1,3-Dinitrobenzene Induces Age- and Region-Specific Oxidation to Mitochondria-Related Proteins in Brain

Abstract

Regions of the brain with high energy requirements are especially sensitive to perturbations in mitochondrial function. Hence, neurotoxicant exposures that target mitochondria in regions of high energy demand have the potential to accelerate mitochondrial damage inherently occurring during the aging process. 1,3-Dinitrobenzene (DNB) is a model neurotoxicant that selectively targets mitochondria in brainstem nuclei innervated by the eighth cranial nerve. This study investigates the role of age in the regional susceptibility of brain mitochondria-related proteins (MRPs) to oxidation following exposure to DNB. Male F344 rats (1 month old [young], 3 months old [adult], 18 months old [aged]) were exposed to 10 mg/kg DNB prior to mitochondrial isolation and histopathology experiments. Using a high-throughput proteomic approach, 3 important region- and age-related increases in DNB-induced MRP oxidation were determined: (1) brainstem mitochondria are ×3 more sensitive to DNB-induced oxidation than cortical mitochondria; (2) oxidation of brainstem MRPs is significantly higher than in cortical counterparts; and (3) MRPs from the brainstems of older rats are significantly more oxidized than those from young or adult rats. Furthermore, lower levels of DNB cause signs of intoxication (ataxia, chromodacryorrhea) and vacuolation of the susceptible neuropil in aged animals, while neither is observed in DNB-exposed young rats. Additionally, methemoglobin levels increase significantly in DNB-exposed adult and aged animals, but not young DNB-exposed animals. This suggests that oxidation of key MRPs observed in brainstem of aged animals is necessary for DNB-induced signs of intoxication and lesion formation. These results provide compelling evidence that environmental chemicals such as DNB may aid in the acceleration of injury to specific brain regions by inducing oxidation of sensitive mitochondrial proteins.

The nearly 60-year-old free radical theory of aging postulates that the aging process is related to the accumulation of oxidative damage to macromolecules (Harman, 1956). During the physiological aging process, the brain accumulates oxidative damage that is known to vary by region (reviewed by Abd El Mohsen et al. [2005]; Dubey et al. [1996]; and Halliwell [1992]). Additionally, there is regional, cellular, and organellar heterogeneity in accrued oxidative modifications (Vaishnav et al., 2007). The heterogeneity in age-related accumulation of oxidative protein modification provides for the possibility that there are latent inherent regional vulnerabilities that are revealed following exposure to environmental chemicals that challenge energy homeostasis.

Mitochondria are acutely sensitive to oxidative damage during aging by virtue of their biochemistry that produces reactive oxygen species as a metabolic by-product (Adam-Vizi and Chinopoulos, 2006; Muller, 2000; Turrens, 2003). Further compounding the situation, antioxidant concentrations decrease in brain mitochondria over time (Imam et al., 2006; Perluigi et al., 2010). These factors, coupled with regional variations in antioxidant levels in aging rodent (Edrey et al., 2014) and human brains (Venkateshappa et al., 2012a,b), point to selective regional vulnerability of key mitochondrial proteins to oxidation over time. Because the mitochondrial proteome in the brain as a whole accumulates oxidative modifications during aging (Perluigi et al., 2010), oxidative modifications to mitochondrial proteins may be more injurious in regions with higher energy requirements. The interaction between aging processes in brain and exposure to environmental chemicals that induce energy deprivation syndromes is largely unknown.

In rodents, exposure to 1,3-dinitrobenzene (DNB), an environmental nitroaromatic contaminant used in the production of dyes, plastics, and explosives, causes the development of vacuolated lesions in brainstem nuclei that form the ascending sensory pathway of the vestibulocochlear nerve. These lesions correspond with ataxia reminiscent of exposure to antimetabolites that interfere with local energy metabolism (Cavanagh, 1993; Philbert et al., 1987). DNB exposure causes inhibition of mitochondrial enzymes (Miller et al., 2011; Phelka et al., 2003), induces the mitochondrial permeability transition in brainstem astrocytes (Tjalkens et al., 2003), and oxidation of mitochondrial proteins in immortalized astrocytes in vitro (Steiner and Philbert, 2011). This study probes the hypothesis that exposure to neurotoxic levels of DNB accelerates mitochondrial proteome oxidation in susceptible regions of the brainstem.

It is of note that DNB exposure causes diminution of proprioceptive function, manifested as ataxia, and that normal physiological aging processes produce time-dependent degradation of audiovestibular function. Epidemiological studies support the link between age-related loss of function of the sensory pathways of the vestibulocochlear nerve: 63% of people in the United States over the age of 70 years have hearing loss (Lin et al., 2011), and 40% of people over the age of 65 years living in the United States living at home will fall at least once per year, with 75% of deaths due to falls occurring in this age group (Rubenstein, 2006). A recent study investigated the link between incidence of falls and hearing loss in older adults in the United States, reporting that the transition from normal hearing to mild hearing loss is associated with a 3-fold increased odds of reporting a fall within the last year (Lin and Ferrucci, 2012). These clinical and epidemiological findings in humans are supported by experimental studies in rodents. This study provides evidence of region-specific and mitochondria-specific vulnerabilities to neurotoxicant-induced oxidative damage in rodents, targeting specific nuclei in the brain which manifest age-related functional decline in humans.

