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Citric Acid Protects Dopaminergic Cells against Rotenone-Induced Neurodegeneration

Omar M.E. Abdel-Salam1, Safaa M. Youssef Morsy2, Eman R. Youness2, Noha N. Yassen3, Nermeen Shaffie3, and Amany A. Sleem4 

1Department of Toxicology and Narcotics, National Research Centre, Cairo, Egypt; 2Department of Medical Biochemistry, National Research Centre, Cairo, Egypt; 3Department of Pathology, National Research Centre, Cairo, Egypt; 4Department of Pharmacology, National Research Centre, Cairo, Egypt 

Correspondence: omasalam@hotmail.com (O.M.A-S.) 

Abdel-Salam OM et al. Reactive Oxygen Species 9(27):118–135, 2020; ©2020 Cell Med Press

http://dx.doi.org/10.20455/ros.2020.819

(Received: January 7, 2020; Revised: February 2, 2020; Accepted: February 4, 2020) 

ABSTRACT | We investigated the effect of citric acid on rotenone-induced oxidative stress and neuro- and hepato-toxicity. Swiss mice received subcutaneous injections of rotenone at a dose of 1.5 mg/kg once every other day for two weeks either alone or in combination with citric acid at 200 or 400 mg/kg given orally. The control group was treated with the vehicle dimethyl sulfoxide. The brain and liver levels of the lipid peroxidation product malondialdehyde (MDA), nitric oxide (NO), total antioxidant capacity (TAC), and paraoxonase-1 (PON-1) activity were measured and as well as brain striatal concentrations of nuclear factor-kappa B (NF-kB) and tyrosine hydroxylase. Histopathology of the brain and liver tissue was also performed. Results indicated that MDA and NO content were increased whereas TAC and PON-1 decreased in the brain and liver tissue following rotenone injection. There was also increased NF-kB and decreased tyrosine hydroxylase concentrations in the brain of rotenone-treated mice. Rotenone-treated mice showed decreased striatal cell size and pyknotic nuclei. There were also interstitial hemorrhage and focal inflammatory cells infiltration in cerebral cortex, and degenerated neurons in cortex and hippocampus. The liver exhibited inflammatory cell infiltration and fibrosis. The administration of citric acid protected against rotenone-induced histopathological changes, decreased MDA, NO, and increased TAC and PON-1 activity in the brain and liver. It also reduced NF-kB and increased tyrosine hydroxylase in the brain of rotenone-treated mice. These data indicate that citric acid prevents rotenone-induced neuronal and liver damage via a decrease in oxidative stress. These findings suggest that supplementation with citric acid could be of value in the prevention of neuronal cell death in Parkinson’s disease. 

KEYWORDS | Citric acid; Neurodegeneration; Nuclear factor-kappa B; Parkinson’s disease; Rotenone; Total antioxidant capacity 

ABBREVIATIONS | ATP, adenosine 5′-triphosphate; MDA, malondialdehyde; NF-κB, nuclear factor-kappa B; NO, nitric oxide; NOS, nitric oxide synthase; PD, Parkinson’s disease; PON-1, paraoxonase-1; SNPc, substantia nigra pars compacta; TAC, total antioxidant capacity 

