Parkinson’s disease (PD) is a neurodegenerative disorder and one of the most common neurological disorders affecting humans. PD results from particular defects in a specific part of the brain (substantia nigra pars compacta - SNpc), involving the loss of the dopamine-containing (dopaminergic) neurons. The prevalence of PD is overwhelmingly associated with increasing age, but it is generally agreed that the development of PD involves both genetic and environmental factors.
An experimental animal model for PD was developed after MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and its toxic metabolite MPP+ (1-methyl-4-phenylpyridinium ion) were shown to cause parkinsonism in drug addicts in California, USA in the 1980s (MPTP was a contaminant in certain batches of illicit drugs). In the later 1980s paraquat started to attract the interest of researchers due to its perceived structural similarity to MPTP. The first study to investigate epidemiological evidence of a potential association between exposure to paraquat and increased risk for PD was published soon afterwards (Hertzman, 1990). Since then, a large number of animal and epidemiology studies have been published on this research topic.
Syngenta has undertaken a major research program using animal models to investigate the alleged link between paraquat and Parkinson’s disease. The research work has been, and will continue to be, published in peer-reviewed scientific journals and the results communicated to relevant regulatory agencies. The key finding is that paraquat, even at the maximum tolerated dose, does not cause dopaminergic neuronal cell loss in the SNpc, the area of the brain associated with Parkinson’s disease.
The majority of publications using animal models have been conducted in the C57BL/6 mouse – a mouse strain known to be particularly sensitive to the effects of MPTP. McCormack et al. (2002) reported that repeated intraperitoneal (i.p.) paraquat injections had led to the loss of dopaminergic neurons in the SNpc. However, there was no significant depletion of striatal dopamine following paraquat administration. Later publications have also reported that paraquat could induce dopaminergic cell loss in the SNpc of this mouse strain.
Within the research community the relevance of the work conducted with a range of chemicals in the C57BL/6 mouse to the development of Parkinson’s disease in man is controversial (Miller, 2007). The doses, routes and duration of exposure used in these experimental studies do not mimic potential human exposure scenarios. The doses of paraquat used in the animal studies far exceed normal occupational exposure scenarios; the dosing regimen most typically applied (three intraperitoneal doses of 10 mg paraquat dichloride/kg bodyweight dosed once a week over three weeks) is approximately one-third of a lethal dose. Some of the publications of one of the research groups working with paraquat were found to have been based on fabricated data1.
Syngenta has an active research program to determine whether paraquat has any adverse effects on dopaminergic neuronal systems in critical regions of the brain of sensitive animal models. A summary of the key results of these extensive investigations in the “C57BL/6J mouse i.p. model” has been published (Breckenridge et al., 2013; individual study reports can be made available upon request). In our studies we were unable to consistently detect a significant loss of dopaminergic neurons in paraquat-treated mice through stereological counting, and, most importantly, there was no pathological evidence of neuronal cell death in the SNpc, nor any effect on neurotransmitter levels in the striatum.
The results of this study have recently been confirmed in a comprehensive inter-laboratory investigation in which paraquat administered at maximum tolerated doses did not result in any neuropathology (Smeyne et al., 2016). This research employed three distinct methods of assessment, performed by three groups of investigators who were all ‘blinded’ to treatment, for a series of neuropathological indices evaluated in two ages and two sub-strains of male C57BL/6 mice, housed under different conditions in two laboratories. Paraquat, administered either once or twice weekly to 9- or 16-week old mice from two suppliers, had no effect on the number of dopaminergic neurons in the SNpc, as assessed by two groups, each blinded to treatment, using different stereological methods. In addition, blinded assessments conducted independently by a neuropathologist and a stereologist did not find any evidence of neuronal cell death (neuropathology) or neuroinflammation (neuropathology and stereology). Based on the scope and comprehensiveness of this study, the authors concluded “that it is implausible that DA neurons could have died without detection in PQ-treated mice”.
The i.p. dosing C57BL/6J mouse model is inappropriate for human risk assessment purposes primarily because ip injection is not a relevant exposure route. Therefore, Syngenta also sponsored a study to investigate the effect of paraquat in the C57BL/6J mouse using a dosing regimen more relevant to potential human exposure scenarios. Male and female mice were continuously exposed to paraquat in their diets for thirteen weeks and potential effects on the nigrostriatal dopaminergic system were assessed using neurochemistry, neuropathology and stereology as toxicological endpoints. No effects of paraquat on dopaminergic neurons were observed in this 13 week dietary study in the C57BL/6J mouse (published in full as Minnema et al., 2014).
