Effects of ultrafine particulate matter on an animal model of Parkinson’s

disease

Presented to the California Air Resources Board April 3, 2019

Arthur K. Cho, William P. Melega, University of California Los Angeles; and Michael T. Kleinman, University of California Irvine

1

http://www.uci.edu/

Introduction

• The project was designed to test the hypothesis that reactive components in ultrafine particles (UFPM) of ambient air could induce the development of Parkinson’s Disease (PD) like behavioral and brain pathology in a strain of transgenic mice that express human alpha synuclein (hα-Syn), a protein marker for the disease.

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In the study, both the transgenic and wild type animals were exposed to concentrated UFPM generated by the Versatile Aerosol Concentration Enrichment System (VACES) for 22 weeks and monitored for behavioral and neurochemical alterations consistent with PD and inflammatory responses.

Tasks and key investigators

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The study involved four interconnected tasks, led by investigators with the relevant expertise. Thus, the exposure task was led by Dr. Michael Kleinman of UCI, the animal work by Dr. William Melega of UCLA and the chemical characterization by Arthur Cho of UCLA who was also the principal investigator. Details of the study are summarized below:

Task Description Investigator/expertise

1. Exposure Exposure of wild type and hα-Syn transgenic mice to concentrated ultrafine particles for 5 months

Michael Kleinman/Air pollution methods, animal exposure

2. Behavior responses Determine motor and cognitive behaviors in the mice before and after the exposure period

William Melega/Neurochemistry, neurotoxicology, behavior

3. Brain pathology Assess neuroinflammation, neurochemical defects and of hα-Syn expression following the exposure period

William Melega/ Arthur Cho

4. Chemical and biological characterization of ambient air

Determine the chemical and biological properties of the air corresponding to the particles to which the animals have been exposed

Arthur Cho/ Biochemical toxicology, air pollution biochemistry

The study involved four interconnected tasks, led by investigators with the relevant expertise. Thus, the exposure task was led by Dr. Michael Kleinman of UCI, the animal work by Dr. William Melega of UCLA and the chemical characterization by Arthur Cho of UCLA who was also the principal investigator. Details of the study are summarized below:

Task

Description

Investigator/expertise

1. Exposure

Exposure of wild type and hα-Syn transgenic mice to concentrated ultrafine particles for 5 months

Michael Kleinman/Air pollution methods, animal exposure

2. Behavior responses

Determine motor and cognitive behaviors in the mice before and after the exposure period

William Melega/Neurochemistry, neurotoxicology, behavior

3. Brain pathology

Assess neuroinflammation, neurochemical defects and of hα-Syn expression following the exposure period

William Melega/ Arthur Cho

4. Chemical and biological characterization of ambient air

Determine the chemical and biological properties of the air corresponding to the particles to which the animals have been exposed

Arthur Cho/ Biochemical toxicology, air pollution biochemistry

Task 1 Michael Kleinman

Exposure

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Versatile Aerosol Concentration Enrichment System (VACES)• Particulate matter (PM) and gaseous

constituents are pulled in from the ambient environment immediately above the lab

• The air stream enters a saturator which allows the particles to clump together and grow in size

• The air then enters a condensing tree where the particles solidify

• The air then flows through a virtual impactor which separates out the gaseous component from the PM and delivers 10x concentrated ambient particles (CAPs) downstream

• CAPs were monitored for particle number and mass

• An Aerosol Mass Spectrometer was run during the first 2 months of the exposure as part of a separate project

• Teflon and Quartz filters collected weekly and used for gravimetric, carbon, and metals analyses

• Purified Air was delivered as a control

5

1.00E-01

1.00E+00

1.00E+01

1.00E+02

1.00E+03

Cohort 1 Cohort 2 Cohort 3 Cohort 4

Mas

s C

once

ntra

tion

(ug/

m3)

Particle Mass Concentrations

UFAmbientFA

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

Cohort 1 Cohort 2 Cohort 3 Cohort 4

Num

ber C

once

ntra

tion

(p/c

m3)

