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. 2021 Jul 12;10(4):810–816. doi: 10.1093/toxres/tfab065

Quercetin fail to protect against the neurotoxic effects of chronic homocysteine administration on motor behavior and oxidative stress in the adult rat’s cerebellum

Mohaddeseh Thaimory, Iran Goudarzi ✉, Taghi Lashkarbolouki 1, Kataneh Abrari 2
PMCID: PMC8403605  PMID: 34484672

Abstract

Homocysteine (Hcy) is an excitatory amino acid that contains thiol group and derives from the methionine metabolism. It increases vulnerability of the neuronal cells to excitotoxic and oxidative damage. This study aimed to investigate the hyperhomocysteinemia (hHcy) effects on rat cerebellum and the possible protective role of quercetin administration in Hcy-treated rats, using behavioral and biochemical analyzes. To this end, the adult male rats were divided randomly into the control group that received vehicle, Hcy group received Hcy (400 μg/kg), Hcy + Que group received Hcy + quercetin (50 mg/kg), quercetin group received quercetin for 14 days. On Day 14 after the final treatment, lipid peroxidation level, the superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities were evaluated in the cerebellum. After completion of treatment, the rat’s performance on rotarod and locomotor activity was evaluated. The results showed that Hcy treatment elicited cerebellar lipid peroxidation, impaired locomotor activity and increased latency to fall on the rotarod. Quercetin failed to attenuate significantly motoric impairment, increased significantly the cerebellar lipid peroxidation and GPx activity in the Hcy + Que group. Our results suggest that Hcy induced cerebellar toxicity and quercetin had no significant protective effects against Hcy toxicity in the cerebellum of adult rats.

Keywords: homocysteine, quercetin, oxidative stress, rat, cerebellum

Introduction

Homocysteine (Hcy) is a sulfur-containing amino acid, which derives from methionine metabolism [1]. Hcy is converted to methionine by methionine synthase that needs to cofactors such as folic acid and vitamin B12 via the re-methylation pathway and to cysteine and taurine via the trans-sulfuration pathways.

Hcy reacts with serine via vitamin B6-dependent cystathionine β-synthase to form cystathionine in the trans-sulfuration pathway. Then, cystathionine via vitamin B6-dependent cystathionine γ-lyase hydrolyzes to yield cysteine and α-ketobutyrate and finally to taurine [2, 3].

Metabolism of Hcy is dependent on the level of vitamins B6, B12, and folic acid [4]. Deficiency of folic acid, vitamins B2, B6, B12, and errors of the following enzymes: methylenetetrahydrofolate reductase, cystathionine β-synthase and methionine synthase, causes Hcy metabolic disturbances, and increase of Hcy in human tissues, known as hHcy [5]. hHcy may appear as mild, moderate, or severe form [6].

Positive correlation between the Hcy concentrations and the rate of brain atrophy has been reported in a randomized controlled study [7]. The potential role of Hcy in neurodegenerative disorders has been demonstrated in studies that evaluated the relation between concentrations of plasma Hcy in Parkinson’s disease or Alzheimer’s disease patients [8].

Patients with homocystinuria have mutations in methylenetetrahydrofolate reductase, 9] that show developmental delay, motor and gait dysfunctions, psychiatric disturbances, hypotonia, and seizures.

Rats born to mothers fed with a methyl donor deficient diet pregnancy and lactation [10, 11], rats showed significant Hcy accumulation and elevated apoptosis in selective brain regions, especially in the cerebellum on postnatal day (PD) 21 [10]. Also, rats showed cognitive and motor defects [10]. In our previous study, postnatal administration of Hcy on Day 4 until 25 resulted in cerebellar toxicity, motor incoordination in the rats [12].

The mechanism underlying the neurotoxic effects of Hcy is unknown. There are evidence that showed Hcy elicits oxidative stress via of glutamate receptors activation, and reactive species production [13–17], autoxidation to Hcy and other disulphides releasing O2—and H2O2 [13, 14, 18, 19], or by decreasing antioxidant defenses and elevating lipid peroxidation [20–23].

