Skip to main content
NCBI home page
EPA Author Manuscripts logoLink to EPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Jan 1.
Published in final edited form as: Toxicol Appl Pharmacol. 2020 Nov 27;410:115351. doi: 10.1016/j.taap.2020.115351

Ozone-induced changes in oxidative stress parameters in brain regions of adult, middle-age, and senescent Brown Norway rats

Prasada Rao S Kodavanti a,*, Matthew Valdez a, Judy E Richards b, Datonye I Agina-Obu a, Pamela M Phillips a, Kimberly A Jarema a, Urmila P Kodavanti b
PMCID: PMC7775355  NIHMSID: NIHMS1652927  PMID: 33249117

Abstract

A critical part of community based human health risk assessment following chemical exposure is identifying sources of susceptibility. Life stage is one such susceptibility. A prototypic air pollutant, ozone (O3) induces dysfunction of the pulmonary, cardiac, and nervous systems. Long-term exposure may cause oxidative stress (OS). The current study explored age-related and subchronic O3-induced changes in OS in brain regions of rats. To build a comprehensive assessment of OS-related effects of O3, a tripartite approach was implemented focusing on 1) the production of reactive oxygen species (ROS) [NADPH Quinone oxidoreductase 1, NADH Ubiquinone reductase] 2) antioxidant homeostasis [total antioxidant substances, superoxide dismutase, γ-glutamylcysteine synthetase] and 3) an assessment of oxidative damage [total aconitase and protein carbonyls]. Additionally, a neurobehavioral evaluation of motor activity was compared to these OS measures. Male Brown Norway rats (4, 12, and 24 months of age) were exposed to air or O3 (0.25 or 1 ppm) via inhalation for 6 h/day, 2 days per week for 13 weeks. A significant decrease in horizontal motor activity was noted only in 4-month old rats. Results on OS measures in frontal cortex (FC), cerebellum (CB), striatum (STR), and hippocampus (HIP) indicated life stage-related increases in ROS production, small decreases in antioxidant homeostatic mechanisms, a decrease in aconitase activity, and an increase in protein carbonyls. The effects of O3 exposure were brain area-specific, with the STR being more sensitive. Regarding life stage, the effects of O3 were greater in 4-month-old rats, which correlated with horizontal motor activity. These results indicate that OS may be increased in specific brain regions after subchronic O3 exposure, but the interactions between age and exposure along with their consequences on the brain require further investigation.

Keywords: Aging, Oxidative stress, Ozone, Air pollution, Protein carbonyls, Antioxidants, Neurotoxicity, Susceptibility

1. Introduction

The assessment of human health risks related to environmental toxicant exposure is composed of many factors that affect susceptibility, such as different life stages. As advances in medicine and human health care have extended life expectancy, the number of aged Americans has increased. This aged group represents a potentially susceptible demographic one which is not customarily included in toxicity assessments. In 2010, 13% of the population was ≥65 years of age, with a projected increase to 20.6% by 2030 (Statistics, 2016). Numerous animal models have been established to understand the aging process (Nandon, 2007) and its role in the toxicity of environmental toxicants (Elder et al., 2000; LeMoine et al., 2006; Snow et al., 2016). Environmental contaminants, including air pollution, may play a role in this process by inequitably affecting the aged population compared to the younger (Peterson and Calderon, 2003; Park et al., 2005).

Ozone (O3) is a major component of smog and is a ubiquitous air pollutant. O3 has been shown to have detrimental effects on pulmonary and central nervous system (CNS) function. Mumaw et al. (2016) showed activated microglia and B-amyloid toxicity in the CNS following O3 exposure, concomitant with a lung immune response that was not associated with pulmonary spillover of cytokines, but of macrophage-1 antigen mediated microglia proinflammatory priming leading to neurotoxicity (Mumaw et al., 2016). This suggests an existing lung-brain axis and gives insight as to how air pollution may not just have major effects on lung physiology, but on the CNS physiology as well.

Oxidative stress (OS) is an imbalance in the production of reactive oxygen species (ROS), predominantly by cellular biological processes, and the biological systems ability to abate said increases, usually by the production of antioxidants and initiation of various repair mechanisms (Samet and Wages, 2018). Since the 1950s, OS has been a major focus in aging research (Harman, 2009; Son and Lee, 2019). The biological consequences imparted by OS include direct damage of intracellular macromolecules such as proteins, lipids and DNA, and the activation of inflammatory processes along with cytokine production that can lead to further release of ROS (Kodavanti, 1999), culminating in a general loss of cellular function. Unsurprisingly, the activation of OS generating processes have been documented in adverse outcome pathways (AOP) for various environmental toxicants (Bondy, 1994; Kodavanti, 1999; Wei et al., 2020). We hypothesize that the addition of any chemical challenge (i.e. O3) to a potentially already compromised aging population could exacerbate the toxicity of that environmental chemical.

Exposure to O3 has been shown to activate regions of the brain that regulate stress (Gackiere et al., 2011) and induce changes in OS markers (Valdez et al., 2018). However, little is known about age-related sensitivities of the brain to O3. Studies in rats have shown that O3 exposure at older ages can have a negative impact on physiological measures such as temperature regulation, heart rate, pulmonary function and metabolic homeostasis (Bass et al., 2013; Gordon et al., 2013; Snow et al., 2016). Ground-level O3 concentrations can reach to 0.2–0.3 ppm in hot tropical climate combined with elevated levels of volatile organic compounds (U.S. EPA, 2020). O3 concentration of 0.25 ppm was chosen to represent such scenarios, whereas the high concentration of 1.0 ppm O3 was chosen to produce a consistent and measurable pulmonary injury response (Gordon et al., 2013). To represent the episodic pattern of exposure, we chose to model our exposures 2 days per week for 13 consecutive weeks. We proposed that O3 exposure on 2 consecutive days/week for multiple weeks could lead to a better understanding of the susceptibility in older animals. To understand the age-dependent effects of O3, this study employed one of the most common models in aging research, the Brown Norway rat (Lipman et al., 1996). We measured oxy-radical production, antioxidant capacity, and oxidative damage in frontal cortex (FC), striatum (STR), hippocampus (HIP), and cerebellum (CB) at three different life stages (young [4 months], middle-age [12 months], and senescent [24 months] rats). In addition, motor activity was assessed to determine functional consequences OS effects on brain regions such as FC or CB with age and O3 exposure. We postulated high metabolic levels of ROS generation and reduction due to the inherently high biological activity of brain tissue (Yu et al., 2018). The STR, and HIP also play important roles in cognition, the loss of which is often associated with aging (Nicolle et al., 2001; Droge and Schipper, 2007). There were two objectives for this study, firstly we sought to examine O3-induced increases in brain OS markers to elucidate the potential adverse outcome pathway and secondly to examine this phenomenon across different age groups in order to understand the sensitivity of life-stage on environmental chemical-induced adverse effects on the nervous system.

2. Materials and methods

2.1. Animals and O3 exposure

Male Brown Norway rats (4-month [adult], 12-month [middle-age], and 24-month [senescent] old) were obtained from Charles River Laboratories (Kingston, NY, or Partage, MI). The 24-month group originated from purchased retired breeders and were allowed to grow older in the USEPA animal facility in Research Triangle Park, NC. All three ages of rats were acclimated for ≥ two weeks in the animal facility (AAALAC approved) before O3 exposure and testing. All animals received standard Purina 5001 rat chow (Brentwood, MO) and water ad libitum. Animals were housed individually in polycarbonate cages with wood shaving bedding at an ambient temperature of 21–23 °C, relative humidity of 50–55%, and under a 12:12 L:D photoperiod (lights on at 6 AM). All animal protocols were approved by the USEPA, HEALTH Institutional Animal Care and Use Committee (IACUC) before initiation of the study.

