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Mechanism of the association between pesticides and Parkinsons - PS2608

Parkinson’s Disease (PD) is diagnosed in 20 of every 100,000 people in the United Kingdom each year; there are an estimated 150,000 persons living with PD in the UK today. Although a few of those cases are based on a genetic mutation, the vast majority are of unknown cause. The herbicide paraquat closely resembles a compound that is known to cause chemically-induced PD, and because it is used for agriculture, paraquat has been examined as a potential cause for PD cases that are apparently not genetically based. There has been little conclusive evidence that paraquat causes PD, although some human and animal studies support that theory.

Over the last 5 years, one laboratory has published multiple papers that strongly suggest that mice developmentally exposed to paraquat or the fungicide maneb lose neurons in the region of the brain that degenerates during PD in humans. Subsequent adult exposure appears to exacerbate the neuron loss. Concurrent exposure of paraquat and maneb, while not likely according to usual agronomic practices, may still provide useful mechanistic information. Replicating these observations could be very important, both in defining the likelihood that this model is relevant to the English population, and in creating a human PD laboratory model that could be used to test drugs and other interventions.

However, these studies have drawbacks. One important concern is that the doses that produce neuron loss in mice are far in excess of human exposures to pesticides during field use, and even farther in excess of human exposures through residues in the food supply. This model must be tested at levels that are relevant to the exposures humans encounter, because biological systems have protection and repair mechanisms that could be relevant at low environmental doses but could be overwhelmed at high laboratory doses. Therefore, low doses relevant to human exposures may not produce effects similar to the doses used in this model.

A second drawback is that humans are exposed by inhaling droplets of pesticide as it is sprayed, by swallowing residues in the throat after inhalation, by absorption from spray landing on the skin, or from swallowing it as a food residue. The current paraquat/maneb mouse model requires that compounds be injected into the body, which is not a realistic human exposure route. The effects from injecting a compound are almost always far more severe than the effects from the same amount of compound when it is inhaled or ingested. Therefore, an initial experiment will be performed to create a model for how the different routes of exposure compare to each other after exposure to these pesticides.

Thirdly, although neurons are lost in the mouse model, the mice were not allowed to live into old age, the stage of life when humans develop PD. The relevance of this mouse model would be strengthened if the animals lived long enough for age-related symptoms to appear. Therefore, a study to examine whether exposure to paraquat and maneb, alone or in combination, is a risk for developing PD requires human-relevant doses, equivalency to the inhalation/oral exposure route, and maintenance of the animals into old age.
The aim of the project is to determine whether neuronal degeneration subsequent to developmental or adult exposure to a combination of paraquat (PQ) and maneb (MB) results in selective neurodegeneration of the regions of the brain that degenerate in PD. A mouse model has been developed in the laboratory of Dr. Cory-Slechta at Rutgers University in the United States. This laboratory model indicates that neurons of the substantia nigra are selectively lost after early postnatal exposure to either pesticide, and that loss is increased after exposure to both pesticides combined. Adult exposure to the combination may also cause neuron loss, but to a lesser degree. However, the greatest number of neurons are lost with exposure to the combination of PQ + MB during postnatal life and again as young adults. This research plan would replicate the Cory-Slechta study design but choose dosages and exposure routes relevant to humans, and allow the mice to live into old age to determine whether neurons are lost at doses approximating human exposure levels, and whether the loss continues over time. The specific objectives are listed below.
1) Toxicokinetic Study
The objective is to model the distribution of the pesticides into the body during various exposure routes relevant to human exposure and the established animal model. Supervisors of this section of the project will be Chris Mackay, Ph.D, and Laura McIntosh, Ph.D, DABT, and advised by Simon Warren, DABT, DIBT, Dip.R.C.Path.
a. This objective is expected to take 1012 weeks for treatment and analysis of the animals used to determine penetration and distribution of 14-C labelled PQ with and without the presence of MB into tissues under four possible exposure conditions (oral, dermal, intra-nasal instillation (rodent model of human inhalation), and intraperitoneal injection). This objective includes animals 4 months of age, so may take longer depending on animal availability for that age.
b. The deliverables are:
i. Two physiologically based pharmacokinetic (PBPK) models that predict the penetration and distribution of pesticide within various compartments of the body and brain according to the route of exposure. The first PBPK model will be of young adult mice approximately 4 months old, and will model tissue distribution and elimination parameters of PQ with and without the presence of MB after oral, injection, inhalation, and dermal doses. This will be modelled as a 10-compartment PBPK simulation. The purpose is to establish internal dose equivalency between human exposure routes (inhalation, oral, and dermal) and injection exposure as used in the mouse model. These models will ultimately predict how much pesticide reaches the brain in PND5 and young adult mice, and how that relates to human doses after typical routes of exposure. The second PBPK model will be of day 5 mice (PND5), using only oral and injection doses. The purpose is to determine equivalency of these routes of exposure and doses to human conditions and to each other. The dose equivalents generated here will be used to determine the applicable human doses for the lifespan mouse test (Objective 2).
ii. The animals treated by intranasal instillation in 1bi will be used to determine whether there is a detectable alternative transport mechanism independent of the circulatory system from the nasal surface into the brain (e.g. via the olfactory bulb). Examination of PQ concentrations in the brain and those applied to the nasal epithelium, relative to central compartment concentrations, will hopefully allow the resolution of the kinetics of any independent transport pathway. At the very least, it will allow the assignment of practical bounds of detection that will indicate the magnitude of possible direct transport to the brain via this route.