Several studies reported various beneficial effects of quercetin on human health such as cardiovascular protective, anticancer, antiviral, and anti-inflammatory activities [24, 25]. It is found in consumable foods such as apples, berries, onions, tea, and brassica vegetables [25]. Also, several studies demonstrated the quercetin effect against cognitive impairment in animal models [24, 26–28]. Also, quercetin prevents oxidant damage by scavenging oxygen radicals, lipid peroxidation protection, and chelating metal ions [28–32].

Considering that neonatal Hcy administration induced cerebellar damage and motor impairments in our previous investigation, we decided to examine the effect of Hcy on motor behavior in order to determine whether Hcy induces oxidative stress and cerebellar toxicity in adult rats. Further, we evaluated the effect of quercetin on motor behavior and oxidative stress in the adult rat’s cerebellum.

Results

Behavioral results

Effect of Hcy and quercetin administration on initial motoric capacity

Administration of Hcy significantly decreased the initial motoric capacity, as the Hcy group showed a significantly shorter latency on rotarod in comparison to the control group (P < 0.001). Quercetin could not significantly elevate initial motoric capacity in the Hcy + Que group. Moreover, no significant difference was observed in the initial latency between quercetin and the control groups (Fig. 2A).

Figure 2.

Figure 2

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The rotarod performances following Hcy and quercetin administration in rats. (A) Hcy administration significantly increased latency on rotarod than the control group (P < 0.001), whereas quercetin treatment could not improve it. (B) Rotarod performances on all sessions significantly were impaired following Hcy treatment and administration of quercetin could not improve it. Values are mean ± SEM of 8–9 rats. ***P < 0.001 compared with the control group.

Effect of Hcy and quercetin administration on subsequent latencies to fall from the rotarod

The latency to fall from the rotarod in the Hcy group was shorter than that of the control group (P < 0.001) in all sessions compared with control group. Quercetin did not significantly increase the latency of the Hcy + Que group at all sessions when compared with the Hcy group. Also, administration of quercetin alone significantly increased latency to fall than the control group in session 4 (P < 0.05, Fig. 2B).

Effect of Hcy and quercetin administration on open field test

Findings in the open field test indicated that the number of crossings significantly decreased in the Hcy group that states a reduction in locomotor activity (P < 0.01, Fig. 3A). Also, the number of rearing and grooming significantly reduced in the Hcy group than the control group (Fig. 3B and C, P < 0.001). Quercetin significantly improved the number of rearing in the Hcy + Que group when compared with the Hcy group (Fig. 3B, P < 0.05). Moreover, the number of crossings, rearing, and grooming in the Que group did not show a significant difference in comparison to the control group.

Figure 3.

Figure 3

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Effects of Hcy and quercetin treatment on the locomotor activity in rats. Number of square crossings, rearings, and grooming events in 4 groups was shown in the (A), (B), and (C), respectively. Hcy administration significantly reduced the number of squares crossed, the number of rearing and grooming than the control (P < 0.01 and P < 0.001, respectively). Quercetin significantly improved the number of rearings in the Hcy + Que group (P < 0.05). Values are mean ± SEM of 8–9 rats. **P < 0.01 and ***  P < 0.001 than the control group. †P < 0.05 than the Hcy group.

Biochemical studies

Hcy and quercetin influences on activity of antioxidant enzymes and lipid peroxidation levels

Cerebellar malondialdehyde (MDA) levels significantly elevated following Hcy treatment when compared with the control (Fig. 4A, P < 0.05). Following quercetin administration cerebellar MDA levels significantly increased in the Hcy + Que group in comparison to the Hcy group (Fig. 4A, P < 0.05). There was no significant difference in MDA levels between quercetin and the control groups.

Figure 4.