CONTENTS 

  1. Introduction
  2. Materials and Methods

2.1. Animals

2.2. Drugs and Chemicals

2.3. Study Design

2.4. Biochemical Analyses

2.4.1. Determination of Lipid Peroxidation

2.4.2. Determination of Nitric Oxide

2.4.3. Determination of Total Antioxidant Capacity

2.4.4. Determination of Paraoxonase-1

2.4.5. Quantification of Nuclear Factor-kappa B

2.4.6. Quantification of Tyrosine Hydroxylase

2.5. Histopathological Assessment

2.6. Quantitative Assessment of Neuronal Damage

2.7. Statistical Analysis

  1. Results

3.1. Brain Parameters

3.1.1. Oxidative Stress

3.1.2. NF-κB

3.1.3. PON-1

3.1.4. Tyrosine Hydroxylase

3.2. Liver Parameters

3.2.1. Oxidative Stress

3.2.2. PON-1

3.3. Histopathological Results of the Brain

3.4. Quantitative Assessment of Neuronal Damage

3.5. Histopathological Results of the Liver

  1. Discussion

1. INTRODUCTION 

Parkinson’s disease (PD) is a chronic and progressive neurodegenerative disorder of movement control which affects approximately 1% of the population over the age of 65 years [1]. The classic motor symptoms in PD are those of rigidity, bradykinesia/akinesia, resting tremor, gait disturbances, and postural instability [2]. Non-motor symptoms also occur and include cognitive decline, behavioral disorders, depression, apathy, sleeping disturbances, and autonomic impairment [3]. The main neuropathological finding in PD is the loss of the pigmented dopaminergic neurons of the substantia nigra pars compacta (SNPc) of midbrain basal ganglia which project to the striatum with consequent depletion of dopamine in the nigrostriatal pathway [4]. This results in disturbed functioning of basal ganglia circuits, whose function is to modulate cortical motor activity [5]. 

The mechanism underlying the selective death of dopaminergic cells in SNPc is not fully understood but accumulating evidence strongly supports a role for oxidatively mediated damage indicated by the presence of increased levels of lipid peroxidation products [6], protein carbonyls [7], and 8-hydroxyguanosine [8] in the brain of PD subjects post-mortem. Normally, the cell is equipped with a number of antioxidant mechanisms in order to cope with the reactive oxygen species (ROS) generated during cellular metabolism. These include both enzymatic means such as superoxide dismutase, catalase, and glutathione peroxidase and non-enzymatic antioxidants including, reduced glutathione, ascorbic acid, α-tocopherol, and uric acid. Oxidative stress develops when these antioxidant defenses are deficient/consumed or ROS are produced at a rate exceeding the cell’s antioxidant capacity [9]. In this context, the PD brain appears to be exposed to high levels of ROS, resulting, for instance, from mitochondrial dysfunction and the consequent increased formation of superoxide (O2˙ˉ), dopamine metabolism, the presence of high level of the redox transition metal iron, and from decreased glutathione content, possibly due to deficient synthesis [10]. 

Presently, the treatment of PD is largely directed towards replacing the dopamine deficit by the administration of L-3,4-dihydroxyphenylalanine or levodopa (L-DOPA) which provides symptomatic relief and remains the cornerstone of therapy. Other drugs aiming to increase dopaminergic neurotransmission include the dopaminergic receptor agonists (e.g., bromocriptine and pergolide), the monoamine oxidase B inhibitors (e.g., rasagiline and selegiline), and the catechol-O-methyl transferase inhibitors [11]. Improvement of symptoms follows the administration of these drugs; however, because of the progressive nature of the disease, by time fewer cells will be available for dopaminergic stimulation and hence drugs become less effective with the emergence of the side effects of L-DOPA including dyskinesia and on-off fluctuations [12]. This indicates the need for finding new add-on treatments that would reduce oxidative stress and hence the rate of cell loss in the disease. 

Citric acid (2-hydroxy-1,2,3-propane-tricarboxylic acid) is an important intermediate in the citric acid cycle (also termed tricarboxylic acid or Krebs cycle) in the mitochondria which is the final common pathway for the oxidation of carbohydrates, fatty acids, and amino acids and provides adenosine 5′-triphosphate (ATP) for the metabolic requirements of the cell [13]. Citric acid is also found in citrus fruits and juices such as lemon, orange, grapefruit, and tangerine and these constitute rich sources of citric acid in human diet [14]. It is also used in food as a natural preservative due to its antioxidant action [15] and to give a sour taste to food and soft drinks [16]. The daily intake of citric acid is estimated to be ~4 g/day [17]. Citric acid has therapeutic applications such as the prevention of urinary calcium oxalate crystallization and stone formation [18]. Citric acid has been shown to exert antioxidant and anti-inflammatory actions, attenuating the degranulation of polymorphonuclear cells and platelets and release of myeloperoxidase and interleukin-1beta (IL-1β) during hemodialysis [19, 20]. It also reduced oxidative stress and protected against brain and liver damage due to lipopolysaccharide endotoxin [21], malathion [22], or carbon tetrachloride [23] in experimental animals. 

In this study, we aimed to examine the effect of citric acid administration on the neurodegeneration in the rotenone-induced PD in mice. Pesticideshave been implicated in the increased risk for developing PD [24]. Rotenone is a pesticide of plant origin which reproduces many features of PD when injected into rodents and thus is widely used to model human PD [25, 26]. Since the pesticide was shown to affect the liver [27], this study was extended to include the liver tissue. The effect of citric acid was investigated at the levels of biochemical and histological changes. 