The paradigm for generation and exposure to O3 has been previously described (Gordon et al., 2016). Concisely, a silent arc discharge generator (OREC, Phenix, AZ) was used to generate O3 from oxygen, and its entry into the Rochester style Hinner chambers was controlled by mass flow controllers. Monitoring of O3 concentration within the chambers was accomplished via photometric O3 analyzers (API Model 400, Teledyne, San Diego, CA). Within the chamber, stainless steel wire exposure cages (27.3 cm long X 14.6 cm width x 7.75 cm height) were used to individually house rats during exposure. The air temperature and relative humidity inside the four chambers (two control and two O3 chambers) was monitored hourly. All the air entering the control and O3 chambers was filtered with HEPA 0.3-μm filters rated at 99.97% efficient. Animals were exposed to air or O3 (0.25 and 1 ppm) for 6 h/day, 2 days/week (Tuesday and Wednesday) for 13 consecutive weeks (Fig. 1).

Fig. 1. Episodic Ozone (O3) exposure paradigm and experimental plan.

Fig. 1.

Open in a new tab

Male Brown Norway rats at ages 4-, 12-, and 24-months old were exposed to ozone (O3) at 0 (air), 0.25, and 1 ppm for 6 h per day, two days per week for 13 weeks. Horizontal Motor activity was measured at 1, 4, 8, and 12 weeks after exposure while all oxidative stress parameters were measured in selected brain regions at 24 h following 13 weeks of exposure.

2.2. Motor activity

24 h after the two-day O3 exposure (see Fig. 1), neurobehavioral effects were assessed by monitoring motor activity. Data was collected using six photocell devices (Motron Electronic Motility Meter, Stockholm, Sweden) at weeks 1, 4, 8, and 12. 40 photodetectors, arranged on each platform in a 5 × 8 matrix and illuminated by a single overhead incandescent (30 W) bulb. Horizontal activity counts were defined as any movement that triggered a photodetector. To measure vertical activity a bank of six infrared LEDs and detectors were oriented in a horizontal plane above the platform. The platform, which sat on a scissor elevator, was adjusted based on the rearing height of the animal being tested.

A Plexiglas chamber (33x21x26 cm) with a removable lid that had holes for ventilation was placed over the platform to contain the rat. Each device was housed in a larger light and sound attenuating ventilated chamber in a separate room dedicated to this testing. The six boxes were stacked at two high and three across. Data were recorded using a Windows PC programmed with MED-PC software and hardware (Med Associates, St. Albans VT). Baseline activity levels were determined during a single 30 min session of 5 intervals at 6 min each. This baseline testing was done for each squad (contain all three age groups) 1 week prior to the first O3 exposure. The scheduled testing was 1 day after two day/week O3 exposure. The three squads of animals consisted of adult (4-month), middle-age (12-month), and senescent (old age, 24 months) with all three O3 exposure levels. Each squad had a total number of 26 to 27 rats. There were 5 runs per squad. Each run in the squad was balanced and contained no more than one of each age/ O3 level to assure all ages/exposure levels rotated through the six test boxes and runs. Each squad was tested thirteen times during the study ending with the last data collection one week before the end of exposure and necropsy.

2.3. Necropsy and tissue isolation

Approximately 18 h (the next day) following the final O3 exposure, rats were euthanized with an overdose of sodium pentobarbital (Virbac AH, Inc., Fort Worth, TX; 50–100 mg/kg, i.p.) and decapitated. Brains were quickly harvested and brain regions (frontal cortex, cerebellum, hippocampus, and striatum) were dissected on ice (Glowinski and Iversen, 1966), then quick frozen on dry ice, and stored at −80 °C until analyzed.

2.4. Tissue preparation for OS measures

The brain region samples were homogenized with a polytron in 20 mM cold Tris-HCl buffer (pH 7.4) at 50 mg/ml, and centrifuged at 8000 g for 20 min at 4 °C. All assays were modified and adapted for use on the KONLAB clinical chemistry analyzer (Thermo Clinical Lab Systems, Espoo, Finland) with the exception of Coomassie Plus Protein Assay Kit (Pierce, Rockford, IL) and BSA standards from Sigma Chemical Co. (St. Louis, MO) used to determine protein concentration.

2.5. Markers of ROS production

To evaluate ROS production, the activities of NAD(P)H:quinone oxidoreductase (NQO1) and NADH-Ubiquinone reductase (UBIQ-RD) were measured. NQO1 activity was calculated from the difference in reaction rates of the NADH and menadione-dependent dicumarol-inhibitable reduction of cytochrome C obtained with and without dicumarol. An extinction coefficient of 18.5 mM-1 cm-1 was used in calculations of specific activity (Bello et al., 2001; Lind et al., 1990).

The UBIQ-RD activity was assayed by monitoring the oxidation of NADH + H+, with the ultimate reduction of ubiquinone to ubiquinol (Cormier et al., 2001). The rate of UBIQ-RD activity was measured as rotenone-sensitive rate of NADH oxidation at 37 °C and 340 nm.

2.6. Markers of cellular antioxidant homeostasis

Total antioxidant status was measured with a kit from RANDOX Laboratories (Crumlin, Co. Antrim, UK). Briefly, ABTS® (2,2′-Azino-di-[3-ethylbenzthiazoline sulphonate]) was incubated with both a peroxidase (metmyoglobin) and H2O2 to produce the free radical cation ABTS®+, which yields a stable blue-green colour (absorbance at 600 nm). In proportion to their concentration, the amount of antioxidants in the sample suppresses the formation of this colour (Miller et al., 1993). Superoxide dismutase (SOD) activity, which catalyzes the reaction of superoxide radicals to oxygen and hydrogen peroxide, was measured using a kit, RANSOD (Randox Laboratories, Oceanside, CA). Xanthine and xanthine oxidase are used to form superoxide radicals, which react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium (INT) to form a red dye. SOD activity was determined by the degree to which this reaction was inhibited. γ-Glutamylcysteine synthetase (γ-GCS) activity was determined from the rate of ADP formation and calculated from the change in absorbance at 340 nm (Seelig and Meister, 1984).

2.7. Markers of oxidative damage

Both aconitase activity and protein carbonyl formation were used to assess oxidative damage. Total aconitase activity was assayed using commercial kits (OXIS International Inc. Portland, OR) based on the formation of NADPH from NADP+. Aconitase catalyzes the conversion of citrate to isocitrate, followed by oxidative decarboxylation catalyzed by isocitrate dehydrogenase and becomes α-ketoglutarate. NADP+ is reduced to NADPH during the last step of this reaction, which consequently spectrophotometrically at 340 nm absorbance and is proportional to aconitase activity.

Protein carbonyls were assayed using commercial kits from Cayman Chemical Company (Ann Arbor, MI). The amount of protein-hydrozone produced is quantified spectrophotometrically at an absorbance between 360 and 385 nm. following the reaction of 2,4,-dinitrophenylhy-drazine (DNPH).

2.8. Statistical analysis

The values represented in all figures are mean ± SE of 6 rats. In most measures, the data were analyzed by two-way ANOVA with dose and age as factors, using SigmaStat software, version 3.5 (SigmaStat Software, Inc., Point Richmond, CA). Horizontal motor activity data was analyzed within each age group with week of O3exposure and the dose as two factors. In the case of a significant main effect or interaction, step-down ANOVAs were used to test for main effects of age or O3. Pair-wise comparisons between groups were made using the Fisher’s LSD test. The accepted level of significance was p ≤ 0.05.