Exposure to environmental neurotoxicants has been shown to cause age-dependent, regional variation in elevated oxidative stress (as noted in rodent exposure to toluene, e.g., Kodavanti et al. [2011]). However, there is very little evidence of an age-related increase in mitochondrion-specific, region-selective accumulation of oxidized proteins in response to neurotoxicant exposure. The primary objective of this investigation was to identify mitochondrial proteins in the aging brainstem that are also selectively susceptible to oxidation upon exposure to an environmental neurotoxicant, DNB. This study uses a high-throughput proteomic approach to provide novel evidence of DNB-induced mitochondrial proteome oxidation localized to the brainstem; advanced age of the animal exacerbates the extent and specific peptide loci of oxidation to the mitochondrial proteome.

MATERIALS AND METHODS

Animals

Male Fischer 344 rats (1 month old, 3 months old, and 18 months old) were purchased from Harlan Laboratories and housed in groups of 3 in an AAALAC-accredited facility at the University of Michigan. Rats were fed a commercial rodent diet and watered ad libitum and housed on a 12-h light/dark cycle. This study was approved by the University of Michigan’s University Committee on Use and Care of Animals.

Study design

A total of 78 rats were utilized for this study. Rats of 3 different ages were used (1 month old, 3 months old, and 18 months old); rats were separated by age in different cages, with 3 rats per cage in the 1-month-old group, 2–3 rats per cage in the 3 months old group, and 2 rats per cage in the 18 months old group. Groups of rats numbered 24–8 per age group (24 1 month old, 26 3 months old, and 28 18 months old). Rats in each age group were randomly assigned to exposure or control groups. Rats from each age group assigned to the exposure group were exposed by intraperitoneal injection to 10 mg/kg DNB (Sigma Aldrich) in dimethylsulfoxide (DMSO) (Sigma Aldrich) at a volume of 1 µl/g at 0, 4, and 24 h (1 month old [n = 12]; 3 months old [n = 13]; and 18 months old [n = 15]). Rats from each age group serving as control rats were administered a vol/vol equivalent of DMSO as a vehicle control at the same time points, 0, 4, and 24 h, (1 month old [n = 12]; 3 months old [n = 13]; and 18 month old [n = 13]). Exposed and control rats were further divided into subgroups for proteomics experiments (1 month old [n = 3 exposed and n = 3 control]; 3 months old [n = 4 exposed and n = 4 control]; and 18 months old [n = 6 exposed and n = 4 control]) and pathological findings (signs of intoxication, histopathology, and methemoglobinemia (n = 18 for each age group)): (1 month old [n = 9 exposed and n = 9 control]; 3 months old [n = 9 exposed and n = 9 control]; and 18 month old [n = 9 exposed and n = 9 control]). Endpoints for proteomics and pathological findings occurred at 12 h and 24 h following the last dose. Brainstem was chosen for the experiments in this study because it is one of the primary sites of lesion development (Philbert et al., 1987), increased glucose consumption (Hu et al., 1997), and onset of astrocytic mitochondrial permeability transition pore (Tjalkens et al., 2003) in DNB exposure. Cortex was used as an “internal control” to compare mitochondrial oxidation in an area of the brain where lesions do not develop within the brain. The cortex was chosen because (1) DNB is distributed through the cortex and causes an increase in glucose consumption, much like brainstem (Hu et al., 1997) but it is a region in which lesions do not develop and (2) the study would then be comparable to other studies examining mitochondrial function in response to DNB using brainstem and cortical astrocytes (Tjalkens et al., 2003). Deep cerebellar nuclei histology data illustrate that there is DNB-induced, region-specific, age-related damage in within a vulnerable region of the brain that typically develops lesions in DNB exposure (Philbert et al., 1987).

Histology

At study endpoint, rats were anesthetized with isoflurane, followed by opening of the abdominal and thoracic cavity and perfusion by normal saline, followed by 4% paraformaldehyde. Directly after perfusion fixation, the rat was decapitated via guillotine. The brain was then carefully removed from the skull and further immersion fixed in 4% PFA overnight. Fixed brains were placed in a rat brain matrix (Harvard Apparatus, Holliston, Massachusetts) for trimming per published guidelines for general evaluation of neurotoxicity (Bolon et al., 2013). Tissues were cassetted and processed to paraffin by routine histological methods. Four micron thickness sections were cut on a rotary microtome and stained with hematoxylin and eosin. Histology procedures were performed at the ULAM in vivo Animal Core (University of Michigan, Ann Arbor). Pathology interpretation was made by a board-certified veterinary pathologist blinded to the animal groups at the time of evaluation.

