Rotenone, Paraquat, and Parkinson’s Disease

We identified 170 suspect cases and 644 potential controls and screened 156 (92%) and 542 (84%) of these, respectively (Figure 1). We conducted study evaluations in 137 (88%) of eligible suspect cases (including 4 initially indentified as potential controls who self-reported PD at screening) and 383 (71%) of eligible potential controls. Final diagnoses in suspect cases were PD (115), essential tremor/other tremor disorder (12), no neurologic diagnosis (5), dystonia (2), multiple system atrophy (2), and atypical parkinsonism not fulfilling any diagnostic criterion (1), the latter three cases subsequently termed “atypical parkinsonism.”

The analyses reported here include 110 PD cases and 358 controls who provided complete information on pesticide use and application practices. Cases and controls were similar for most characteristics assessed (Table 2), although cases were more likely to have impaired smell recognition or to have a family history of PD, and were less likely to smoke.

To validate our approach to pesticide exposure assessment, we compared information on use of four pesticides collected at enrollment in the AHS with information collected for FAME. Despite an average lapse of 9 years, agreement was good [DDT (dichlorodiphenyltrichloroethane) 79%, 2,4-D (2,4-dichlorophenoxyacetic acid) 84%, paraquat 85%, rotenone 93%].

Ninety-eight percent of men and 44% of women had ever used pesticides. Information on 18 pesticides used by > 10 subjects is presented in Supplemental Material, Table 2 (doi:10.1289/ehp.1002839). Results from analyses in men were similar to those in men and women combined (Supplemental Material, Table 2). Information on pesticides linked to complex I inhibition or oxidative stress, regardless of the number of users, is presented in Table 3. Most of the pesticides in the oxidative stressor and mitochondrial inhibitor groups were used infrequently, limiting individual analyses; but in general, use of these chemicals was more common in cases. Use of paraquat (OR = 2.5; 95% CI, 1.4–4.7) or any of the group of oxidative stressors (OR = 2.0; 95% CI, 1.2–3.6) was associated with PD (Table 3). Similarly, use of rotenone (OR = 2.5; 95% CI, 1.3–4.7) or any of the group of complex I inhibitors (OR = 1.7; 95% CI, 1.0–2.8) was associated with PD (Table 3).

Associations between ever use of paraquat or rotenone and PD were similar when exposures were truncated at 5, 10, or 15 years before the diagnosis or reference date [paraquat: 5 years prior OR = 2.7 (95% CI, 1.4–4.9), 10 years OR = 2.9 (95% CI, 1.6–5.5), 15 years OR = 3.1 (95% CI, 1.6–5.8); rotenone: 5 years prior OR = 2.3 (95% CI, 1.2–4.4), 10 years OR = 2.4 (95% CI, 1.3–4.6), 15 years OR = 2.4 (95% CI, 1.3–4.6]. Exposures to rotenone and paraquat were not correlated (r = 0.004), and ORs from a model that included both pesticides (paraquat OR = 2.6; 95% CI, 1.4–4.9; rotenone OR = 2.9; 95% CI, 1.5–5.5) were comparable with those from models without mutual adjustment (Table 3). For men who used paraquat plus any dithiocarbamate (7 cases, 16 controls), the OR compared with use of neither was 2.2 (95% CI, 0.79–5.9). Compared with never users, associations with PD were generally stronger among those who had used pesticides for more than the median number of lifetime days than among those who had used pesticides for fewer than the median number of days [Supplemental Material, Table 3 (doi:10.1289/ehp.1002839)].

Results from analyses stratified by race/ethnicity, cigarette use, state, or duration of disease (in cases) were not appreciably different between subgroups (data not shown). In an analysis restricted to 18 cases and 22 controls (28 men and 12 women) with a first-degree relative with PD, PD was not associated with ever exposure to rotenone (OR = 0.98) or paraquat (OR = 1.1). Adjustment for education or overall pesticide use did not appreciably alter effect estimates, nor did exclusion of 28 cases and 6 controls with proxy interviews or inclusion of 3 atypical parkinsonism cases.

We compared clinical features of cases who did or did not use paraquat or any oxidative stressor, or rotenone or any complex I inhibitor (Table 4). PD was diagnosed at a younger age among those who used oxidative stressors (mean age = 59 vs. 64 years, p = 0.02). Although postural reflex impairment appeared to be less frequent in those using either paraquat or rotenone, these differences were not statistically significant. Clinical features did not otherwise differ between groups. We were unable to determine whether exposure to both paraquat and rotenone was associated with unique clinical features because only five cases reported use of both pesticides (data not shown).