Particle Number Concentrations

UFAmbient

Mass and number concentrations for the UCLA exposure looking at the effect of quasi-ultrafine concentrated ambient particulate matter (CAPs). • The concentration of CAPs (UF) was about 8-10x that of ambient particles as measure by both mass and number using a

real time condensation particle counter (CPC 3022 A; TSI) and aerosol mass monitor (DustTrak DRX, TSI)• CAPs exposure for all cohorts were comparable to each other• The mass of PM measured in the FA atmosphere was routinely below 1 ug/m3• Each cohort was exposed for 22 weeks, 4 days/week, 5 hours/day

Particle Concentration Information

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Element Concentration (ug/m3) SEM

B(11) 4.55 0.62

Na(23) 72.21 0.97

Mg(24) 9.06 0.76

Al(27) 81.21 2.49

P(31) 10.00 0.93

V(51) 3.32 0.83

Cr(52) 4.55 0.86

Mn(55) 4.86 0.77

Fe(57) 153.62 3.07

Co(59) 1.00 0.78

Ni(60) 4.00 0.31

Cu(63) 10.73 0.41

Zn(66) 11.02 0.97

As(75) 0.30 0.55

Se(77) 0.49 0.33

Rb(85) 0.17 0.20

Sr(88) 0.61 0.27

Ag(107) 2.75 0.28

Cd(111) 14.04 0.53

Ba(136) 4.90 0.07

Pb(208) 5.18 0.04

Organic and Elemental Carbon ConcentrationsA) Concentration of metals in CAPs across all exposure cohorts measure by

ICP-MSB) OCEC Data separated by cohort; all values in μg/m3

• Average OC and EC values consistent across all 4 cohorts• Values are mean ± standard error

A BParticle Constituent Analyses

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Alcohols (A and C) and carbonyls (B and D) appear to generally correlate with UF and accumulation mode mass• changes in the presence of these compounds

is probably more evenly split between the larger and smaller particle modes than the more oxygenated compounds

Aerosol Constituent Analysis

UF Mass: ≤ 0.1 μm • (Fraction of UF present/100)*(CAPs Mass Concentration)Accumulation mode Mass: 0.1 μm < particle size < 2.5 μm• (100-Fraction of UF present)*(CAPs Mass Concentration)

Data acquired by Aerosol Mass Spectrometer (Aerodyne Inc.) courtesy of Lisa Wingen (AirUCI; University of California, Irvine)

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The mass concentration of UF particles (A) and the concentrations of carbonyl (B) and alcohol (C) species in UF particles are strongly related to ambient temperature• i.e the inhaled dose of ambient UF particles and reactive

organic compounds is potentially greater on warmer days than on cooler days.

A B

C

Data acquired by Aerosol Mass Spectrometer (Aerodyne Inc.) courtesy of Lisa Wingen (AirUCI; University of California, Irvine)

Aerosol Constituent Correlations with Ambient Temperature

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Tasks 2 and 3. William MelegaBehavior responses and brain pathology

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Hypothesis - reactive chemical species in AP mixtures are responsible for adverse health effects by interacting with cellular targets to elicit responses such as inflammation, neurochemical content, and accumulation of human alpha-synuclein in the transgenic mice

Task 2, 3: In Vivo Responses (William Melega)

Animal model - a strain of transgenic mice (C57BL/6) expressing human alpha-synuclein (hα-Syn), a protein marker that accumulates in PD- mice were exposed to VACES-concentrated UFPM during a period in their development in which hα-Syn accumulation in brain is associated with behavioral and neurochemical deficits (particularly evident for the increases in hα-Syn levels in the hippocampus and striatum regions that are present during the age/time period of the study - 4 to 9 months)

- wild-type littermates of the same mouse strain were included in the study design to provide insights into the effects of the AP exposure without a hα-Syn contribution

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Motor and cognitive behavior assessments:Behaviors monitored: Motor (pole descent, beam traversal, nestlet shredding) and cognitive (Y maze) tests

Brain assessments:Inflammation (TNFα, IL-6, heme oxygenase-1)Neurochemistry (dopamine, norepinephrine)

Task 2, 3: In Vivo Responses (William Melega)

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Temporal Pattern of Human Alpha-Synuclein (hα-Syn) Expression in Brains of Transgenic Mouse