Mitochondrial isolation

Mitochondria were isolated according to the protocol (Method C) by Sims and Anderson (2008), with minor modifications. Briefly, the rat was anesthetized under isoflurane, decapitated with a small animal guillotine, and the brain was rapidly removed. Brainstem and cortex were dissected from the rest of the brain in cold isolation buffer (100 mM Tris, 10 mM potassium-EDTA, 960 mM sucrose, pH 7.4). Brainstem and cortex were then weighed and minced in cold isolation buffer. Minced tissue was then homogenized using a glass Dounce homogenizer on ice; this step was performed while being flushed with argon. Subsequent differential centrifugation steps using a Percoll gradient followed, according to the protocol (Sims and Anderson, 2008). Prior to the Percoll gradient centrifugation step, digitonin was added to disrupt synaptosomal membranes. Mitochondria were stored at −80°C.

Detection and identification of mitochondria-related proteins and oxidative modification sites

The volume of each submitted mitochondrial sample was reduced 50% and 20 µl of the concentrated sample was processed by SDS-PAGE on a 4%–12% Bis-Tris Mini-gel (Invitrogen) using the MOPS buffer system and non-reducing Laemmli buffer. Each gel lane was excised into 10 equal bands and these were processed by in-gel digestion using a robot (ProGest, DigiLab): bands were washed with 25 mM ammonium bicarbonate followed by acetonitrile, reduced with 10 mM dithiothreitol at 60°C followed by alkylation with 50 mM iodoacetamide at room temperature, digested with trypsin (Promega) for 4 h at 37°C, and quenched with formic acid and the supernatant was analyzed directly without further processing. Samples were then analyzed by nano LC/MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive mass spectrometer. Peptides were loaded on a trapping column and eluted over a 75-µm analytical column at 350 nl/min; both columns were packed with Jupiter Proteo resin (Phenomenex). The mass spectrometer was operated in data-dependent mode, with MS and MS/MS performed at 70 000 and 17 500 FWHM resolution, respectively. The 15 most abundant ions were selected for MS/MS and proteins with oxidized amino acid residues, as determined by M + 16 spectra indicating methionine sulfoxide, were considered to be oxidized proteins. This method was used for the determination of all mitochondrial samples from control and DNB-exposed animals (all age groups, both brainstem and cortex). Relative differences in DNB-exposed and control sample protein oxidation were calculated as %Oxidation (unweighted spectral count of oxidized peptides for a protein/total unweighted spectral count for that same protein). Thresholds for significance were set at %Oxidation <0.25-fold or >4.0-fold compared with controls for each protein. The criteria used for quantitative analysis of data are based on empirical observations of many large datasets (Zybailov et al., 2006). The 4-fold change is deliberately conservative to reduce the number of false positive leads and to concentrate efforts on those changes most likely to be significant in the target system. Proteins were identified by reference to the UniProt rat database (forward and reverse appended with common contaminant proteins) using the Mascot search engine (Matrix Science, UK). Results were displayed in Scaffold software (Version 3.6.5, Proteome Software, Inc).

Pathway analysis

A pathway analysis was performed to denote commonalities in protein function among proteins identified as oxidized by DNB. UniProtID numbers for proteins <0.25-fold or >4.0-fold oxidized, as determined by %Oxidation, were uploaded to the Database for Annotation, Visualization, and Integrated Discovery (Huang da et al., 2009). Pathways were identified in the DAVID database by selecting Gene Ontology Term (GOTerm) and searching the protein lists by Molecular Function. Options for pathway determination were set to: 3 proteins to constitute a pathway, and 0.05 as a significance level for P-values and Benjamini values.

Methemoglobin analysis

Cardiac blood was taken from control- and DNB-exposed animals of each age group using the 12 h endpoint time after the last of the 3 intraperitoneal injections. Whole blood was stored less than 1 week at 4°C in tubes containing EDTA to prevent coagulation. Samples were submitted to AniLytics, Inc (Gaithersburg, Maryland) for analysis of methemoglobin percentage.

Statistical analysis

Prior to oxidation analysis on mitochondria-related proteins (MRPs), the false-discovery rate (FDR) for identified proteins (1823 detected by LC/MS/MS) was calculated in Scaffold software, (FDR = 0.3%). Regional oxidation is shown as an average fold change and compared using Student’s t test, with P < 0.05 considered statistically significant (Fig. 4) (Prism software, version 5.0, GraphPad). Data for the comparison of oxidation of MRPs in brainstem and cortex in control animals across age (Fig. 5) were analyzed using 2-way ANOVA with Bonferroni’s multiple comparisons test to assess significance between age groups and between the 2 regions. Data for the methemoglobin assay (Fig. 6) were analyzed using 1-way ANOVA with Tukey’s multiple comparisons test to assess significance between controls and exposed rats within each age group and across all 3 age groups (Prism, Version 5.0, GraphPad).

FIG. 4.

