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Pharmacy Faculty Publications Wegmans School of Pharmacy

3-2012

Environmental Toxins Linked toNeurodegeneration and Autism Activate the Brain’sImmune SystemJ. L. Hill

C. Teijelo

H. M. To

A. Meyer

E. S. Micca

See next page for additional authors

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Publication InformationHill, J. L.; Teijelo, C.; To, H. M.; Meyer, A.; Micca, E. S.; Vaughn, N.; and Lull, Melinda E., "Environmental Toxins Linked toNeurodegeneration and Autism Activate the Brain’s Immune System" (2012). Pharmacy Faculty Publications. Paper 26.http://fisherpub.sjfc.edu/pharmacy_facpub/26Please note that the Publication Information provides general citation information and may not be appropriate for your discipline. Toreceive help in creating a citation based on your discipline, please visit http://libguides.sjfc.edu/citations.

Environmental Toxins Linked to Neurodegeneration and Autism Activatethe Brain’s Immune System

AbstractMicroglia are the primary immune cells of the central nervous system and become activated in response tonoxious stimuli, leading to a cycle of inflammation and cell death that has been implicated in the developmentof Parkinson’s disease and autism. This study examines the effects of environmental toxins, at levels commonlyfound in humans, on microglial cell survival and activation. The toxins used in this study includepolybrominated diphenyl ether (PBDE) flame retardants, the food additive propionic acid (PPA), and theorganochlorine pesticide dieldrin. These chemicals have been linked to neuronal damage, although theireffects on microglial cells have not been fully studied. Our results indicate that microglial cell survival could bedecreased by as much as 50% due to exposure to these toxins, without the production of certain cytokinesproduced by lipopolysaccharide (LPS)-induced activation. These effects are significant as furtherunderstanding of the role of microglia in neuronal damage could provide a pharmacologic target for futuredrug development as well as elucidate the pathology of neurodegenerative diseases.

DisciplinesPharmacy and Pharmaceutical Sciences

CommentsPoster presented at the American Pharmacists Association Annual Meeting in New Orleans, Louisiana, inMarch 2012.

AuthorsJ. L. Hill, C. Teijelo, H. M. To, A. Meyer, E. S. Micca, N. Vaughn, and Melinda E. Lull

This poster presentation is available at Fisher Digital Publications: http://fisherpub.sjfc.edu/pharmacy_facpub/26

Environmental Toxins Linked to Neurodegeneration and Autism Activate the Brain’s Immune System

Abstract:

Microglia are the primary immune cells of the central nervous system and

become activated in response to noxious stimuli, leading to a cycle of

inflammation and cell death that has been implicated in the development of

Parkinson’s disease and autism. This study examines the effects of

environmental toxins, at levels commonly found in humans, on microglial cell

survival and activation. The toxins used in this study include polybrominated

diphenyl ether (PBDE) flame retardants, the food additive propionic acid

(PPA), and the organochlorine pesticide dieldrin. These chemicals have

been linked to neuronal damage, although their effects on microglial cells

have not been fully studied. Our results indicate that microglial cell survival

could be decreased by as much as 50% due to exposure to these toxins,

without the production of certain cytokines produced by lipopolysaccharide

(LPS)-induced activation. These effects are significant as further

understanding of the role of microglia in neuronal damage could provide a

pharmacologic target for future drug development as well as elucidate the

pathology of neurodegenerative diseases.

J.L. Hill1, C. Teijelo1, H.M. To1, A. Meyer1, E.S. Micca2, N. Vaughn1, M.E. Lull3

1Pharm.D. Candidate, Wegmans School of Pharmacy, St. John Fisher College, Rochester, NY 14618 USA

2Biology Major, Science Scholars, St. John Fisher College, Rochester, NY 14618 USA 3Department of Pharmaceutical Sciences, Wegmans School of Pharmacy, St. John Fisher College, Rochester, NY 14618 USA Abstract ID: 298

Methods:

An immortalized rat microglial cell line (HAPI) was subjected to

physiologic concentrations of PBDEs, dieldrin, or PPA, in vitro.

Effects on microglia are measured via cell death, cytokine (TNFα),

and nitrite production. Cell survival and nitrite production were

measured at 24 hours post-treatment, while TNF alpha was

measured 3 hours post-treatment. Statistically significant changes

for each measure were determined using one-way ANOVA with a

Bonferroni post-hoc test; p-values < 0.05 were considered

significant.

MTT Assay The MTT assay was used to measure the metabolic viability of

cultured microglia. Thiazole blue (MTT) is applied to the cell

cultures and cell survival is based on a color change.8

TNFα ELISA The production and release of TNFα was measured using an

enzyme-linked immunosorbent assay (ELISA) according to

manufacturer protocols (R & D Systems, Minneapolis, MN) and as

published previously.8

Nitrite Assay The amount of nitrite in the culture was determined using the

Griess reagent method and measuring color change as previously

described.8

Introduction:

In the central nervous system (CNS), microglia are the innate immune cells.

Microglia are spread throughout the brain and when resting appear ramified,

or branched (See Fig. 1). These branches extend into the surrounding tissue.

Microglia wait in this resting state until contact is made with environmental

triggers: damaged cells, foreign matter, plaques or other inflammatory

triggers (e.g., pathogens or toxic chemicals).1 After contact, microglia

change into an activated ameboid form, where they phagocytize cellular

debris and release pro- or anti- inflammatory factors and reactive oxygen

species (ROS). Microglia can, however, become over-activated and damage

neurons in a process known as reactive microgliosis (see Fig. 2). Diseases

such as Alzheimer's disease and Parkinson’s disease have been associated

with reactive microgliosis and activated microglia. Environmental toxins, such

as pesticides and flame retardants, are also known to activate microglia and

may play a role in the development of neurodegenerative diseases.

Dieldrin is an organochlorine pesticide that persists heavily in the

environment. Widely used in the U.S. until the 1980’s, exposure occurs via

contaminated food.2 Dieldrin induces neurotoxicity in dopaminergic neurons

through oxidative stress. Dieldrin has also been shown to induce microglia to

produce ROS, which can activate apoptosis in dopaminergic neurons.2

Propionic acid (PPA), a natural component of butter and some cheeses,

has been used as a pesticide due to its fungicidal and bactericidal properties.

It is also an endogenous metabolite in humans, and as such is exempt from

legal residue limits when applied to crops. Moreover, it does not have to

undergo typical pesticide toxicology data and testing. PPA is readily able to

cross the blood brain barrier and enter the CNS, where it has been linked to

autism, and has been proven to activate microglial cells in vivo.3 The

molecular effects of this activation are unknown.

PBDE flame retardants are commonly used in many household items, from

computers and televisions to carpeting and furniture. They’ve also been

found to be increasingly prevalent in the environment, with high levels

recorded in soil, animals, and human serum and milk.4 Studies in rats and

mice have shown low concentrations of PBDEs to cause irreversible

neuronal damage.5 PBDE-47, a common PBDE homolog, has been shown

to induce ROS formation in human neutrophil granulocytes and primary rat

hippocampal neurons in vitro.6

All three toxins are common environmental exposures that have been linked

to neurodegenerative or neurodevelopmental disease, but the role of

microglia in these processes has not been fully explored. Thus, the objective

of this study is to examine the effects of these environmental toxins from a

number of categories on the activation of microglial cells in vitro.

References: 1.Lull ME, Block ML. 2010. Microglial activation and chronic neurodegeneration. Neurotherapeutics; 7(4): 354-365.

2.Song C, Kanthasamy A, Anantharam V, Sun F, Kanthasamy AG. 2010. Environmental neurotoxic pesticide increases histone

acetylation to promote apoptosis in dopaminergic neuronal cells: relevance to epigenetic mechanisms of neurodegeneration. Mol

Pharmacol; 77: 621-632.

3.Thomas RH, Foley KA, Mepham JR, et al. Altered brain phospholipids and acylcarnitine profiles in propionic acid infused rodents:

further development of a potential model of autism spectrum disorders. Journal of Neurochemistry 2010;113:515-529.

4.Sjodin A, Jones RS, Focant JF, et al. Retrospective time-trend study of polybrominated diphenyl ether and polybrominated and

polychlorinated biphenyl levels in human serum from the United States. Environ Health Perspect. 2004 May;112(6):654-8.

5.Branchi I, Alleva E, Costa LG. Effects of perinatal exposure to a polybrominated diphenyl ether (PBDE 99) on mouse

neurobehavioural development. Neurotoxicology. 2002 Sep;23(3):375-84.

6.He P, He W, Wang A, et al. PBDE-47-induced oxidative stress, DNA damage and apoptosis in primary cultured rat hippocampal

neurons. Neurotoxicology. 2008 Jan;29(1):124-9.

7.Stence N, Waite M, Dailey ME. Dynamics of microglial activation: a confocal time-lapse analysis in hippocampal slices. Glia. 2001

Mar 1;33(3):256-66.

8.Lull ME, Levesque S, Block ML. Chronic apocynin treatment attenuates beta-amyloid plaque deposition and microglia in hAPPSL TG

mice. In revision for PLOS One, March 2011.44

FIG. 2. Reactive microgliosis drives chronic neuron damage. Both stimulation of microglia

with pro-inflammatory triggers (e.g., lipopolysaccharide [LPS]) and direct neuron damage

(e.g., glutamate excitotoxicity) result in microglial activation causing the release of

neurotoxic factors, such as nitrite (NO2⁻), tumor necrosis factor-alpha (TNFα), superoxide

(O2·⁻), and hydrogen peroxide (H2O2). Subsequently, after damage with either a pro-

inflammatory trigger or a direct neurotoxin, the neuron releases microglial activators such

as µ calpain, α-synuclein, and neuromelanin, which activate microglial cells and

propagate the cycle. This self-perpetuating cycle of neurotoxicity is known as reactive

microgliosis. (LPS lipopolysaccharide; MPTP 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine) Figure slightly modified from Lull and Block (2010)1 FIG. 3. HAPI microglia cell survival in response to differing concentrations of the

pesticide dieldrin. Treatment of HAPI cells with dieldrin at 10, 20, 30, and 40 µM

concentrations produced statistically significant decreases in cell survival. *p<0.05

FIG. 4. HAPI microglia cell survival in response to differing concentrations of

polybrominated diphenyl ether (PBDE-47) flame retardant . Reduction in cell survival

at a PBDE concentration of 60µM was statistically significant. *p<0.05

FIG. 5. HAPI microglia cell survival in response to differing concentrations of the pesticide

propionic acid (PPA). A decreasing trend in cell survival is seen becoming statistically

significant at a PPA concentrations of 1.5 mM and 3.0mM. *p<0.05

Results:

Dieldrin (FIGURE 3) significantly reduces microglial cell survival at doses of 10, 20, 30 and 40 uM, with

a dose-dependant response.

PBDE-47 (FIGURE 4) caused a statistically significant reduction in survival of microglia at a dose of 60

uM.

PPA (FIGURE 5) exposure also caused a significant decrease in microglial cell survival at

concentrations of 1.5 and 3.0 mM.

Neither dieldrin, PBDE-47, nor PPA activate microglia to release the cytokine TNF-α or nitrite, both

typical markers of microglial activation.

Conclusions:

Microglia play a major role in immunity for the CNS, and chronic

activation due to environmental toxins or neuronal damage has

been shown to result in neuronal death and inflammation. This

study shows that the environmental toxins dieldrin, PBDE-47 and

PPA can cause microglial cell death at physiologically relevant

levels, instead of the release of inflammatory mediators normally

seen in response to threatening stimuli in the CNS (e.g., LPS and

air pollution). This implies the presence of a separate mechanism

of action of these toxins, resulting in cell death. This also implies

that microglia may be compromised, and be unable to serve

protectively when exposed to certain toxins, leaving neurons

vulnerable.

Future directions of study involve exploring the mechanisms of

toxin-induced microglial cell death. This includes the

measurement of the activity of histone acetyl transferase (HAT)

proteins. Histone acetylation allows for DNA to become unwound

from histones and free for gene transcription. Preliminary studies

have indicated that HAT inhibitors may help protect against toxin-

induced microglial cell death. Elucidating the pathways of

neurodegeneration due to environmental toxins will offer new

targets for investigational drug research.

PBDE-47: PPA:

*

*

*

0

20

40

60

80

100

120

Control Dieldrin 10µM Dieldrin 20µM Dieldrin 30µM Dieldrin 40µM

Cel

l S

urv

iva

l (%

)

0

20

40

60

80

100

120

Control PPA 0.375 PPA 0.75 PPA 1.5 PPA 3

Cel

l S

urv

iva

l (%

)

0

20

40

60

80

100

120

Control PBDE 2 uM PBDE 20 uM PBDE 40 uM PBDE 60 uM

Cel

l S

urv

iva

l (%

)

FIG. 1. Microglial morphological changes in response to cell activation. Figure from

Stence et al (2001)7.

Dieldrin:

*

*

*

*

Soluble Neuron-Injury Factors

μ calpain, α-synuclein, & Neuromelanin

TNF-α, No2-, O2

·-, & H202

Neurotoxic Factors

Cell

Surv

ival (%

) C

ell

Surv

ival (%

)

Cell

Surv

ival (%

)

Control Dieldrin 10μM Dieldrin 20 μM Dieldrin 30 μM Dieldrin 40 μM

Control PBDE 2 μM PBDE 20 μM PBDE 40 μM PBDE 60 μM Control PPA 0.375 mM PPA 0.75 mM PPA 1.5 mM PPA 3 mM

* *

*