2. MATERIALS AND METHODS 

2.1. Animals 

Male Swiss albino mice (20–25 g: National Research Centre, Cairo, Egypt) were used in the experiments. Mice were housed under a standard 12-h light/dark cycle and had free access to food and water. Animal procedures were performed in accordance with the Ethics Committee of the National Research Centre (Cairo, Egypt) and followed the recommendations of the U.S. National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985). 

2.2. Drugs and Chemicals 

Rotenone and citric acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). Rotenone was dissolved in 100% dimethyl sulfoxide (DMSO). Citric acid was diluted in physiological saline to obtain the necessary doses. Other chemicals and reagents were purchased from Sigma-Aldrich and were of analytical grade. The doses of citric acid were selected based on previous studies [21, 23]. 

2.3. Study Design 

Mice were randomly allocated into four equal groups, six mice each. Mice received subcutaneous injections of rotenone 1.5 mg/kg every other day for two weeks combined with either saline (group 1: positive control) or citric acid at doses of 200 or 400 mg/kg orally (groups 2 and 3). Saline or citric acid was given at time of rotenone injection. A 4th group received only the vehicle i.p. (no rotenone) and served as a vehicle control. Thereafter, mice were euthanized by cervical decapitation under light ether anesthesia. The brain and liver of each mouse were then quickly removed, washed with ice-cold phosphate-buffered saline (PBS, pH 7.4), weighed, and stored at −80oC until the biochemical analyses were carried out. The tissues were homogenized in 0.1 M phosphate-buffered saline at pH 7.4 to give a final concentration of 10% (w/v) for the biochemical assays. 

2.4. Biochemical Analyses 

2.4.1. Determination of Lipid Peroxidation 

The lipid peroxidation product, malondialdehyde (MDA) was quantified in tissue homogenates as thiobarbituric acid reactive substances (TBARS) [28]. In the assay, TBARS react with thiobarbituric acid to form TBA-MDA adduct which can be measured colorimetrically at 532 nm. 

2.4.2. Determination of Nitric Oxide 

Nitric oxide (NO) was determined using a colorimetric assay, where nitrate is converted to nitrite via nitrate reductase. The Griess reagent reacts with nitrite to form a deep purple azo compound that can be measured using a spectrophotometer [29]. 

2.4.3. Determination of Total Antioxidant Capacity 

Total antioxidant capacity (TAC) in the supernatants was measured using an assay kit (Biodiagnostics, Cairo, Egypt)). This assay measures antioxidant capacity by the reaction of antioxidants in the sample with a defined amount of exogenously provided hydrogen peroxide. The antioxidants eliminate a certain amount of peroxide. The residual peroxide is determined colorimetrically by an enzymatic reaction which involves the conversion of 3,5-dichloro-2-hydroxybenzenesulfate to a colored product [30]. 

2.4.4. Determination of Paraoxonase-1 

The arylesterase activity of paraoxonase-1 (PON-1) was determined by a colorimetric method using phenyl acetate as a substrate. In this assay, PON-1 catalyzes the cleavage of phenyl acetate resulting in phenol formation. The rate of phenol formation was measured by monitoring the increase in absorbance at 270 nm at 25°C. One unit of arylesterase activity is equivalent to 1 μmol of phenol formed per min. Enzyme activity expressed as kU/L is calculated based on the extinction coefficient of 1310 M–1 cm–1 for phenol at 270 nm, pH 8.0, and 25°C [31]. 

2.4.5. Quantification of Nuclear Factor-kappa B 

The level of nuclear factor-kappa B (NF-kB)was determined using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit purchased from NOVA (Bioneovan, Beijing, China) according to the manufacturer’s instructions. The concentration of NF-kB in a sample was determined by interpolation from the standard curve. 

2.4.6. Quantification of Tyrosine Hydroxylase 

Tyrosine hydroxylase was determined using an ELISA kit from NOVA (Bioneovan) according to the manufacturer’s instructions. The concentration of tyrosine hydroxylase in a sample was determined from the standard curve. 

2.5. Histopathological Assessment 

The liver and brain from all groups were dissected immediately after euthanasia. The specimens were then fixed in 10% neutral-buffered formalin saline for at least 72 h. Specimens were washed in tap water for 30 min and then dehydrated in ascending grades of alcohol, cleared in xylene, and embedded in paraffin. Serial sections of 5 μm thick were cut and stained with hematoxylin and eosin (H&E) for the histopathological study. Images were examined and photographed under a digital camera (DP70, Tokyo, Japan) and processed using Adobe Photoshop version 8.0 (San Jose, CA, USA). 

