TY - JOUR
AU - Cannon, Jason, R.
AB - Abstract Parkinson's disease (PD) is the second most common neurodegenerative disease. Much data has linked the etiology of PD to a variety of environmental factors. The majority of cases are thought to arise from a combination of genetic susceptibility and environmental factors. Chronic exposures to dietary factors, including meat, have been identified as potential risk factors. Although heterocyclic amines that are produced during high-temperature meat cooking are known to be carcinogenic, their effect on the nervous system has yet to be studied in depth. In this study, we investigated neurotoxic effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a highly abundant heterocyclic amine in cooked meat, in vitro. We tested toxicity of PhIP and the two major phase I metabolites, N-OH-PhIP and 4′-OH-PhIP, using primary mesencephalic cultures from rat embryos. This culture system contains both dopaminergic and nondopaminergic neurons, which allows specificity of neurotoxicity to be readily examined. We find that exposure to PhIP or N-OH-PhIP is selectively toxic to dopaminergic neurons in primary cultures, resulting in a decreased percentage of dopaminergic neurons. Neurite length is decreased in surviving dopaminergic neurons. Exposure to 4′-OH-PhIP did not produce significant neurotoxicity. PhIP treatment also increased formation of oxidative damage markers, 4-hydroxy-2-nonenal (HNE) and 3-nitrotyrosine in dopaminergic neurons. Pretreatment with N-acetylcysteine was protective. Finally, treatment with blueberry extract, a dietary factor with known antioxidant and other protective mechanisms, prevented PhIP-induced toxicity. Collectively, our study suggests, for the first time, that PhIP is selectively toxic to dopaminergic neurons likely through inducing oxidative stress. Parkinson's disease, PhIP, neurotoxicity, heterocyclic amines Parkinson's disease (PD) affects ∼5 million people worldwide (Dorsey et al., 2007). Approximately 90% of PD cases are thought to be sporadic, arising from unknown causes, with ∼10% arising from purely genetic factors (Cannon and Greenamyre, 2013). The loss of dopaminergic neurons in the substantia nigra and resultant striatal dopamine depletion results in the cardinal motor symptoms of the disease, which is characterized by a combination of bradykinesia, resting tremor, postural instability, and rigidity (Cannon and Greenamyre, 2010; Shulman et al., 2011). The etiology of PD is not completely understood but has been linked to numerous diverse risk factors, including genetics and environmental exposures to pesticides, solvents and a variety of heavy metals, but none have been definitively implicated as a major cause of the disease (Cannon and Greenamyre, 2011). However, much data suggests that nigral dopamine neurons are selectively sensitive to a variety of insults in part due to anatomical features related to long, poorly myelinated processes, differences in cell signaling related to selective expression of specific calcium channels, and a high oxidative stress burden due to the production and metabolism of dopamine (Braak et al., 2004; Hastings and Zigmond, 1994; Surmeier et al., 2011). Dietary factors have not been examined to the same extent as environmental toxicants as potential etiological factors in PD. Such factors, including toxicants present in foods, may be encountered in significantly higher doses and to a larger percentage of the population than many rarely or regionally encountered environmental toxicants. Furthermore, diet has a critical role in other disease states, such as cancer and other inflammatory diseases (Barbaresko et al., 2013; Tete et al., 2012). Connections between cancer and neurodegeneration have started to emerge, showing that dopamine has tumor-protective effects by influencing growth and apoptosis (Inzelberg et al., 2012; Rubi and Maechler, 2010), and mutations in several cancer genes cause PD in humans (Rub et al., 2002). Additionally, diets that are high in saturated and animal fat have been linked to an elevated risk for developing PD (Chen et al., 2003; Logroscino et al., 1998). Meat is a clear source of saturated fat, yet dietary toxicants produced during the cooking process of meat have not been investigated as a contributing factor for the development of PD. Meat preparation that involves cooking at high temperatures and resultant charring can produce several toxic compounds. Specifically, heterocyclic amines are produced during this process (Felton et al., 1984). There is limited knowledge on the neurological effects of heterocyclic amines. However, two known compounds, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) affect dopamine catabolism by inhibiting monoamine oxidase (Ichinose et al., 1988; Kojima et al., 1990; Maruyama et al., 1994). Further, Trp-P-2 has also been reported to inhibit aromatic l-amino acid decarboxylase (dopamine synthesis) (Ota et al., 1990). Elevated blood levels of harmane (a β-carboline alkaloid that is also a heterocyclic amine) have also been linked to tremor severity in humans and more recently blood levels have been found to be increased in PD patients (Louis et al., 2008, 2011, 2014). Thus, specific heterocyclic amines modify dopamine metabolism and have been linked to neurological phenotypes, including PD. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine that may account for up to ∼75% of the genotoxic material isolated from the crust of cooked meat (∼15 ppb of the original weight of uncooked meat) (Felton et al., 1986a,b). Therefore, PhIP could potentially be consumed in high doses through diets rich in meat. In a mouse model, PhIP and a primary metabolite, N-OH-PhIP, were shown to cross the blood-brain-barrier (BBB) 0.5 h postinjection (Teunissen et al., 2010). Interestingly, in this study, the highest tissue levels of N-OH-PhIP were found in the brain. Moreover, PhIP and several phase I metabolites have a pyridine ring, and the metabolites have a potentially reactive hydroxyl group, sharing structural features of dopamine and other known dopaminergic neurotoxicants including, 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridinium (MPP+), and paraquat (Fig. 1) (Chen et al., 2007). Finally, several of us observed (Turteltaub and Director-Myska, unpublished personal observations) in high-dose genotoxicity studies using PhIP that C57BL/6 female mice exhibited freezing and tremors in the highest dose animals. Fig. 1. Open in new tabDownload slide PhIP and select phase I metabolites contain a pyridine ring, and PhIP metabolites have a potentially reactive hydroxyl group. Structures of dopamine, known dopaminergic toxicants, PhIP, and select phase I PhIP metabolites. Potentially reactive hydroxyl groups are shown in bold and pyridine ring structure is shown (dashed box). Fig. 1. Open in new tabDownload slide PhIP and select phase I metabolites contain a pyridine ring, and PhIP metabolites have a potentially reactive hydroxyl group. Structures of dopamine, known dopaminergic toxicants, PhIP, and select phase I PhIP metabolites. Potentially reactive hydroxyl groups are shown in bold and pyridine ring structure is shown (dashed box). To the best of our knowledge, detailed investigations into PhIP neurotoxicity and a potential role in neurodegeneration have not been examined. For the first time, in this study, we sought to test the hypothesis that dopaminergic neurons are selectively sensitive to PhIP and two of the most prevalent phase I PhIP metabolites. Primary mesencephalic neuronal cultures containing both dopaminergic and nondopaminergic neurons were utilized to test acute neurotoxicity in vitro. The results from this study present the first data on PhIP-induced selective toxicity in primary dopaminergic neurons and suggest that this compound should be further evaluated as a putative PD toxicant. MATERIALS AND METHODS Preparation of primary mesencephalic cultures Day 17 embryos were harvested from pregnant Sprague Dawley rats (Harlan, Indianapolis, IN) and primary midbrain cultures were prepared from the isolated brains using a method similar to that previously described by members of our group (Cooper et al., 2006; Liu et al., 2008; Strathearn et al., 2014). Briefly, the cells were plated on 48-well plates or on glass coverslips at a density of 1250 cells/mm2. On the fifth day, the cell cultures were treated with 20μM cytosine arabinofuranoside (AraC) for 48 h to restrict the growth of glial cells (removed prior to experimental treatments). Cultures were treated immediately after the AraC treatment, at 7 days in vitro (AraC treatment was from 5 to 7 days in vitro). This treatment resulted in a glial cell density accounting for about 50% of the overall cell population. In our experience, although this glial density may be less than that which occurs in vivo, it represents an optimal environment for neuronal viability in cell culture. This preparation typically results in a neuronal population that is ∼3–5% dopaminergic; the majority of neurons are GABAergic (Cooper et al., 2006; Liu et al., 2008; Strathearn et al., 2014). All studies were approved by the Purdue Animal Care and Use Committee. Preparation of blueberry (BB) extract To test whether a dietary factor could prevent PhIP-induced neurotoxicity, a BB extract was prepared. The rationale for testing whether a BB extract would be protective was: (1) BB and other anthocyanin- and proanthocyanidin-rich extracts are known to have antioxidant effects (Hakkinen et al., 1999), and we know that PhIP triggers an increase in oxidative stress (Fig 4); (2) anthocyanin- and proanthocyanidin-rich extracts (other than BB) have been shown to have neuroprotective activity in PD models; and (3) we recently found that a BB extract alleviates neurotoxicity elicited by a PD-related environmentally relevant insult that triggers oxidative stress in primary midbrain cultures (Strathearn et al., 2014). Fig. 2. Open in new tabDownload slide PhIP and select phase I metabolites induce selective dopaminergic toxicity in primary midbrain cultures. Primary midbrains neurons were treated with PhIP, N-OH-PhIP, or 4′-OH-PhIP for 24 h (0–1μM). (A) Representative confocal microscopy images obtained after immunocytochemistry with antibodies raised against MAP2 and TH after PhIP (1μM), N-OH-PhIP (1μM), or 4′-OH-PhIP (1μM) treatment. Scale bar represents 25 μm. (B) PhIP and N-OH-PhIP induce selective loss of TH+ neurons (minimum of 500 total neurons in ten fields of view quantified for each treatment; expressed as %TH+, relative to total neurons). 