Brainstem and cortex MRP oxidation in 18 months rats. Average fold-change in oxidation of MRPs were calculated as: %Oxidation values of DNB-exposed brainstem divided by %Oxidation values of control brainstem (Brainstem) compared with %Oxidation values of DNB-exposed cortex divided by %Oxidation values of control cortex (Cortex) (Top). Since >4-fold %Oxidation was only detected in 18 months samples, these data reflect DNB-induced oxidation in aged animals (Cortex n = 4, Brainstem n = 20). Percentage of oxidized proteins that were oxidized >4-fold were tabulated (Bottom). Data were analyzed using Student’s t test (Prism, Version 5.0, GraphPad).

FIG. 5.

Region-specific oxidation of brainstem and cortex mitochondria in control F344 rats. Mitochondria isolated from 1 month old, 3 months old, and 18 months old brainstem and cortex were submitted for mass spectrometry analysis for protein and posttranslational modification identification and quantification. Proteins with oxidized spectra (47 detected) were analyzed for %Oxidation (number of oxidized peptide spectra/total unweighted peptide spectra specific to that protein). Mean percentages for each age and region were calculated. A 2-way ANOVA with Bonferroni’s multiple comparisons test was performed using Prism software (GraphPad, version 5.0).

FIG. 6.

DNB exposure increases methemoglobin percentage and is exacerbated by age. Percent methemoglobin detected in cardiac blood from 1 month old, 3 months old, and 18 months old DNB-exposed and control rats. Rats were exposed to 10 mg/kg DNB or DMSO via intraperitoneal injection at 0, 4, and 24 h with cardiac blood collection occurring 12 h after the last exposure. Samples were collected within 1 week, stored at 4°C in tubes containing EDTA to prevent coagulation, and methemoglobin percentage was determined (n = 3). A 2-way ANOVA with Bonferroni’s post hoc test was performed using Prism software (GraphPad, version 5.0) (* p < 0.05; ** p < 0.01; ***p < 0.001).

RESULTS

Effects of Age on Severity of Lesions

Aged animals had increased severity and incidence of the behavioral signs and histopathological signs of DNB intoxication. In animals exposed to DNB at 0, 4, with a 24-h period after the last dose to sacrifice, the oldest rats (18 months old) exhibited signs of intoxication including ataxia (8/9 rats) and chromodacryorrhea (i.e., pigmented ocular discharge indicative of stress, 9/9 rats); this group also exhibited moribund condition necessitating euthanasia (3/9 rats) (Table 1). No signs of intoxication were observed in the 1-month-old group, and only 2/7 of the 3 months old DNB exposed animals were ataxic (Table 1).

TABLE 1.

Conditions and Mortality Rates of Male F344 Rats Sacrificed 24 h After Last Exposure

Animal Age (months)	Exposure	Hunched	Ataxia	Chromodacryorrhea	Mortality
1	VC	0% (0/6)	0% (0/6)	0% (0/6)	0% (0/6)
1	DNB	0% (0/6)	0% (0/6)	0% (0/6)	0% (0/6)
3	VC	0% (0/7)	0% (0/7)	0% (0/7)	0% (0/7)
3	DNB	42.8% (3/7)	28.5% (2/7)	0% (0/7)	0% (0/7)
18	VC	0% (0/7)	0% (0/7)	0% (0/7)	0% (0/7)
18	DNB	100% (9/9)	88.8% (8/9)	100% (9/9)	33.3% (3/9)

Notes: Number of animals affected out of total number of animals observed in parentheses. VC, vehicle control (DMSO); DNB, 10 mg/kg 1,3-dinitrobenzene given at 0, 4, and 24 h; sacrificed 24 h later. These animals were used for mitochondrial isolation and subsequent proteomics experiments.

Because of the observed mortality rate in the 18 months old exposure group, the duration of time between the third exposure and sacrifice was halved (i.e., sacrifice at 12 h after last dose) for subsequent experiments. Interestingly, even at this earlier endpoint, the oldest rats (18 months old) still exhibited signs of intoxication with DNB exposure including ataxia (6/6 rats) and chromodacryorrhea (6/6 rats). However, all of the animals except one survived this shortened post-exposure duration (1/6 rats) (Table 2). Signs of intoxication were not observed in DNB-exposed 1 month old, and only one 3 months old DNB-exposed rat was ataxic at this time point (Table 2), demonstrating a clear association between advanced age and likelihood of gross neurotoxic effects of DNB.

TABLE 2.

Conditions and Mortality Rates of Male F344 Rats Sacrificed 12 h After Last Exposure

Animal Age (months)	Exposure	Hunched	Ataxia	Chromodacryorrhea	Mortality
1	VC	0% (0/3)	0% (0/3)	0% (0/3)	0% (0/3)
1	DNB	0% (0/3)	0% (0/3)	0% (0/3)	0% (0/3)
3	VC	0% (0/4)	0% (0/4)	0% (0/4)	0% (0/4)
3	DNB	75% (3/4)	25% (1/4)	0% (0/4)	0% (0/4)
18	VC	0% (0/3)	0% (0/3)	0% (0/3)	0% (0/3)
18	DNB	100% (6/6)	100% (6/6)	100% (6/6)	60% (1/6)

Notes: Number of animals affected out of total number of animals observed in parentheses. VC, vehicle control (DMSO); DNB, 10 mg/kg 1,3-dinitrobenzene given at 0, 4, and 24 h; sacrificed 12 h later. These animals were used for histopathology experiments.

