Neurotoxicity of Diesel Exhaust Particles

Abstract

Background:

Air pollution particulate matter (PM) is strongly associated with risks of accelerated cognitive decline, dementia and Alzheimer’s disease. Ambient PM batches have variable neurotoxicity by collection site and season, which limits replicability of findings within and between research groups for analysis of mechanisms and interventions. Diesel exhaust particles (DEP) offer a replicable model that we define in further detail.

Objective:

Define dose- and time course neurotoxic responses of mice to DEP from the National Institute of Science and Technology (NIST) for neurotoxic responses shared by DEP and ambient PM.

Methods:

For dose-response, adult C57BL/6 male mice were exposed to 0, 25, 50, and 100μg/m³ of re-aerosolized DEP (NIST SRM 2975) for 5 h. Then, mice were exposed to 100μg/m³ DEP for 5, 100, and 200 h and assayed for amyloid-β peptides, inflammation, oxidative damage, and microglial activity and morphology.

Results:

DEP exposure at 100μg/m³ for 5 h, but not lower doses, caused oxidative damage, complement and microglia activation in cerebral cortex and corpus callosum. Longer DEP exposure for 8 weeks/200 h caused further oxidative damage, increased soluble Aβ, white matter injury, and microglial soma enlargement that differed by cortical layer.

Conclusion:

Exposure to 100μg/m³ DEP NIST SRM 2975 caused robust neurotoxic responses that are shared with prior studies using DEP or ambient PM0.2. DEP provides a replicable model to study neurotoxic mechanisms of ambient PM and interventions relevant to cognitive decline and dementia.

Keywords

Air pollution Alzheimer’s disease brain dementia diesel exhaust particle neurotoxicity

INTRODUCTION

Air pollution exposure is a recognized risk of elderly for accelerated cognitive decline and Alzheimer’s disease (AD) and related dementia [1 –4]. Meta-analyses show strong association of dementia risk with late-life exposures to fine particles, but less consistent association with gas pollutants [5]. The mechanisms of air pollution neurotoxicity are usually studied with rodent models exposed directly to traffic related air pollution particles (TRAP), the sub-fraction of particulate matter (PM), or the gas pollutants.

TRAP from local sources exhibits a diverse chemical composition and toxic potency [6 –8]. Although TRAP represents local sources for the defined collection duration, other industrial sources may also contribute to down-wind local particle pollution [9]. In addition, TRAP collected from the same site can vary in toxicity. For instance, we observed declining neurotoxicity of “nPM”, a nano-sized sub-fraction of TRAP from urban Los Angeles after 2018 [10], in exposure paradigms with the same mouse and cell models, and the same exposure doses and durations since 2011 [11]. This diversity of sources and toxic potency could potentially confound the replicability of experimental studies of mechanisms and interventions.

Diesel engine exhaust, as a single source component of TRAP, may increase the experimental replicability of PM toxicity. Diesel exhaust is a major component of air pollution at near-freeway locations [12, 13] and near diesel engines used for shipping transfers or power generation. Rodent diesel exhaust exposure paradigms include direct diesel exhaust containing particles and volatiles, and fresh diesel exhaust particles (DEP) collected from truck engines. Exposure to DEP from various sources could cause damages to brain neurons and glia, cerebrovascular beds, lung, and promote atherosclerosis in parallel with ambient PM [14, 15] (Supplementary Table 1). The various sources of DEP, however, raises similar concerns of variations in toxic potency and consistency as that for ambient PM.

For more than a decade, DEP from the National Institute of Science and Technology (NIST) has been used to study PM toxicity. The well characterized batch of NIST DEP (SRM 2975) was obtained from a single diesel engine [9] thus has the potential to be a model PM for neurotoxicity studies. In current study we further characterized the neurotoxicity of DEP NIST SRM 2975 with experimental paradigms used for nPM neurotoxicity. Dose response (20, 50, and 100μg/m³) and time course response studies (100μg/m³ for 5 h, 100 h, and 200 h) showed DEP effects on several neurotoxic responses related with dementia and AD pathogenesis, including white matter injury, neuroinflammation, increased levels of soluble amyloid-β peptides and microglia activation. These results are compared with our prior findings on nPM from Los Angeles downtown area. We conclude that DEP NIST SRM 2975 shares many of the neurotoxic effects caused by ambient nPM and DEPs from other sources.