4’-OH-PhIP appears to be less toxic to dopaminergic neurons (n = 3). **p < 0.01, ***p < 0.001; square root transformation, one-way ANOVA, followed by Tukey's multiple comparisons post hoc test. (C) Neurite lengths after PhIP and PhIP metabolite exposure. A total of 694 neurites were analyzed. The data are presented as the mean ± SEM. Fig. 2. Open in new tabDownload slide PhIP and select phase I metabolites induce selective dopaminergic toxicity in primary midbrain cultures. Primary midbrains neurons were treated with PhIP, N-OH-PhIP, or 4′-OH-PhIP for 24 h (0–1μM). (A) Representative confocal microscopy images obtained after immunocytochemistry with antibodies raised against MAP2 and TH after PhIP (1μM), N-OH-PhIP (1μM), or 4′-OH-PhIP (1μM) treatment. Scale bar represents 25 μm. (B) PhIP and N-OH-PhIP induce selective loss of TH+ neurons (minimum of 500 total neurons in ten fields of view quantified for each treatment; expressed as %TH+, relative to total neurons). 4’-OH-PhIP appears to be less toxic to dopaminergic neurons (n = 3). **p < 0.01, ***p < 0.001; square root transformation, one-way ANOVA, followed by Tukey's multiple comparisons post hoc test. (C) Neurite lengths after PhIP and PhIP metabolite exposure. A total of 694 neurites were analyzed. The data are presented as the mean ± SEM. Fig. 3. Open in new tabDownload slide PhIP does not compete with [3H]-dopamine for dopamine transporter (DAT)-mediated transport. Human SH-SY5Y cells were treated in the presence of [3H]-dopamine or with [3H]-dopamine in combination with either bupropion (DAT inhibitor) at 10μM, or various concentrations of PhIP (1, 10, 25, and 50μM) for 30 min (n = 3/group). The amount of [3H]-dopamine uptake was measured using a scintillation counter and plotted as counts per min (cpm). ***p < 0.001; one-way ANOVA, followed by Dunnett's post hoc test. Fig. 3. Open in new tabDownload slide PhIP does not compete with [3H]-dopamine for dopamine transporter (DAT)-mediated transport. Human SH-SY5Y cells were treated in the presence of [3H]-dopamine or with [3H]-dopamine in combination with either bupropion (DAT inhibitor) at 10μM, or various concentrations of PhIP (1, 10, 25, and 50μM) for 30 min (n = 3/group). The amount of [3H]-dopamine uptake was measured using a scintillation counter and plotted as counts per min (cpm). ***p < 0.001; one-way ANOVA, followed by Dunnett's post hoc test. Fig. 4. Open in new tabDownload slide PhIP produces oxidative stress in dopaminergic neurons and antioxidant treatment is protective. Primary midbrain cultures were pretreated with control media or NAC at 1mM. After 72 h, cultures were cotreated with control media, 1μM PhIP, or 1μM PhIP and 1mM NAC for 24 h. Treatment with H2O2 was at 150μM for 30 min. (A) Representative confocal microscopy images obtained after immunocytochemistry with antibodies raised against HNE, NT, and TH. Scale bar represents 20 μm. (B) NAC prevented PhIP-induced HNE production in TH+ neurons. (C) NAC prevented PhIP-induced NT production in TH+ neurons. For (B) and (C), n = 134–234 ROIs analyzed/group, from three to five separate experiments. The data are presented as the mean ± SEM; *p < 0.05, ***p < 0.001 compared with control; ###p < 0.001 compared with PhIP-treated cultures (one-way ANOVA, followed by Tukey's multiple comparisons post hoc test). Fig. 4. Open in new tabDownload slide PhIP produces oxidative stress in dopaminergic neurons and antioxidant treatment is protective. Primary midbrain cultures were pretreated with control media or NAC at 1mM. After 72 h, cultures were cotreated with control media, 1μM PhIP, or 1μM PhIP and 1mM NAC for 24 h. Treatment with H2O2 was at 150μM for 30 min. (A) Representative confocal microscopy images obtained after immunocytochemistry with antibodies raised against HNE, NT, and TH. Scale bar represents 20 μm. (B) NAC prevented PhIP-induced HNE production in TH+ neurons. (C) NAC prevented PhIP-induced NT production in TH+ neurons. For (B) and (C), n = 134–234 ROIs analyzed/group, from three to five separate experiments. The data are presented as the mean ± SEM; *p < 0.05, ***p < 0.001 compared with control; ###p < 0.001 compared with PhIP-treated cultures (one-way ANOVA, followed by Tukey's multiple comparisons post hoc test). The BB extract was prepared as previously described (Joseph et al., 2003; 2014, Strathearn et al., 2014). Briefly, BBs were homogenized in water (1:1, wt/vol) using a blender. After centrifugation, the supernatant was collected and lyophilized. Treatment of primary mesencephalic cultures To test the effects of PhIP and the phase I PhIP metabolites on neurotoxicity, primary midbrain cultures were treated with PhIP (Toronto Research Chemicals, Toronto, Canada), N-OH-PhIP (MRIGlobal Chemical Carcinogen Repository, Kansas City, MO), or 4′-OH-PhIP (Toronto Research Chemicals) for 24 h at a final concentration of 200nM or 1μM in DMSO (≤0.1%). Doses used in the study were based upon results of a pilot study of doses ranging from 100nM to 10μM. To determine if N-acetylcysteine (NAC) pretreatment would be protective, the primary cells were pretreated with NAC (1mM) beginning at 7 days after plating. After 72 h, the cells were cotreated with NAC (1mM) and PhIP (1μM) for 24 h. Control cells were incubated in fresh media. Quantification of oxidative damage was then performed as described below. To assess whether BB extract is protective against PhIP and N-OH-PhIP toxicity, the primary cells were pretreated after 7 days in vitro with blueberry extract (10 ng/ml) for 72 h. The cells were then cotreated with fresh media containing blueberry extract (10 ng/ml) and PhIP or N-OH-PhIP at 1μM for an additional 24 h. We tested the ability of the BB extract to protect against neurotoxicity of these two compounds as well as to prevent neurite length decreases (as described below). Immunocytochemistry and measurement of primary dopaminergic cell