The pathogenesis of PD is thought to involve several critical abnormalities, each of which can be the result of genetic or environmental factors. Chief among these abnormalities are dysfunction of the mitochondrial respiratory chain, particularly complex I, and the production of reactive oxygen species (Henchcliffe and Beal 2008). To our knowledge, we have performed the first analysis of pesticides classified by presumed mechanism, rather than by functional categories (e.g., herbicides) or chemical class (e.g., organochlorines). We found significant associations of PD with use of groups of pesticides classified as complex I inhibitors or as oxidative stressors, providing support in humans for findings from decades of experimental work. In particular, PD was strongly associated with rotenone and paraquat, two individual pesticides used extensively to model PD in the laboratory.

This study provides strong evidence of an association between rotenone use and PD in humans. PD developed 2.5 times as often in those who reported use of rotenone compared with nonusers, and an association of similar magnitude was observed even when exposure was truncated up to 15 years before PD diagnosis. In our prior analysis of self-reported PD in the AHS, information on rotenone was available for a small subgroup, and nonsignificant association with PD (OR = 1.7; 95% CI, 0.6–4.7) was observed (Kamel et al. 2007), whereas in a multicenter, clinic-based case–control study distinct from the AHS, only two individuals were exposed and no association was observed (Tanner et al. 2009). In the only other report of rotenone-like compounds and PD, use of organic pesticides such as rotenone in the previous year was determined for PD clinic attendees, who reported current use more often than did cases with other neurologic diseases (Dhillon et al. 2008). This information cannot be used to assess etiology, because the study evaluated associations with rotenone use that occurred after PD had been diagnosed. In contrast, in the present population-based study, we evaluated rotenone use before PD diagnosis in cases and during a comparable time period in neurologically healthy controls.

Rotenone is a plausible cause of PD because of its mechanism of action. Like 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a toxicant known to cause parkinsonism in humans, rotenone directly inhibits mitochondrial complex I (Langston et al. 1983; Sherer et al. 2007). In experimental models, both MPTP and rotenone cause selective injury of dopaminergic neurons in the substantia nigra, a key pathological feature of PD (Greenamyre et al. 1999; Langston et al. 1984). Because rotenone is believed to have a relatively short environmental half-life and limited bioavailability, a relationship to human disease has been questioned (Hatcher et al. 2008; Li et al. 2005). However, recent work in rodent models indicated that a temporally limited exposure to rotenone later caused progressive functional and pathologic changes in the enteric nervous system of rodents, mimicking changes found in human PD; as in PD, these enteric nervous system changes preceded central nervous system pathology (Abbott et al. 2001; Braak et al. 2006; Drolet et al. 2009; Greene et al. 2009; Pan-Montojo et al. 2010). Chronic rotenone exposure in the laboratory has been reported to have additional effects associated with PD pathogenesis, including ones similar to changes observed in monogenic forms of PD (Henchcliffe and Beal 2008). Rotenone toxicity, therefore, provides a conceptual bridge, suggesting shared mechanisms for both sporadic and inherited forms of PD.

Although we report here findings for agricultural use of rotenone, the ubiquitous use of rotenone in both work and home settings that occurred until recently suggests that many people may have been exposed. Humans have used rotenone-containing plants as pesticides for centuries (Cabras et al. 2002). Because rotenone is plant derived, it has been considered an organic pesticide and was commonly used as a household insecticide in home gardening and agriculture, and to kill fish. For example, the California Department of Pesticide Regulation (2007) reported that almost 15,000 pounds of rotenone were used in 2007, not including home use. Rotenone was withdrawn from use in the European Union in 2007 (Schapira 2010), after which time most uses were voluntarily cancelled in the United States (U.S. EPA 2007). Other agents associated with mitochondrial complex I inhibition remain in common use. For example, permethrin is used in nonagricultural settings as an insect repellant, including use of permethrin-impregnated fabric for military uniforms and recreational clothing (Armed Forces Pest Management Board 2010).

Our study also extends prior research on paraquat. Experimentally, paraquat produces subcellular changes associated with PD, including increased production of reactive oxygen species, alpha-synuclein aggregation, and selective nigral injury (Dinis-Oliveira et al. 2006; Kuter et al. 2010; McCormack et al. 2002). Previously, we found an association between paraquat use and PD in prevalent but not incident self-reported cases in the AHS (Kamel et al. 2007) and a nonsignificant association between PD and occupational paraquat use in a multicenter case–control study (Tanner et al. 2009). Cumulative use was not assessed in either study. Only a few other studies have assessed associations between PD and paraquat use (Table 1). A study of 120 cases and 240 controls conducted in Taiwan (Liou et al. 1997) reported an OR of 3.22 (95% CI, 2.41–4.31) for PD in paraquat users compared with nonusers. Cumulative exposure was associated with greater risk, but paraquat use in the Taiwanese study was highly correlated with use of other herbicides. Although the inconsistency of findings in human populations has been used as a basis for suggesting that paraquat is not associated with PD (Li et al. 2005; Miller 2007), an alternative explanation is that few studies have had adequate size and sufficiently detailed exposure information to allow the association to be observed. Our findings, considered together with earlier results, suggest that paraquat use plays a role in human PD. Because paraquat remains one of the most widely used herbicides worldwide (Frabotta 2009), this finding potentially has great public health significance.