MATERIALS AND METHODS

Animal procedures and exposure design

All animal procedures were reviewed and approved by the University of Southern California Institutional Animal Care and Use Committee (IACUC) and were in accordance with the Guide for the Care and Use of Laboratory Animals (NIH). Jackson Laboratories supplied C57BL/6 male mice aged 10 weeks. After 1-week acclimation, mice were assigned randomly to filter air (FA) or DEP exposure groups. Mice were housed on a 12-h light dark cycle (6 am to 6 pm) with free access to food and water at facilities supervised by the USC Department of Animal Resources. When exposure to DEP was completed, mice were humanely euthanized within 18-72 h by cardiotomy with saline perfusion under deep anesthesia with isoflurane. Brains were chilled for dissection, followed by storage at -80°C; tissues for immunohistochemistry were stored in 4% cold paraformaldehyde.

Diesel exhaust exposure

DEP (NIST SRM 2975) was suspended in ultrapure Milli-Q water (200μg/ml) by 30 min sonication. The DEP suspension was re-aerosolized and animal exposure conducted using procedures reported for nPM exposure [11]. In brief, DEP suspension was re-aerosolized by HOPE jet nebulizer (Model 1131; B&B Medical Technologies, Carlsbad, CA) connected to HEPA-filtered compressed air. The aerosolized stream was mixed with clean air filtered by HEPA (1214; Pall Laboratory, Port Washington, NY). The re-aerosolized DEP was drawn through a silica gel diffusion dryer (Model 3620, TSI Inc., USA), followed by Po-210 neutralizers (Model 2U500, NRD Inc., USA) to remove the excess water content and electrical charges of the particles, respectively. The air stream was then entered into animal exposure chambers at flow rate of 2.5 lpm for the exposure. Variations in particle size and mass concentration were measured continuously (size distribution each 5 min) by TSI DustTrak (Model 8520, TSI Inc., USA) in parallel with the exposure chambers. The variability of mass concentration was < 10% throughout the exposure. Animal exposures were conducted in sealed whole-body chamber with adequate ventilation. Chamber size is 52 cm long, 16 cm high, and 32 cm wide. Each chamber has 9 separate meshed boxes that can house up to 27 mice (3 mice per box) from same exposure group. For dose response exposures, mice of the same group were housed in one exposure chamber designated for either FA or DEP for 5 h. Exposure to different doses (20, 50, and 100μg/m³) were done on sequential days. For the time course response exposures, mice of same group were housed in the exposure chamber designated for FA or DEP and exposed to either filtered air or DEP (100μg/m³) for 5 h/day for durations of 1 day (5 h), 4 weeks (100 h), or 8 weeks (200 h). Figure 1 shows the size distributions of DEP suspension after sonication and a representative DEP sample collected from the re-aerosolization system. After sonication, DEP suspensions had mean size of 57 nm (Fig. 1A), and after re-aerosolization, 69% particles were < 100 nm (Fig. 1B). For further details on DEP re-aerosolization system and characterization see [16]. DEP quality was maintained with a standardized procedure of DEP preparation, which includes storage of DEP suspension as aliquots in a -20°C freezer, using the same sonication condition, measurement of DEP size before daily exposure, and monitoring and modulating DEP concentration during the exposure.

Fig. 1

Size distribution of DEP SRM2975. A) Suspension after 30 min sonication. Black dots represent samples prepared at different times, mean±SD, N = 9. B) DEP after re-aerosolization at 100μg/m³ (Methods above).

Reagents

TRIzol reagent was from Thermal Fisher Scientific (Rockford, IL); reverse transcription reagent and qPCR master mixture were from BioPioneer Inc. (San Diego, CA); antibodies for immunochemistry of β-actin, Iba1, Cd68, and dMBP from Abcam (Cambridge, MA), Abeta peptides were from Biolegend (San Diego, CA); SRM 2975 DEP was from National Institute of Standards and Technology (NIST, Gaithersburg, MD); other reagents were from Sigma-Aldrich (St. Louis, MO).

Klüver-Barrera histochemistry for white matter injury

Klüver-Barrera (KB) staining used paraffin-embedded slides. After deparaffining and dehydration using graded alcohol, sections were immersed in Luxol Fast Blue and incubated at 56°C overnight; then sections were differentiated with lithium carbonate solution and subsequently counterstained with Cresyl Violet. Sections were analyzed at 40x by microscopy and visual scale ratings were evaluated by two independent observers.

