Parkinson’s disease (PD) is a neurodegenerative disease characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta region of the midbrain [1]. The etiology of PD possibly involves environmental and genetic factors. However, <5% of the total cases of PD have been attributed to genetic factors [2].

Among the risk factors of PD, pesticide exposure has been associated with an increased incidence of PD among agricultural workers in rural environments [3]. Specific pesticides, such as paraquat [4], and maneb [5] have been associated with PD and another pesticide rotenone [6] can produce neurodegeneration when presented in conjunction with another toxicant. As a widely used broad-spectrum herbicide in many regions worldwide, atrazine (ATR) can persist in ground and surface water for long periods because this substance is moderately volatile and water-soluble [7].

Toxicological studies have focused primarily on the effects of ATR on the endocrine and reproductive systems [8–10]. Studies on the potential neurotoxicity of ATR have also been conducted, showing that ATR exposure can alter striatal neurochemistry; as a result, striatal dopamine (DA) levels are decreased and a loss in tyrosine hydroxylase (TH)-positive dopaminergic neurons is observed in the substantia nigra of rats [11] and mice [12]. Animal studies have also indicated that male offspring elicit anti-androgenic effects after their mothers have been exposed to ATR during pregnancy [13].

Although many PD cases are observed later in life, the pathological mechanism begins at early stages before this disease has progressed to the point at which it is diagnosed [14]. The presence of a long preclinical phase and the association of environmental exposures with an increased risk of PD have indicated that early life exposure to environmental toxicants may enhance dopaminergic neurodegeneration; for this reason, the risk of PD increases. Therefore, we considered the following questions: whether or not the toxic effect of ATR is observed on dopaminergic neurons; whether or not such toxic effects occur during the early formation and development of dopaminergic neurons; and whether or not the dopaminergic neurons of progeny are damaged if their mothers are exposed to ATR during pregnancy or lactation.

Studies on twins and relatives have shown that the susceptibility to PD may be a result of prenatal predisposition [15]. This finding suggests that genes may be involved in controlling the development and differentiation of DA neurons. Among these genes, orphan nuclear hormone (Nurr1) is considered as the most important. Studies have further indicated that Nurrl is essential for the developmental differentiation and survival of dopaminergic neurons [16]. Defects or altered expression of Nurrl in the substantia nigra (SN) is possibly associated with PD [17,18]. Hence, Nurrl is considered as a candidate gene in the etiology of PD. Therefore, we aimed to determine whether or not the appearance of PD-like symptoms is influenced by Nurr1 in rats exposed to ATR during developmental growth. We also aimed to investigate whether or not this gene functions in the neurotoxicity of ATR.

ATR is frequently detected in ground and surface water in agricultural regions because this substance exhibits limited solubility in water [19]. ATR is also one of the most frequently detected pesticides in fresh water sources in the US [20,21]. Furthermore, this herbicide has been frequently detected in rain water [22,23], fog, arctic ice, and seawater at great distances from agricultural areas [24] In living organisms, ATR is not completely removed from the body within 24 h; for instance, the major metabolites of ATR can be detected in the urine of mice at 48 h after administration [25]. Therefore, the toxic effects of ATR may be manifested only after organisms have been repeatedly exposed because this chemical has accumulated and exceeded the critical threshold.

For a short-term exposure (15 to 21 days) of a mixer-loader-tender applicator in California, the absorbed daily dose (ADD) of ATR ranges from 1.8 to 6.1 μg/kg/day [26,27]. By comparison, a higher ADD of ATR is predicted for farmers and commercial applicators in developing countries because of inappropriate personal protective equipment and unintentional excessive application. The families of ATR applicators are also at risk of high-level ATR exposure [28]. Our experimental doses administered to rats ranged from 25 to 50 mg/kg. These doses were based on the concentrations used in previous studies [13,29] and similar to 70 mg/kg/day, which is used to calculate the lowest observed adverse effect level [30].

