that are thought to be heavily influenced by genetics.

    

   View all comments by Jason Richardson

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References

News Citations

1. Does DDT’s Toxic Legacy Include Alzheimer’s Disease? 29 Jan 2014
2. New Microarray Data Offer Grist for AD Hypothesizing Mills 8 Feb 2004
3. A New Link Between Pesticides and Parkinson's Disease 22 Nov 2002

Paper Citations

1. Rappaport SM. Genetic Factors Are Not the Major Causes of Chronic Diseases. PLoS One. 2016;11(4):e0154387. Epub 2016 Apr 22 PubMed.
2. Johnson FO, Atchison WD. The role of environmental mercury, lead and pesticide exposure in development of amyotrophic lateral sclerosis. Neurotoxicology. 2009 Sep;30(5):761-5. Epub 2009 Jul 24 PubMed.
3. Haley RW. Excess incidence of ALS in young Gulf War veterans. Neurology. 2003 Sep 23;61(6):750-6. PubMed.
4. Horner RD, Kamins KG, Feussner JR, Grambow SC, Hoff-Lindquist J, Harati Y, Mitsumoto H, Pascuzzi R, Spencer PS, Tim R, Howard D, Smith TC, Ryan MA, Coffman CJ, Kasarskis EJ. Occurrence of amyotrophic lateral sclerosis among Gulf War veterans. Neurology. 2003 Sep 23;61(6):742-9. PubMed.
5. Weisskopf MG, Cudkowicz ME, Johnson N. Military Service and Amyotrophic Lateral Sclerosis in a Population-based Cohort. Epidemiology. 2015 Nov;26(6):831-8. PubMed.
6. Yu Y, Su FC, Callaghan BC, Goutman SA, Batterman SA, Feldman EL. Environmental risk factors and amyotrophic lateral sclerosis (ALS): a case-control study of ALS in Michigan. PLoS One. 2014;9(6):e101186. Epub 2014 Jun 30 PubMed.
7. Kamel F, Umbach DM, Munsat TL, Shefner JM, Hu H, Sandler DP. Lead exposure and amyotrophic lateral sclerosis. Epidemiology. 2002 May;13(3):311-9. PubMed.
8. Callaghan B, Feldman D, Gruis K, Feldman E. The association of exposure to lead, mercury, and selenium and the development of amyotrophic lateral sclerosis and the epigenetic implications. Neurodegener Dis. 2011;8(1-2):1-8. Epub 2010 Aug 4 PubMed.
10. Wu J, Basha MR, Brock B, Cox DP, Cardozo-Pelaez F, McPherson CA, Harry J, Rice DC, Maloney B, Chen D, Lahiri DK, Zawia NH. Alzheimer's disease (AD)-like pathology in aged monkeys after infantile exposure to environmental metal lead (Pb): evidence for a developmental origin and environmental link for AD. J Neurosci. 2008 Jan 2;28(1):3-9. PubMed.
11. Bakulski KM, Rozek LS, Dolinoy DC, Paulson HL, Hu H. Alzheimer's disease and environmental exposure to lead: the epidemiologic evidence and potential role of epigenetics. Curr Alzheimer Res. 2012 Jun;9(5):563-73. PubMed.
12. Eid A, Zawia N. Consequences of lead exposure, and it's emerging role as an epigenetic modifier in the aging brain. Neurotoxicology. 2016 Sep;56:254-261. Epub 2016 Apr 8 PubMed.
13. Durrenberger PF, Fernando FS, Kashefi SN, Bonnert TP, Seilhean D, Nait-Oumesmar B, Schmitt A, Gebicke-Haerter PJ, Falkai P, Grünblatt E, Palkovits M, Arzberger T, Kretzschmar H, Dexter DT, Reynolds R. Common mechanisms in neurodegeneration and neuroinflammation: a BrainNet Europe gene expression microarray study. J Neural Transm (Vienna). 2015 Jul;122(7):1055-68. Epub 2014 Aug 13 PubMed.
14. Voineagu I, Wang X, Johnston P, Lowe JK, Tian Y, Horvath S, Mill J, Cantor RM, Blencowe BJ, Geschwind DH. Transcriptomic analysis of autistic brain reveals convergent molecular pathology. Nature. 2011 May 25;474(7351):380-4. PubMed.
15. Saeed M, Siddique N, Hung WY, Usacheva E, Liu E, Sufit RL, Heller SL, Haines JL, Pericak-Vance M, Siddique T. Paraoxonase cluster polymorphisms are associated with sporadic ALS. Neurology. 2006 Sep 12;67(5):771-6. Epub 2006 Jul 5 PubMed.
16. Slowik A, Tomik B, Wolkow PP, Partyka D, Turaj W, Malecki MT, Pera J, Dziedzic T, Szczudlik A, Figlewicz DA. Paraoxonase gene polymorphisms and sporadic ALS. Neurology. 2006 Sep 12;67(5):766-70. Epub 2006 Jul 5 PubMed.
17. Cronin S, Greenway MJ, Prehn JH, Hardiman O. Paraoxonase promoter and intronic variants modify risk of sporadic amyotrophic lateral sclerosis. J Neurol Neurosurg Psychiatry. 2007 Sep;78(9):984-6. PubMed.

External Citations

1. ALSGene

Further Reading

News

1. Potential Guamian ALS Toxin Also Scourge of the Baltic Sea? 18 Jun 2010
2. Dietary Toxins and Neurodegenerative Diseases—Guam Revisited 10 Nov 2003
3. Twin Study Suggests Epigenetic Differences in AD 21 Aug 2009