Immunohistochemistry and immunofluorescence

Paraffin sections (5μm) were stained overnight with primary antibodies for degraded myelin basic protein (dMBP), ionized calcium binding adaptor molecule 1 (Iba-1), complement C5, C5a, and CD88, 8-hydroxydeoxyguanosine (8-OHdG), and 4-hydroxynonenal (4HNE). After antigen retrieval and blocking, sections were incubated with goat anti-Iba-1 for immunohistochemistry or donkey antisera to dMBP, C5, C5α, CD88, 8-OHdG, and 4HNE for immunofluorescence. Secondary antibody was matched to primary antibody and stained for 1 h at room temperature. The slides were imaged by fluorescent microscopy at 40x and analyzed by ImageJ. For all the fluorescent stains, total fluorescent density in the region of interest was quantified with an average from two independent observers. For non-fluorescent morphology stains, visible cells were counted per region of interest area. All visible Iba-1 positive microglia cells were manually traced for the measurement of soma area.

qPCR assay

Tissues were extracted by TRIzol reagent for reverse transcription and qPCR with primers used previously [17].

Western and dot blots

Frozen mouse cerebral cortex (30 mg/1 ml) was homogenized in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors (Sigma, St. Louis, MO) and phosphatase inhibitors (Thermofisher Scientific, Waltham, MA) [2 –4]. Supernatants were collected after 10,000 g/1 h at 4°C. For western blots, 20μg of total protein was boiled and loaded onto 26 well 4–20% Criterion gels and transferred for 1 h using Criterion blotter (Biorad, Hercules, CA). For Dot blots 100μg of protein was loaded by gravity filtration. Transfers used 0.5μm PVDF membranes (Millipore, Burlington, MA). Dot blots were stained with Revert 700 (Licor, Lincoln, NE) for total protein measurement before blotting. After blocking for 1 h in Intercept blocking buffer (Licor, Lincoln, NE), membranes were incubated overnight at 4°C with primary antibodies for β-Actin, Cd68, Aβ₃₈, Aβ₄₀, and Aβ₄₂ (M78; kindly provided by Professor Charles Glabe, University of California at Irvine). Blots were washed, incubated 1 h at room temperature with Licor secondary antibodies diluted (1 : 20,000) in TBS-T+5% milk, and imaged by Licor 9120 (Licor, Lincoln, NE). Densitometry was analyzed by ImageJ and normalized to actin (western blot) or total protein (dot blot).

Statistical analysis

Data are shown as mean±standard deviation, with significance at 2-sided alpha of 0.05. The immunofluorescence and immunohistochemistry data for dose response and time course response were analyzed by general linear model with main effects of DEP exposure (compared to FA). General linear model analysis with repeated measures was used for analysis of cortical layer responses to DEP. For qPCR data analysis, the data for FA group from two independent experiments, one evaluating 5-h response and another evaluating 200-h response, were pooled. qPCR data for time course response were analyzed by three-way ANOVA, treating exposure to FA or DEP and time points as fixed effects and experiment year of pooled exposure as a random factor. Pairwise comparisons with Fisher LSD adjustment were used for multiple comparisons, and Student t-test was used for two group comparisons. Statistical analyses used SPSS (version 28) and GraphPad Prism (version 9).

RESULTS

Dose response (20, 50, and 100μg/m³) to 5-h DEP exposure

Prior studies on DEP toxicity used a 10-fold range of concentrations, 35μg/m³-300 mg/m³ (Supplementary Table 1). To establish the lowest dose of DEP for repeatable brain responses, we examined 5-h DEP exposure at 20, 50, and 100μg/m³ on the brain and lung relative to FA controls, followed by tissue collection 18 h later. In corpus callosum, mice exposed to 100μg/m³ DEP had 50% higher Iba-1, a microglia-specific calcium-binding protein, than FA group, but Iba1 did not respond to lower doses (Fig. 2E). Compared to FA group, mice exposed to DEP at 100μg/m³ also had significantly higher dMBP level by 40% (Fig. 2C) and higher complement component C5a by 20% in corpus callosum, but not for mice exposed to lower DEP concentrations (Fig. 2A, C). In addition, DEP exposure at 100μg/m³ increased 4HNE conjugates, a product of lipid peroxidation, and 8-OHdG, a marker of DNA oxidation, by 30% and 50% respectively in corpus callosum, but not for lower DEP concentrations (Fig. 2D, F). In lung, Tnfa mRNA was increased by 60% by 100μg/m³ DEP but not by lower concentrations of DEP (Fig. 2I), while in the olfactory bulb, cortex and hippocampus it was not altered by 5 h-DEP at any concentration (Fig. 2G, H). Together these data suggest that at 100μg/m³, DEP exposure causes a robust response in the lung and corpus callosum. Thus 100μg/m³ DEP was used for the following studies.