Our study focused on the effects of ATR exposure on the development of Mesencephalic dopaminergic (MesDA) neurons. MesDA neurons control voluntary movement; as such, PD occurs when these neurons are degenerated. MesDA neurons begin to develop at embryonic day 10 (E10); at E10.5, Nurr1 expression begins shortly before TH is formed (E11.5). At E12.5, numerous MesDA neurons are formed, and development of the MesDA system continues until the postnatal period [31,32]. Ross et al. reported that ATR could cross the blood brain barrier and enter the brain through an unknown mechanism [25]. Rayner et al. [33] reported that administration of atrazine to lactating dams resulted in delayed vaginal opening in nursing litters. This study suggested that milk-derived factors might have effect on the offspring. Similar studies (Stoker et al., 1999; Rayner et al., 2007) [29,34] reported that exposure of pregnant and early postpartum dams to atrazine at 100 mg/kg/d resulted in preputial separation delays in the male offspring and resulted in increased prostate inflammation in the adult males. Stoker et al. also found that 14C-atrazine can be detected in the anterior and posterior hypothalamus and striatum of nursing rat when administration of 14C-atrazine a single dose of 2 or 4 mg/kg by gavage to lactating dams [35]. The above studies indicated that ATR could enter the infant brain through breast milk. In our study, the offspring were not directly treated with ATR; instead, these offspring were exposed to ATR by maternal gavage from GD5 to PND22. Neither the offspring nor the dams were treated with ATR after PND22. Hence, the duration of exposure began at the critical period of MesDA neuron development.

The present study is based on the following hypotheses: (1) exposure to ATR at early stages of life may lead to disease and dysfunction in later years [36] and (2) exposure to environmental factors during the critical periods of development can lead to adult expression of the disease [37].

DA is one of the primary neurotransmitters in the central nervous system. As such, a remarkable decrease in DA in the striatum is a sign of PD. In vivo [11,12] and in vitro [38–40] studies have shown that exposure to ATR can induce dopaminergic neurotoxicity manifested by decreased DA levels. We assessed the DA levels in the striatum of the offspring after one year of exposure of their mothers to ATR during gestation and lactation. Our studies showed that ATR can decrease the DA content in the striatum. This suggests that exposure to ATR during development can induce dopaminergic neurotoxicity.

The metabolism of DA in the brain is a complicated process involving synthesis, release, degradation, and reuptake. Dopaminergic cells in the brain use TH to convert tyrosine to levodopa (L-DOPA). L-DOPA is then converted to DA by aromatic amino acid decarboxylase (AAAD). TH is the rate-limiting enzyme for DA synthesis. Intra-neuronal DA is then transported and stored in synaptic vesicles by VMAT-2. Under normal circumstances, neuronal activation promotes the vesicular release of DA into the synaptic cleft and elicits a physiological effect via the combination of DA and DA receptors (DR). When released, DA undergoes rapid reuptake to terminate activity and maintain DA homeostasis. Reuptake is accomplished in two ways: (1) synaptosomal uptake via a DA transporter (DAT), which transports DA from the extracellular space into the cytosol; and (2) vesicular uptake by VMAT-2, which stores DA in synaptic vesicles. Free DA in the synaptic cleft is converted to homovanillic acid (HVA) by catechol-O-methyl transferase (COMT) and monoamine oxidase (MAO). DA in the cytosol is converted to 3,4-dihydroxyphenylacetic acid (DOPAC) by MAO [41]. If any stage of DA metabolism is disrupted, the quantity of DA is decreased, causing PD. In addition, other transcription factors, including Pitx3, Lmx1b, Nurr1, and Wnt (Figure 7), possibly affect the synthesis and metabolism of DA. Among these factors, Nurr1 was investigated in this study.

The possible reason for the decrease in striatum DA levels reported in the present studies could be an inhibition of expression of the rate-limiting enzyme for DA synthesis TH [41]. We assessed the mRNA and protein levels of TH in the ventral midbrain of the offspring after one year of exposure of their mothers to ATR during gestation and lactation. However, our studies showed that TH was not significantly affected by ATR, This is consistent with the findings of previous studies done by Filipov et al. [40]. Similarly, the expression of TH was not significantly affected by ATR in PC12 cells [39]. Also, TH protein levels in the striatum of mice exposed to ATR were not different among treatments at a time when DA levels were decreased [12]. This result suggested that TH was not a major target of ATR.