Many epidemiology studies investigating a possible association between paraquat exposure and PD have been published. These studies are characterized by weaknesses in their study design, particularly the assessment of past exposure, and provide an inconsistent picture. Syngenta has investigated former production workers at a paraquat manufacturing site and has found no evidence of an increased risk of PD-related fatalities. Overall, no conclusion can be drawn from the available epidemiology studies on a possible association between paraquat exposure and PD.
Since the publication of Hertzman et al. (1990) many epidemiology studies have investigated a potential association between PD and exposure to pesticides in general as well as paraquat in particular. Brown et al. (2006) comprehensively reviewed the epidemiologic literature. They concluded that there does appear to be a relatively consistent relationship between pesticide exposure and PD. Particular classes of pesticides found to be associated with PD included herbicides and insecticides. However, Brown et al. also concluded that the epidemiologic studies were limited by methodological weaknesses, e.g. in the selection of cases and controls, statistical power, consideration of confounding factors, or exposure assessment.
The US Agricultural Health Study (AHS) is a prospective cohort study of farmers and their families in Iowa (IA) and North Carolina (NC) (Alavanja et al., 1996). Amongst other objectives the study was designed to evaluate non-cancer health endpoints and disease risk from farm exposures among family members (spouses and children). Kamel et al. (2007) analyzed the AHS data on self-reported lifetime pesticide use obtained at enrollment. Odds ratios and 95% confidence intervals for both prevalent (i.e. diagnosed before enrollment in the study) and incident (i.e. diagnosed after enrollment in the study) PD cases were presented for 45 specific pesticides. For “ever-use” of paraquat, the odds ratios were 1.8 (95% CI: 1.0, 3.4) for prevalent PD cases and 1.0 (95% CI: 0.5, 1.9) for incident PD cases. This pattern of results (i.e. an elevated odds ratio for prevalent but not for incident cases) would arise if cases diagnosed prior to enrollment were more likely to enroll if they were exposed than if they were not exposed. There is no way to know if this occurred, but analysis of incident cases reduces the likelihood of such bias (Gray et al., 2000). On the basis of consideration of the incident PD cases there was no association between exposure to paraquat and the development of PD in this study.
In 2007, a multidisciplinary group of 29 independent experts met to assess what is known about the contribution of different environmental factors to Parkinson’s disease (Bronstein et al., 2009). Depending on the evidence of an association to PD, they assigned the alleged factors to one of four possible groups:
- “Sufficient evidence of a causal relationship”
- “Sufficient evidence of an association”
- “Limited suggestive evidence of an association” and
- “Inadequate/insufficient evidence to determine whether an association exists”.
Paraquat was assigned to the group of factors for which there is “Inadequate/insufficient evidence to determine whether an association exists”. The group considered the results of the Kamel et al. (2007) study in reaching their overall conclusion.
Syngenta monitors the developments in the scientific literature in order to keep updated on health issues related to our products. This includes evaluating advice from experts related to paraquat. Syngenta has consulted with several independent medical and epidemiological scientists under the guidance of Professor Sir Colin Berry (Emeritus Professor at Queen Mary, University of London, and former member of the UK Medical Research Council) and Professor Pierluigi Nicotera (Founding Director of the German Center for Neurodegenerative Diseases, Bonn) to learn their views on the hypothesis that there might be a causal link between exposure to paraquat and Parkinson’s disease. They concluded that the evidence available from epidemiological studies was fragmentary and insufficient to establish whether herbicides and paraquat in particular increase the risk for PD. They further concluded that the overall epidemiological evidence from combined exposure studies and those limited to paraquat alone did not support the existence of a specific association between paraquat and PD. The analysis was subsequently published (Berry et al., 2010).
Tanner et al. (2011) undertook a case-control study (the Farming & Movement Evaluation, FAME study) nested within the existing US Agricultural Health Study (AHS) previously reported in Kamel et al. (2007) to determine whether pesticides that cause mitochondrial dysfunction or oxidative stress are associated with PD. Based on any use of paraquat by 23 subjects before the reference date, the adjusted odds ratio and 95% confidence intervals for paraquat were 2.5 (1.4, 4.7).