Amschl D, Neddens J, Havas D, Flunkert S, Rabl R, et al. 2013. Time course and progression of human alpha-synuclein accumulation in transgenic mice. BMC Neuroscience 14:6

Background Reference:

For our study, we began the study with animals at 4 months of age and to be exposed to AP or filtered air for ~5 months. We hypothesized that the largest potential for AP-induced increases in hα-Syn may occur during the 6 – 9 month time window 13

ARB UFPM Study DesignCohort # 1 2 3 4

dob (ave) 5-Mar 15-Mar 12-Apr 28-Apr

Transfer 23-JunTransfer 30-Jun

Behav 30-JunBehav 7-Jul

(17 wks) (T - F) Transfer 21-Jul

Exp-st Wk 1 7-Jul (16 wks) (T - F)2 14-Jul Exp-st Wk 1 14-Jul Behav 27-Jul Tues-Fri

3 21-Jul 2 21-Jul Transfer 11-Aug4 28-Jul 3 28-Jul (16 wks) (T - F)5 4-Aug 4 4-Aug Exp-st Wk 1 4-Aug Behav 17-Aug6 11-Aug 5 11-Aug 2 11-Aug7 18-Aug 6 18-Aug 3 18-Aug (16 wks) (T - F)8 25-Aug 7 25-Aug 4 25-Aug Exp-st Wk 1 25-Aug9 1-Sep 8 1-Sep 5 1-Sep 2 1-Sep

10 8-Sep 9 8-Sep 6 8-Sep 3 8-Sep11 15-Sep 10 15-Sep 7 15-Sep 4 15-Sep12 22-Sep 11 22-Sep 8 22-Sep 5 22-Sep13 29-Sep 12 29-Sep 9 29-Sep 6 29-Sep14 6-Oct 13 6-Oct 10 6-Oct 7 6-Oct15 13-Oct 14 13-Oct 11 13-Oct 8 13-Oct16 20-Oct 15 20-Oct 12 20-Oct 9 20-Oct17 27-Oct 16 27-Oct 13 27-Oct 10 27-Oct18 3-Nov 17 3-Nov 14 3-Nov 11 3-Nov19 10-Nov 18 10-Nov 15 10-Nov 12 10-Nov20 17-Nov 19 17-Nov 16 17-Nov 13 17-Nov21 22-Nov 20 22-Nov 17 22-Nov 14 22-Nov Sun-Wed22 30-Nov 21 30-Nov 18 30-Nov 15 30-Nov

Behav 7-Dec 22 7-Dec 19 7-Dec 16 7-Dec Mon-ThursSacrifice 10-Dec Behav 14-Dec 20 14-Dec 17 14-Dec

Sacrifice 17-Dec 21 21-Dec 18 21-Dec22 28-Dec 19 28-Dec

Behav 4-Jan 20 4-JanSacrifice 7-Jan 21 11-Jan

22 18-JanBehav 25-Jan

Sacrifice 28-Jan

Grouping into multiple cohorts and staggering of the initiation of each cohort exposure by 2 – 3 wks allowed for animals to be age-matched both within and across cohorts

Each cohort contained 4 subgroups (hα-Syn +/-AP exposure and wild type +/-AP exposure). Exposures began on July 7, 2015 and continued until January 18, 2016. Each cohort was exposed for 5h/d for 4d/wkfor 22 wks (~ 5 mo)

Significant overlap across all 4 exposure periods, indicating that all exposed mice were exposed to essentially the same AP content- practical time limitations in the number of animals that could be processed for behavioral assessment and brain acquisition at the end of each exposure period

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Three motor tests • vertical pole descent: used to assess aspects of basal ganglia function that have been

associated with movement disorders in PD

• challenging beam traversal: used to assess fine motor coordination and balance;considered sensitive to varying degrees of nigrostriatal dopaminergic dysfunction in genetic mouse models

• nestlet shredding/nest building: used to assess fine motor skills for a species-typical behavior

One cognitive test• Y maze spontaneous alternations response: used to assess measures the willingness of

mice to explore new environments; represents a measure of short term spatial memory capacities, chiefly in the hippocampus and associated structures

Behavioral Assessments:

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Behavioral Responses After AP Exposure - Pole Test

Results: No significant differences were observed in air and exposed animals from either WT or hα-Syn groups

Comments: We suggest that in our study the absence of such motor deficits was primarily related to the absence of significant AP-associated striatum dopamine deficits as determined from the post-mortem brain analysis 16

Videotapes were viewed and rated for errors, number of steps made by each animal, and time to traverse across five trials

Behavioral Responses After AP Exposure - Beam Traversal Test

Results: Paired Student’s t-tests were used to analyze pre- and post-exposure effects in ha-Syn and WT groups; Significant increases in back limb foot slips were observed for both groups post-exposure (final); ha-Syn/exp, p = .0345; WT/exp, p = .0385. Back limb slips (final) for ha-Syn/exp (0.1994 ± 0.02, mean, SE) were significantly increased relative to ha-Syn/air (0.1371 ± 0.019); Student’s t-test, p = .0425

Comments:The observed motoric deficit is considered a behavioral marker for detection of early onset motor disorders in animal models of neurodegeneration

paired analysis mean/groupanalysis

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At 2 and 18 h, manipulation of the nestletand built nest were rated by a five-point Likert scale and nonparametric analysis;

Comments: The absence of deficits in the nestlet response may be attributable to absence of significant alterations detected in hippocampus for NE, and inflammatory markers IL-6 and TNFa

Results: All groups showed increases (mean ±SEM; n = 16-20 mice/group) nestlet building from 2 to 18h; significant differences were not observed between wild type and hα-Syn groups at either time point (Mann–Whitney U-test (p >.05) )

Behavioral Responses After AP Exposure - Nestlet Response

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Behavioral Responses After AP Exposure - Y maze Spontaneous Alternations

Results: Significant differences ± AP exposure were not observed for ‘Fraction’ (# of consecutive entries in three different arms)/total visits), and total number of visits into any of the three arms (means ±SEM; 16- 20 animals /group); Student’s t-test; p >.05

Comments: All groups showed similar results of ~ 60% for the percentage of alternations; results are within range of typical alternation rates, suggesting that experimental methods factors did not mask any differences between groups- It appears that the neither the exposure conditions or hippocampus hα-Syn expression affected performance

Methods: Both percentage alternations (exploring different arms (ABC) nonrepetitive in sequence (e.g., alternation = BAC; not AAB or ABB etc), and the total # of arm visits were measured

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Analyte Marker function MethodTNFa, IL-6 Inflammation ELISA

HO-1 Adaptation/anti-inflammation

ELISA

human alpha-synuclein

Pathologydevelopment

ELISA

HippocampusNorepinephrine

Neurotransmitterdeficit

HPLC-ECD

Striatum Dopamine

Neurotransmitterdeficit

HPLC-ECD

TNFa: tumor necrosis factor alpha; IL6: interleukin 6; HO-1: heme oxygenase-1; HPLC-ECD: High performance liquid chromatography-electrochemical detection

Brain Responses After AP Exposure

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Toxicokinetics

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Site Events Direct actions Indirect

Lungs Reactive species enter lungs, interact with lung lining fluid components and cells Prooxidants generate reactive oxygen species, electrophiles bind to proteins

Cell-reactive species interaction causes release of inflammatory cytokines TNFα, interleukins .

Circulation Reactive oxygen species affect membranes (lipid peroxidation), prooxidants and electrophiles circulate

Cytokine concentration in blood stream increases

Blood brain barrier

Lipid peroxidation lyses membranes, increases permeability to reactive species

Induce inflammatory response, alter permeability

Brain Reactive species enter brain promote inflammatory response through oxidative and electrophilic stress

Promote inflammatory response, increase in TNFα, IL6

Site

Events

Direct actions

Indirect

Lungs

Reactive species enter lungs, interact with lung lining fluid components and cells

Prooxidants generate reactive oxygen species, electrophiles bind to proteins

Cell-reactive species interaction causes release of inflammatory cytokines TNFα, interleukins .