2.6. Quantitative Assessment of Neuronal Damage 

Ten fields for each section of cerebral cortex and hippocampus were investigated and counted for total cells in the fields and cells that show degenerative signs such as pyknotic nuclei. The number of cells and percentage of damaged cells were calculated. 

2.7. Statistical Analysis 

Results are expressed as mean ± SE. Data were statistically analyzed using one-way analysis of variance followed by Duncan’s multiple range test for group comparison. Statistical significance was considered at a probability value of less than 0.05. The Statistical Package for Social Sciences (SPSS) software (SAS Institute, Cary, NC, USA) was used.

3. RESULTS 

3.1. Brain Parameters 

3.1.1. Oxidative Stress 

Mice treated with only rotenone showed significant increase in brain MDA by 75.1% compared to the corresponding controls (29.6 ± 1.7 vs. 16.9 ± 0.3 nmol/g tissue) (Figure 1A). The level of NO increased by 52.8% compared with the vehicle group (27.2 ± 1.5 vs. 17.8 ± 0.8 µmol/g tissue) (Figure 1B). Meanwhile, TAC was significantly decreased 81.5% by rotenone treatment (0.12 ± 0.004 vs. 0.65 ± 0.03 µmol/g tissue) (Figure 1C). The administration of citric acid at 400 mg/kg resulted in significant decreases in MDA and NO levels by 32.1% (20.1 ± 1.2 vs. 29.6 ± 1.7 nmol/g tissue) and 31.2% (18.7 ± 1.0 vs. 27.2 ± 1.5 µmol/g tissue), respectively (Figure 1A and 1B). There were also significant increments in TAC by 95.8% and 217.5% after citric acid at 200 and 400 mg/kg, respectively (0.235 ± 0.01 and 0.381 ± 0.05 vs. 0.12 ± 0.004 µmol/g tissue) (Figure C).

 

FIGURE 1. Effect of citric acid on the rotenone-induced biochemical changes in the brain of mice. Shown are data of (A) malondialdehyde (MDA), (B) nitric oxide, (C) total antioxidant capacity (TAC), (D) nuclear factor kappa B (NF-kB), (E) paraoxonase-1 (PON-1) activity, and (F) tyrosine hydroxylase. *, p < 0.05 vs. vehicle; +, p < 0.05 vs. rotenone control; #, p < 0.05 vs. rotenone + citric acid 200 mg/kg. 

3.1.2. NF-kB 

NF-kB level was significantly increased by 353.3% in mice treated with only rotenone administration (1.36 ± 0.04 vs. 0.30 ± 0.01 ng/ml). A significant decrease in NF-kB by 29.4% was seen in mice treated with citric acid at 400 mg/kg (0.96 ± 0.05 vs. 1.36 ± 0.04 ng/ml) (Figure 1D). 

3.1.3. PON-1 

A significant decrease in PON-1 activity by 50.0% was observed in rotenone-treated mice compared to the control group (5.72 ± 0.31 vs. 11.45 ± 0.70 kU/L). PON-1 activity increased by 58.1% by 400 mg/kg citric acid compared with the rotenone only group (9.1 ± 0.34 vs. 5.72 ± 0.31 kU/L) (Figure 1E). 

3.1.4. Tyrosine Hydroxylase 

Rotenone caused a significant decrease in the level of striatal tyrosine hydroxylase by 52.7% (768.7 ± 40.8 vs. 1625 ± 33.0 pg/ml). Tyrosine hydroxylase showed significant increase by 50% following the administration of citric acid at 400 mg/kg compared with the rotenone only group (1153 ± 34.6 vs. 768.7 ± 40.8 pg/ml) (Figure 1F). 