Figure 4

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Effect of Hcy and quercetin treatment on oxidative stress marker in the cerebellum. (A) Cerebellar MDA levels, (B) SOD activity, and (C) GPx activity. Hcy treatment significantly increased MDA levels in the cerebellum but it did not elicit significant alteration in SOD and GPx activities. Quercetin administration significantly increased MDA levels and GPx activity in the Hcy + Que group. Values are mean ± SEM of 6–7 rats. *P < 0.05, **P < 0.01 than the control group. +P < 0.05, ††P < 0.001 than the Hcy group.

Statistical analysis of cerebellar SOD activity demonstrated that no significant difference was observed among experimental groups (Fig. 4B).

In addition, there was no significant difference in GPx activity between Hcy and control group (Fig. 4C). Quercetin significantly elevated GPx activity in the Hcy + Que group in comparison to Hcy and the control group (P < 0.01). No significant difference was observed in the cerebellar GPx activity between quercetin and control groups (Fig. 4C).

Discussion

The present investigation, for the first time, showed that Hcy treatment in adult rats caused motor behavior impairment and cerebellar oxidative stress.

The data of initial latency show unlearned potential motoric capacity since animals learn training in the rotarod task in the initial session. The shorter initial latency time observed in the Hcy group suggesting that Hcy reduces motor performance before and after learning in rats. The rotarod performance comparison shows a significant motor impairment in all of sessions in the Hcy group. Also, Hcy-treated group compared with control had significant reduction in the number of crossing, rearing, and grooming in the open field test. These results suggest impairment of motor coordination and locomotor activity by Hcy in the adult rats. Our results are consistent with our previous investigation in which Hcy administration from PD 4 until 25 days in pups induced oxidative stress in the cerebellum and motor incoordination [12]. Also, Rats born to mothers fed with a methyl donor deficient diet in pregnancy and lactation periods [10, 11], they showed significant Hcy accumulation and elevated apoptosis in selective brain regions, especially the cerebellum on PD 21 with cognitive and motor impairments [10].

Although the neurotoxic effect mechanisms of Hcy are unclear, reports suggest the contribution of oxidative stress in Hcy neurotoxicity. For this reason and for unraveling the pathology of Hcy, we investigated oxidative stress markers in the cerebellum. Significant increase in cerebellar MDA level by Hcy administration indicates that oxidative stress has occurred in the cerebellum of rats since the level of MDA is an indicator of lipid peroxidation.

There is evidence that shows Hcy induces oxidative stress by activation of glutamate receptors, and then reactive oxygen production (ROS; [16, 17]) or by autoxidation to Hcy and other disulphides releasing O2.− and H2O2 [14, 18]. In addition, Hcy can produce ROS such as superoxide, hydroxyl radicals, and hydrogen peroxide and start lipid peroxidation [18, 33].

Our result is in line with other reports that indicated Hcy administration induces oxidative stress in the brain [34–36]. Some evidences show Hcy reduces antioxidant defenses and increases lipid peroxidation [21, 23, 37]. However, in this study, no significant difference was observed in cerebellar SOD and GPx following Hcy administration. Considering the enhancement of the MDA level by Hcy administration and since MDA is a biomarker of oxidative stress-induced lipid peroxidation, it seems that Hcy probably increased the generation of ROS, but without alteration in antioxidant enzyme activity, as oxidative stress was occurred in the cerebellum. Also, no observed change in cerebellar SOD and GPX activity following administration of Hcy maybe relate to compensatory response to increased levels of ROS during 14 days Hcy administration.

Matte’ et al. showed that SOD activity did not alter following Hcy administration in the parietal cortex that is in accordance with our results [34]. Considering that oxidative stress is involved in the pathophysiology of Hcy effects in the cerebellum, we studied the quercetin effects, a flavonoid with antioxidant and neuroprotective properties, in Hcy induced cerebellar toxicity.