viability Primary cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton-X100, and blocked in permeabilization and blocking buffer (0.3% Triton-X100, 10% fetal bovine serum (FBS) , 1% bovine serum albumin (BSA), in phosphate-buffered saline (PBS) (136mM NaCl, 0.268mM KCl, 10mM Na2HPO4, 1.76mM KH2PO4, pH 7.4) as described (Cooper et al., 2006). The cells were then incubated with the following primary antibodies (overnight at 4°C): chicken antimicrotubule-associated protein 2 (MAP2) (1:2000; EnCor Biotechnology Inc.), a pan-neuronal marker that clearly stains cell-bodies and processes; and rabbit antityrosine hydroxylase (TH) (1:500; Millipore; AB152) to identify dopaminergic neurons (Tapias et al., 2014). The cells were then washed three times with PBS before the secondary antibodies, Alexa Fluor goat antichicken 594 (1:1000; Life Technologies; A11042) and Alexa Fluor goat antirabbit 488 (1:1000; Life Technologies; A11008), were added. The cells were incubated for 1 h at room temperature and then washed again with PBS. Dopaminergic cell quantification was performed as previously described (Liu et al., 2008). Briefly, MAP2- and TH-immunoreactive neurons were blindly counted in at least 10 fields of view (∼500 total neurons) chosen randomly for each condition using a Nikon TE2000-U inverted fluorescence microscope (Nikon Instruments, Melville, NY) using a ×20 objective. Random fields of view were chosen systematically to provide representation throughout the well, avoiding sampling near the edges, where density is highly variable. To be deemed a morphologically viable dopaminergic neuron, only cells with a clearly visible nucleus and definitive TH+ staining were counted (Cannon et al., 2009). The data were expressed as a percent ratio of TH+ neurons to MAP2+ neurons to correct for variations in cell density. Each experiment was repeated three to five times on cultures isolated from different pregnant Sprague Dawley rats. Neurite length measurements Neurite length measurements were performed on the identical primary midbrain cultures that were used to determine dopaminergic cell viability (assessed at 1μM toxicant doses only). Images were taken blindly with a Nikon A1R confocal microscope from three individual experiments. Neurite lengths for ∼90 dopaminergic neurons (TH+) for each condition were assessed. Each neurite that was clearly attached to a viable cell body was measured using Nikon NIS Elements software to measure MAP2+ processes in TH+ neurons. High quality neurite length images were also taken with a Nikon A1R confocal microscope from primary cells grown on coverslips using a ×20 objective with a ×2.5 optical zoom. Radioactive competition assay Due to the chemical structure of PhIP and preferential neurotoxicity observed with PhIP and N-OH-PhIP (Fig. 1), we sought to determine if PhIP might be a substrate for the dopamine transporter (DAT), a potential route of toxicant entry into dopaminergic neurons. In order to test this hypothesis, we performed a radioactive competition assay in which human neuroblastoma SH-SY5Y cells (a dopaminergic cell line) (ATCC, Manassas, VA) were treated with [3H]-dopamine, [3H]-dopamine and the known DAT inhibitor, bupropion (10μM), or [3H]-dopamine and various concentrations of PhIP (1, 10, 25, and 50μM). Cells were plated on 12-well plates at a seeding density of 250,000 per well. After 24 h, the cells were washed with PBS, pH 7.4 and treated with RPMI media (supplemented with 0.2 mg/ml ascorbic acid, 120mM NaCl, and 3mM KCl) containing either 12.5nM (40 Ci/mM) [3H]-dopamine (Fisher Scientific) alone or with PhIP or bupropion and incubated at 37°C for 30 min. The radioactive media was then removed and the cells were washed with PBS, pH 7.4. The cells were lysed by the addition of 1% sodium dodecyl sulfate (SDS) and the mixture was added to scintillation vials containing a liquid scintillation cocktail (15 ml, Ecolite Scintillation Cocktail). Radioactive dopamine uptake was assessed using a Beckman Coulter LS6500 Multi-Purpose Scintillation Counter. Counting was conducted using a predefined tritium protocol for 1 min (expressed as counts/minute, cpm). Each experiment was repeated three times. 4-Hydroxy-2-nonenal (HNE) and 3-nitrotyrosine (NT) quantification HNE is an α,β-unsaturated hydroxylalkenal that is produced by lipid peroxidation in cells as a result of oxidative stress. Another indicator of oxidative/nitrosative stress is the production of reactive nitrogen species (RNS), such as peroxynitrite. RNS may nitrate tyrosines in cells, causing increased levels of nitrotyrosines. To assess oxidative damage as a result of PhIP treatment, primary cells plated and treated on coverslips were fixed as described above. In this experiment, a known reactive oxygen species H2O2 (150μM, 30 min) was utilized to induce oxidative stress. The following primary antibodies were used for immunocytochemistry: mouse anti-HNE (1:500; Percipio Biosciences; 24327), rabbit anti-NT (1:500; Millipore; 06-284) and sheep anti-TH (1:2000; Millipore; AB1542). Primary antibody incubation occurred overnight at 4°C. The coverslips were then washed three times with PBS before the secondary antibodies, Alexa Fluor donkey antimouse 647 (1:500; Jackson IR Laboratories; 715-606-151), Cy3 donkey antirabbit (1:500; Jackson IR Laboratories; 711-165-152) and Alexa Fluor donkey antisheep 488 (1:500; Jackson IR Laboratories; 713-545-147), were added. Secondary antibody incubation occurred for 1 h at room temperature