Age-Related Regional Susceptibility to DNB

Lesions were not observed in deep cerebellar nuclei in 1 month old (Fig. 1) or 3 months old (Fig. 2) rats exposed to DNB with a 12-h period after the last exposure. However, vacuolation of the neuropil in 18 months old DNB exposed rats was observed in 1 of the 3 rats assessed for histopathology at this time point (Fig. 3). The vacuolation was limited to the deep cerebellar roof nuclei and was consistent with previously published observations with respect to cellular and regional targets. However, the time to onset of the lesion was markedly more rapid than that seen in younger rats at the same dose (Philbert et al., 1987; Romero et al., 1991). In DNB-associated testicular toxicity, the severity of vacuolated lesion development in seminiferous epithelia is positively associated with advanced rat age (Brown et al., 1994), further demonstrating that despite the occurrence of an age-associated increased severity of lesion in target tissues, the lesions remain confined to specific regions within the brain and testis.

FIG. 1.

Deep cerebellar nuclei in 1-month-old male F344 rats exposed to DNB. Hematoxylin and eosin staining of deep cerebellar nuclei in 1-month-old male F344 rats showing no lesions in rats exposed to DNB in comparison to vehicle controls. See methods for dose and dose regimen. (N = Neuron, V = Blood Vessel, A with arrow = Astrocyte). Scale bar: 100 μm at x100; 20 μm at x400.

FIG. 2.

Deep cerebellar nuclei in 3 months old male F344 rats exposed to DNB. Hematoxylin and eosin staining of deep cerebellar nuclei in 3 months old male F344 rats showing no lesions in rats exposed to DNB in comparison to vehicle controls. See methods for dose and dose regimen. (N = Neuron, V = Blood Vessel, A with arrow = Astrocyte). Scale bar: 100 μm at x100; 20 μm at x400.

FIG. 3.

Deep cerebellar nuclei in 18 months old male F344 rats exposed to DNB. Hematoxylin and eosin staining of deep cerebellar nuclei in 18 months old male F344 rats showing lesions (vacuolation indicated by bold arrow) in rats exposed to DNB in comparison to vehicle controls. See methods for dose and dose regimen. (N = Neuron, V = Blood Vessel, A with arrow = Astrocyte). Scale bar: 100 μm at x100; 20 μm at x400.

Proteomic identification of MRPs with oxidized methionine residues revealed that in young rats (1 month old), DNB caused a reduction in oxidation (<0.25-fold change compared with control) in one MRP in brainstem (acetyl-CoA acetyltransferase, mitochondrial) and did not induce any changes in oxidation in any cortical MRPs (Table 3). Similarly, DNB did not produce significant changes in MRP oxidation in brainstem or cortex of 3 months old rats (Table 3). However, DNB caused the oxidation of 7 MRPs (>4.0-fold compared with control) in brainstem from 18 months old animals, and none in cortex (Table 3). There were no differences in MRP oxidation observed in 3 months old brainstem or cortex when compared with 1 month old MRP oxidation; however, DNB caused the oxidation of 4 MRPs in cortex and 13 MRPs in brainstem (>4.0-fold compared with 1 month old) in 18 months old animals (Table 4). The degree to which DNB oxidizes MRPs is significantly higher in brainstem than in cortex (Fig. 4), and brainstem MRPs are approximately ×3 more sensitive to oxidation than cortical counterparts (Fig. 4). These data establish a clear link between subsets of the mitochondrial proteome that become more vulnerable to oxidation during the physiological aging process and regional susceptibility to mitochondrial proteomic oxidation caused by exposure to an environmental neurotoxicant.

TABLE 3.

Effects of DNB on Regional MRP Oxidation

Rat Age (months)	Cortex	Brainstem	Protein Identification
1	0	1	Acetyl-CoA acetyltransferase, mitochondrial*
3	0	0	—
18	0	7	Cytochrome c oxidase subunit 6C-2
			Glyceraldehyde-3-phosphate dehydrogenase
			NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 6
			RCG58449, isoform CRA_a
			Synaptosomal-associated protein 25
			Syntaxin-binding protein 1
			Tubulin beta-2A chain

Notes: LC-MS/MS identified proteins containing peptides with methionine sulfoxide residues (the oxidized form of methionine). The number of methionine sulfoxide spectra for each protein was divided by the number of peptide spectra detected for that protein (%Oxidation). Fold-change of %Oxidation was calculated for DNB-exposed animals compared with control (= %OxidationDNB/%OxidationVC) for brainstem and cortex within each age group of rats. Fold changes <0.25 or >4.0 shown above (bold text denotes >4.0-fold %Oxidation; * denotes <0.25-fold %Oxidation) out of the total number of proteins identified as being oxidized in cortical samples (106) and in brainstem samples (99).