Our findings demonstrated that quercetin significantly did not increase the Hcy + Que group latency at all sessions as compared with the Hcy group. In addition, no significant difference was observed in the number of crossings and grooming following Hcy treatment, suggesting quercetin treatment could not improve motor incoordination and reduction of locomotor activity induced by Hcy administration in the adult’s rats. These results are inconsistent with our previous findings that indicated the administration of antioxidants could improve motor behavior defects and oxidative stress elicited by the increase of plasma Hcy levels in the cerebellum [12, 37]. This discrepancy may relate to Hcy administration time, Hcy administration route, animal age, and treatment time.

Quercetin is a powerful antioxidant, which can prevent oxidative damage via chelating metal ions, scavenging oxygen radicals, and lipid peroxidation protection [14]. Additionally, quercetin reduces ROS generation, elevates Mn-SOD (manganese superoxide dismutase) activity, and levels of glutathione.

We have expected, quercetin as an antioxidant improves oxidative stress induced by Hcy administration. Our findings showed that cerebellar GPx activity significantly increased in the Hcy + Que group in comparison to Hcy group and no change in SOD activity was observed, but MDA levels significantly increased in the Hcy + Que group compared with the Hcy group.

Based on these results, it seems that quercetin alone was not able to increase the GPx activity or MDA, then it is not the only one responsible for the effect observed in the Hcy + Que group. Thus, what happened probably was a synergistic effect.

Lipid peroxidation is an indicator of toxicity following several xenobiotics and causes by oxidative stress started when the balance between peroxidant and antioxidant mechanism is impaired that finally results in the loss of membrane integrity and function [38].

Kaur et al. reported that chlorpyrifos exposure induced oxidative stress in the cerebrum and cerebellum and quercetin co-administration could improve it. They suggested that quercetin can be used to prevent neurotoxicity induced by chlorpyrifos [39].

Adedara et al. showed that co-administration with quercetin prevented locomotor and motor defects induced by manganese exposure. Furthermore, quercetin improved reduction in antioxidant enzyme activities and the increase in activity of acetylcholinesterase, hydrogen peroxide production, and levels of lipid peroxidation induced by manganese in the rat’s hypothalamus, cerebrum and cerebellum [40].

Results of our previous study have revealed that folic acid treatment improved motor coordination deficit and oxidative stress induced by postnatal Hcy treatment in rats cerebellum [41]. The folic acid protective actions on motor incoordination and cerebellar oxidative stress probably be related to its antioxidant activity and enhancement of GPx activity.

Taken together, our results were not in accordance with reports stating that quercetin had a neuroprotective effect against oxidative stress in the cerebellum [39, 40] and hippocampus [28, 42].

Although quercetin functions as scavenger of free radicals in vitro, but considering its chemical structure as well as mechanism of action, it seems that quercetin might have pro-oxidant effects in certain conditions. Enzymatic oxidation of quercetin which leads to the production of ortho-semiquinone and ortho quinone/quinone methide intermediates and its auto-oxidation is related to the pro-oxidant activity of quercetin and might be involved in ROS generation and depletion of GSH [44, 45].

Martins et al. showed that 21 days’ treatment with methylmercury and quercetin induced a significant motor deficit in mice. Also, treatment with quercetin and methylmercury resulted in a higher oxidative injure in the cerebellum than the individual exposures. Methylmercury + quercetin caused a higher lipid peroxidation in the cerebellum when compared with methylmercury or quercetin alone. Their findings indicated that quercetin and methylmercury induce additive pro-oxidative effects in the cerebellum that is related to the motor defects [46].

Casuso  et al. showed that administration of quercetin impaired exercise-induced adaptations in cerebellar tissue [47].

In conclusion, quercetin treatment does not prevent motor deficits and oxidative stress-induced by Hcy administration. Also, quercetin increased toxicity when co-administration with Hcy that might be due to the pro-oxidative effects of the quercetin and/or its quinone metabolite(s) combined with Hcy toxicity. Therefore, we propose that probably synergic effects of Hcy and quercetin administration could be responsible for the observed results, however need to more studies in future.

Materials and Methods

Animals

Protocol of the experiment was approved by the Research and Ethics Committee of Damghan University. Male Wistar rats (200 ± 20 g) were purchased from Shahid Beheshti University of Iran. Animals were kept in well-ventilated rooms with a 12-h light/dark cycle and freely had access to water and food throughout the experiments.