3. Results

3.1. Effects of ozone and aging on motor activity

In order to understand O3 effects on the neurobehavior of rats, we measured motor activity (horizontal and Vertical) in 4-, 12- and 24- month old rats. In the absence of O3 exposure, baseline horizontal motor activity was low in 4-month old rats during week 1 compared to 12- and 24-month old rats (Fig. 2bar graphs). However, horizontal motor activity increased over time of air exposure and low dose O3 in the 4-month-old rats while such an increase was not observed in 12 or 24-month-old rats (Fig. 2). Analysis of horizontal motor activity data revealed that there was no significant interaction of O3 exposure and week of exposure in any of the age groups, however there was a significant effect of O3 concentration (F2,108 = 8.063; p = 0.001) only in 4 month old rats. Following O3 exposure, there was no significant difference in horizontal motor activity in any of the age groups up to 4 weeks of exposure (2 days/week). Horizontal motor activity was not affected by low concentration O3 (0.25 ppm) exposure in any age group. However, high concentration O3 (1 ppm) exposure resulted in a significant decrease in horizontal motor activity in 4-month old rats, but not in 12- or 24-month old rats, indicating an increased susceptibility toward O3 mediated morbidities in younger animals (Fig. 2). There were no discernable differences across age, week nor O3 exposure in the vertical activity (data not shown).

Fig. 2. Effects of subchronic Ozone exposure on Horizontal Motor Activity of 4-, 12-, and 24-month-old Brown Norway rats.

Fig. 2.

Open in a new tab

Motor activity was measured at 1, 4, 8, and 12 weeks after ozone exposure (doses shown in inserts). Each value is a mean ± SE of 6 rats. *Significantly different from 0 ppm Ozone within that age group. $Significantly different from 4 months within that treatment. @Significantly different from 24 months within that treatment.

3.2. Effects of ozone and aging on the production of reactive oxygen species

There was no significant interaction of age and O3 exposure in any of the brain regions studied as indicated by analysis of NQO1 data. However, there was a significant age effect in the STR (F2,44 = 7.042; p = 0.002), FC (F2,44 = 3.699; p = 0.033), and HIP (F2,44 = 5.191; p = 0.009). Step-down multiple comparisons indicated that NQO1 activity of FC in 12 month old rats was significantly different from 4 (p = 0.016) and 24 (p = 0.038) month old rats while in STR, 4 month old are significantly different from 12 (p = 0.017) and 24 (p = 0.001) month old rats. In the HIP, NQO1 activity was significantly different only between 4 and 12 (p = 0.003) month old rats (Fig. 3).

Fig. 3. Effects of subchronic Ozone exposure on NAD(P)H:Quinone oxidoreductase (NQO1) activity in striatum, hippocampus, frontal cortex and cerebellum of 4, 12, and 24-month-old Brown Norway rats.

Fig. 3.

Open in a new tab

Each value is a mean ± SE of 6 rats. *Significantly different from 0 ppm Ozone within that age group. $ Significantly different from 4 months within that treatment. @Significantly different from 24 months within that treatment. Refer to Table 1 for baseline values of NQO1.

UBIQ-RD data indicated a significant interaction of age and O3 exposure only in the CB (F4, 44 = 10.603; p ≤0.001) and HIP (F4, 44 = 18.883; p ≤0.001). The step-down analyses indicated that O3 decreased UBIQ-RD activity of CB in 4 (0 to 0.25 ppm, p = 0.006; 0 to 1 ppm, p ≤0.001) while there was as significant increase in 24 month old rats (p < 0.001) (Fig. 4). In the HIP, O3 decreased UBIQ-RD activity in 12-month-old rats (p ≤0.001) while increasing in 24-month-old rats (p = 0.004). While there is no significant interaction of O3 and age in the STR or FC, there was a significant effect of age (F2, 44 = 10.971; p ≤0.001) in the STR and FC (F2, 44 = 31.086; p < 0.001).

Fig. 4. Effects of subchronic Ozone exposure on NADH Ubiquinone reductase activity (UBIQ-RD) in striatum, hippocampus, frontal cortex and cerebellum of 4, 12, and 24-month-old Brown Norway rats.

Fig. 4.

Open in a new tab

Each value is a mean ± SE of 6 rats. *Significantly different from control within that age group. $Significantly different from 4 months within that treatment. @Significantly different from 24 months within that treatment. Refer to Table 1 for baseline values of UBIQ-RD.

3.3. Effects of ozone and aging on antioxidant homeostasis

Analysis of TAS indicated a significant interaction of age and O3 exposure in the FC (F4,44 = 2.990; p < 0.029), CB (F4,44 = 3.115; p = 0.024), and HIP (F4,44 = 8.761; p < 0.001). The step-down analyses indicated a significant decrease (p = 0.002) in TAS following 1 ppm O3 exposure in the FC in 24-month-old rats relative to age-matched controls. In the CB, O3 at 1 ppm increased (p ≤0.001) TAS levels only in 4-month-old rats. In the HIP, O3 at 0.25 ppm decreased TAS levels in 12-month-old rats (p < 0.001), however levels were increased (p < 0.005) in 24-month-old rats (Fig. 5).

Fig. 5. Effects of subchronic Ozone exposure on Total Antioxidant substances (TAS) in striatum, hippocampus, frontal cortex and cerebellum of 4, 12, and 24-month-old Brown Norway rats.

Fig. 5.

Open in a new tab

Each value is a mean ± SE of 6 rats. *Significantly different from control within that age group. $Significantly different from 4 months within that treatment. @Significantly different from 24 months within that treatment. Refer to Table 1 for baseline values of TAS.

Analysis of SOD activity revealed a significant interaction of age and O3 exposure in the HIP (F4,44 = 7.945; p < 0.001) only. The step-down analysis showed a significant decrease (p ≤0.001) at 0.25 ppm O3 in SOD activity in 12-month-old rats. In the CB, there was a significant age effect (F2,44 = 6.585; p = 0.003) where 4 and 12 months are different from 24-month-old rats (Fig. 6).

Fig. 6. Effects of subchronic Ozone exposure on Superoxide Dismutase (SOD) activity in striatum, hippocampus, frontal cortex and cerebellum of 4, 12, and 24-month-old Brown Norway rats.

Fig. 6.

Open in a new tab

Each value is a mean ± SE of 6 rats. *Significantly different from control within that age group. $Significantly different from 4 months within that treatment. @Significantly different from 24 months within that treatment. Refer to Table 1 for baseline values of SOD.

With regard to O3 exposure on glutathione (GSH) metabolism, the 2-way ANOVA on γ-GCS indicated a significant interaction of age and O3 exposure in the CB (F4,44 = 3.976; p = 0.008) where O3 decreased γ-GCS in 12 month old rats (p = 0.018) while increasing (p = 0.037) in 24 month old rats (Fig. 7). In the STR, there was a significant O3 effect (F2,44 = 5.056; p = 0.011) with increases in 12-month-old rats at 1 ppm and at both concentrations in 24-month-old rats (Fig. 7).

Fig. 7. Effects of subchronic Ozone exposure on γ-Glutamylcysteine synthetase (γ-GCS) in striatum, hippocampus, frontal cortex and cerebellum of 4, 12, and 24-month-old Brown Norway rats.

Fig. 7.

Open in a new tab

Each value is a mean ± SE of 6 rats. *Significantly different from control within that age group. $Significantly different from 4 months within that treatment. @Significantly different from 24 months within that treatment. Refer to Table 1 for baseline values of γ-GCS.

3.4. Effects of ozone and aging on oxidative damage

Total aconitase activity in STR exhibited an increase while decreased in the HIP with increasing age. (Fig. 8). The two-way ANOVA indicated a significant interaction (F4,44 = 38.846; p < 0.001) of age and O3 exposure on the total aconitase activity of the HIP, and main effects of age in the FC (F2,44 = 3.372; p = 0.043) and CB (F2,44 = 6.300; p = 0.004) and O3 exposure on total aconitase activity of STR (F2,44 = 7.505; p = 0.002) and FC (F2,44 = 3.404; p = 0.020). In the FC, O3 exposure at 1 ppm significantly increased total aconitase activity in the 4 (p = 0.017) and 12 (p = 0.043) month old rats. A similar increase was also seen in the STR following O3 exposure at both concentrations in 4-month-old rats (p = 0.005 and p = 012, respectively) and an increase only at 0.25 ppm concentration in 12-month-old rats (p = 0.028). No significant effect of O3 was evident on aconitase activity in the cerebellum (Fig. 8).