Parkinsonism in humans due to high-dose exposure to toxicants such as carbon monoxide or manganese has characteristic clinical features including less prominent tremor, more prominent postural instability, symmetric distribution of signs, and poor response to dopaminergic therapy (Tanner 1992). We did not observe such features in our cases. Cases who did or did not use rotenone, paraquat, or groups of pesticides with similar mechanisms were generally similar, suggesting that PD associated with these agents is clinically typical and indirectly supporting a role for pesticide exposure in the etiology of typical PD. We did note an earlier age at diagnosis in users of oxidative stressors and a suggestion of this in paraquat users specifically. Early age at onset is also a characteristic of genetic parkinsonism in which oxidative stress is a presumed pathophysiologic mechanism (alpha-synuclein, PINK-1, DJ-1, and PARKIN mutations) (Henchcliffe and Beal 2008; Klein et al. 2009).

In FAME, pesticide exposure was not associated with PD in individuals with a family history, although numbers were small. Interestingly, Hancock et al. (2008) similarly found pesticide exposure to be associated with PD risk only in those without an affected first-degree relative.

Our study has some limitations. First, because most participants were exposed to many pesticides, we cannot confidently exclude effects of agents other than those studied or rule out the possibility that our results are attributable to combined exposures. However, the associations that we observed remained after adjustment for overall pesticide use, and estimated effects of rotenone and paraquat were comparable after mutual adjustment. Future investigations of combinations of pesticides and of other mechanistic groups will be important. Second, we could not use laboratory measures of pesticides or their metabolites to estimate exposure. Such measures are not available for many of the pesticides we studied, and when available, they are poor predictors of past or long-term use. Thus, although we recognize that retrospective exposure assessment has limitations, it is often the best approach for studying lifelong exposure in an adult population in connection with a rare disease. Third, we included prevalent cases already diagnosed but still living at enrollment in the AHS; therefore, survivor bias is possible. However, our results were similar when only those with shorter disease duration were analyzed. Additionally, we were able to investigate only persons willing to participate. Thus, PD cases or controls in this study may not have been fully representative of the entire population. However, participation was good, partially allaying this concern. Finally, we selected pesticides presumed to act through specific toxic mechanisms, but for most pesticides there is very little information regarding toxic effects in humans, as most studies are directed toward effects on plant or animal pests. It is likely that we have misclassified some pesticides with regard to mechanism. However, the likely effect of any misclassification would be to attenuate an association with pesticides grouped according to a common mechanism.

Strengths include the size of the study; the focus on an agricultural cohort with many exposed individuals and wide variability in exposure; the quality of diagnosis, which was based on in-person assessment and agreement of movement disorders experts; and the completeness and reliability of the pesticide exposure information. An additional strength is the nested case–control design with an internal control group who had similar exposure opportunities as the cases and similar demographic and lifestyle characteristics, reducing the likelihood of bias or confounding. Use of pesticides in general was ubiquitous, of course, in applicators and relatively common among their spouses, and all participants may have had additional passive pesticide exposure. However, these features would all be likely to lower the chance of identifying any effect.

Our study helps connect the dots between basic research and human populations. Rotenone and paraquat have been linked experimentally to pathophysiological mechanisms implicated in human PD. Groups of pesticides linked to the mechanisms of mitochondrial dysfunction or oxidative stress were also associated with PD in our study, thus extending experimental work to provide strong evidence that these mechanisms play a role in PD in humans. Importantly, the potential for exposure to many of these pesticides, including rotenone and paraquat, extends well beyond the occupational setting. Many persons with nonoccupational pesticide exposures may be unaware of the presence of pesticides in their environments (Centers for Disease Control and Prevention 2009). The potential for exposure to other toxicants with similar mechanisms is even greater. To continue the interplay between human and experimental studies, future mechanistic studies of these pesticides should model exposure conditions similar to those occurring in humans, including chronic low-dose exposure, exposure to multiple agents, and assessment of gene–exposure interactions. Such work could provide new insights into the pathogenesis and ultimately the prevention of PD.

Supplemental Material is available online (doi:10.1289/ehp.1002839 via http://dx.doi.org/).

The authors thank the participants, the research team, T. Greenamyre for manuscript review, and J. Wright for editorial assistance.

This study was supported in part by the Intramural Research Program of the National Institutes of Health [National Institute of Environmental Health Sciences (NIEHS) grants Z01-ES044007 and Z01-ES049030, National Cancer Institute grant Z01-CP010119], NIEHS grant R01-ES10803, and James and Sharron Clark.