3.2. Liver Parameters 

3.2.1. Oxidative Stress 

The level of MDA was significantly increased by 59.4% in the liver of rotenone-treated mice compared with the vehicle control value (34.6 ± 1.2 vs. 21.7 ± 1.6 nmol/g tissue) (Figure 2A). There was also a significant increase in NO level by 72.1 (41.3 ± 1.8 vs. 24.0 ± 0.8 µmol/g tissue) and decreased TAC by 47.7% (1.83 ± 0.06 vs. 3.5 ± 0.18 µmol/g tissue) in the rotenone only group (Figure 3B and 3C). In rotenone-treated mice, citric acid given at 200 and 400 mg/kg resulted in 19.6% and 32.9% decrease in MDA (27.8 ± 1.3 and 23.2 ± 1.1 vs. 34.6 ± 1.2 nmol/g. tissue), respectively (Figure 2A). NO decreased by 27.4% by 400 mg/kg citric acid (30.0 ± 1.0 vs. 41.3 ± 1.8 µmol/g tissue) (Figure 2B). Meanwhile, TCA showed 26.8% and 69.4% increases after treatment with citric acid at 200 and 400 mg/kg, respectively (2.32 ± 0.16 and 3.1 ± 0.17 vs. 1.83 ± 0.06 µmol/g tissue) (Figure 2C).

 

FIGURE 2. Effect of citric acid on the liver (A) malondialdehyde (MDA), (B) nitric oxide, (C) total antioxidant capacity (TAC), and (D) paraoxonase-1 (PON-1) activity in rotenone-treated mice. *, p < 0.05 vs. vehicle; +, p < 0.05 vs. rotenone control; #, p < 0.05 vs. rotenone + citric acid 200 mg/kg.

 

FIGURE 3. Representative photomicrographs of striatum sections from different groups. (A) Vehicle-treated group with 2 different magnifications, showing 2 types of cells: large cells (arrow) and small cells (arrowhead). (B) Rotenone only-treated group showing small cells only; no large cells are noticed. Some small cells show pyknotic nuclei. (C) Rotenone and 200 mg/kg citric acid group showing a few large cells (arrow) among small cells. (D) Rotenone and 400 mg/kg citric acid group showing striatum area close to normal. 

3.2.2. PON-1 

PON-1 activity showed a significant decrease by 89.1% after rotenone injection compared with the vehicle-treated control (32.9 ± 2.2 vs. 17.4 ± 1.2 kU/L). The administration of citric acid resulted in 31.6% and 63.2% increases in PON-1 activity compared with the rotenone only group (22.9 ± 1.3 and 28.4 ±1.8 vs. 17.4 ± 1.2 kU/L) (Figure 2D). 

3.3. Histopathological Results of the Brain

Rotenone caused marked reduction of large cells in the striatum as compared to the normal structure in vehicle-treated animals (Figure 3A and 3B). The administration of citric acid resulted in a dose-dependent amelioration of the rotenone-induced changes (Figure 3E–3H). Sections of the cerebral cortex from rotenone-treated mice showed interstitial hemorrhage in some areas with focal inflammatory cells infiltration and many dark neurons (Figure 4C and 4D) in contrast to normal tissue structure (Figure 4A and 4B). Interstitial hemorrhage disappeared after treatment with 200 mg/kg citric acid, while neurons with signs of degeneration were still observed (Figure 4E and 4F). Citric acid at 400 mg/kg restored the cerebral tissue to normal structure (Figure 4G and 4H).

 

FIGURE 4. Representative photomicrographs of cerebral cortex sections from different groups. (A and B) Vehicle-treated group with 2 different magnifications. (C) Rotenone only-treated group showing interstitial hemorrhage (arrow) with focal inflammatory cells infiltration (arrowhead). (D) A higher magnification for the same section in C showing many dark neurons. (E) Rotenone and 200 mg/kg citric acid group showing no hemorrhage or inflammatory cells infiltration. (F) The same section with higher magnification showing some dark neurons with pyknotic nuclei (arrow) among normally appeared neurons. (G and H) Rotenone and 400 mg/kg citric acid group showing quite normal cerebral cortical tissue. 

Figure 5A and 5B shows the normal architecture of hippocampus in vehicle-treated animals, while Figure 5C and 5D shows the toxic effect of rotenone on this area in the form of many small dark neurons especially in CA3 area. A slight reduction of these affected neurons was observed in sections obtained from 200 mg/kg citric acid-treated mice (Figure 5E and5F).Noticeableameliorationofthe rotenoneeffect was detected by administering 400 mg/kg citric acid, despite the presence of a few dark neurons among healthy cells (Figure 5G and 5H).