2) Lifespan Mouse Test
The second objective is to test the current mouse model for PQ and MB-induced neurodegeneration in a mouse lifespan experiment. The published PD mouse model will be replicated using the high laboratory doses used by the Cory-Slechta group1 during their creation of the model, and compared against lower human-exposure-derived doses as determined by the PBPK model established in the first objective. Supervisors of this section of the project will be Abby Li, Ph.D., and Laura McIntosh, Ph.D, DABT, advised by Simon Warren, DABT, DIBT, Dip.R.C.Path.
a. This objective is expected to take up to 2 years.
b. The deliverables are:
i. Generation of sets of animals exposed to different doses of a combination of PQ and MB, and sacrificed at two intervals throughout the 2 year mouse lifespan (see Figure 1)
ii. Perfusion of those sets of animals according to the protocols required for immunoneurohistology. Perfused brains will be sent to NeuroScience Associates, a specialty neurohistology laboratory, as they are generated (see Objective 3).

3) Brain Tissue Preparation
Neuroscience Associates, a neurohistology laboratory, specializes in sectioning and staining brains for detection of neurotoxic responses to bioactive compounds. A battery of histological stains will allow determination of the extent, specificity, and mechanism of neurodegeneration throughout the brain. Supervisor of this section of the project will be Dr. James O’Callaghan, Head of the Molecular Neurotoxicology Laboratory at the United States Centers for Disease Control in the National Institute for Occupational Safety and Health.
a. This objective is expected to take 46 weeks from receipt of perfused brains
b. The deliverables are 50 µm sections of the brain and olfactory bulb stained with a selection of these stains according to appropriateness for the time point:
i. Nissl stain for cell bodies and nuclei
ii. Glial fibrillary acidic protein (GFAP), a glial protein marker for neurotoxicity; see O’Callaghan (2005)
iii. Tyrosine hydroxylase (TH+) antibody to detect dopaminergic (DA) neurons
iv. Silver staining to detect recent neurodegeneration
v. 8-Hydroxy-2'-deoxyguanosine (oh8dG) antibody to indicate whether the mechanism of action is oxygen radical damage as hypothesized.