TABLE 4.

Effects of Age on Regional MRP Oxidation

Rat Age (months)	Cortex	Brainstem	Protein Identification
3	0	0	—
18	4	13	Glutathione S-transferase kappa 1
			Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial
			Succinyl-CoA:3-ketoacid-coenzyme A transferase 1, mitochondrial
			Trifunctional enzyme subunit beta, mitochondrial
			10 kDa heat shock protein, mitochondrial
			4-aminobutyrate aminotransferase, mitochondrial
			Acetyl-CoA acetyltransferase, mitochondrial
			Cytochrome c oxidase subunit 6C-2
			Cytoplasmic dynein 1 heavy chain 1
			NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 6
			Plasma membrane calcium-transporting ATPase 2
			RCG58449, isoform CRA_a
			Synaptosomal-associated protein 25
			Syntaxin-binding protein 1
			Tubulin beta-2A chain
			Uncharacterized protein
			Uncharacterized protein

Notes: The number of methionine sulfoxide spectra (as detected by LC-MS/MS) for each protein was divided by the number of peptide spectra detected for that protein (%Oxidation). Fold change of %Oxidation was calculated for DNB-exposed animals compared with control (=%OxidationDNB/%OxidationVC) for brainstem and cortex as a fold change of 1 month old rats. Fold changes >4.0 shown above (grayscale fields denote cortical proteins) out of the total number of proteins identified as being oxidized in cortical samples (106) and in brainstem samples (99).

A pathway analysis was performed using the UniProtID numbers for MRPs oxidized >4.0-fold by DNB. Five GOTerm Molecular Function pathways contained MRPs oxidized by DNB in brainstem (cation transmembrane transporter activity, nucleoside-triphosphatase activity, pyrophosphatase activity, hydrolase activity acting on acid anhydrides in phosphorous-containing anhydrides, and hydrolase activity acting on acid anhydrides).

Interestingly, 4 out of the 5 identified pathways contain the same 3 proteins (cytoplasmic dynein 1 heavy chain 1, tubulin beta-2a chain, and plasma membrane calcium-transporting ATPase 2) (Table 5). Cytoplasmic dynein is chiefly responsible for retrograde transport of mitochondria and other organelles down the axon (Schnapp and Reese, 1989); microtubules, with which mitochondria are transported throughout the cell, are composed mainly of tubulin heterodimers, and plasma membrane transporting ATPase regulates intracellular calcium levels that, when imbalanced, cause uptake of mitochondrial calcium and disrupts mitochondrial transport (Kiedrowski and Costa, 1995). Dysfunction of isoforms of all of these proteins have been linked to disruption of mitochondrial trafficking in age-related neurodegeneration (Berrocal et al., 2009; Boutte et al., 2006; El-Kadi et al., 2010) and demonstrate that mitochondrial transport is an underlying age-related mechanism likely contributing to the observed DNB-induced lesions in the brainstem.

TABLE 5.

Pathways Significantly Oxidized by DNB in Brainstem Identified by the Database for Annotation, Visualization, and Integrated Discovery (DAVID)

Molecular Function Pathway	Proteins in Molecular Function Pathway
Cation transmembrane transporter activity	Synaptosomal-associated protein 25
	Cytochrome c oxidase, subunit 6-c2
	Plasma membrane calcium-transporting ATPase 2
Nucleoside-triphosphatase activity	Cytoplasmic dynein 1 heavy chain 1
	Tubulin beta-2A chain
	Plasma membrane calcium-transporting ATPase 2
Pyrophosphatase activity	Cytoplasmic dynein 1 heavy chain 1
	Tubulin beta-2A chain
	Plasma membrane calcium-transporting ATPase 2
Hydrolase activity, acting on acid anhydrides, in phosphorous-containing anhydrides	Cytoplasmic dynein 1 heavy chain 1
	Tubulin beta-2A chain
	Plasma membrane calcium-transporting ATPase 2
Hydrolase activity, acting on acid anhydrides	Cytoplasmic dynein 1 heavy chain 1
	Tubulin beta-2A chain
	Plasma membrane calcium-transporting ATPase 2

Notes: UniProtID numbers for proteins <0.25-fold or >4.0-fold oxidized, as determined by %Oxidation, were uploaded to DAVID (Huang da et al., 2009). Proteins were grouped by DAVID into pathways based on molecular function (as defined by Gene Ontology Term Molecular Function within the database).Pathways distinct from control brainstem are shown.