Another possible reason for the decrease in striatum DA levels could be the effect on transportation and storage of DA by ATR. Thus we assessed the mRNA and protein levels of DAT, and VMAT2 in the ventral midbrain of the offspring after one year of exposure of their mothers to ATR during gestation and lactation. The major finding in the present study indicated that exposure to ATR during a critical period of dopaminergic neuron development decreased VMAT2 expression in the ventral midbrain. This is similar to previous studies [14,42] that showed exposure of pregnant mice to the organochlorine pesticide heptachlor can increase the vulnerability to developing PD by influencing the expression of VMAT2 and DAT. However the difference is that the organochlorine pesticide heptachlor can upregulate the dopamine transporter (DAT) and the vesicular monoamine transporter 2 (VMAT2) in their offspring at 12 weeks of age. The possible reason is that exposure of pregnant mice to heptachlor may effect VMAT2 and DAT, and increase their compensatory expression in the adult stage, thus increasing the susceptibility to later stimulation. Results of this study showed that, with the increase of age, the aging caused the loss of expression compensatory capacity and expression downregulation, leading to DA neuron injury. The effect of ATR on the offspring in adulthood was not examined in this study.

We hypothesize that the decrease of neuronal activity in dopamine neurons by ATR may be responsible for alterations in transcription, so we determined mRNA and protein levels of the nuclear transcription factor Nurr1, known to regulate DAT and VMAT2 [43,44]. We found dose-dependent decreases in mRNA levels of Nurr1 transcription in the offspring. This indicated that, ATR could damage the DA neurons in senile rats through its early effect on Nurr1, thus changing the DAT and VMAT2 expression.

The low mRNA expression of VMAT2 reduced transport capacity and inhibited DA uptake into striatal synaptic vesicles. ATR did not exhibit a significant influence on the mRNA expression of DAT, although this expression possibly increased by 10%. Such inhibition could decrease vesicular DA levels and increase cytosolic DA levels; as a result, free DA excessively accumulates in the cytosol. Increased cytosolic DA then induces oxidative stress by producing free radicals and reactive metabolites [45,46]. Therefore, ROS-mediated toxicity occurs in DA neurons [47]. In addition, high concentrations of cytosolic DA inhibit mitochondrial respiration and can cause cytotoxicity [48].

To further confirm the effect of Nurr1, TH, DAT and VMAT2 on DA neurons, their protein expressions were also observed (Figure 6). Western blots showed that, the expression of Nurr1 and VMAT2 were down regulated with the increasing concentrations of ATR (0, 25 and 50 mg/kg), which had the same tendency with mRNA expression. VMAT2 mRNA decrease is only significant at 50 mg/kg while VMAT2 protein decreased significantly at 25 and 50 mg/kg. The expression of DAT was upregulated with the increasing concentrations of ATR (0, 25 and 50 mg/kg). This change tendency was also the same with mRNA, but DAT protein increased significantly at 50 mg/kg. It is possible that VMAT2 protein is lost at 25 mg/kg before a mRNA decrease is measurable. The expression of DAT protein is increased before mRNA increase is measurable.

Our findings suggested a vulnerability to developing PD, in which exposure to ATR during the critical period of dopaminergic neuron development subsequently altered the dopamine system. As a result, DA levels in the striatum decreased.

A possible reason for this decrease in striatal DA levels was attributed to the decreased mRNA expression of Nurr1. This decrease may result in reduced Nurr1 activity and downregulate the mRNA expression of VMAT2. ATR would then function by interfering with the vesicular storage or reuptake of DA.

Thus, our findings suggested that early life exposure to ATR leads to persistent changes in the developing dopaminergic system by regulatory Nurr1 expression. This condition may increase the susceptibility to PD. Other possible ATR targets, particularly AAAD, MAO, and Pixt3, should be further investigated (Figure 7).