4) Histological Examination
Detection of any neurodegeneration resulting from PQ/MB exposure needs to be quantified and localized to specific neuronal tracts to determine if the substantia nigra and striatum are selectively targeted by the pesticides, as would be required in a valid PD model. The laboratories of Dr. James O’Callaghan, Dr. Robert Switzer at Neuroscience Associates, and Dr. Daryl Thake at Seventh Wave Pathology and Biotechnical Solutions have extensive experience evaluating the neurotoxic response produced by many different compounds. This battery of neurohistological biomarkers, accompanied by stereology, will provide a thorough examination of any neurodegeneration resulting from pesticide exposure.
a. This objective is expected to take 4 months from receipt of prepared brain sections.
b. The deliverables are:
i. Quantitative measurement of neurons within the substantia nigra, and other brain areas as needed, determined by stereology. Other areas could be analysed if degeneration stains indicate areas outside the substantia nigra are also targeted by PQ/MB. Dr. Jim O’Callaghan
ii. Localization of degeneration and astroglial activation; determination whether degeneration is limited to specific areas or widespread throughout the brain. Dr. Daryl Thake and Dr. Jim O’Callaghan
iii. Determination of loss of TH+ neurons for comparison with results published by the Cory-Slechta laboratory. Dr. Daryl Thake and Dr. Jim O’Callaghan
iv. Test of the theory of oxidative stress as the mechanism for PQ/MB toxicity; the use of an antibody against oxidatively damaged DNA will indicate whether there is an increase in oxidative damage to brain cells within the treatment groups, and whether it is elevated between high and low dose treatment groups. Dr. Daryl Thake

5) Data Analysis
Data will be analysed for consistency with the theory that PQ and MB cause selective neurodegeneration of TH+ neurons within the substantia nigra and striatum by a mechanism of oxidative stress at doses approximating human exposure levels, especially when those doses are encountered during a developmental stage of life. Supervisor of the statistics section of this project will be chosen from the Exponent Data Analysis Group. Co-supervisors of the histological data analysis will be James O’Callaghan, Ph.D., Abby Li, Ph.D., and Laura McIntosh, Ph.D., DABT.
a. This objective is expected to take 12 months after scoring of tissue from the histological examination.
b. The deliverables are:
i. A detailed analysis of each point of the theory set out above, i.e., do human-magnitude doses result in detectable neurodegeneration? If neurodegeneration is seen is it selective for TH+ neurons, do PQ and MB cause neuron loss in the substantia nigra after developmental exposure, etc.
ii. A comparison to results published by the Cory-Slechta laboratory.
iii. A limited representation of the dose-response relationship.

6) Presentation of Results
The data will be available for presentation in multiple public forums. Presenters could include any of the supervisors of the project, most likely Dr. James O’Callaghan, Dr. Abby Li, Dr. Chris Mackay, and Dr. Laura McIntosh.
a. This objective is expected to begin after the completion of the PBPK model, and extend to 1 year after the termination of the experimental work.
b. The deliverables are:
i. First Year—Presentation of the PBPK models to the Society of Environmental Toxicology and Chemistry (SETAC), including the first test of retrograde transport from the nasal epithelium to the brain, by Dr. Chris Mackay.
ii. Second Year (or when available)—Presentation of the data collected on the young adult animals to the Society of Toxicology by Dr. McIntosh (Dr. Li and Dr. O’Callaghan would be co-presenters but would not receive funding from Defra).
iii. Third Year (or when available)—Presentation of all data generated through termination of the study to the Society of Toxicology by Dr. McIntosh (Dr. Li and Dr. O’Callaghan would be co-presenters but would not receive funding from Defra).
iv. Third Year (or when available)—Presentation of all data generated through study termination to Defra, and to a European meeting designated by Defra (Dr. McIntosh and Dr. Mackay).
v. Three peer-reviewed research papers reflecting the presentations above.
vi. Annual reports to Defra as outlined in the contract
Project Documents
• EVID4 - Final project report : PS2608 final report   (227k)
Time-Scale and Cost
From: 2006

To: 2008

Cost: £906,552
Contractor / Funded Organisations
Exponent International Ltd
Food Safety              
Health Effects              
Pesticide use              
Public Health              
Fields of Study
Pesticide Safety