Aging alone causes an increase in MRP oxidation in brainstem, but not cortex, in control animals (Fig. 5), demonstrating that mitochondria in the aged brainstem are more susceptible to oxidative damage than cortex. Aging alone does not significantly increase the percentage of methemoglobin in F344 rats, with no statistical difference between control animals across all 3 age groups (Fig. 6). However, exposure to DNB causes a significant increase in methemoglobin percentage in 3 months old and 18 months old rats compared with controls. Additionally, there is a significant increase in %Methemoglobin in DNB-exposed animals across the age groups (1 month old = 2.4%, 3 months old = 7.3%, and 18 months old = 15.10%, respectively), demonstrating that advanced age of the animal exacerbates the risk of DNB-induced methemoglobinemia (Fig. 6).

DISCUSSION

There are likely multiple environmental exposures occurring over time that cause oxidative stress and contribute to a net increase in risk of developing adverse neurological outcomes during aging. This study gives evidence of increasing age exacerbating mitochondrial toxicity in the brainstem during exposure to an environmental neurotoxicant, DNB. Clinically, severity of signs of DNB intoxication in this study was positively correlated with age. Exposure to DNB resulted in severe signs of intoxication in the 18 months old animals (Tables 1 and 2), whereas there were very few cases of ataxia in the 3 months old animals (1 of 6 exposed animals at 12 h) and no instances of ataxia, or any other signs of intoxication, in 1 month old DNB-exposed animals. Lesion development as a result of DNB was not seen in 1 month or 3 months old vulnerable brain regions (Figs. 1 and 2). However, focal vacuolation within the deep cerebellar nuclei in the brainstem of an aged rat was observed (Fig. 3), though the evaluated post-exposure time point of sacrifice (12 h) was likely too early for severe lesion development. The observed lesion development in the 18 months old rat is consistent with previous observations in rats exposed to DNB (Philbert et al., 1987). The specific cause of vacuolated lesions in DNB-exposed animals is not currently known. One possibility is that the vacuolation represents swollen foot processes of damaged astrocytes. DNB is known to cause toxicity to astrocytes in vitro and similar vacuolation of the neuropil has been attributed to toxicant-related swelling of astrocyte foot processes (Garman, 2011). Since astrocytes form a vital component of the blood brain barrier, this may support vascular leakage and local edema as a cause for the vacuolation. There is evidence demonstrating that astrocyte swelling is observed as a result of increased ROS and imbalances in osmotic pressure (Schliess et al., 2004), and that astrocyte swelling is observed in cases of ataxia caused by exposure to mycotoxin in rats (Cavanagh et al., 1998). Thus, the increase in reactive oxygen species (ROS) associated with DNB exposure may similarly be causing the observed signs of intoxication.

Evidence in the literature suggests a correlation between increasing age and increased severity of adverse neurological effects upon exposure to other neurological toxicants, including paraquat exposure and dopamine depletion in mice (Liang et al., 2013), cypermethrin exposure and dopaminergic neurodegeneration in rats (Singh et al., 2012), lead exposure and decreased dexterity in humans (Grashow et al., 2013), and lead exposure and decreased brain volume in humans (Stewart et al., 2006). To our knowledge, however, this is the first study to report a mitochondrial-specific and region-specific age-related susceptibility to a neurotoxicant. Mitochondria were investigated as a mechanistic linchpin of DNB-induced neurotoxicity as a function of age because (1) the lesions that occur as a result of DNB exposure mimic those observed in acute energy deprivation syndromes (Philbert et al., 1987), likely due to futile redox cycling in mitochondria (Cavanagh, 1993), (2) DNB causes mitochondrial protein oxidation in immortalized astrocytes in vitro (Steiner and Philbert, 2011), and (3) mitochondrial protein oxidation in brain is positively correlated with age (Imam et al., 2006; Perluigi et al., 2010).

Significant changes in the oxidation of MRP were not noted in the brainstem of 3 months old animals, however, in 18 months old animals, 7 MRPs were over 4-fold oxidized by DNB in the brainstem (Tables 3 and 4). Significant changes in cortical MRP oxidation were not detected in any age group, which supports the hypothesis that there is regional variation in MRP susceptibility to oxidation. Additionally, within the MRPs that were found to be oxidized by proteomic analysis, brainstem MRPs had 3 times the amount of MRPs oxidized >4.0-fold than cortical MRPs (Fig. 4). Furthermore, in MRPs that were oxidized >4.0-fold, the average level of oxidation was statistically significantly higher in brainstem than in cortex (P < 0.05) (Fig. 4). This evidence suggests that brainstem mitochondria were more sensitive to oxidation by DNB.