Fig. 2

Dose Response to DEP for 5 h. Mice were exposed to DEP at 20, 50, and 100μg/m³ for 5 h; after 18 h, mice were euthanized. Panels A-F are immunofluorescence assays: A) complement C5a; B) complement receptor CD88; C) white matter marker dMBP; D) oxidative damage marker 4HNE; E) microglia marker Iba-1; F) oxidative damage marker 8-OHdG. Panels G-I are Tnfa mRNAs measured by qPCR: G) cortex; H) hippocampus; I) lung. *p<0.05 compared with Filter (Air), N = 8-16 mice per group.

Time course response to DEP exposure

To define the time-course of neurotoxic response, mice were exposed to DEP for 5, 100, and 200 h, representing acute, sub-acute, and chronic exposures. Inflammatory genes and oxidative damage in brain and lung were assayed by qPCR and immunohistochemistry in corpus callosum, cerebral cortex, hippocampus, and lung (Figs. 3–7).

Fig.3

Time-course response of corpus callosum to DEP exposure with representative images. Mice were exposed to DEP at 100μg/m³ for 5, 100, and 200 h; brains were collected 18 h after the last exposure. White bars are filter air (FA); grey bars, DEP. Panels A1-C2, E1-E2, and G1-H2 are immunofluorescence assays. A1 and A2, complement C5; B1 and B2, complement C5a; C1 and C2, dMBP; E1 and E2, Iba-1; G1 and G2, 4HNE; H1 and H2, 8-OHdG. Panels D1, D2, F1 and F2 are immunohistochemical assays; D1 and D2, Klüver-Barrera (KB); F1 and F2, Iba-1 microglial soma area. Space bar 50μm. *p<0.05, **p<0.01; N = 8-16.

Fig. 4

Time-course response of cerebral cortex to DEP exposure. For protocols, see Fig. 3 legend. White bars are filter air (FA); grey bars, DEP. Panels A-E are mRNAs (qPCR): A) Il1b; B) Il6; C) Nfkb1; D) Rela; E) Tnfa. Panels F-G are data from immunohistochemical assays: F) Iba-1 Cell Count; G) Iba-1 soma Area. H) Aβ₃₈; I) Aβ₄₀ (p < 0.18); J) Aβ⁴²; K) CD68 (western blot). * p < 0.05 and ** p < 0.01, compared to FA for same exposure time, N = 8-16 mice per group.

Fig. 5

Time-course response of hippocampus mRNA to DEP exposure. White bars are filter air (FA); grey bars, DEP. Exposure and tissue collection are described in Fig. 3 legend: A) Il1b; B) Il6; C) Nfkb1; D) Rela; E) Tnfa. * p < 0.05 DEP compared with FA of same exposure time, N = 8-16 mice per group.

Fig. 6

Time-course response of lung mRNA to DEP exposure. DEP exposure and tissue collection as described in Fig. 3 legend. White bars are filter air (FA); grey bars, DEP group. A) Il1b; B) Il6; C) Nfkb1; D) Rela; E) Tnfa. * p < 0.05 DEP compared with FA of same exposure time, N = 8-16 mice per group.

Fig. 7

Response of cerebral cortex layers to DEP exposure for inflammatory responses (complement C5, microglial Iba1) and oxidative damage (4HNE). White bars are filter air (FA); grey bars, DEP. All data are immunohistochemical staining. For panels A1-C3, mice were exposed to DEP at 100μg/m³ for 200 h (8 weeks): A1) complement C5 in the whole cortex; A2) complement C5 by cortical layer; B1) 4HNE in whole cortex; B2) 4HNE by cortical layers; C1) Iba1 in whole cortex; C2) Iba1 by cortical layers; C3) representative image for Iba1. For panels D1-E3, mice were exposed to DEP at 100μg/m³ for 5, 100, and 200 h: D1-D3) Iba1-positive cell count for 5 h-, 100 h-, and 200 h-DEP exposure, respectively; E1-E3) Iba1 soma area for 5 h-, 100 h-, and 200 h-DEP exposure, respectively; DEP compared with FA for same time. Space bars, 100μm. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; N = 8-16.

