In this population-based case-control study, agricultural application of both maneb and paraquat within 500 m of a residence during the period 1974–1999 greatly increased the risk of developing PD, especially when exposure occurred between 1974 and 1989 or when PD was diagnosed at a younger age (≤60 years). Exposure to both pesticides during the earlier time window (1974–1989) also doubled the risk for older cases. Associations were particularly strong for younger-onset patients (≤60 years), who would have been children, teenagers, and young adults during the exposure period: Among those exposed in the earlier time window, risk was increased more than 4-fold with exposure to both pesticides and more than 2-fold with exposure to just 1 of the pesticides. Consistent with some theories regarding the progression of PD pathology (25
), these data suggest that the critical window of exposure to toxicants may be years before the onset of motor symptoms which lead to diagnosis.
Pesticide and herbicide exposures have previously been implicated in idiopathic PD. Paraquat is structurally similar to the toxic metabolite (MPP+) of the 1-methyl-4-phenylpyridinium ion (a metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), an agent known to induce Parkinsonian symptoms in humans that has been widely used to study Parkinsonism in animal models (26
). MPP+ is believed to cause cell death by interfering with mitochondrial respiration (27
), because it concentrates in mitochondria and inhibits complex I of the electron transport chain (28
). Many lines of evidence point to possible mitochondrial dysfunction in PD. Several genes have been identified in familial forms of PD that are linked to mitochondrial function (PINK1
), and in sporadic cases of PD, pathologic free radical reactions that damage mitochondria and decrease electron transport activity have been described (29
). Impaired electron transport hampers adenosine triphosphate production and leads to the diversion of electrons from their normal electron transport recipients and, thus, further formation of damaging free radicals (29
Although paraquat is also used to induce Parkinsonism in some animal models, the mechanism by which it produces symptoms is not yet understood (30
). Recent mammalian and yeast-cell experiments suggest that mitochondria take up paraquat actively across their membranes, where complex I reduces it to the paraquat radical cation that subsequently produces mitochondria-damaging superoxide (31
). It has also been suggested that maneb may inhibit the ubiquitin proteasome system, thereby damaging the dopaminergic neuron (24
). Additionally, maneb has been linked to Parkinsonism in mice also exposed to paraquat. In 3 recent studies, investigators reported that only when mice were exposed to a combination of the fungicide maneb and the herbicide paraquat (paraquat + maneb), not to either pesticide alone, did they exhibit increased neuronal pathology (7
), age-dependent motor degeneration and progressive reductions in dopamine metabolites and dopamine turnover (8
), and reduced tyrosine hydroxylase and dopamine transporter immunoreactivity (9
The fungicide maneb and the herbicide paraquat are both used in the Central Valley of California and are often used on the same crops, including potatoes, dry beans, and tomatoes. The average amount of maneb applied near the homes of these study subjects was relatively stable throughout both time windows; however, annual paraquat exposure increased during the later (1990–1999) time window. Persons living near fields sprayed with maneb and paraquat may also be exposed to a host of other agricultural chemicals. When we controlled for the influence of other groups of pesticides suspected a priori to be risk factors for PD in our study, the odds ratios for combined maneb and paraquat exposure and PD in the younger subjects were still in the 3- to 6-fold range and statistically significant; however, our precision decreased, probably because of correlated exposures. Correlation between pesticides is an inherent problem when assessing the effects of human exposure. However, since adjustment for other pesticides did not remove the association for maneb and paraquat, our data provide compelling evidence that these 2 pesticides may in fact affect PD risk in humans, as has been suggested by animal experiments.
Paraquat and maneb are applied by ground, aerial, and backpack methods; however, paraquat has a much longer field half-life of 1,000 days (33
), as compared with only 12–36 days for maneb (34
). Both chemicals bind strongly to soil, though, and are not thought to be a threat to groundwater (35
). Such strong binding could result in contaminated soil getting blown or tracked into homes by wind, pets, and shoes, thereby increasing exposure for persons who live closer to agricultural application sites (3
In a previous validation study, our prediction model for a serum measure of dichlorodiphenyldichloroethylene (DDE) explained 47% of the biomarker's variance (39
). Additionally, our GIS-derived measure of organochlorine exposure identified persons with high serum DDE levels reasonably well (specificity of 87%) (39
Although our GIS model allowed us to calculate the number of pounds of each active ingredient applied per acre within a 500-m buffer, these quantities are not comparable across pesticides. That is, a pound of active ingredient does not represent the same human neurotoxicity across pesticides, and no information currently exists that would allow us to standardize these measures. Thus, while we believe that our model provided us with an accurate indicator of any pesticide exposure from applications close to a residence, our exposure measure cannot be considered quantitative beyond a crude rank ordering of low/medium likelihood of exposure and high likelihood of exposure. Since we hypothesized that coexposure to 2 pesticides, maneb and paraquat, would increase the risk of PD, we also lacked the statistical power to perform extensive categorical analyses (note that only 3 cases and 1 control were exposed solely to maneb). We conducted additional analyses after dichotomizing pounds per acre at their median and mean levels and found that exposure to both pesticides at the highest level was associated with PD, especially in persons aged ≤60 years; however, wide confidence intervals surrounding our point estimates rendered these results generally uninformative (results not shown).
In only 1 previous analysis, conducted within the Agricultural Health Study cohort (40
), did researchers assess the effects of maneb and paraquat exposures. Statistical power was limited by the small number (n
= 78) of incident cases identified during follow-up and the very small number (n
= 4–10) of cases exposed to maneb/mancozeb (OR
2.1) and paraquat (OR
1.4). In a small Taiwanese study, the only case-control study to date with sufficient statistical power to examine exposure to the herbicide paraquat, Liou et al. (41
) reported a 4- to 6-fold increase in PD risk among long-term applicators. In a case-control study from the Mayo Clinic (Rochester, Minnesota), Brighina et al. (42
) presented associations between self-reported pesticide exposure and PD in subjects younger than 60 years only (for all pesticides, OR
1.80, 95% CI: 1.12, 2.87; for herbicides, OR
2.46, 95% CI: 1.34, 4.52).
Our exposure estimates did not depend on the subject's recall of pesticide exposure and are therefore unlikely to have been biased by differential exposure misclassification. Since all of our PD diagnoses were clinically confirmed, we expect disease misclassification to have been minimal. Nondifferential exposure misclassification is a possibility in our study and may have attenuated our effect estimates.
Our results may be biased if cases and controls selected themselves into our study according to their potential for pesticide exposure, but our subjects were not asked to self-report environmental exposures and probably were unaware of their true historical exposures. There is no reason to suspect that cases and controls would have chosen to participate on the basis of their historical residence near certain agricultural plots. We saw no difference in estimated effects when we restricted analyses to only those subjects with more (≥12 years) or less (<12 years) education. Similarly, we saw no difference in our results when we restricted the sample to persons whose addresses had been mapped with high precision in the tricounty area during the period 1974–1999 (363 cases, 336 controls).
Our analysis has confirmed 2 previous observations from animal studies: 1) exposure to multiple chemicals may potentiate the effect of each chemical (of interest, since humans are often exposed to more than 1 pesticide in the environment) and 2) the timing of exposure is important. To our knowledge, this is the first epidemiologic study to provide strong evidence that 2 specific pesticides, suggested by animal research as potentially acting synergistically to become neurotoxic, strongly increase the risk of PD in humans, especially given combined exposure and when encountered earlier in life.