and then the cells were washed again with PBS and finally mounted on microscope glass slides using mounting media. Oxidative damage in dopaminergic neurons was quantified similar to our previous report (Cannon et al., 2013). Briefly, images were taken blindly with a Nikon A1R confocal microscope using a ×60 objective from five separate experiments. Regions of interest (ROIs) were drawn around dopaminergic neurons (TH+). ROIs were drawn around the border of dopaminergic neurons with clear evidence of TH staining and a visible nucleus. Mean HNE and NT intensities of each ROI (individual dopaminergic neurons) were quantified using Nikon NIS Elements software and normalized to mean of control level in each experiment. Each ROI served as a distinct data point with a rationale for analysis similar to that previously described (Horowitz et al., 2011). Statistical analysis Dopaminergic neuron content and radioactivity experiments were subjected to ANOVA analysis. Where the ANOVA was significant, post hoc analysis was conducted to identify significant differences between groups (Prism 6, GraphPad, La Jolla, CA). For comparison only to control, Dunnett's post hoc test was used. For comparison between all groups, Tukey's multiple comparisons post hoc test was used. In analyzing percentage data by ANOVA, we conducted square root transformation to conform to ANOVA assumptions. Our analysis suggested that transformation did not impact the outcome of statistical testing. Neurite length data were subjected to a more robust analysis to account for the potential of multiple neurites arising from a single cell and comparison across experiments conducted on different days. Neurite lengths for four treatment groups (toxicity experiments) or six treatment groups (toxicity and BB-mediated neuroprotection) were compared using a general linear model implemented in the GLM procedure of SAS Version 9.3 followed by the Tukey's multiple comparisons post hoc test (Cary, NC). Immunofluroescence intensity for markers of oxidative stress was normalized to the mean control value for a given plate and analyzed by ANOVA. For comparison between all groups, Tukey's multiple comparisons post hoc test was used. Raw data (not normalized) was also subjected to the GLM procedure with day used as a blocking variable. The Tukey's multiple comparisons post hoc test was used for multiple comparisons among treatments. This analysis produced identical conclusions to one-way ANOVA with post hoc analysis. Thus, straightforward ANOVA analysis is presented. For all tests, p < 0.05 was deemed significant. RESULTS PhIP and N-OH-PhIP are Selectively Toxic to Dopaminergic Neurons Both PhIP and N-OH-PhIP induced a dose-dependent decrease in the percentage of TH+ neurons [at 1μM, 37% decrease for PhIP (2.001 ± 0.136% vs. 3.193 ± 0.132%; mean% DA neurons ± SEM; PhIP vs. control, p < 0.01) and 49% decrease for N-OH-PhIP (1.642 ± 0.252; p < 0.001)] indicating selective toxicity to dopaminergic neurons (Figs. 2A and 2B). Interestingly, 4’-OH-PhIP did not produce detectable neurotoxicity to dopaminergic neurons, with levels of TH+ neurons similar to control (n = 3/group) (Fig. 2A and 2B). Presenting the data as TH+ neurons relative to total neurons (MAP2+) best accounts for differences in cell density that invariably occur in primary neuronal culture. Nonetheless, we have examined differences in absolute neuronal counts, similarly to our previously published report (Liu et al., 2008). The nearly 40% decrease in the relative number of TH+ neurons upon treatment with 1μM PhIP resulted primarily from a decrease in the absolute number of TH+ neurons, without a consistent change in the absolute number of MAP2+ neurons. Effects of PhIP and N-OH-PhIP on Neurite Length in Primary Dopaminergic Neurons Morphologic examination revealed an apparent decrease in neurite length upon treatment (Fig. 2A). However, GLM procedure analysis did not reveal a significant treatment effect (Fig. 2C) (p = 0.09) in this dataset, which examined a total of 694 neurites, suggesting increased statistical power was required, as was evident in (Fig. 5). The data in Figure 2 suggest a trend toward decreased neurite length in cultures treated with PhIP or N-OH-PhIP versus untreated cultures, and this trend was confirmed with the more statistically robust dataset shown in Figure 5. It should be noted that both PhIP and N-OH-PhIP treatment of primary neurons yielded a considerable increase in the amount of dopaminergic neurons that presented with zero neurites, compared with the untreated cells. Fig. 5. Open in new tabDownload slide Blueberry extract protects dopaminergic neurons from PhIP neurotoxicity. Primary midbrain cultures were pretreated with blueberry extract (BB) (10 ng/μl) for 72 h followed by a cotreatment with BB and either PhIP or N-OH-PhIP at 1μM for an additional 24 h. (A) Representative confocal microscopy images obtained after immunocytochemistry with antibodies raised against MAP2 and TH after PhIP (1μM) or N-OH-PhIP (1μM) with or without BB 10 (ng/μl) treatment. Scale bar represents 25 μm. (B) BB extract treatment prevented PhIP-induced, and showed a trend toward attenuating N-OH-PhIP-induced, selective loss of TH+ neurons (minimum of 500 total neurons in ten fields of view quantified for each treatment) (n = 3). *p < 0.05, **p < 0.01; square root transformation, one-way ANOVA, followed by Tukey's multiple comparisons post hoc test. (C) BB extract treatment prevented PhIP- and N-OH-PhIP-induced