Circulation

Reactive oxygen species affect membranes (lipid peroxidation), prooxidants and electrophiles circulate

Cytokine concentration in blood stream increases

Blood brain barrier

Lipid peroxidation lyses membranes, increases permeability to reactive species

Induce inflammatory response, alter permeability

Brain

Reactive species enter brain promote inflammatory response through oxidative and electrophilic stress

Promote inflammatory response, increase in TNFα, IL6

Results: TNFα Minimum of a 1:4 dilution of the homogenate was required for recovery of added analyte and indicating that this dilution was required to insure an accurate analysis of TNFα

Comments: the dilution reduced TNFα concentrations in the sample to levels below the assay sensitivity - TNFα brain levels could not be detected

Brain Responses After AP ExposureTNFα Interleukin-6 (IL-6)

Interleukin-6 (IL-6) A similar problem was encountered with IL-6, i.e., minimally detectable brain levels were that showed a trend towards significanceLeft panel - individual tissues from 5 WT and 14 hα-syn mice (means ± SEM); Right panel - pooled (5 mice in each group) tissues performed in duplicate). Differences between exposed and filtered air were not significant Student’s t-test; p >.05

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Brain Responses After AP ExposureHeme oxygenase (HO-1)

Results: means ± SEM for 14 mice/group were not significantly different, Student’s t-test; p >.05

Comments: The unchanged HO-1 regional brain content suggests that an inflammatory response was not associated with the protocol of 22 weeks of AP exposure

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Brain Responses After AP ExposureStriatal Dopamine Hippocampal Norepinephrine

Results: Groups (n = 12 -15) ± AP were not significantly different, Student’s t-test; p >.05

Results: Groups (n = 12 -15) ± AP were not significantly different, Student’s t-test; p >.05

Comments: The primary outcome measures were DA content in striatum and NE content in hippocampus because those analytes have been extensively used and validated at markers of neurotransmitter system integrity in those brain regions which was unaffected by the AP exposure

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Results: Significant differences for hα-Syn protein levels in hippocampus and striatum were not measured between air and AP-exposed hα-Syn mice, Student’s t-test; p >.05

Brain Responses After AP Exposure -hα-Syn Content in Striatum and Hippocampus

Comments: The primary outcome measures were DA content in striatum and NE content in hippocampus because those analytes have been extensively used and validated at markers of neurotransmitter system integrity in those brain regions and was unaffected by the AP exposure25

Conclusions – In Vivo Responses The results generally did not show significant changes reflective of development of PD-like behavioral and brain pathology

Although exposure to a more ‘toxic’ CAPS may have produced changes of greater magnitude, conclusions on their relevance to the human condition would need to be determined. Assessing the effects of human AP exposure conditions in animal models may require the use of subtle behavioral tests that are reflective of changes in functional brain activity rather than changes in content of brain inflammatory markers or neurotransmitters

Although the exposure period of 22 weeks was considerably longer than that used in prior studies using VACES concentrated particles, the cumulative effect of exposure to low level toxins may not have affected the target organs because of their ability to reduce chemical challenges with enzymatic detoxification procedures

After AP exposure, deficits observed in the challenging beam/rear limb task for both WT and hα-Syn transgenic mice suggest development of an early onset motor disorder

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Task 4 Arthur ChoChemical and biological characterization of ambient airConclusions

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Chemical and biological properties of ambient PM2.5

The particles of 2.5 microns or less (PM2.5) were collected in parallel to the VACES-generated UFPM using a Tisch sampler with a Teflon filter to collect particles

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Reactive chemical species

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Assays:Prooxidants: DTT +/- DTPA

Electrophiles: GAPDH

Anti-inflammation: HO-1Inflammation : TNFα; IL6

Reactive species Target Product Example Effect

H2O2

OH

Protein-S-

Protein-SOH Keap-1 Nrf2/ARE activation(HO-1)

Anti-inflammation

H H H O Fatty acid Membrane permeability Cell disruption

OR Protein-S-

Protein-S

OR Keap-1 Anti-inflammation

IκBNFkB activation (TNFα)

Inflammation

IκBInflammation

GSH/NADPH

O

Response

Nrf2/ARE activation(HO-1)

NFkB activation (TNFα)

Prooxidant

Electrophile

Chemical properties of PM2.5Prooxidants, or compounds that generate reactive oxygen species (superoxide, hydrogen peroxide and hydroxyl radical) by electron transfer reactions with oxygen are assayed by the DTT assay The organic prooxidants are assayed by measuring DTT activity in the presence of the metal chelator diethylenetriamine pentaacetic acid (DTPA) Electrophiles are assayed by measuring the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity following its irreversible inhibition The assays are performed on an aqueous suspension of the particles and the results normalized to m3

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Range of prooxidant content in different locales

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City/PM2.5 DTT activity/m3 DTT + DTPA activity % non metal DTT Beijing 3.5 0.19 5% Mexico City (Salinas et al.)