FIGURE 5. Representative photomicrographs of hippocampal area sections from different group. (A and B) Vehicle-treated group with 2 different magnifications. (C) Rotenone only-treated group showing many neurons with degenerative signs. (D) A higher magnification for the same section in C showing many dark neurons that appear smaller than normal neurons. (E) Rotenone and 200 mg/kg citric acid group showing that some degenerated neurons are still observed. (F) The same section in E with higher magnification showing some dark neurons with pyknotic nuclei in between normal neurons. (G) Rotenone and 400 mg/kg citric acid group showing that a few dark neurons are still observed. (H) A higher magnification for the same section in G showing that most of neurons are normal in size and shape with a few neurons displaying signs of degeneration in between. 

3.4. Quantitative Assessment of Neuronal Damage 

Quantitative results are presented in Table 1 and Figure 6. Table 1 shows the number of neurons (normal and degenerated neurons) and the % of degenerated neurons in each group. Sections of the cerebral cortex and hippocampus from saline-treated mice revealed that only 1.18% and 1.15% of counted cells showed variable damage compared with 31.7% and 36.7% damaged neurons in the rotenone control group. Citric acid administered at 200 and 400 mg/kg to rotenone-treated mice resulted in a dose-dependent decrease in the number of degenerated neurons (Table 1). Figure 6 shows the number of degenerated neurons (mean ± SEM) in each group. 

TABLE 1. Number of normal and degenerated neurons in rotenone-treated mice and the effect of treatment with citric acid
Treatment group Cerebral cortex Hippocampus
Degenerated neurons Normal neurons %Degenerated. neurons Degenerated neurons Normal neurons %Degenerated neurons
Vehicle 7 583 1.18% 6 514 1.15%
Rotenone 221 477 31.66% 300 518 36.67%
Rotenone + citric acid 200 mg/kg 173 467 27.03% 202 585 25.66%
Rotenone + citric acid 400 mg/kg 118 525 18.35% 148 606 19.62%

  

FIGURE 6. Number of degenerated neurons in different treatment groups. *, p < 0.05 vs. vehicle; +, p < 0.05 vs. rotenone control; #, p < 0.05 vs. rotenone + citric acid 200 mg/kg. 

3.5. Histopathological Results of the Liver 

Figure 7A and 7B shows the normal liver tissue structure in vehicle-treated mice. Rotenone affected the liver tissue causing severe dilatation of blood vessels in portal area with massive fibrosis and inflammatory cells infiltration around (Figure 7C and 7D). These damaging effects were reduced by citric acid in a dose dependent manner (Figure 7E–7H).

FIGURE 7. Representative photomicrographs of liver tissue sections from different treatment groups. (A and B) Vehicle-treated group with 2 different magnifications. (C) Rotenone only-treated group showing severe dilatation of blood vessels in portal area with massive fibrosis around. (D) A higher magnification for the same section in C showing inflammatory cells infiltration in fibrotic tissue around portal area components. (E) Rotenone and 200 mg/kg citric acid group showing reduction of blood vessels’ dilatation and fibrosis. (F) The same section in E with higher magnification showing restriction of inflammatory cells infiltration to a focal aggregation (arrow) with mild dilatation of central vein. (G) Rotenone and 400 mg/kg citric acid group showing marked amelioration of the rotenone-induced damage signs. (H) A higher magnification for the same section in G showing slight dilatation of main blood vessels with no fibrosis or inflammatory cells infiltration. Hepatocytes are normal. 

4. DISCUSSION 

The findings in the present study provide the first evidence that the administration of citric acid was able to inhibit neurodegeneration and protect striatal, cortical and hippocampal neurons against the rotenone neurotoxicity. The neuroprotective effect of citric acidwasassociatedwithasignificantdecreaseinlipid peroxidation, NO, and NF-kB levels and an increase in TAC in the brain of rotenone-treated animals. Moreover, citric acid attenuated the decrease in striatal tyrosine hydroxylase, suggesting conservation of the dopaminergic neurons in SNPc. Quantitative assessment of neuronal damage in the cerebral cortex and hippocampus indicated that citric acid significantly decreased the number of degenerated neurons in these regions. In addition, the oxidative stress and histopathological changes caused by rotenone in liver tissue were alleviated by administering citric acid. 

