Involvement of oxidative stress and mitochondrial mechanisms in air pollution-related neurobiological impairments

Abstract

Background: Vehicle exhaust emissions are known to be significant contributors to physical and psychological stress. Vehicle exhaust-induced stress and associated respiratory and cardiovascular complications are well-known, but the impact of this stress on the brain is unclear. Simulated vehicle exhaust exposure (SVEE) in rats causes behavioral and cognitive deficits. In the present study, the underlying mechanisms were examined. Our postulation is that SVEE, a simulation of physiologically relevant concentrations of pro-oxidants (0.04% carbon dioxide, 0.9 ppm nitrogen dioxide, 3 ppm carbon monoxide) creates a toxic stress environment in the brain that results in an imbalance between production of reactive oxygen species and the counteracting antioxidant mechanisms. This impairs mitochondrial function in the high bioenergetic demand areas of the brain including the hippocampus (HIP), amygdala (AMY) and the prefrontal cortex (PFC), disrupting neuronal network, and causing behavioral deficits. Mitochondria-targeted antioxidant Mito-Q protects against these impairments.

Methods: Sprague Dawley rats were provided with Mito-Q (250 μM) in drinking water for 4 weeks followed by SVEE 5 h/day for 2 weeks, followed by behavioral and biochemical assessments.

Results: SVEE resulted in anxiety- and depression-like behavior, accompanied with increased oxidative stress, diminished antioxidant response and mitochondrial impairment reflected from electron transport chain (ETC) disruption, reduced oxygen consumption, low adenosine tri-phosphate (ATP) synthesis and an alteration in the mitochondrial biochemical dynamics assessed via protein expression profiles of mitochondrial fission marker, dynamin-related protein-1 and fusion markers, mitofusin-1/2 in the HIP, AMY and the PFC. Mito-Q treatment prevented SVEE-induced behavioral deficits, attenuated rise in oxidative stress and also prevented SVEE-induced mitochondrial impairment.

Conclusion: This study demonstrates a causal mechanism mediating SVEE-induced behavioral deficits in rats. We further established that SVEE is a toxicological stressor that induces oxidative stress and results in mitochondrial impairment, which by disrupting neural circuitry impairs cognitive and behavioral functions.

Keywords: Behavioral health; Mitochondria; Oxidative stress; Toxicological stress; Traffic pollution.

Graphical abstract

Fig. 1

LC-MS estimation of Mito-Q in rat brain. (A) A linear curve with an equation of y = 0.0733x – 0.0161, r² = 0.9993 was plotted with x-axis representing ratio of analyte (Mito-Q) concentration to IS (d₁₅-Miito-Q) concentration and y-axis representing ratio of analyte (Mito-Q) area to IS (d₁₅-Miito-Q) area. (B) Retention time of d₁₅-Miito-Q (first peak) and Mito-Q (second peak) have been listed along the peaks. (C) Tabular representation of average Mito-Q concentration. n = 5 rats/group.

Fig. 2

Examination of anxiety-like behavior in the (A) open field test (OFT), (B) Elevated Plus Maze Test, (C) Light-Dark Test and (D) Marble Burying Test in rats exposed to normal air/simulated vehicle exhaust with/without Mito-Q pre-treatment. Examination of depression-like behavior in the Force Swim Test by measuring (E) Immobility time and (F) Active behavior time in rats exposed to normal air/simulated vehicle exhaust with/without Mito-Q pre-treatment. (*) p < 0.05, (**) p < 0.01 significantly different from CON + VEH; (#) p < 0.05, (##) p < 0.01, (###) p < 0.001 significantly different from EXP + VEH; Values are mean ± SEM, n = 13 rats/group.

Fig. 3

Examination of oxidative stress levels. Measurement of 8-isoprostane levels in (A) PFC, (B) Hippocampus (C) Amygdala; Measurement of Total Antioxidant Capacity in (D) PFC, (E) Hippocampus (F) Amygdala in rats exposed to normal air/simulated vehicle exhaust with/without Mito-Q pre-treatment. (*) p < 0.05, (**) p < 0.01 significantly different from CON + VEH; (#) p < 0.05, significantly different from EXP + VEH; Values are mean ± SEM, n = 6–8 rats/group.

Fig. 4

Measurement of Mn SOD protein expression in (A) PFC, (B) Hippocampus (C) Amygdala; and Measurement of SOD activity in (D) PFC, (E) Hippocampus (F) Amygdala in rats exposed to normal air/simulated vehicle exhaust with/without Mito-Q pre-treatment. Figures indicate cropped images of gels. (*) p < 0.05, (**) p < 0.01 significantly different from CON + VEH; (#) p < 0.05, (###) p < 0.001 significantly different from EXP + VEH; Values are mean ± SEM, n = 8–9 rats/group.

Fig. 5

Measurement of mitochondrial oxygen consumption. Representative oxygraphs showing respiratory activity in (A) healthy and (B) damaged mitochondria. The rate of oxygen consumption is shown in pmol/sec*mL and represented as % control in mitochondria isolated from (C) PFC, (D) Hippocampus (E) Amygdala of rats exposed to normal air/simulated vehicle exhaust with/without Mito-Q pre-treatment. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 significantly different from CON + VEH; (#) p < 0.05 significantly different from EXP + VEH; Values are mean ± SEM, n = 6 rats/group.

Fig. 6

Measurement of ATP synthesis in (A) PFC, (B) Hippocampus (C) Amygdala; Measurement of mitochondrial membrane potential in (D) PFC, (E) Hippocampus (F) Amygdala in rats exposed to normal air/simulated vehicle exhaust with/without Mito-Q pre-treatment. (*) p < 0.05, (***) p < 0.001 significantly different from CON + VEH; (#) p < 0.05, (##) p < 0.01 significantly different from EXP + VEH; Values are mean ± SEM, n = 6–8 rats/group.

Fig. 7

Examination of mitochondrial fission and fusion. Measurement of Mitofuin-1 protein expression in (A) PFC, (B) Hippocampus (C) Amygdala; Mitofusin-2 protein expression in (D) PFC, (E) Hippocampus (F) Amygdala; DRP-1 protein expression in (G) PFC, (H) Hippocampus (I) Amygdala, in rats exposed to normal air/simulated vehicle exhaust with/without Mito-Q pre-treatment. Figures indicate cropped images of gels. (*) p < 0.05 significantly different from CON + VEH; (#) p < 0.05 significantly different from EXP + VEH; Values are mean ± SEM, n = 9 rats/group.