In addition to region-specific DNB-induced MRP oxidation, there is an aging effect on cortical and brainstem MRP oxidation. While cortical MRPs showed no DNB-induced alterations in the degree of oxidation across age groups, brainstem MRPs were sensitive to DNB exposure as a function of age (Tables 3 and 4). In 1 month old animals, one MRP was significantly less oxidized in brainstem (acetyl-CoA acetyltransferase, mitochondrial), which is active in ketone body metabolism by mitochondria (Middleton, 1973). Mitochondrial ketone body metabolism provides acetyl CoA from acetoacetyl CoA and allows for the production of energy while circumventing the need for pyruvate dehydrogenase (Veech, 2004); this is likely to be important in the mitochondrial response to DNB exposure, as DNB has been shown to render pyruvate dehydrogenase inactive (Miller et al., 2011). This suggests that significantly lower oxidation of mitochondrial acetyl-CoA acetyltransferase may contribute to the role of ketone metabolism in younger animals and may be part of a protective mitochondrial mechanism in DNB exposure, as no lesions were observed in the youngest animals. This, however, might only be a significant part of the mitochondrial response to DNB in the youngest group of animals and not in the 3 months old animals, as lesions were not observed in the 3 months old group. Thus, these animals might have a differential mitochondrial response to DNB exposure not involving mitochondrial acetyl-CoA acetyltransferase.

The increased oxidation of MRPs having activity in specific pathways (cation transmembrane transporter, nucleoside-triphosphatase, pyrophosphatase, and hydrolase activity) provides cues as to the molecular mechanisms of selective pathogenesis in brainstem (Table 5). Three MRPs significantly oxidized in brainstem constituted 4 of the 5 pathways identified in this analysis: cytoplasmic dynein, tubulin, and a plasma membrane calcium-transporting ATPase. Cytoplasmic dynein is one of the linker proteins that bind to the microtubule and mitochondrion, and along with kinesin-1, facilitates fast transport of the mitochondrion down the microtubule in axons (Pilling et al., 2006); the microtubule is composed of a tubulin heterodimer that polymerizes and depolymerizes, rendering the microtubule a dynamic component of the cytoskeleton. The plasma membrane calcium-transporting ATPases are responsible for the maintenance of intracellular (Jensen et al., 2004) and extracellular (Talarico et al., 2005) calcium concentrations by pumping out calcium. Aberrant high intracellular calcium concentrations have been shown to affect mitochondrial transport in the brain by causing an increase in mitochondrial calcium uptake (as is seen in glutamate toxicity) (Kiedrowski and Costa, 1995), which can cause induction of the mitochondrial permeability transition pore (Kristal and Dubinsky, 1997) and in turn disrupt the efficiency of mitochondrial trafficking throughout the cell (Guo et al., 2013). The potentially synergistic effects of the observed DNB-induced oxidation of these 3 particular proteins are likely to significantly disrupt mitochondrial transport in the brainstem of older animals, contributing to the observed DNB-induced signs of intoxication in older animals. The observed age-associated increase in %Methemoglobin in DNB-exposed animals (Fig. 6) suggests that the age of the animal is inversely related to its ability to cope with DNB-induced oxidative stress. This, combined with the region-specific difference in MRP oxidation related directly to age in control animals (Fig. 5) suggests that even though there is a general decline in the ability to counteract oxidative stress induced by DNB exposure during aging, differential regional susceptibility to MRP oxidation exists, with brainstem being inherently more vulnerable than cortex. This is despite global distribution of DNB throughout the brain during exposure (Hu et al., 1997).

There are fundamental functional differences between the brainstem and cortex. One difference of note is the functional composition of subpopulations of astrocytes within the brainstem that differ from those in the cortex. Brainstem astrocytes release ATP in response to cellular stresses, such as decreases in physiological pH, whereas cortical astrocytes do not (Kasymov et al., 2013). This potentially puts brainstem astrocytes at risk of further energetic deficit, as DNB causes mitochondrial damage in astrocytes. Additionally, chemically induced phenotypic differences in astrocytes exist between cortex and brainstem: the degree to which astrocytic stellation (the transition from an epithelial phenotype to one in which processes are evident) occurs in response to chemical stimulation differs between brainstem and cortex (Davis-Cox et al., 1994). The differential regional response to DNB is likely related to differing compensatory mechanisms in subpopulations of astrocytes in the brainstem, as DNB targets astrocytes in vivo (Philbert et al., 1987) and elicits responses from brainstem astrocytes in vitro (Tjalkens et al., 2003). A limitation of this study is that region-specific and age-specific MRP oxidation cannot be delineated into cell-specific mitochondrial responses to DNB exposure. Thus, the relative contribution of mitochondrial oxidation to astrocyte-specific mechanisms of DNB toxicity in brainstem in vivo is unknown, but is supported by in vitro studies (Steiner and Philbert, 2011; Steiner et al., 2013).

This study shows evidence of a clear correlation between age of the exposed animal, an increase in the signs of intoxication and elevated %Methemoglobin with concomitant increases in MRP oxidation in vulnerable brainstem regions. Future investigations will need to determine whether or not oxidation of key mitochondrial proteins is necessary and/or sufficient for precipitating the formation of vacuolar lesions in the auditory and vestibular pathways of the rat or are merely reflections of a highly stressed neural pathway that loses its compensatory capacity with age.

FUNDING

National Institutes of Health (2R01 ES008846 to M.A.P.) and the Environmental Toxicology and Epidemiology Program from the National Institutes of Environmental Health Science (2T32 ES007062).