Consistent with previous studies [32, 33], rotenone caused marked increase in brain oxidative stress evidenced by the increase in the level of the lipid peroxidation end product MDA, suggesting oxidative damage of membrane lipids by the increase in ROS. Rotenone is a highly lipophilic compound which readily crosses the blood-brain barrier and inhibits the mitochondrial complex I or NADH-ubiquinone oxidoreductase, resulting in the increased generation of superoxide (O2˙ˉ) or hydrogen peroxide (H2O2). The activation of microglia by rotenone is another source of O2˙ˉ and also hypochlorous acid [34]. Rotenone thus increases intracellular ROS and induces neuronal apoptosis [35] which is preventable by antioxidants such as glutathione or its precursor N-acetylcysteine, ascorbic acid, and α-tocopherol both in vitro and in vivo [36, 37]. The occurrence of oxidative stress in the brain following rotenone exposure was further supported by the marked depletion of TAC. The latter is a frequently used test in biological studies to assess all antioxidant in the sample, such as tissue or plasma and is considered to give more information than assessing individual antioxidants. It has been suggested that a decrease in TAC could indicate the presence of oxidative stress or alternatively an increase in vulnerability to oxidative damage [38]. TAC is decreased in the serum of PD patients [39] and in substantia nigra of PD brain post-mortem [40]. It also showed marked decrease in the brain of rats after exposure to rotenone [41]. In this study, the administration of citric acid resulted in significant and dose-dependent increase in TAC in the brain of rotenone-treated mice, suggesting an antioxidant action for citric acid and sparing of cellular antioxidants. 

We also demonstrated an increased level of the transcription factor NF-κB in the brain tissue after rotenone injection which is in agreement with other studies [42]. NF-κB regulates the transcription of different genes involved in cellular immune and inflammatory response. NF-κB is present inactivated in the cytoplasm by binding to an inhibitory IκB proteins (IκBs) and is activated by cytokines, oxidative stress, and bacterial lipopolysaccharide endotoxin. NF-κB dimers (p50 and p65) are then released from the IκB-NF-κB complex, translocate to the nucleus inducing the expression of genes encoding proinflammatory cytokines (IL-1β, IL-2, IL-6, IL-8, IL-12, and TNF-α), cycloxygenase-2, inducible nitric oxide synthase (iNOS), leucocyte adhesion molecules, chemoattractant protein-1 (MCP-1), acute-phase proteins, and growth factors [43]. NF-κB is redox-sensitive and its activation by TNF-α or H2O2 could be inhibited by glutathione peroxidase overexpression [44]. In this study, we demonstrated that the administrationofcitricacidwasassociatedwithsignificant decrease in brain tissue level of NF-κB, suggesting reduced NF-κB activation, possibly due to a lower level of oxidative stress. 

Our findings indicated that rotenone resulted in marked increase in brain NO which is consistent with previous observations [32, 37, 41]. The signaling molecule NO is synthesized from L-arginine via the action of the enzyme nitric oxide synthase (NOS) which exists in constitutive endothelial (eNOS) and neuronal (nNOS) isoforms and a third inducible isoform (i.e., iNOS) activated by ROS, cytokines, and bacterial lipopolysaccharide. There is evidence that the increase in NO is linked to neurodegeneration through the formation of more reactive nitrogen species, such as peroxynitrite (ONOOˉ) capable of oxidation and nitration of protein tyrosine residues and nitrosylation of thiols. In this context, iNOS is the source of excessive NO release by microglia and astrocytes during brain inflammation, infection, and trauma [45]. Studies indicated increased expression of iNOS in the substantia nigra and striatum of rodents following rotenone administration, thereby suggesting a role for iNOS-derived NO in neuronal cell death caused by the toxicant [46, 47]. Moreover, rotenone neurotoxicity is reduced by an iNOS-specific inhibitor [48]. NO derived from nNOS also mediated neuronal injury since in rotenone-treated rats, the administration of a selective nNOS inhibitor was neuroprotective [49]. The present study showed that the administration of citric acid was associated with decreased brain (and liver) NO. In a previous study, oral administration of citric acid was found to inhibit the strong expression of iNOS in the liver of mice treated with lipopolysaccharide endotoxin [21]. It is thus suggested that inhibition of iNOS and the decrease in NO release could be involved in neuroprotective and hepatic protective effects of citric acid against the rotenone toxicity. 