3.3 0.63 19%

Los Angeles (110 Frwy) (Verma, Pakbin et al.)

2.5 ND ND

San Bernardino 0.65 0.00 0 Irvine 0.23 0.081 35% Irvine times 12* 2.76 0.97

Samples of particles were analyzed by the DTT assay in the presence and absence of DTPA. The Irvine values is multiplied by 12 to show the effect of VACES concentration.

ND Not determined.

City/PM2.5

DTT activity/m3

DTT + DTPA activity

% non metal DTT

Beijing

3.5

0.19

5%

Mexico City

(Salinas et al.)

3.3

0.63

19%

Los Angeles (110 Frwy)

(Verma, Pakbin et al.)

2.5

ND

ND

San Bernardino

0.65

0.00

0

Irvine

0.23

0.081

35%

Irvine times 12*

2.76

0.97

Samples of particles were analyzed by the DTT assay in the presence and absence of DTPA. The Irvine values is multiplied by 12 to show the effect of VACES concentration.

ND Not determined.

SB

IR

CM

LB

Comparison of data with other sites in LA Basin

Sites of collections and analyses made in a SCAQMD funded railyard ambient air study. Irvine is shown for comparison

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PM2.5 prooxidants in LA Basin

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Near railyards

Background site (> 1 mi)

Diesel truck loading

Results of DTT assays performed on PM2.5 from the SCAQMD study sites compared to Irvine at different weeks during the study

Right panel shows total and the left panel the organic based prooxidants

Note that a significant proportion of the Irvine prooxidants were organic in contrast to the other sites

Summary and Conclusions Hypothesis

- VACES concentrated UFPM exposure was appropriate; a concentration factor of 12 raised prooxidant content to 2.8 DTT units/m3 - comparable to the 3 DTT units/m3 determined for Mexico City

- Exposure to concentrated UFPM / Irvine, CA would effect progression of Parkinson’s disease-like behavioral and neurochemical changes in transgenic/hα-Syn mice

Results

- Changes in inflammatory, neurochemical (DA-striatum, NE- hippocampus) and hα-Syn content, and in most motoric/cognitive tasks were not observed

Conclusions

- Chemical properties of the PM2.5 from Irvine qualitatively different from other LA Basin sites;Irvine - higher levels of organic prooxidants and lower levels of metal prooxidant per unit DTT activity

- Challenging beam traversal task -increases in hind limb foot slips for both WT and transgenic mice suggestive of AP induced subtle deficits in motor skills / balance

Future Directions

- Data from prior studies suggest metal content to be important in eliciting inflammation- In this study, the higher proportion of organic components may have attenuated the overall responses by stimulation of an anti-inflammatory response

- Measurement of metal content in ambient air samples is relevant for interpretation of AP analyses because of their ability to generate the proinflammatory stimulus – the highly reactive hydroxyl radical

Effects of ultrafine particulate matter on an animal model of Parkinson’s diseaseIntroductionTasks and key investigatorsTask 1 Michael Kleinman�Slide Number 5Slide Number 6Slide Number 7Slide Number 8Slide Number 9Tasks 2 and 3. William MelegaSlide Number 11Slide Number 12Temporal Pattern of Human Alpha-Synuclein (hα-Syn) Expression in Brains of Transgenic MouseSlide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20ToxicokineticsSlide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Task 4 Arthur Cho�Chemical and biological properties of ambient PM2.5Reactive chemical speciesChemical properties of PM2.5Range of prooxidant content in different localesComparison of data with other sites in LA BasinPM2.5 prooxidants in LA BasinSummary and Conclusions