Fig. 8. Effects of subchronic Ozone exposure on Total Aconitase activity in striatum, hippocampus, frontal cortex and cerebellum of 4, 12, and 24-month-old Brown Norway rats.

Fig. 8.

Open in a new tab

Each value is a mean ± SE of 6 rats. *Significantly different from control within that age group. $Significantly different from 4 months within that treatment. @Significantly different from 24 months within that treatment. Refer to Table 1 for baseline values for total aconitase.

Hydrogen peroxide (H2O2) was used as a positive control in the assessment of protein carbonyl levels. H2O2 has been shown to increase protein carbonyls in cortical tissue in vitro in a concentration-dependent manner (data not shown and published previously; (Kodavanti et al., 2011)). Unfortunately, due to a lack of tissue, we were unable to analyze protein carbonyls in the STR and HIP. In FC and CB, there was an increase of protein carbonyls with age, though no interaction was found. Two-way ANOVA indicated a significant main effect of age (F2,44 = 10.812; p < 0.001) in the FC. Similar effects of age (F2,44 = 5.360; p = 0.008) and O3 exposure (F2,44 = 5.563; p = 0.007) were obtained in the CB. Although O3 exposure increased protein carbonyls in both brain regions and in all age groups, step-down analyses indicated O3 effects were statistically significant in 4-month-old rats at both concentrations and only at 1 ppm in 24-month-old rats (Fig. 9).

Fig. 9. Effects of subchronic Ozone exposure on Protein Carbonyl content in the frontal cortex and cerebellum of 4, 12, and 24-month-old Brown Norway rats.

Fig. 9.

Open in a new tab

Each value is a mean ± SE of 6 rats. *Significantly different from control within that age group. $Significantly different from 4 months within that treatment. @Significantly different from 24 months within that treatment.

4. Discussion

Findings from the current study showed that aging significantly altered several OS parameters and subchronic O3 exposure enhanced these changes resulting in increased cellular damage as suggested by increased protein carbonyl content (protein damage) and changes in aconitase activity. These effects were significant only in youngest (4-month-old) rats, but not in older (12 /24-month-old) rats. Concomitant with these findings, we observed that O3 produced dose-related decreases in motor activity (% decrease over respective controls), which again were only significant in 4 month old rats but not in 12 or 24 month old rats, demonstrating an association between oxidative stress and altered nervous system function in adult rats. Previous studies have shown that O3 exposure reduced spontaneous motor activity in a concentration-related manner in mice (Murphy et al., 1964; Dorado-Martinez et al., 2001) and 6-month old rats (Konigsberg and Bachman, 1970; Weiss et al., 1981). In mice, O3 has been reported to increase escape behavior indicating its aversive properties (Tepper et al., 1985). O3 has been shown to cause oxidative stress in the lung and other organs such as kidney and liver in Wistar rats. The effect of O3 was ameliorated in aged (14 months) rats compared to adult (3 months) rats as indicated by increased lipid peroxidation, protein carbonyls, and lipofuscin with decreased levels of GSH (Safwat et al., 2014). In addition, O3 exposure increased circulating levels of corticosterone along with increases in glucocorticoid-inducible genes such as GILZ (glucocorticoid-inducible leucine zipper) and SDK (glucocorticoid-inducible kinase) in several organs including cerebral hemispheres suggesting the activation of hypothalamic pituitary axis (HPA) pathway (Bass et al., 2013; Thomson et al., 2013; Henriquez et al., 2019).

Alterations in the various OS parameters measured in this study across different brain regions illustrate that life stage alone potentially affects the capacity of brain to respond to OS. Our data show (see Figs. 3 and 4) that markers of reactive oxygen species (ROS) production, in general, increase with age of the animal while the degree and timing of increase seems to be brain region specific. Previous reports indicate similar regional differences in the brains of 7 week old Sprague-Dawley male and female rats (Stakhiv et al., 2006). Initially, we examined the status of ROS production. NQO1 (EC 1.6.99.2; DT-diaphorase) is a cytosolic flavoenzyme with antioxidant properties that is ubiquitously present in the tissues of nearly all animal species (Jaiswal, 2000; Siegel and Ross, 2000). In the brain, NQO1 is highly expressed in astrocytes and endothelial cells (van Muiswinkel et al., 2004). NQO1 catalyzes the reduction of quinones thus precluding their contribution in redox cycling (Cadenas, 1995; Siegel et al., 2004). OS activation of the antioxidant response element (ARE) at the 5′ region of the NQO1 gene (Jaiswal, 1994; Dhakshinamoorthy and Jaiswal, 2000) mediates its expression. NQO1 expression and activity play a role in neurodegenerative diseases such as Alzheimer’s disease (Raina et al., 1999), Parkinson’s disease (van Muiswinkel et al., 2004), and multiple sclerosis (van Horssen et al., 2006) and have recently been implicated in the regulation of psychomotor behaviors common in various psychiatric disorders (Go et al., 2020). In the current study, NQO1 activity significantly increased in the STR of aged rats compared to young adults suggesting a higher rate of ROS production in aged rats. O3 exposure further increased NQO1 activity in the FC and STR of 12-month-old rats suggesting increased ROS production by subchronic O3. O3 exposure also increased NQO1 activity in the STR of 24-month-old rats. Consistent with these results, Ni et al. (2010) reported methylmercury induced increases in NQO1 mRNA levels in primary microglial cells while Liu et al. (2010) reported similar increases in liver NQO1 expression subsequent to exposure to 1-bromopropane in rats. Results from this study suggest that ROS production is greater in some brain regions of aged rats and that subchronic O3 exposure exacerbates this effect.

UBIQ-RD, often referred to as mitochondrial complex I enzyme, couples the oxidation of NADH with the reduction of ubiquinone to generate and maintain the proton gradient utilized during ATP synthesis (Weiss et al., 1991). This enzyme is perhaps the most susceptible component of the electron transport chain to impairment by ROS and its dysfunction has been suggested as one of the major causes of aging (Balaban et al., 2005). UBIQ-RD has long been implicated in various neurodegenerative disorders (Schapira, 1998) and is now a novel therapeutic target for such disorders (Hyun, 2019). In select brain regions (FC and STR) of 24-month-old rats, UBIQ-RD activity was higher compared to 4 and 12-month-old rats implying higher levels of ROS production in old rats. O3 exposure increased UBIQ-RD in all brain regions, especially in STR and CB of 24-month-old rats at 1 ppm exposure. In accordance with NQO1 results, data on UBIQ-RD are suggestive of greater ROS production is select brain regions of aged rats which is further heightened after O3 exposure. In agreement with an O3-induced increase in ROS production, literature reports indicate that O3 exposure caused a significant decrease in motor activity and also produced lipid peroxidation, morphological alterations, loss of fibers, and cell death of the dopaminergic neurons (Rivas-Arancibia et al., 2003; Pereyra-Munoz et al., 2006). These changes were also associated with increases in DARPP-32, iNOS, and SOD expression following repetitive O3 exposure at 0.25 ppm for 15 days and 30 days (Pereyra-Munoz et al., 2006).

Antioxidants are cellular defense molecules to offset the production of ROS. Organisms have several ROS defense systems, each having its varied efficacy and deficiencies across different organs. One of the key cellular defense systems against OS is the glutathione (GSH) pathway which participate in the removal of oxidized proteins, redox recycling of peroxides, and removal of xenobiotics via conjugation. GSH is a ubiquitous tripeptide present in relatively high concentrations (0.1 to 10 mM) in mammalian tissues (Kodavanti, 1999). In this study, we measured total antioxidant substances (TAS) as well as activities of γ-glutamylcysteine synthetase (γ-GCS), the rate limiting enzyme in the synthesis of GSH and superoxide dismutase (SOD), which breakdown superoxide radicals (Kodavanti, 1999).