This study also showed that the administration of rotenone caused marked decrease in brain PON-1, which is in agreement with previously reported studies [37, 41, 47]. The importance of this finding derives from the following: (i) the enzyme PON-1 acts to hydrolyze the active metabolites of some organophosphate insecticides [50]; (ii) there is an increasingly accumulating evidence that links exposure to these compounds in agriculture and in the household with an increased risk for developing PD, likely in genetically susceptible individuals [24, 51]; (iii) the catalytic efficiency of PON-1 has been shown to modulate the susceptibility to the toxic effects of some organophosphate insecticides in vivo and in vitro [52]; (iv) in man, the genetic variation in the catalytic activity of this enzyme alters the susceptibility to organophosphates [53]; and (v) PON-1 displays antioxidative and anti-inflammatory actions [54]. It follows that the decrease in enzyme activity following exposure to rotenone (or other insecticides) will compromise the capacity of the cell to deal with oxidativestressandwouldrenderthecellvulnerable to further oxidative insults. In this study, we showed that administering citric acid was associated with significant increase in PON-1 activity. Since the enzyme is inhibited by oxidative stress, this effect of citric acid could be attributed to a decreased oxidative burden. 

In PD, tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis is progressively deceased in the substantia nigra and striatum and is considered a reliable marker of dopaminergic neuronal death [4, 55]. Previous studies using tyrosine hydroxylase immunohistochemistry showed a significant decrease in tyrosine hydroxylase immunostaining in the substantia nigra and striatum of rotenone-treated rats [33, 46]. In this study, the level of tyrosine hydroxylase measured by ELISA was markedly decreased in the striatum after rotenone injection, indicating the loss of dopaminergic neurons, but increased following treatment with citric acid together with the decrease in neurodegeneration, confirming a protective effect for citric acid on SNPc dopaminergic neurons. 

A bioenergetic defect might be involved in the rotenone neurotoxicity. In human neuroblastoma cells in vitro, rotenone causes concentration-dependent ATP depletion as well as oxidative damage and neuronal death [25]. In vivo, rotenone treatment resulted in decreased ATP levels in mice brain and dopaminergic cell death [56]. The energy required for neuronal functioning is derived from both glycolysis and citric acid cycle. Pyruvate, the end product of glycolysis is oxidatively decarboxylated to form acetyl-CoA which condenses with oxaloacetic acid to yield citric acid. Citrate then enters the citric acid cycle within the mitochondrial matrix and the energy released in the electron transport chain is stored in the form of ATP [13]. Studies showed that administering citric acid cycle substrates such as α-ketoglutarate or pyruvate was able to protect against neuronal death due to the activation of poly (ADP-ribose) polymerase-1 (PARP1) and the consequent impairment of glycolysis [57]. In rat hippocampal slices, the N-methyl-D-aspartate (NMDA) excitotoxic neuronal damage was prevented by the application of pyruvate which restored ATP levels [58]. In a PD model in mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), enhancing glycolysis by increasing the activity of phosphoglycerate kinase 1 activity was reported to increase the level of pyruvate, ATP, and dopamine and improve motor dysfunction [59]. In the same way, the administration of citric acid might be able to alleviate the rotenone-induced neuronal degeneration through increasing the activity of citric acid cycle, thereby counteracting the bioenergetic defect caused by the pesticide. 

In this study, the administration of rotenone led to increased hepatic lipid peroxidation and NO along with decreased TAC and PON-1 activity, similar to that observed in brain tissue. Moreover, the liver histopathology demonstrated the presence of marked fibrosis and inflammatory cell infiltration. Other studies showed fatty changes in the liver following systemic rotenone injection in rats [27]. The pesticide thus causes liver oxidative stress and tissue damage which, in the present study, could be alleviated by treatment with citric acid. 

In summary, the present study suggests a neuroprotective effect for orally given citric acid against rotenone-induced neurotoxicity. Citric acid also alleviated the hepatic injury caused by the pesticide. These effects of citric acid involve decreased oxidative stress. Citric acid might also inhibit neurodegeneration by decreasing NO release and enhancing citric acid cycle, thereby alleviating a rotenone-induced bioenergetic defect. The dietary intake of citric acid in humans is ~4 g [17]. Following oral ingestion, citrate is rapidly absorbed (96 to 98% within 3h) [60]. In this study, the doses of citric acid used correspond to human doses of ~2.2 and 4.4 g, respectively, which are comparable with the human daily intake of citrate. Interestingly, orange and lemon juices contain 7.6–19.7 g/L and 40–50 g/L of citric acid, respectively [61, 62] beside their rich content of the antioxidant ascorbic acid and therefore could be a source of dietary intervention in PD. 

ACKNOWLEDGMENTS 

This works was not supported by research grants. The authors declare no conflicts of interest. 

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