In corpus callosum (Fig. 3), DEP exposure for 100 h (4 weeks) and 200 h (8 weeks) caused 40% elevations of complement C5a above FA control (Fig. 3B1). 200-h DEP also increased C5 by 200% (Fig. 3A), but none of C5 and C5a responded to 5-h DEP exposure. 4HNE and 8-OHdG were increased by DEP exposure at all three times; 4HNE increased by 70%, 60%, and 80%, respectively, and 8-OHdG by 70%, 90%, and 110%, respectively (Fig. 3G1, H1). In addition, DEP exposure for 100 and 200 h increased dMBP fluorescent density by 30% and 70% respectively (Fig. 3C1). The response of Klüver-Barrera (KB) staining to DEP was slower, and only increased by 200-h (240%), but not by 5 h or 100 h DEP (Fig. 3D1). The microglial marker Iba-1, measured as total fluorescent intensity, progressively increased during DEP exposure for 5 h (30%), 100 h (50%), and 200 h (100%); however, the microglial soma area had larger increases by 100-h and 200-h than 5-h DEP exposure (Fig. 3F1).

In cerebral cortex (Fig. 4), 5-h DEP increased mRNAs of Tnfa and Rela by 40% and 20% respectively, while there was no response for Il1b, Il6, and Nfkb1. None of the inflammatory genes (Tnfa, Il1b, Il6, Rela, and Nfkb1) responded to 100-h and 200-h DEP exposures except Nfkb1 mRNA, which was decreased by 200-h DEP exposure (Fig. 4A-E). Soluble amyloid peptides Aβ₃₈ and Aβ₄₂ were increased > 50% by 200 h DEP; Aβ₄₀ also trended to increase (Fig. 4H-J). Microglia marker Cd68 increased 20% by 200-h DEP (Fig. 4K). Aβ peptides and Cd68 in the cortex were not measured for shorter exposures.

In hippocampus, 5-h DEP exposure decreased Nfkb1 mRNA by 30% relative to FA controls but did not alter mRNA of other inflammatory genes. In addition, DEP exposures for 100 h and 200 h did not change mRNAs of Tnfa, Il1b, Il6, Rela, and Nfkb1 in hippocampus (Fig. 5). This data suggest that DEP exposure may not induce pro-inflammatory genes in the hippocampus.

The inflammatory response in the lung was asynchronous (Fig. 6): Tnfa mRNA was increased only by 5-h DEP exposure by 45%, but was back to baseline at 100-h and 200-h DEP. Contrarily, Il1b mRNA was increased by 100% only at 100-h DEP. No inflammatory gene in the lung responded to 200-h DEP exposure.

Response of cortical layers to DEP exposure

The responses of microglia and complement and oxidative damage were analyzed in whole cortex and cerebral cortex cell layers (Fig. 7). Upon 200-h DEP exposure, C5 and 4HNE were increased in the whole cortex by 40% and 10% (Fig. 7A1, B1), respectively, paralleled by increases in each cortical layer (Fig. 7A2, B2). Iba-1 fluorescent intensity was increased by 90% in whole cortex, and in all cortical layers (Fig. 7C1, C2). Iba-1 positive cell counts were higher by 40% in cortical Layer 4, while the microglial soma area was increased in Layers 3-5 by 200-h DEP, but not by shorter exposures (Fig. 7D1, E3). Taken together, these data show that DEP exposure for 200 h caused oxidative damage, inflammatory response and microglia activation in the overlaying cerebral cortex, with microglia activation differing between cortical layers.

DISCUSSION

DEP caused rapid mouse brain responses at 100μg/m³, a level lower than the widely used > 200μg/m³ DEP or direct diesel exhaust in the literature (Supplementary Table 1). First, we describe these data and then compare them with our prior findings with nPM, a nanoscale sub-fraction of ambient urban air PM, and with DEP from other laboratories. We then consider how these DEP differ from those directly emanated by diesel engines and DEP components in TRAP.

Current studies used DEP collected by the National Institute of Science and Technology (NIST) from a single engine (NIST SRM-2975) that has been widely used (Supplementary Table 1). Corpus callosum white matter responded to 5 h exposure of DEP at 100μg/m³ (assayed 18 h later, Fig. 2A-F). The largest responses were 50% elevations of Iba1-1 (microglial calcium binding protein) and 8-OHdG (DNA oxidation), with smaller increases of dMBP (degraded myelin basic protein, 30%), 4HNE (protein oxidative modification, 30%), and complement C5a (activated complement, 20%). Tnfa mRNA only responded in lung to 5 h DEP (Fig. 2I). Lower DEP doses of 20 and 50μg/m³ were insufficient to cause significant response of examined markers in acute exposure (5 h). This is the first evidence that DEP can induce oxidative and inflammatory responses in white matter within 1 day of exposure. These rapid responses allow convenient testing of mechanisms and drug protection.