γ-GCS activity decreased slightly with age in the STR and CB (primarily from 4 to 24 months), demonstrating an age-related decrease in GSH synthetic capacity in the brain (Fig. 7). Consistent with this finding, comparable age-related decreases in γ-GCS have been reported in the STR, cortex, midbrain and CB of Sprague-Dawley rats between 4 and 17 months of age (Zhu et al., 2006). These types of decreases in antioxidant defense capacities could represent an age-related reduction in detoxication capability in these key functional brain regions. Although levels of γ-GCS decreased in some brain regions, corresponding levels of TAS and SOD were not altered much with age (Fig. 5-6). In the current study, O3 exposure significantly decreased TAS levels in the FC while increasing them in the HIP and CB in 24-month-old rats. This increasing trend was also seen with γ-GCS in the STR and CB in both 12 and 24-month-old rats suggesting differential effects of O3 with age. Collectively, these findings imply that brains of older animals have less antioxidant defense capabilities as corroborated in the literature (Sandhu and Kaur, 2002), and that some antioxidant systems may be stimulated following exposure to O3 as the constitutive levels are low in aged rats compared to adult rats.

An imbalance in the production of ROS and cellular antioxidant defense mechanisms result in oxidative damage (Stefanatos and Sanz, 2018). We measured both total aconitase and protein carbonyls as indicators of oxidative damage in the cellular components. While there exist two types of aconitase, mitochondrial aconitase makes up approximately 80% of total aconitase activity in the cell (Meyron-Holtz et al., 2004). This type of aconitase is susceptible to ROS, thus decreased mitochondrial aconitase activity is a good indicator of oxidative stress (Yan et al., 1997; Cantu et al., 2009) and correlates with the destabilization of mitochondrial DNA (Chen et al., 2005; Shadel, 2005). The second type of aconitase, cytosolic aconitase, exists in two forms with different physiological functions. The holo [4Fe-4S] form has aconitase activity and the iron free apo form known as iron regulatory protein (IRP-1) that mediates the expression of several proteins important in regulation of iron metabolism via the binding iron-responsive elements (IRE’s). Furthermore, it has been demonstrated that aconitase plays a key role in OS-mediated cell death and neurodegeneration (Fariss et al., 2005).

Our data demonstrate how total aconitase levels decrease with age within the HIP (4 to 12 to 24 months) and CB (primarily 4 to 24 months) but are only slightly altered with age in the STR and FC. Interestingly, total aconitase activity increased in the STR and FC while it decreasing in the HIP and CB subsequent to O3 exposure. The increased aconitase activity could be attributed to changes in iron homeostasis imparted by aging and O3. In a related study, cardiac cytosolic aconitase activity increased with age and was accompanied by increases in ferritin protein levels (Gordon et al., 2009) suggesting an Fe overload in aged rats. This outcome has also been observed in aging humans (Saito et al., 2003). Moreover, O3 exposure has been associated with a disruption of Fe homeostasis and the resulting increased availability of Fe potentially contributes to oxidative stress and altered pulmonary function (Ghio et al., 2014).

As a second measure of oxidative stress-induced cellular damage, protein carbonyls were measured. Primarily formed by the metal-catalyzed oxidation of lysine, arginine, proline or threonine protein residues (Friguet et al., 2000), protein carbonyls can also arise from sugar glycation reactions and conjugation with aldehydes that are produced during lipid peroxidation (Stadtman, 1992; Shringarpure and Davies, 2002). We found that protein carbonyl levels in both FC and CB increased approximately 2-fold with age from 4 to 24-month-old rats. These results are consistent with reports from other species, including mice (Dubey et al., 1996; Abdul et al., 2008), gerbils (Stadtman, 1992), D. melanogaster (Das et al., 2001), houseflies (Sohal et al., 1993; Agarwal and Sohal, 1994) and humans (Smith et al., 1991; Stadtman, 1992) as well as rats (Agaiwal and Sohal, 1996; Aksenova et al., 1998; Navarro and Boveris, 2004). A number of factors may contribute to the accumulation of protein carbonyls with age (Levine, 2002) such as increased level of oxidizing species, decreased activity of ROS scavenging enzymes, increased protein transcriptional or translational errors leading to greater susceptibility (Dukan et al., 2000) or a decrease in the removal of damaged proteins. Typically a 2-step process is involved in the removal of oxidized proteins involving ubiquitination and then degradation (Vernace et al., 2007) by the 26S proteasome complex which is susceptible to oxidation and demonstrates age-related loss of activity (Keller et al., 2000; Sitte et al., 2000; Vernace et al., 2007). Therefore, age-related loss of proteolytic capacity could contribute to the accumulation of damaged proteins in the aging brain. Protein carbonyls in both FC and CB, in all age groups, were increased by O3 but statistical analyses indicated that subchronic O3 effects were only significant in 4 month old rats in both brain regions at 1 ppm and in the CB of 24 month old rats. These results suggest that younger (4-month-old) rats are more susceptible to changes in OS parameters caused by O3 exposure.

5. Conclusions

Current results highlight that some oxidative stress parameters are altered significantly in brain tissue, and these effects were further enhanced by exposure to O3. The effect of age appears to have a greater efficacy when compared to subchronic O3 exposure. Both mitochondrial (UBIQ-RD) and cytosolic (NQO1) markers for ROS production were impacted, and antioxidant homeostasis mechanisms were compromised which lead to oxidative damage. The effects of O3 exposure only on protein carbonyl in 4-month-old rats suggests that O3 effects are age-dependent. Moreover, horizontal motor activity decreased following O3 exposure only in 4-month-old rats signifying a clear role for oxidative stress in O3-induced motor deficits.

Table 1.

Baseline enzymatic activity values in different brain regions for 0 ppm controls in young (4-month old), middle-age (12-month old) and senescent (24-month old) rats.

ACONITASE
mU/mg		 UBIQ-RD
mU/mg		 gGCS
uM/mg		 NQO1
nM/mg		 SOD
U/mg		 TAS
umol/mg
Age (M)
Striatum 4 1.186 ± 0.083 17.525 ± 1.009 0.043 ± 0.003 327.065 ± 22.36 8.372 ± 0.543 0.213 ± 0.015
12 1.442 ± 0.055 23.796 ± 1.504 0.04 ± 0.003 362.532 ± 8.034 7.158 ± 0.207 0.212 ± 0.011
24 1.395 ± 0.08 24.839 ± 1.253 0.033 ± 0.002 411.717 ± 15.466 7.44 ± 0.179 0.203 ± 0.008
Hippocampus 4 3.596 ± 0.127 27.248 ± 0.775 0.032 ± 0.002 150.871 ± 5.826 10.821 ± 0.376 0.244 ± 0.012
12 3.216 ± 0.11 29.221 ± 0.905 0.029 ± 0.001 134.278 ± 4.055 10.504 ± 0.277 0.24 ± 0.01
24 3.069 ± 0.122 28.508 ± 1.509 0.033 ± 0.002 142.008 ± 7.84 10.429 ± 0.36 0.255 ± 0.007
Frontal Cortex 4 1.471 ± 0.064 25.445 ± 1.133 0.044 ± 0.002 389.985 ± 11.133 6.392 ± 0.184 0.261 ± 0.008
12 1.358 ± 0.085 27.337 ± 1.912 0.045 ± 0.002 413.117 ± 13.31 6.645 ± 0.152 0.254 ± 0.008
24 1.366 ± 0.083 33.116 ± 1.715 0.046 ± 0.001 380.936 ± 25.506 6.819 ± 0.238 0.285 ± 0.012
Cerebellum 4 1.189 ± 0.023 84.412 ± 2.197 0.056 ± 0.003 452.052 ± 10.41 11.946 ± 0.239 0.154 ± 0.007
12 1.074 ± 0.029 75.49 ± 1.242 0.058 ± 0.004 441.156 ± 31.098 12.877 ± 0.655 0.127 ± 0.007
24 0.937 ± 0.063 77.966 ± 2.243 0.051 ± 0.002 453.294 ± 13.137 13.509 ± 0.549 0.147 ± 0.009
Open in a new tab

Acknowledgments

The authors thank Drs. Jan Dye and Cina Mack of PHITD, CPHEA and Dr. Susan Hester at CCTE of USEPA for their helpful comments on an earlier version of this manuscript. The research described in this article has been reviewed by the Center for Public Health and Environmental Assessment, US Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

Funding information

Ms. Datonye I. Agina-Obu is funded by a contract from USEPA (Award # EP-11-D-000477).