Drugs and chemicals

Hcy, 2-Thiobarbituric acid (TBA), 1,1,3,3 tetramethoxypropan, Nitro blue tetrazolium (NBT), and quercetin (Sigma–Aldrich Co., St. Louis, MO) were purchased from Sigma-Aldrich Chemicals.

Hcy was dissolved in saline and buffered to pH 7.4. Hcy was injected (dose 400 μg/kg) daily in the vena caudalis for 2 weeks [48]. Quercetin was suspended in ethanol (1%) in saline and rats received it intraperitonally with a dose 50 mg/kg 1 h before Hcy administration for 2 weeks.

Considering the protective effect of quercetin with a dose 50 mg/kg on memory impairment and oxidative stress in the hippocampus following stress in our previous investigation, we administrated this dose to rats [28].

Experimental groups

Rats were divided into four groups.

Control group

Control rat received quercetin vehicle [ethanol (1%) in saline] for 2 weeks.

Hcy group

Hcy was administrated to rats via vena caudalis in a dose 400 μg/kg for 2 weeks.

Hcy + quercetin group

Rats received intraperitoneally quercetin (50 mg/kg) 1 h before Hcy administration for 2 weeks.

Quercetin group

Quercetin was injected to rats for 2 weeks (Que).

On Day 14 after the final treatment, 6–7 animals in each group were decapitated, and the cerebellum dissected and used for biochemical analysis. Rotarod and locomotor activity were performed after completion of treatments in the rest of the animals (n = 8–9 in each group). Figure 1 show experimental timeline.

Figure 1.

Figure 1

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Timeline of experiments.

Behavioral study

Open field test

Each rat was placed at the center of a clean homemade open field apparatus (40 × 40 × 15 cm, divided into nine squares). First, rats were placed to the open field apparatus for habituation for 1 min. The parameters including the number of squares crossed (locomotor activity), rearing (number of times the rat stood completely erect on its hind legs), and grooming (number of times the rat scratched its face with its forepaws) were recorded for 5 min by two blind observers [49].

Accelerating rotarod assay

From rotarod apparatus (Hugo Sachs Electronik, Germany) were used to measurement of motor coordination as described in our previous study [49].

Biochemical study

Cerebellar homogenate preparation

On Day 14, rats were sacrificed by decapitation, the cerebellum was removed and homogenized in cold sodium phosphate buffer (50 mM, pH 7.0) comprising 0.1-mM EDTA. The homogenates were centrifuged at 6000 × g for 10 min at 4°C and then the supernatants were stored at −80°C.

Thiobarbituric acid-reactive substances

Thiobarbituric acid-reactive substances were assayed based on Ohkawa et al. [50] as described in our previous work by Soleimani et al. [51].

SOD assay

Total SOD activity was evaluated based on Becana et al. [52] following the inhibition of the photochemical reduction of NBT as described in our previous work by Soleimani and colleagues [51].

GPx assay

Measurement of GPx activity was performed based on the Wendel method with tertbutyl hydroperoxide as substrate [53] as described in our previous work by Soleimani et al. [51].

Protein assay

Cerebellar homogenates protein content was measured using bovine serum albumin as standard [54].

Statistical analysis

Data were expressed as the mean ± standard error of the mean (SEM). One-way analysis of variance and then Tukey test was performed as post hoc for comparison of the means in the behavioral study and LSD for biochemical data. Difference was considered significant if P < 0.05.

Acknowledgments

We acknowledge Damghan University for supporting this work.

Contributor Information

Taghi Lashkarbolouki, Faculty of Biology, Damghan University, Cheshme-Ali, Damghan 3671641167, Iran.

Kataneh Abrari, Faculty of Biology, Damghan University, Cheshme-Ali, Damghan 3671641167, Iran.

Conflict of interest statement

The authors declare that they have no conflict of interest.

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