Mandel et al. (2012) concluded that the FAME study by Tanner et al. (2011) is difficult to interpret due to important methodological issues, including: different eligibility criteria for cases and controls, ambiguous and non-standard selection of controls, exclusion of controls because they subsequently developed PD, low participation rates among those eligible, much higher proportion of proxy respondents among cases than controls, substantial differences in participation rate between cases and controls and the absence of estimates based only on the exposure reported at enrollment. Mandel et al. (2012) also provide a critical review of other epidemiological studies investigating any association between paraquat and PD.
Chang et al. (2014) provides a more specific critical review of the use of geographic proximity as a surrogate for actual data on exposure. In the absence of supporting environmental or biological monitoring data for specific pesticides, geographically modelled estimates of pesticide exposure cannot be assumed to be valid surrogates of personal exposure to pesticides. The authors conclude that geographic exposure models must be rigorously constructed and validated if they are to be relied upon to produce credible epidemiological research.
Bradford Hill’s criteria are widely accepted as useful guidelines for investigating causality in epidemiological studies (Hill, 1965). Breckenridge et al., (2016), conducted an assessment of the weight of the evidence according to Bradford Hill’s viewpoints and concluded that there is an inadequate basis to draw an inference of causality between PD and paraquat. The authors evaluated all published epidemiological studies on paraquat and determined that in only two of the twenty studies was there both individual reporting of whether they were exposed to paraquat and medical confirmation of diagnosis of Parkinson’s disease confirmed by examination by a medical expert, i.e. studies with incident PD cases classified according to clinical data for diagnostic confirmation and individual-level exposure assessment. In both of these studies (Firestone, 2005 and 2010), the association between exposure to paraquat and the diagnosis of Parkinson’s disease was not statistically significant. In the most recent update (Firestone, 2010) on this cohort of subjects, the relative risk ratio was near unity (RR= 0.90; 95% CI= 0.14-5.43).
A publication by Tomenson and Campbell (2011) concluded that there was no evidence of an increased incidence of PD among paraquat production workers based on mentions of PD on the death certificates of workers. A strength of this study is the likely higher exposure (level and duration) of workers engaged in paraquat production during the 1960s, 1970s and 1980s than many of the subjects in case-control studies classified as exposed to paraquat.
High-doses of MPTP very quickly produce parkinsonism in man. Brent and Schaeffer (2011) assessed whether confirmed high-dose paraquat exposure is associated with the development of parkinsonism. They carried out a systematic review of all published cases of paraquat toxicity meeting a case-definition of paraquat poisoning and who either recovered or lived for at least 30 days (primary analysis) or lived for 15 to 30 days (secondary analysis). Cases were included if they contained sufficient information to determine whether or not they had signs of parkinsonism. This analysis found no connection between high-dose paraquat exposure in humans and the development of parkinsonism.
Published laboratory studies using the C57BL/6J mouse model with intraperitoneal administration do not provide unequivocal evidence of an association between paraquat exposure and effects on the dopaminergic system. No effects of paraquat on dopaminergic neurons were found in a dietary study in C57BL/6J mice which involves a more appropriate exposure scenario for exposure of humans to paraquat and accordingly for human risk assessments. Most importantly there was no evidence of dopaminergic cell death in this study.
The epidemiologic studies on the association between paraquat exposure and PD continue to be inconclusive. A key weakness of many studies is their lack of a robust exposure assessment. The biological plausibility of such an association is also reduced by the absence of neuropathological evidence for dopaminergic cell loss. Two publications which investigated the onset of PD (Tomenson and Campbell, 2011) or signs of parkinsonism (Brent and Schaeffer, 2011) after confirmed long-term or high-dose exposure to paraquat indicate that exposure to paraquat does not result in the development of PD in humans.
Considering both the animal and the human epidemiologic studies, the evidence linking paraquat to Parkinson’s disease is fragmentary and does not support the existence of a causal association between paraquat and PD.
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1.On June 28th 2012, the United States Department of Health and Human Services’ Office of Research Integrity (ORI) announced findings that Neuroscientist Dr. Mona Thiruchelvam, a former assistant professor at the University of Medicine and Dentistry, New Jersey (UMDNJ) used fabricated data in published studies claiming to examine how pesticides influence neuronal mechanisms involved in Parkinson’s disease (PD). The falsified data included the results of 13 experiments that supposedly counted nigrostriatal neurons in the brains of mice and rats following exposure to atrazine, paraquat and paraquat & maneb in combination. FR Notice Volume 77, No. 125 Thursday, June 28, 2012, 38632 -38633: http://www.gpo.gov/fdsys/pkg/FR-2012-06-28/html/2012-15887.html.