Abbreviations:

OS

oxidative stress

O3

ozone

NQO1

NAD(P)H:Quinone oxidoreductase

UBIQ-RD

NADH Ubiquinone reductase

TAS

total antioxidant substances

GSH

glutathione

γ-GCS

γ-glutamylcysteine synthetase

SOD

superoxide dismutase

Footnotes

Disclosures

No conflict of interest, financial or otherwise, are declared by the authors.

Declaration of Competing Interest

All authors have declared no existing conflicts of interest.

References

  1. Abdul HM, Sultana R, St Clair DK, Markesbery WR, Butterfield DA, 2008. Oxidative damage in brain from human mutant APP/PS-1 double knock-in mice as a function of age. Free Radic. Biol. Med 45, 1420–1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Agarwal S, Sohal RS, 1994. Aging and proteolysis of oxidized proteins. Arch. Biochem. Biophys 309, 24–28. [DOI] [PubMed] [Google Scholar]
  3. Agarwal S, Sohal RS, 1996. Relationship between susceptibility to protein oxidation, aging, and maximum life span potential of different species. Exp. Gerontol 31, 365–372. [DOI] [PubMed] [Google Scholar]
  4. Aksenova MV, Aksenov MY, Carney JM, Butterfield DA, 1998. Protein oxidation and enzyme activity decline in old brown Norway rats are reduced by dietary restriction. Mech. Ageing Dev 100, 157–168. [DOI] [PubMed] [Google Scholar]
  5. Balaban RS, Nemoto S, Finkel T, 2005. Mitochondria, oxidants, and aging. Cell 120, 483–495. [DOI] [PubMed] [Google Scholar]
  6. Bass V, Gordon CJ, Jarema KA, MacPhail RC, Cascio WE, Phillips PM, Ledbetter AD, Schladweiler MC, Andrews D, Miller D, Doerfler DL, Kodavanti UP, 2013. Ozone induces glucose intolerance and systemic metabolic effects in young and aged Brown Norway rats. Toxicol. Appl. Pharmacol 273, 551–560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bello RI, Gomez-Diaz C, Navarro F, Alcain FJ, Villalba JM, 2001. Expression of NAD(P)H:quinone oxidoreductase 1 in HeLa cells: role of hydrogen peroxide and growth phase. J Biol Chem 276, 44379–44384. [DOI] [PubMed] [Google Scholar]
  8. Bondy S, 1994. Induction of oxidative stress in the brain by neurotoxic agents In: Chang LW (Ed.), Principals of Neurotoxicology. New York, pp. 563–569. [Google Scholar]
  9. Cadenas E, 1995. Antioxidant and prooxidant functions of DT-diaphorase in quinone metabolism. Biochem. Pharmacol 49, 127–140. [DOI] [PubMed] [Google Scholar]
  10. Cantu D, Schaack J, Patel M, 2009. Oxidative inactivation of mitochondrial aconitase results in iron and H2O2-mediated neurotoxicity in rat primary mesencephalic cultures. PLoS One 4, e7095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chen XJ, Wang X, Kaufman BA, Butow RA, 2005. Aconitase couples metabolic regulation to mitochondrial DNA maintenance. Science 307, 714–717. [DOI] [PubMed] [Google Scholar]
  12. Cormier A, Morin C, Zini R, Tillement J-P, Lagrue G, 2001. In vitro effects of nicotine on mitochondrial respiration and superoxide anion generation. Brain Res. 900, 72–79. [DOI] [PubMed] [Google Scholar]
  13. Das N, Levine RL, Orr WC, Sohal RS, 2001. Selectivity of protein oxidative damage during aging in Drosophila melanogaster. Biochem. J 360, 209–216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dhakshinamoorthy S, Jaiswal AK, 2000. Small maf (MafG and MafK) proteins negatively regulate antioxidant response element-mediated expression and antioxidant induction of the NAD (P) H: Quinone oxidoreductasel gene. J. Biol. Chem 275, 40134–40141. [DOI] [PubMed] [Google Scholar]
  15. Dorado-Martinez C, Paredes-Carbajal C, Mascher D, Borgonio-Perez G, Rivas-Arancibia S, 2001. Effects of different ozone doses on memory, motor activity and lipid peroxidation levels, in rats. Int J Neurosci 108, 149–161. [DOI] [PubMed] [Google Scholar]
  16. Droge W, Schipper HM, 2007. Oxidative stress and aberrant signaling in aging and cognitive decline. Aging Cell 6, 361–370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dubey A, Forster MJ, Lal H, Sohal RS, 1996. Effect of age and caloric intake on protein oxidation in different brain regions and on behavioral functions of the mouse. Arch. Biochem. Biophys 333, 189–197. [DOI] [PubMed] [Google Scholar]
  18. Dukan S, Farewell A, Ballesteros M, Taddei F, Radman M, Nystrom T, 2000. Protein oxidation in response to increased transcriptional or translational errors. Proc. Natl. Acad. Sci. U. S. A 97, 5746–5749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Elder AC, Finkelstein J, Johnston C, Gelein R, Oberdorster G, 2000. Induction of adaptation oo inhaled lipopolysaccharide-E in young and old rats and mice. Inhal. Toxicol 12, 225–243. [DOI] [PubMed] [Google Scholar]
  20. Fariss MW, Chan CB, Patel M, Van Houten B, Orrenius S, 2005. Role of mitochondria in toxic oxidative stress. Mol. Interv 5, 94–111. [DOI] [PubMed] [Google Scholar]
  21. Friguet B, Bulteau AL, Chondrogianni N, Conconi M, Petropoulos I, 2000. Protein degradation by the proteasome and its implications in aging. Ann. N. Y. Acad. Sci 908, 143–154. [DOI] [PubMed] [Google Scholar]
  22. Gackiere F, Saliba L, Baude A, Bosler O, Strube C, 2011. Ozone inhalation activates stress-responsive regions of the CNS. J. Neurochem 117, 961–972. [DOI] [PubMed] [Google Scholar]
  23. Ghio AJ, Soukup JM, Dailey LA, Richards JH, Duncan KE, Lehmann J, 2014. Iron decreases biological effects of ozone exposure. Inhal. Toxicol 26, 391–399. [DOI] [PubMed] [Google Scholar]
  24. Glowinski J, Iversen LL, 1966. Regional studies of catecholamines in the rat brain. I. the disposition of [3H]norepinephrine, [3H]dopamine and [3H]dopa in various regions of the brain. J. Neurochem 13, 655–669. [DOI] [PubMed] [Google Scholar]
  25. Go J, Ryu YK, Park HY, Choi DH, Choi YK, Hwang DY, Lee CH, Kim KS, 2020. NQO1 regulates pharmaeo-behavioral effects of d-amphetamine in striatal dopaminergic system in mice. Neuropharmacology 170, 108039. [DOI] [PubMed] [Google Scholar]
  26. Gordon CJ, Gottipolu RR, Kenyon EM, Thomas R, Schladweiler MC, Mack CM, Shannahan JH, Wallenborn JG, Nyska A, MacPhail RC, Richards JE, Devito M, Kodavanti UP, 2009. Aging and susceptibility to toluene in rats: a pharmacokinetic, biomarker, and physiological approach. J. Toxic. Environ. Health A 73, 301–318. [DOI] [PubMed] [Google Scholar]
  27. Gordon CJ, Jarema KA, Lehmann JR, Ledbetter AD, Schladweiler MC, Schmid JE, Ward WO, Kodavanti UP, Nyska A, MacPhail RC, 2013. Susceptibility of adult and senescent Brown Norway rats to repeated ozone exposure: an assessment of behavior, serum biochemistry and cardiopulmonary function. Inhal. Toxicol 25, 141–159. [DOI] [PubMed] [Google Scholar]
