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
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