The time course of response to 100μg DEP/m³ (5 h/d) was examined at exposures of 5 h/d after one day, 4 weeks (100 h) and 8 weeks (200 h). Some markers that responded to single 5 h exposure increased progressively with longer exposure (C5a, 4HNE, 8-OHdG), while C5 increased only after 8 weeks in corpus callosum and cerebral cortex. Corpus callosum dMBP fluorescent density was increased by up to 70% after 4 weeks and 8 weeks DEP exposures, while the response of Klüver-Barrera (KB) myelin degeneration was delayed to 8-week exposure (240%). Microglial cell soma area was enlarged > 2-fold by 4-week and 8-week DEP exposures. The soluble amyloid-β peptides Aβ₃₈ and Aβ₄₂ were increased in cerebral cortex by 8-week DEP exposure. Hippocampus and lung showed few inflammatory mRNA responses after 8-week DEP exposure. DEP exposure for 5 h increased Tnfa mRNA in lung, while the Tnfa response in the cortex was inconsistent between the initial (Fig. 2 G) and repeat exposure (Fig. 4E).

Cerebral cortex was analyzed in more detail in this first analysis of responses to air pollution components by cortical layers 1-6 (Fig. 7). Complement C5 and 4HNE were increased in all cortical layers after 8 weeks of DEP exposure (Fig. 7A1-B2). Microglial Iba-1 had the most robust increase as measured by Iba1-positive cell count in Layer 4 and by microglial soma area in Layers 2-5; these responses were only observed after 200 h DEP exposure but not earlier (Fig. 7C1-E3). However, Iba-1 intensity was increased by DEP exposure for as short as 5 h in corpus callosum (Fig. 3E1-F2). Taken together, these data show that white matter microglia are more rapidly activated by DEP exposure than microglia in the overlaying cerebral cortex. Cortical Layer 4 is the direct target of sensory inputs coming from the subcortical structures, e.g., thalamus [18]. Similarly, cortical pyramidal neurons in Layers 5 and 6 contain long distance projections connecting cortex with other subcortical structures [19]. The microglial activation in Layers 2-5 (Fig. 7D3, E3) thus may be more involved in the inflammatory response of white matter than the increase of C5 increase in Layer 1 (Fig. 7A1). Furthermore, the layer-specific responses to DEP may be reciprocally linked to the corpus callosum, which receives more axonal projections from Layers 2, 3, and 5 than from Layer 1 [20, 21]. Future studies on air pollution neurotoxicity should examine the effects by brain regions including cortical layers, corpus callosum, and anterior-posterior regions that represent different functional domains [22].

The responses to DEP exposure as observed in current study are generally shared with other DEP neurotoxicity studies (Supplementary Table 1). Rats exposed directly to diesel exhaust 6 h/day for 6 months exhibited increase in Tnfa mRNA and Aβ₄₂ peptide in cerebral cortex at the highest dose 992μg/m³ [23]. Similarly, amyloid plaques were enlarged in 5X-FAD-tg mice exposed to 950μg/m³ direct diesel engine exhaust for 3 weeks [24]. In addition, prior studies with rodents showed microglial activation and inhibition of adult neurogenesis within 1 day exposure to direct diesel exhaust at 250μg/m³ [25], and increased brain Aβ peptides within 1 day of nickel particle inhalation PM [26]. Prior DEP studies with NIST SRM2975 increased reactive oxidants in cell cultures [27], altered blood-brain barrier function and induced inflammation in the brain [23, 28], caused lung inflammation [29 –31] and behavioral changes in rodents [32, 33]. These neurotoxic effects are similar to those caused by nPM collected from urban traffic area (Table 1) and DEP from other sources (Supplementary Table 1). Together, these data support the use of NIST SRM2975 as a reliable and replicable paradigm to study PM neurotoxicity.