  28. Gordon CJ, Phillips PM, Beasley TE, Ledbetter A, Aydin C, Snow SJ, Kodavanti UP, Johnstone AF, 2016. Pulmonary sensitivity to ozone exposure in sedentary versus chronically trained, female rats. Inhal. Toxicol 28, 293–302. [DOI] [PubMed] [Google Scholar]
  29. Harman D, 2009. Origin and evolution of the free radical theory of aging: a brief personal history, 1954-2009. Biogerontology 10, 773–781. [DOI] [PubMed] [Google Scholar]
  30. Henriquez AR, House JS, Snow SJ, Miller CN, Schladweiler MC, Fisher A, Ren H, Valdez M, Kodavanti PR, Kodavanti UP, 2019. Ozone-induced dysregulation of neuroendocrine axes requires adrenal-derived stress hormones. Toxicol. Sci 172 (1), 38–50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hyun DH, 2019. Plasma membrane redox enzymes: new therapeutic targets for neurodegenerative diseases. Arch. Pharm. Res 42, 436–445. [DOI] [PubMed] [Google Scholar]
  32. Jaiswal AK, 1994. Antioxidant response element. Biochem. Pharmacol 48, 439–444. [DOI] [PubMed] [Google Scholar]
  33. Jaiswal AK, 2000. Regulation of genes encoding NAD(P)H:quinone oxidoreductases. Free Radic. Biol. Med 29, 254–262. [DOI] [PubMed] [Google Scholar]
  34. Keller JN, Huang FF, Markesbery WR, 2000. Decreased levels of proteasome activity and proteasome expression in aging spinal cord. Neuroscience 98, 149–156. [DOI] [PubMed] [Google Scholar]
  35. Kodavanti P, 1999. Reactive oxygen species and antioxidant homeostasis in neurotoxicology Neurotoxicology (Tilson HA, Harry G, eds). Target organ toxicology series. Philadelphia: Taylor and Francis, 157–178. [Google Scholar]
  36. Kodavanti PR, Royland JE, Richards JE, Besas J, Macphail RC, 2011. Toluene effects on oxidative stress in brain regions of young-adult, middle-age, and senescent Brown Norway rats. Toxicol. Appl. Pharmacol 256, 386–398. [DOI] [PubMed] [Google Scholar]
  37. Konigsberg A, Bachman C, 1970. Ozonized atmosphere and gross motor activity of rats. Int. J. Biometeorol 14, 261–266. [DOI] [PubMed] [Google Scholar]
  38. LeMoine CM, McClelland GB, Lyons CN, Mathieu-Costello O, Moyes CD, 2006. Control of mitochondrial gene expression in the aging rat myocardium. Biochem. Cell Biol 84, 191–198. [DOI] [PubMed] [Google Scholar]
  39. Levine RL, 2002. Carbonyl modified proteins in cellular regulation, aging, and disease. Free Radic. Biol. Med 32, 790–796. [DOI] [PubMed] [Google Scholar]
  40. Lind C, Cadenas E, Hochstein P, Ernster L, 1990. [30] DT-diaphorase: purification, properties, and function, methods in enzymology. Elsevier, pp. 287–301. [DOI] [PubMed] [Google Scholar]
  41. Lipman RD, Chrisp CE, Hazzard DG, Bronson RT, 1996. Pathologic characterization of brown Norway, brown Norway x Fischer 344, and Fischer 344 x brown Norway rats with relation to age. J. Gerontol. A Biol. Sci. Med. Sci 51, B54–B59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Liu F, Ichihara S, Valentine WM, Itoh K, Yamamoto M, Sheik Mohideen S, Kitoh J, Ichihara G, 2010. Increased susceptibility of Nrf2-null mice to 1-bromo-propane-induced hepatotoxicity. Toxicol. Sci 115, 596–606. [DOI] [PubMed] [Google Scholar]
  43. Meyron-Holtz EG, Ghosh MC, Iwai K, LaVaute T, Brazzolotto X, Berger UV, Land W, Ollivierre-Wilson H, Grinberg A, Love P, Rouault TA, 2004. Genetic ablations of iron regulatory proteins 1 and 2 reveal why iron regulatory protein 2 dominates iron homeostasis. EMBO J. 23, 386–395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Miller NJ, Rice-Evans C, Davies MJ, Gopinathan V, Milner A, 1993. A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin Sci (Lond) 84, 407–412. [DOI] [PubMed] [Google Scholar]
  45. Mumaw CL, Levesque S, McGraw C, Robertson S, Lucas S, Stafflinger JE, Campen MJ, Hall P, Norenberg JP, Anderson T, Lund AK, McDonald JD, Ottens AK, Block ML, 2016. Microglial priming through the lung-brain axis: the role of air pollution-induced circulating factors. FASEB J. 30, 1880–1891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Murphy SD, Ulrich CE, Frankowitz SH, Xintaras C, 1964. Altered function in animals inhaling low concentrations of ozone and nitrogen dioxide. Am. Ind. Hyg. Assoc. J 25, 246–253. [DOI] [PubMed] [Google Scholar]
  47. Nandon NL, 2007. Animal Models in Gerontology Research. Elsevier Academic Press INC. [DOI] [PubMed] [Google Scholar]
  48. Navarro A, Boveris A, 2004. Rat brain and liver mitochondria develop oxidative stress and lose enzymatic activities on aging. Am J Physiol Regul Integr Comp Physiol 287, R1244–R1249. [DOI] [PubMed] [Google Scholar]
  49. Ni M, Li X, Yin Z, Jiang H, Sidoryk-Wegrzynowicz M, Milatovic D, Cai J, Aschner M, 2010. Methylmercury induces acute oxidative stress, altering Nrf2 protein level in primary microglial cells. Toxicol. Sci 116, 590–603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Nicolle MM, Gonzalez J, Sugaya K, Baskerville KA, Bryan D, Lund K, Gallagher M, McKinney M, 2001. Signatures of hippocampal oxidative stress in aged spatial learning-impaired rodents. Neuroscience 107, 415–431. [DOI] [PubMed] [Google Scholar]
  51. Park SK, O’Neill MS, Vokonas PS, Sparrow D, Schwartz J, 2005. Effects of air pollution on heart rate variability: the VA normative aging study. Environ. Health Perspect 113, 304–309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Pereyra-Munoz N, Rugerio-Vargas C, Angoa-Perez M, Borgonio-Perez G, Rivas-Arancibia S, 2006. Oxidative damage in substantia nigra and striatum of rats chronically exposed to ozone. J. Chem. Neuroanat 31, 114–123. [DOI] [PubMed] [Google Scholar]
  53. Peterson CA, Calderon RL, 2003. Trends in enteric disease as a cause of death in the United States, 1989-1996. Am. J. Epidemiol 157, 58–65. [DOI] [PubMed] [Google Scholar]
  54. Raina AK, Templeton DJ, Deak JC, Perry G, Smith MA, 1999. Quinone reductase (NQO1), a sensitive redox indicator, is increased in Alzheimer’s disease. Redox Rep. 4, 23–27. [DOI] [PubMed] [Google Scholar]
  55. Rivas-Arancibia S, Dorado-Martinez C, Colin-Barenque L, Kendrick KM, de la Riva C, Guevara-Guzman R, 2003. Effect of acute ozone exposure on locomotor behavior and striatal function. Pharmacol. Biochem. Behav 74, 891–900. [DOI] [PubMed] [Google Scholar]
  56. Safwat MH, El-Sawalhi MM, Mausouf MN, Shaheen AA, 2014. Ozone ameliorates age-related oxidative stress changes in rat liver and kidney: effects of pre- and postageing administration. Biochemistry (Mosc) 79, 450–458. [DOI] [PubMed] [Google Scholar]