neurite length decreases in surviving dopaminergic neurons. A total of 587 neurons were examined and the lengths of 1213 neurites were measured. The data are presented as the mean ± SEM. ***p < 0.001; Tukey's multiple comparisons post hoc test after general linear model implementation. Fig. 5. Open in new tabDownload slide Blueberry extract protects dopaminergic neurons from PhIP neurotoxicity. Primary midbrain cultures were pretreated with blueberry extract (BB) (10 ng/μl) for 72 h followed by a cotreatment with BB and either PhIP or N-OH-PhIP at 1μM for an additional 24 h. (A) Representative confocal microscopy images obtained after immunocytochemistry with antibodies raised against MAP2 and TH after PhIP (1μM) or N-OH-PhIP (1μM) with or without BB 10 (ng/μl) treatment. Scale bar represents 25 μm. (B) BB extract treatment prevented PhIP-induced, and showed a trend toward attenuating N-OH-PhIP-induced, selective loss of TH+ neurons (minimum of 500 total neurons in ten fields of view quantified for each treatment) (n = 3). *p < 0.05, **p < 0.01; square root transformation, one-way ANOVA, followed by Tukey's multiple comparisons post hoc test. (C) BB extract treatment prevented PhIP- and N-OH-PhIP-induced neurite length decreases in surviving dopaminergic neurons. A total of 587 neurons were examined and the lengths of 1213 neurites were measured. The data are presented as the mean ± SEM. ***p < 0.001; Tukey's multiple comparisons post hoc test after general linear model implementation. PhIP does not Compete with [3H]-Dopamine for the Dopamine Transporter (DAT) As expected, bupropion blocked [3H]-dopamine uptake (6584 ± 520 cpm vs. 16653 ± 1235 cpm; mean ± SEM; bupropion vs. control, p < 0.0001, Dunnett's test) by ∼60% (Fig. 3). However, a significant decrease in [3H]-dopamine uptake by SH-SY5Y cells was not observed with PhIP at any of the concentrations tested (n = 3/group) (Fig. 3). There was a slight decrease in [3H]-dopamine uptake observed at 50μM PhIP, but the same decrease was not observed with treatment of SH-SY5Y cells at 75 and 100μM PhIP in pilot studies (data not shown). Therefore, we conclude that PhIP transport into cells is unlikely to be mediated through DAT uptake. PhIP Produces Oxidative Damage in Dopaminergic Neurons and N-Acetylcysteine (NAC) Pretreatment is Protective HNE production, as measured by quantifying immunofluorescence, was significantly higher (4.5%) in PhIP-treated dopaminergic neurons compared with untreated cells (104.5 ± 1.7% vs. 100.0 ± 1.0%; PhIP vs. control; mean immunofluorescence intensity relative to control ± S.E.M; p < 0.05, n = 134–234 ROIs analyzed/group, from three to five separate experiments) (Figs. 4A and 4B). NAC pretreatment was protective against PhIP-induced HNE production (97.4 ± 1.2 vs. 104.5 ± 1.7; NAC + PhIP vs. PhIP, p < 0.001). H2O2 (150μM) did not alter HNE levels compared with untreated cells. Nitrotyrosine (NT) formation, also measured by quantifying immunofluorescence, increased by about 22.5% with PhIP treatment at 1μM (138.7 ± 2.7 vs. 100.0 ± 1.4; PhIP vs. control; p < 0.001) compared with control cells. H2O2 (150μM) treatment also produced increased NT formation (122.5 ± 2.6 vs. 100.0 ± 1.4; H2O2 vs. control; p < 0.001). NAC pretreatment at 1mM prior to PhIP treatment partially prevented PhIP-induced NT increases (111.2 ± 3.0 vs. 122.5 ± 2.6; NAC + PhIP vs. PhIP; p < 0.001) (Figs. 4A and 4C). Blueberry (BB) Extract Protected Dopaminergic Neurons from PhIP Neurotoxicity and Prevented both PhIP and N-OH-PhIP-induced Neurite Length Decreases In Figure 5A and 5B, treatment of the rat primary midbrain cultures with BB extract was able to protect against PhIP neurotoxicity (4.315 ± 0.312 vs. 2.833 ± 0.146; mean% DA neurons ± SEM; PhIP + BB vs. PhIP, p < 0.01; n = 3/group). Further, the percentage of DA neurons in BB + PhIP cultures was not significantly different from control. Treatment of the cells with BB extract did not provide significant protection from DA cell loss elicited by N-OH-PhIP (Fig. 5B). The statistical model for neurite length analysis included the following factors: Experiment, a blocking factor with three levels, Treatment with 6 levels (BB, BB + N-PhIP, BB + PhIP, N-PhIP, PhIP, and Control), Neuron nested within Experiment and Treatment with 30–36 levels. A total of 587 neurons were examined and the lengths of 1213 neurites were measured. Neurite length analysis indicated that there were differences among the six treatments (p < 0.0001). The Tukey-Kramer multiple comparison procedure identified two groups of treatments: (1) BB, BB + N-OH-PhIP, BB + PhIP, Control, and (2) N-OH-PhIP, PhIP. Within each group no statistically significant differences in mean neurite lengths were found. Higher mean neurite lengths were found for treatments in group (1) than in group (2). Embedded in the design is a 2 × 2 factorial design with factors BB (yes or no) and PhIP (PhIP or N-PhIP). The main effect of PhIP and the interaction of BB and PhIP were not statistically significant (p = 0.72 and p = 0.16, respectively) whereas the main effect of BB indicated that the presence of BB reversed the negative effect of PhIP and N-OH-PhIP (p < 0.001). Thus, the analysis above clearly indicates that PhIP and N-OH-PhIP elicit a reduction in dopaminergic neurite length. Although a trend toward decrease was observed in the “toxicity only” experiment (Fig. 2C), statistical significance was only achieved in this larger experiment also examining neuroprotection, which