Rodent exposure models have generally used inhalation of PM at concentrations of > 100μg/m³ (Supplementary Table 1). These include our studies with a nano-sized aqueous suspension of urban PM (designated as nPM in distinction from total 0.2μ PM) that were used for exposures at 300μg/m³ [11]. Although we did not observe response to 20 and 50μg/m³ DEP after a single 5 h exposure, longer low dose exposures could cause neurodegeneration, as shown in other exposure paradigms. Exposure of rats transgenic for human Alzheimer genes to roadway tunnel PM2.5 at 14μg/m³ for 14 months caused hippocampal damage with neuron loss, and increased brain Aβ deposits [34]. Comparison of these findings with tunnel traffic exhaust and with other studies of ambient roadway air must consider quantitative differences in volatile organic carbons, ozone, and oxides of nitrogen and sulfur, which may differ from ambient roadway air [35]. Nanosized refractive particles were also observed in these brains that warrant our future studies to examine DEP particles in lung, blood, and brain.

Table 1

Data summery

DEP NIST	Aβ₃₈	Aβ₄₀	Aβ₄₂	C5	C5a	CD68	CD88	dMBP	4HNE	Iba-1	KB	IL1B	IL6	NFkB1	NFkBp65	8OHdG	TNFα
Corpus callosum 5 h, 100μg/m³	–	–	–	0	↑	–	↑	↑	↑	↑	0	–	–	–	–	↑	–
Corpus callosum 100 h, 100μg/m³	–	–	–	0	↑	–	↑	↑	↑	↑	0	–	–	–	–	↑	–
Corpus callosum 200 h, 100μg/m³	–	–	–	↑	↑	–	↑	↑	↑	↑	↑	–	–	–	–	↑	–
Cortex 5 h, 100μg/m³	–	–	–	–	–	–	–	–	–	0	–	0	0	0	↑	–	↑
Cortex 100 h, 100μg/m³	–	–	–	–	–	–	–	–	–	0	–	0	0	0	0	–	0
Cortex 200 h, 100μg/m³	↑	0	↑	↑	–	↑	–	–	↑	↑	–	0	0	↓	0	–	0
Hippocampus 5 h, 100μg/m³	–	–	–	–	–	–	–	–	–	–	–	0	0	↓	0	–	0
Hippocampus 100 h, 100μg/m³	–	–	–	–	–	–	–	–	–	–	–	0	0	0	0	–	0
Hippocampus 200 h, 100μg/m³	–	–	–	–	–	–	–	–	–	–	–	0	0	0	0	–	0
Lung 5 h, 100μg/m³	–	–	–	–	–	–	–	–	–	–	–	0	0	0	0	–	↑
Lung 100 h, 100μg/m³	–	–	–	–	–	–	–	–	–	–	–	↑	0	0	0	–	0
Lung 200 h, 100μg/m³	–	–	–	–	–	–	–	–	–	–	–	0	0	0	0	–	0
nPM by our group
*Cortex 5 h	–	–	–	–	–	–	–	–	–	–	–	0	0	↓	0	–	0
*Hippocampus5 h	–	–	–	–	–	–	–	–	–	–	–	0	0	0	0	–	0
*Lung 5 h	–	–	–	–	–	–	–	–	–	–	–	0	0	↓	0	–	↑
Cortex [11]	–	–	–	–	–	↑	–	–	–	–	–	–	–	–	–	–	↑
Corpus callosum [42]	–	–	–	↑	↑	–	↑	–	–	↑	–	–	–	–	–	–	–
Hippocampus [43]	–	–	–	–	–	–	–	–	–	↑	–	–	–	–	–	–	↑
Cortex [44]	–	↑	↑	–	–	–	–	–	↑	–	–	–	–	–	–	–	–
Cortex [17]	–	–	–	–	–	–	–	–	–	–	–	↑	0	–	↑	–	0
Corpus callosum [45]	–	–	–	–	–	–	–	↑	–	↑	–	–	–	–	–	–	–
Corpus callosum [46]	–	–	–	↑	↑	–	0	↑	–	↑	0	–	–	–	–	↑	–
Cortex [10]	–	–	↑	–	–	–	–	–	–	–	–	–	–	↓	–	–	↑
Hippocampus [10]	–	–	–	–	–	–	–	–	–	↑	–	–	–	–	–	–	–
Other DEP rodent inhalation
Liver [32]	–	–	–	–	–	–	–	–	–	–	–	↑	–	–	–	–	–
Cortex [47]	–	–	↑	–	–	–	–	–	–	–	–	–	–	–	–	–	↑
Midbrain [47]	–	–	–	–	–	–	–	–	–	–	–	↑	0	–	–	–	↑
Cortex [47]	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	↑
Midbrain [47]	–	–	–	–	–	–	–	–	–	↑	–	↑	↑	–	–	–	↑
Cortex [23]	–	–	–	–	–	–	–	–	–	↑	–	↑	↑	–	–	–	↑
Midbrain [23]	–	–	–	–	–	–	–	–	–	–	–	↑	↑	–	–	–	↑
Cortex [48]	–	–	–	–	–	–	–	–	↑	–	–	–	–	–	–	–	–
Hippocampus [49]	–	–	–	–	–	–	–	–	–	–	–	–	↑	–	–	–	↑
Hippocampus [50]	–	–	–	–	–	–	–	–	–	–	–	↑	↑	–	–	–	↑

