Epidemiological studies have reported increased risk of PD due to pesticide exposure (Drechsel and Patel, 2008
; Costello et al., 2009
; Hancock et al., 2008
; Ritz and Yu, 2000
), and rodent studies have demonstrated decreased motor activity, decreased tyrosine hydroxylase immunoreactivity in the SNpc, and altered dopamine metabolism in the striatum of PQ- and MB-treated mice (Thiruchelvam et al., 2000
). Additionally, MB has been shown to increase levels of PQ in the brain and slow PQ clearance (Barlow et al., 2003
). For these reasons, we sought to further elucidate if MB could potentiate PQ-mediated oxidative stress in dopaminergic neurons. Through the evaluation of alterations in Trx, Prx, and GSH redox states, the data presented here indicate that PQ and MB cause toxicity via divergent mechanisms involving compartment-specific stress and reactive thiol oxidation and alkylation.
GSH is one of the most central thiol antioxidants and has frequently been characterized as a “redox buffer” to preserve the reduced state of proteins, protecting against cellular insults like ROS, reactive nitrogen species, and electrophiles (Jones and Go 2010). Decreased intracellular GSH is associated with impaired cellular functions including ubiquitin proteasome activity, inflammation, and mitochondrial energy production and is observed in the SNpc of PD patients (Martin and Teismann, 2009
). Here, we report that PQ, at concentrations that caused only moderate toxicity, led to a minor loss of GSH. In contrast, MB increased cellular GSH, an effect also observed by Barlow et al. (2003)
in PC12 cells and in primary mesencephalic neuron cultures. Our data indicate that PQ and MB are working through different mechanisms and that oxidative stress can be considered more than a simple imbalance of oxidants and reductants.
To advance the understanding of the toxic mechanisms of PQ and MB, we investigated the ability of these agents to oxidize other important cellular redox couples, namely Trx and Prx. Trx, along with thioredoxin reductase and NADPH, acts as a general disulfide reductase (Arner and Holmgren, 2000
) and is involved in the catalytic function of ribonucleotide reductase and Prx and in the redox regulation of transcription factors such as NF-κB, Nrf2, and AP-1 (Hansen et al., 2004
; Hirota et al., 1997
; Matthews et al., 1992
; Rhee et al., 2005
). Prx are homodimeric, thiol-specific antioxidant proteins that have a major role in detoxification of mitochondrial ROS (Drechsel and Patel, 2010
; Wood et al., 2003
). The current results showed that PQ oxidized mitochondrial Trx2 and Prx3 but not cytoplasmic Trx1. MB treatment did not result in an oxidation of either Trx or Prx. These results suggest that PQ is acting via a mitochondrial mechanism, supporting the results of the WST-1 assay and the interpretation that PQ stimulates mitochondrial ROS generation (Castello et al., 2007
Trx2 performs a variety of functions related to cellular viability. For example, the mitochondrial permeability transition has been demonstrated to be regulated by Trx2 (He et al., 2008
) and is a key defensive mechanism against oxidant-induced apoptosis (Chen et al., 2006
). Trx2 knockouts cause embryonic lethality during developmental periods of mitochondrial maturation, highlighting the necessity of proper Trx2 function in overall mitochondrial function (Nonn et al., 2003
). Thus, untimely PQ-mediated oxidation or MB modification of this key protein could compromise its function and contribute to a decrease in cell viability.
MB caused little to no oxidation of either Prx or Trx, indicating a mechanism divergent from that of PQ. MB did not cause disulfide formation; however, it could be adducting essential Cys residues. This type of modification may not be evident in a Prx or Trx2 redox Western blot because an intra- or intermolecular disulfide would not be created. Both reduced and MB-adducted Prx or Trx2 could migrate to the molecular weight corresponding to “reduced” Prx or Trx2. Also, due to the low concentration of MB (1.5–4μM) used compared with the concentration of intracellular GSH (~300μM), GSH could be buffering the toxic effects of MB.
Our data suggest that PQ is acting via a mechanism involving mitochondrial ROS production, whereas MB acts via an alkylation mechanism. This is evidenced by the oxidation of Trx2, Prx1, and Prx3, and a significant increase in DCF fluorescence caused by PQ treatment and the lack thiol oxidation or oxidants produced in the MB samples. H2
DCFDA, the probe used to measure ROS production, is thought to mostly detect hydrogen peroxide (Oyama et al., 1994
). In this study, MB treatment actually resulted in decreased DCF fluorescence compared with the control cells. Decreased DCF fluorescence was also seen in yeast cells treated with mancozeb, a mixture zinc and manganese ethylenebis (dithiocarbamate) (Dias et al., 2010
). However, the use of H2
DCFDA to detect ROS is controversial (Chen et al., 2010
), and additional detailed studies will be needed to address possible upstream effects of MB on oxidant generation.
Alteration in tissue redox balance is a common pathological feature of neurodegenerative diseases (de Vries et al., 2008
). The transcription factor Nrf2 is central to the efficient detoxification of reactive metabolites and ROS and is effective in blocking neurotoxicity resulting from GSH depletion, lipid peroxidation, calcium overload, excitotoxins, and mitochondrial dysfunction (Johnson et al., 2008
). Our data show that MB caused a large increase in the abundance of nuclear Nrf2. Additionally, MB increased mRNA for phase II detoxification enzymes under the regulation of Nrf2. These enzymes include γ-glutamylcysteine ligase catalytic subunit (GCLC), NADPH:quinone oxidoreductase-1 (NQO1), glutathione S-transferase, heme oxygenase-1 (HO-1), Trx, and Prx (Zhang, 2006
). In our study, MB was the most potent Nrf2 activator, and this activation could occur by MB alkylation of sensor thiols in Keap-1. Keap-1, a protein involved in the negative regulation of Nrf2 through sequestration of Nrf2 in the cytosol, preventing activation, contains multiple Cys-rich regions believed to sense ROS and electrophiles. Oxidation or alkylation of these residues cause the release of Nrf2 and allows nuclear translocation and transcription of phase II detoxification genes (Zhang, 2006
). Barlow et al. (2003)
also, indirectly, demonstrated MB-mediated Nrf2 activation via increased HO-1 protein and GCLC activity, both Nrf2-mediated genes, in response to MB.
In summary, the data presented here show MB potentiation of PQ neurotoxicity does not occur by enhancing oxidative stress. The data provide little evidence of synergy or potentiation of the toxicity of one by the other. Instead, the data show that PQ and MB act through different toxic mechanisms (). PQ induces ROS production affecting intracellular redox states, especially affecting mitochondria, whereas MB does not. MB was especially potent in activating the transcription of genes controlled by Nrf2 and increasing cellular GSH, indicating the involvement of cytoplasmic targets by an alkylation mechanism. Cell death occurred despite a strong Nrf2 response, suggesting that acute exposures may be more important for loss of cells than chronic exposures. The results underscore the importance of mixtures toxicology, where transient individual exposures can be additive in toxicologic outcome.
FIG. 7. A scheme illustrating the proposed divergent mechanisms of PQ and MB toxicity. PQ, through ROS generation, is able to alter redox status of the Trx/Prx system of mitochondria and cause a minor activation of Nrf2. MB, acting as an alkylating agent, alters (more ...)