included the analysis of more groups and total neurites. BB treatment of the primary neurons significantly alleviated reduction in neurite length elicited by PhIP (62.44 ± 2.73 μm vs. 45.79 ± 2.72 μm; BB + PhIP vs. PhIP; mean ± SEM, p < 0.001) and N-OH-PhIP (69.35 ± 2.61 vs. 42.83 ± 2.76; BB + N-OH-PhIP vs. N-OH-PhIP) (Figs. 5A and 5C). Additionally, BB treatment resulted in neurite lengths of PhIP- and N-OH-PhIP-treated neurons that were not significantly different from control (Figs. 5A and 5C). BB treatment alone at the tested dose had no effect on TH+ neuron density or neurite length (Fig. 5). DISCUSSION Increasing evidence in the literature suggests that there is a strong association between environmental factors and PD. Although exposure to numerous compounds has been linked to PD, many of these factors may be rarely encountered and likely do not account for a significant portion of sporadic PD cases. The goal of this work was to examine whether a dietary factor, PhIP, might be selectively toxic to DA neurons. To the best of our knowledge, the role of PhIP in the nervous system has not been investigated. In this study, for the first time, we report that PhIP is selectively toxic to DA neurons, in a primary mesencephalic culture system. Examination of dietary factors in PD has, to date been very limited. Dietary factors, and heterocyclic amines in specific represent a class of compounds that might be encountered in high and chronic doses. Our results suggest that this class of compounds should receive much more attention as putative DA neurotoxicants and that mechanistic studies are warranted. PhIP is extensively metabolized in several mammalian species, including humans. Detailed metabolic studies have identified primary, phase I metabolites (Chen et al., 2007; Malfatti et al., 1999; Turteltaub et al., 1999). Studies to date on brain penetrance have been limited. However, both PhIP and N-OH-PhIP have been shown to cross the BBB after systemic administration (Teunissen et al., 2010). Given the paucity of data on PhIP neurotoxicity, for this first report, we chose to utilize an in vitro approach that would allow us to test whether PhIP and its two major metabolites were selectively toxic to DA neurons affected in PD. Primary mesencephalic cultures from rat embryos at E17 offer an ideal test system to accomplish this goal, replicating many of the physiological features of mature neurons in vivo (Cooper et al., 2006; Liu et al., 2008). Of note, this system allows us to test both PhIP and PhIP metabolites for toxicity. Currently, cost and availability of these compounds limit wide-ranging in vivo pilot studies. Utilizing primary cell culture also has several distinct advantages over immortalized cell lines. Specifically, the presence of glial cells and postmitotic neurons of various types (TH+ and GABA+) enables us to investigate neurotoxic and neuroprotective mechanisms in a native-like environment and allows selectivity of neurotoxicity to be assessed. Here, we report that both PhIP and a major phase I metabolite, N-OH-PhIP, elicit selective loss of DA neurons and morphological alterations in surviving DA neurons as assessed by quantifying neurite length. Significant differences in neurite length were only evident in the neuroprotection study, where a greater number of total neurites were analyzed. Selectivity of DA cell loss was assessed by expressing DA cell density relative to total neuronal cell density with and without toxicant treatments. At tested doses, 4′-OH-PhIP was not toxic to dopamine neurons. N-OH-PhIP is known to be a precursor to the reactive N-acetoxy-PhIP, which forms a highly reactive nitrenium ion that binds DNA, and likely protein (Nguyen and Novak, 2007; Peng et al., 2012). 4′-OH-PhIP is not genotoxic (Holme et al., 1989). Therefore, dopaminergic neurotoxicity of metabolites could be related to genotoxicity, an area that warrants further examination. Our data suggests that PhIP should be evaluated in vivo as a putative dopaminergic neurotoxicant. Further, nitrenium ion formation should be examined as a potential mechanism of neurotoxicity. MPP+, a metabolite of MPTP that is a well-known dopaminergic neuron toxin, enters dopaminergic neurons via DAT-mediated transport. Given that PhIP shares some structural features with DA and known DA toxicants, we tested whether selective toxicity of PhIP is due to its transport via DAT. Our data showed that PhIP does not compete with tritiated dopamine at much higher doses than the known DAT inhibitor, bupropion (Fig. 3). Thus, it is unlikely that PhIP is a substrate for DAT. Given its structure and lipophilicity, it is possible that PhIP might simply diffuse nonselectively through neuronal membranes. Future experiments utilizing radioactive PhIP and DAT blockers will be able to directly assess uptake into DA neurons. Dopaminergic neurons in the substantia nigra are highly sensitive to oxidative stress due in part to dopamine metabolism and resultant production of reactive oxygen species and catechol formation (Hastings and Zigmond, 1994). Indeed, studies have shown that many toxin-based PD models result in oxidative stress-induced loss of DA neurons (Cannon and Greenamyre, 2010). Thus, we wished to test whether PhIP-elicited neurotoxicity might be mediated through oxidative stress and if a potentially beneficial dietary factor could be protective. Berries, including blueberries, are postulated to be protective, in part due to antioxidant properties (Willis