*Unpublished data, “↑” –increase, “↓”- decrease, “0” –no difference, “-“ –not assayed.

Opinions diverge on the validity of DEP from single engines as models for ambient air pollution. Farahani, Pirhadi, and Sioutas concluded that standardized DEP “cannot represent typical ambient PM nor traffic emission” because of chemical differences [36]. The chemical comparison with PM2.5 from Los Angeles showed DEP NIST SRM 2975 contains negligible inorganic ions (ammonium, nitrate, sulfate) and fewer high molecular weight polycyclic aromatic hydrocarbons (PAH). In contrast, DEP has 20-fold excess elemental carbon (EC). On the other hand, Block and Kodavanti [37] argued that standardized DEP was useful experimental model that allowed multiple labs to independently confirm findings with the same exposure model and note the similarity of toxicity caused by DEP with ambient PM.

Our prior studies with nPM acknowledged the complete lack of PAHs and > 50% diminution of transition metals, inorganic ions, black carbon, and other organic compounds found in ambient PM2.5 at various levels [38, 39]. Nonetheless, nPM and total PM2.5 have a shared core of inflammatory responses in cortex and neurobehavioral changes that do not depend on PAHs [38]. Table 1 shows the substantial overlap of neurotoxic responses to DEP NIST 2975 and nPM with prior studies of DEP. The current DEP was largely nanoscale (69% <PM0.1, Fig. 1b), which approximates the smaller size of DEP emitted by modern diesel engines [40]. The DEP from NIST and local sources lack the volatile organic carbons present in direct diesel exhaust. While we varied the duration of DEP exposures in this study, we did not examine the persistence of change post-exposure.

While DEP may increase replicability of mechanistic studies, future experimental animal studies of air pollution toxicity must include real world ambient air. DEP could have a major role in calibrating variations of air pollution chemistry and toxicity which are changing in many regions, for better or for worse. Additionally, we suggest the collection of PM2.5 from multiple sites as a pooled sample for availability to many labs. Such pooled samples compared to a DEP standard could expand the verifiability of air pollution toxicity studies. As a precedent, the ‘Kentucky reference cigarettes’ developed decades ago [41] enabled expanded tobacco toxicity studies by enabling multiple labs to use the same inhaled materials in replicable findings.

Footnotes

ACKNOWLEDGMENTS

We are grateful for support by grants from the Cure Alzheimer’s Fund (Caleb E. Finch) and the NIH (Caleb E. Finch, R01-AG051521, P50-AG005142; MT, T32-AG000037, Caleb E. Finch, William J. Mack, Hongqiao Zhang, and Constantinos Sioutas P01-AG055367), and DOD Assistant Secretary of Defense for Health Affairs, Parkinson’s Research Program Award W81XWH-17-1-0535, Caleb E. Finch). We appreciate comments on the manuscript from Jiu-Chiuan Chen and Christian Pike, USC. The authors have no conflicts of interest to declare. We thank the Center of Excellence in NanoBiophysics Core Facility and their staff members Dr. X. Chen and Dr. S. Li for their assistance with the dynamic light scattering measurements.

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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Neurotoxicity of Diesel Exhaust Particles

Abstract

Background:

Objective:

Methods:

Results:

Conclusion:

Keywords

INTRODUCTION

MATERIALS AND METHODS

Animal procedures and exposure design

Diesel exhaust exposure

Reagents

Klüver-Barrera histochemistry for white matter injury

Immunohistochemistry and immunofluorescence

qPCR assay

Western and dot blots

Statistical analysis

RESULTS

Dose response (20, 50, and 100μg/m3) to 5-h DEP exposure

Time course response to DEP exposure

Response of cortical layers to DEP exposure

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

Dose response (20, 50, and 100μg/m³) to 5-h DEP exposure