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.

Keywords: 1,3-dinitrobenzene; aging; mitochondria; neurotoxicity; regional susceptibility.

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.

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).