  57. Saito K, Ishizaka N, Mitani H, Ohno M, Nagai R, 2003. Iron chelation and a free radical scavenger suppress angiotensin II-induced downregulation of klotho, an antiaging gene, in rat. FEBS Lett. 551, 58–62. [DOI] [PubMed] [Google Scholar]
  58. Samet JM, Wages PA, 2018. Oxidative stress from environmental exposures. Curr Opin Toxicol 7, 60–66. [PMC free article] [PubMed] [Google Scholar]
  59. Sandhu SK, Kaur G, 2002. Alterations in oxidative stress scavenger system in aging rat brain and lymphocytes. Biogerontology 3, 161–173. [DOI] [PubMed] [Google Scholar]
  60. Schapira AHV, 1998. Human complex I defects in neurodegenerative diseases. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1364, 261–270. [DOI] [PubMed] [Google Scholar]
  61. Seelig GF, Meister A, 1984. Gamma-glutamylcysteine synthetase. Interactions of an essential sulfhydryl group. J. Biol. Chem 259, 3534–3538. [PubMed] [Google Scholar]
  62. Shadel GS, 2005. Mitochondrial DNA, aconitase ‘wraps’ it up. Trends Biochem. Sci 30, 294–296. [DOI] [PubMed] [Google Scholar]
  63. Shringarpure R, Davies KJA, 2002. Protein turnover by the proteasome in aging and disease. Free Radic. Biol. Med 32, 1084–1089. [DOI] [PubMed] [Google Scholar]
  64. Siegel D, Ross D, 2000. Immunodetection of NAD(P)H:quinone oxidoreductase 1 (NQO1) in human tissues. Free Radic. Biol. Med 29, 246–253. [DOI] [PubMed] [Google Scholar]
  65. Siegel D, Gustafson DL, Dehn DL, Han JY, Boonchoong P, Berliner LJ, Ross D, 2004. NAD(P)H:quinone oxidoreductase 1: role as a superoxide scavenger. Mol. Pharmacol 65, 1238–1247. [DOI] [PubMed] [Google Scholar]
  66. Sitte N, Merker K, Von Zglinicki T, Davies KJ, Grune T, 2000. Protein oxidation and degradation during cellular senescence of human BJ fibroblasts: part II–aging of nondividing cells. FASEB J. 14, 2503–2510. [DOI] [PubMed] [Google Scholar]
  67. Smith CD, Carney JM, Starke-Reed PE, Oliver CN, Stadtman ER, Floyd RA, Markesbery WR, 1991. Excess brain protein oxidation and enzyme dysfunction in normal aging and in Alzheimer disease. Proc. Natl. Acad. Sci. U. S. A 88, 10540–10543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Snow SJ, Gordon CJ, Bass VL, Schladweiler MC, Ledbetter AD, Jarema KA, Phillips PM, Johnstone AF, Kodavanti UP, 2016. Age-related differences in pulmonary effects of acute and subchronic episodic ozone exposures in Brown Norway rats. Inhal. Toxicol 28, 313–323. [DOI] [PubMed] [Google Scholar]
  69. Sohal RS, Agarwal S, Dubey A, Orr WC, 1993. Protein oxidative damage is associated with life expectancy of houseflies. Proc. Natl. Acad. Sci. U. S. A 90, 7255–7259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Son JM, Lee C, 2019. Mitochondria: multifaceted regulators of aging. BMB Rep. 52, 13–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Stadtman ER, 1992. Protein oxidation and aging. Science 257, 1220–1224. [DOI] [PubMed] [Google Scholar]
  72. Stakhiv TM, Mesia-Vela S, Kauffman FC, 2006. Phase II antioxidant enzyme activities in brain of male and female ACI rats treated chronically with estradiol. Brain Res. 1104, 80–91. [DOI] [PubMed] [Google Scholar]
  73. Statistics, 2016. Older Americans 2016: Key Indicators of Well-Being Seventh series of report by Federal Interagency Forum on Aging-related Statistics, Washington, DC. [Google Scholar]
  74. Stefanatos R, Sanz A, 2018. The role of mitochondrial ROS in the aging brain. FEBS Lett. 592, 743–758. [DOI] [PubMed] [Google Scholar]
  75. Tepper JS, Weiss B, Wood RW, 1985. Alterations in behavior produced by inhaled ozone or ammonia. Fundam. Appl. Toxicol 5, 1110–1118. [DOI] [PubMed] [Google Scholar]
  76. Thomson EM, Vladisavljevic D, Mohottalage S, Kumarathasan P, Vincent R, 2013. Mapping acute systemic effects of inhaled particulate matter and ozone: multiorgan gene expression and glucocorticoid activity. Toxicol. Sci 135, 169–181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. U.S. EPA, 2020. Integrated Science Assessment (ISA) for Ozone and Related Photochemical Oxidants (Final Report). U.S. Environmental Protection Agency, Washington, DC, EPA/600/R-20/012. [Google Scholar]
  78. Valdez JM, Johnstone AFM, Richards JE, Schmid JE, Royland JE, Kodavanti PRS, 2018. Interaction of diet and ozone exposure on oxidative stress parameters within specific brain regions of male Brown Norway rats. Int. J. Mol. Sci 20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. van Horssen J, Schreibelt G, Bo L, Montagne L, Drukarch B, van Muiswinkel FL, de Vries HE, 2006. NAD(P)H:quinone oxidoreductase 1 expression in multiple sclerosis lesions. Free Radic. Biol. Med 41, 311–317. [DOI] [PubMed] [Google Scholar]
  80. van Muiswinkel FL, de Vos RA, Bol JG, Andringa G, Jansen Steur EN, Ross D, Siegel D, Drukarch B, 2004. Expression of NAD(P)H:quinone oxidoreductase in the normal and parkinsonian substantia nigra. Neurobiol. Aging 25, 1253–1262. [DOI] [PubMed] [Google Scholar]
  81. Vernace VA, Schmidt-Glenewinkel T, Figueiredo-Pereira ME, 2007. Aging and regulated protein degradation: who has the UPPer hand? Aging Cell 6, 599–606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Wei F, Su T, Wang D, Li H, You J, 2020. Transcriptomic analysis reveals common pathways and biomarkers associated with oxidative damage caused by mitochondrial toxicants in Chironomus dilutus. Chemosphere 254, 126746. [DOI] [PubMed] [Google Scholar]
  83. Weiss B, Ferin J, Merigan W, Stern S, Cox C, 1981. Modification of rat operant behavior by ozone exposure. Toxicol. Appl. Pharmacol 58, 244–251. [DOI] [PubMed] [Google Scholar]
  84. Weiss H, Friedrich T, Hofhaus G, Preis D, 1991. The respiratory-chain NADH dehydrogenase (complex I) of mitochondria, EJB reviews 1991. Springer, pp. 55–68. [DOI] [PubMed] [Google Scholar]
  85. Yan LJ, Levine RL, Sohal RS, 1997. Oxidative damage during aging targets mitochondrial aconitase. Proc. Natl. Acad. Sci. U. S. A 94, 11168–11172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  86. Yu Y, Herman P, Rothman DL, Agarwal D, Hyder F, 2018. Evaluating the gray and white matter energy budgets of human brain function. J. Cereb. Blood Flow Metab 38, 1339–1353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  87. Zhu Y, Carvey PM, Ling Z, 2006. Age-related changes in glutathione and glutathione-related enzymes in rat brain. Brain Res. 1090, 35–44. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES