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Developmental exposure to the pesticide dieldrin alters the dopamine system and increases neurotoxicity in an animal model of Parkinson’s disease

Jason R. Richardson^*,,1, W. Michael Caudle^*,, Minzheng Wang^*,, E. Danielle Dean^*,, Kurt D. Pennell^*, and Gary W. Miller^*,,2

^* Center for Neurodgenerative Disease, School of Medicine and

Department of Environmental and Occupational Health, Rollins School of Public Health, Emory University, Atlanta, Georgia, USA; and

School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA

²Correspondence: Center for Neurodengenerative Disease, Emory University, Whitehead Biomedical Research, Bldg., Rm. 505, 615 Michael St., Atlanta, GA 30322, USA. E-mail: gary.miller{at}emory.edu

SPECIFIC AIMS

Epidemiological studies have indicated that pesticides increase the risk of Parkinson’s disease (PD). Although PD is considered a disease of aging, experimental evidence suggests that neurodegeneration begins long before clinical diagnosis of PD. Recent focus on the Barker hypothesis has led to the idea that the etiology of a number of adult diseases may result from alterations occurring during development. In the present investigation, we sought to determine whether developmental exposure to dieldrin causes persistent changes to the dopaminergic system and whether these changes result in increased susceptibility to the parkinsonism-inducing neurotoxin MPTP.

PRINCIPAL FINDINGS

1. Developmental dieldrin exposure alters striatal monoamine transporters
To determine whether developmental exposure to dieldrin alters the dopamine system, we administered 0, 0.3, 1, or 3 mg/kg dieldrin every 3 days to pregnant mice throughout gestation and lactation. Pups were weaned at 3 and at 12 wk of age; we analyzed levels of the dopamine transporter (DAT) and vesicular monoamine transporter 2 (VMAT2) by Western blot. At this time point, DAT protein levels were significantly increased by 30%, 41%, and 52% in the male offspring of dams exposed to 0.3, 1, or 3 mg/kg of dieldrin, respectively. DAT protein levels were also increased in the female offspring by 36%, 42%, and 61% from the same treatment groups. To ascertain whether this increase in DAT protein was the result of increased transcription, we measured DAT mRNA levels by RT-polymerase chain reaction (RT-PCR) in the ventral mesencepahlon of these same mice. DAT mRNA levels were increased in a dose-related manner in the male offspring by 23%, 24%, and 54%. DAT mRNA levels were increased in the female offspring as well by 31%, 32%, and 54%, although this did not reach statistical significance. VMAT2 levels were also increased in the male offspring by 16%, 16%, and 27% and in female offspring by 29%, 38%, and 59%. Concordant with the increase in VMAT2 protein level, VMAT2 mRNA levels were increased by 34%, 72%, and 88% in male offspring and by 27%, 29%, and 47% in female offspring.

The effects of developmental dieldrin exposure appear to preferentially target the dopaminergic system, as we observed no change in striatal GABA transporter levels or in cortical norepinephrine and serotonin transporter levels. In addition, analysis of 1-day-old whole brain mRNA levels, a time before a significant amount of exposure through breast milk is observed, revealed no significant changes in the mRNA levels of DAT or VMAT2. This finding suggests that the effects of dieldrin on the dopaminergic system are most likely the result of lactational exposure. Finally, these effects were observed at a time when there were no detectable levels of dieldrin in the striatum of these animals.

2. Developmental dieldrin exposure alters genes controlling monoamine transporter expression
To elucidate possible mechanisms for the observed increase in DAT and VMAT2, we assessed the mRNA levels of NURR1 and Pitx3, two nuclear transcription factors known to regulate DAT and VMAT2 expression during development. At 12 wk of age, NURR1 mRNA levels were increased in the ventral mesenchephalon of the male offspring by 34%, 72%, and 121% (Fig. 1 A), and by 28%, 50%, and 41% in female offspring (Fig. 1B ). Similar to that observed with DAT and VMAT2, NURR1 levels were not increased at 1 day of age. In contrast to the dose-related increases in NURR1, Pitx3 mRNA levels were only increased in the highest dosage group by 202% in male offspring (Fig. 1C ) and by 106% in the female offspring (Fig. 1D ). To further explore the role of NURR1 in the effects on DAT and VMAT2, we determined the protein and mRNA levels of aromatic amino acid decarboxylase (AADC), a component of the dopaminergic system that is not regulated by NURR1. We found that developmental dieldrin exposure had no significant effect on AADC protein or mRNA levels. These results suggest that the increase in DAT and VMAT2 protein and mRNA levels may be mediated through enhanced expression of NURR1 and that Pitx3 may play a role only at the highest dosage tested.

View larger version (23K): [in this window] [in a new window]   Figure 1. Developmental dieldrin exposure increases NURR1 and Pitx3 mRNA levels. Female mice were administered 0 (control), 0.3, 1, or 3 mg/kg dieldrin throughout gestation and lactation. Levels of NURR1 mRNA were determined in midbrain samples in male (A) and female (B) offspring at 12 wk of age by real-time polymerase chain reaction (PCR) and normalized to the 18S ribosomal subunit. Levels of Pitx3 were determined in the same manner in male (C) and female (D) offspring. Values represent mean ± SE of 4–6 animals each from a different litter. Statistical significance is reported for the *P < 0.05 and **P < 0.01 levels compared with the control group within each sex as determined by 1-way ANOVA, followed by the Student-Newman-Keuls test for post hoc analysis.

3. Developmental dieldrin exposure increases neurotoxicity of MPTP
In addition to disruption of proper dopamine compartmentalization, alterations in the expression and ratio of DAT to VMAT2 can greatly affect the vulnerability of the dopamine neuron to neurotoxins such as the parkinsonism-inducing neurotoxin MPTP. We then sought to determine whether the alterations of DAT and VMAT2 by developmental dieldrin exposure would exacerbate the neurotoxicity of a moderate dose of MPTP. In the offspring of dieldrin-treated animals, MPTP (2x10 mg/kg) caused significantly greater reductions of dopamine in the male offspring (74%, 76%, and 74%) than in the male offspring of controls (62%). However, the female offspring of dieldrin-treated animals did not exhibit significantly greater dopamine loss than in the offspring of control animals (67% for control offspring and 69%, 71%, and 64% in dieldrin-treated offspring). The greater effect of MPTP in the male offspring of dieldrin-treated animals cannot be explained by alterations in MPP+ metabolism, as there were no differences in MPP+ accumulation between control and dieldrin-exposed offspring. However, we found that the DAT to VMAT2 ratio was significantly increased in the male offspring by 18%, 22%, and 22%, while there was no significant alteration in this ratio in the female offspring (Fig. 2 A, B). These data suggest that the DAT-to-VMAT2 ratio is the principal determinant of the enhanced toxicity of MPTP in the male offspring of dieldrin-treated animals.

View larger version (39K): [in this window] [in a new window]   Figure 2. Developmental dieldrin exposure increases the DAT:VMAT2 ratio and exacerbates MPTP neurotoxicity in males but not females. Female mice were administered 0 (control), 0.3, 1, or 3 mg/kg dieldrin throughout gestation and lactation, and striatal levels of DAT and VMAT2 protein were determined by Western immunoblotting. These values were then used to calculate the DAT:VMAT2 ratio in male (A) and female (B) offspring at 12 wk of age. Male and female offspring were then administered 2 x 10 mg/kg of MPTP subcutaneously (s.c.) and striatal levels of (C) glial fibrillary acidic protein (GFAP) and (D) -synuclein levels were determined 48 h after exposure to assess the degree of MPTP-induced neurotoxicity. Only data from male mice are presented since we observed no potentiation of MPTP toxicity in the female mice as assessed by striatal dopamine levels. Levels of -tubulin were determined to ensure equal loading of gels. Values represent mean ± SE of 4–6 animals each from a different litter. Statistical significance is reported for the *P < 0.05, **P < 0.01, and ***P < 0.001 levels compared with the control group within each sex as determined by 1-way ANOVA, followed by the Student-Newman-Keuls test for post hoc analysis.

To determine the neurotoxic damage caused by MPTP, we assessed levels of GFAP, an indicator of astroglial proliferation, and -synuclein, a protein involved in PD, both of which have been shown to be up-regulated and indicative of neuronal damage after MPTP exposure. MPTP significantly increased GFAP levels by 26% in the offspring of control animals (Fig. 2C ). This effect was significantly potentiated in the offspring of dieldrin-treated animals, whose GFAP levels were increased by 54%, 60%, and 73%. MPTP treatment increased -synuclein levels by 27% in the male offspring of control animals (Fig. 2D ), which was significantly potentiated in the offspring of dieldrin-treated animals by 47%, 45%, and 46%. These results suggest that developmental dieldrin exposure results in enhanced neurotoxicity from MPTP treatment, which is demonstrated by both greater loss of striatal dopamine and potentiation of GFAP and -synuclein induction.

CONCLUSIONS AND SIGNIFICANCE

Several epidemiological studies have linked exposure to pesticides, and in particular dieldrin, to increased risk of PD. However, few studies have identified mechanisms by which this may occur. In addition, most studies aimed at determining the pathogenic process in PD have focused on events occurring during adulthood. The results of the present study provide evidence that developmental exposure to dieldrin results in persistent alterations of the dopamine system and enhanced vulnerability of dopamine neurons to the parkinsonism-inducing toxin MPTP. This enhanced vulnerability also follows a gender pattern of increased male susceptibility that is consistent with that observed in the human population affected by PD. The results of these studies also bring to light the idea that exposure to dieldrin during critical periods of development may result in "imprinting" of genes (Nurr1 and Pitx3) that regulate the proper formation and maintenance of function of the dopamine system. These alterations may then induce a "silent" state of dopamine dysfunction and increased vulnerability of dopamine neurons later in life. Taken in concert, the results from this study provide a potential molecular mechanism responsible for the association between dieldrin exposure and increased risk of PD, and suggest that greater attention should focus on the role of early life exposures and the development of PD.

View larger version (28K): [in this window] [in a new window]   Figure 3. Schematic illustrating the hypothesized mechanism by which developmental dieldrin exposure alters the dopamine system and causes enhanced neurotoxicity of MPTP. Dieldrin’s primary mechanism of toxicity is through blockade of chloride flux through GABAA receptors. Inhibition of chloride flux in the dopamine neurons of the substantia nigra pars compacta causes increased neuronal activity and burst firing of dopamine neurons. Increased neuronal activity can then activate Nurr1 transcription, which can target downstream genes such as the DAT and VMAT2, resulting in increased transcription and translation. These alterations of DAT and VMAT2 produce a state in which the ratio of DAT:VMAT2 is increased, thereby increasing the neurotoxicity of MPTP.

FOOTNOTES

¹ Present address: Department of Environmental and Occupational Medicine, University of Medicine and Dentistry-New Jersey/Robert Wood Johnson Medical School and Environmental and Occupational Health Sciences Institute, Piscataway, NJ 08854, USA. E-mail: jricha3{at}eohsi.rutgers.edu

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5864fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5864fjev1 20/10/1695    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Richardson, J. R. Articles by Miller, G. W. Search for Related Content PubMed PubMed Citation Articles by Richardson, J. R. Articles by Miller, G. W.