et al., 2009). Epidemiological evidence suggests that the consumption of berries rich in antioxidants such as anthocyanins and proanthocyanidins (e.g., blueberries and strawberries) may lower PD risk (Gao et al., 2012). BB extracts, in particular, have been shown to have a positive effect on age-related cognition decline in rat models (Rendeiro et al., 2009). Furthermore, several papers have been published demonstrating the ability of BB extracts to reverse deficits in neuronal signaling and cognitive function in aged rats as well as improve the rats’ ability to perform motor tests (Bickford et al., 1999; Joseph et al., 1999, 2003; Weisburger, 2000). Due to the fact that blueberries appear to have many valuable effects on neuronal signaling in rats, protect rat primary midbrain cultures against various PD-related insults including rotenone, paraquat, and α-synuclein-expressing virus (Tambe, Strathearn, and Rochet, unpublished results) (Strathearn et al., 2014) and that PD is an age related disease, we wanted to determine if blueberries could protect against PhIP and N-OH-PhIP induced neurotoxicity and neurite length deficits. Indeed, BB treatment at 10 ng/μl prevented DA neuron loss and decreases in neurite length in PhIP-treated primary cultures (Fig. 5). BB treatment was not able to prevent N-OH-PhIP-induced DA neuron loss, though the BB extract was significantly protective against neurite length decreases in N-OH-PhIP treated cultures. The inability of blueberry treatment to significantly prevent N-OH-PhIP-mediated cell death implies that this metabolic pathway may result in heightened toxicity. Taken together, these findings suggest that PhIP and a primary metabolite might be selectively toxic to DA neurons via induction of oxidative stress. Although blueberries are known to have significant antioxidant capacity, their protective role on neurological function might be due to additional effects. Mice with a BB-containing diet had lower levels of NF-KB compared with mice with a control diet, emphasizing a role in cell signaling regulation (Adams et al., 2011). Another in vivo study has shown reduced caspase-3 activity in the hippocampus of rats receiving BB treatment, suggesting antiapoptotic action (Wang et al., 2005). Thus, we chose to measure whether PhIP produced evidence of oxidative damage in dopaminergic neurons, and whether the use of a compound thought to act primarily as an antioxidant would be protective. Here, NAC, was chosen, given the extensive literature on its role as an antioxidant (Olive et al., 2012; Samuni et al., 2013). HNE and NT are indicators of oxidative damage, which are produced as a result of lipid peroxidation and nitration of tyrosine, respectively (Cannon et al., 2013). Our experiments showed that PhIP induces formation of HNE and NT in primary mesencephalic cultures, compared with control (Fig. 4). In addition, NAC treatment protects DA neurons against oxidative damage. Thus, the protective effects of BB and NAC treatment suggest that PhIP-induced oxidative stress may be a primary mechanism of action related to selective toxicity of dopaminergic neurons. In vivo studies conducted using biologically relevant doses of PhIP will be required in the future to further establish relevance to dopaminergic dysfunction and toxicity. Ongoing studies in our laboratory will examine PhIP-induced neurotoxicity in chronic in vivo models. It is also worth noting that in this report, only PhIP was analyzed for production of oxidative damage (Fig. 4). Although the reactive hydroxyl groups in PhIP metabolites could potentially contribute to oxidative stress, much research on PhIP chemistry will need to be conducted. To date, PhIP and its metabolites have been primarily examined as mutagens. Future research is expected to also elucidate differences between metabolites in oxidative capacity. Here, for the first time, we present data on the selective neurotoxicity of PhIP in dopaminergic neurons using primary mesencephalic cultures. To the best of our knowledge, PhIP toxicity to DA neurons has not been tested. Our results also suggest a potential mechanism. Toxicity is not likely due to selective uptake, because PhIP does not compete with tritiatated dopamine for DAT entry. Oxidative stress is a likely mechanism, given that PhIP produced increases in markers for oxidative/nitrosative damage. Indeed dopaminergic neurons are known to have high basal levels of oxidative stress and to be especially sensitive to oxidative insults (Hastings and Zigmond, 1994; Horowitz et al., 2011). Finally, our data suggest that PhIP should be evaluated as a putative DA neurotoxicant and that follow-up studies examining chronic and in vivo exposures should be conducted. FUNDING National Institute of Environmental Health Sciences at the National Institutes (R00ES019879 and R03ES022819 to J.R.C.); National Institute on Aging at National Institutes of Health (R21 AG039718 to J.-C.R.); Ralph W. and Grace M. Showalter Research Trust (to J.R.C. and J.-C.R.). REFERENCES Adams L. S. Kanaya N. Phung S. Liu Z. Chen S. 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TI - 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) Is Selectively Toxic to Primary Dopaminergic Neurons In Vitro
JF - Toxicological Sciences
DO - 10.1093/toxsci/kfu060
DA - 2014-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/2-amino-1-methyl-6-phenylimidazo-4-5-b-pyridine-phip-is-selectively-6AwZHA0Yqh
SP - 179
EP - 189
VL - 140
IS - 1
DP - DeepDyve
ER -