Effects of Memantine, a Non-Competitive N-Methyl-d-Aspartate Receptor Antagonist, on Genomic Stability
Author for correspondence: Jaqueline Nascimento Picada, Laboratório de Genética Toxicológica, ULBRA: Avenida Farroupilha 8001, Bairro São José, CEP 92425-900, Canoas, RS, Brazil (fax +55 51 34771313, e-mail firstname.lastname@example.org).
Abstract: Memantine is an aminoadamantane drug useful in neurodegenerative diseases, with beneficial effects on cognitive functions. Some studies have shown that memantine protects brain cells, thereby decreasing glutamate excitotoxicity. This study evaluated the genotoxic/antigenotoxic and mutagenic effects of memantine in CF-1 mice, following standardized protocols. Memantine was administered i.p. at 7.5, 15 or 30 mg/kg for three consecutive days. Blood and brain samples were collected to assess DNA damage using the alkaline comet assay. The mutagenic effect was assessed using the bone marrow micronucleus test. In addition, possible antioxidant effects were evaluated measuring the survival of Saccharomyces cerevisiae yeast strains [wild-type (WT) and isogenic mutants lacking superoxide dismutase] to cotreatment of memantine plus hydrogen peroxide. Memantine decreased DNA oxidative damage mainly in brain tissue. This antigenotoxic effect corroborated an increase observed in the survival of S. cerevisiae WT strain against hydrogen peroxide-induced damage. Furthermore, memantine did not increase the micronucleus frequency. The overall results indicate that memantine showed no mutagenic activity, did not cause DNA damage in the blood and brain tissues and showed antigenotoxic effects in brain tissue.
Drugs acting in the central nervous system and in endogenous neurotransmitters can induce DNA damage in brain tissue [1–3]. Glutamate is a neurotransmitter capable of inducing excitotoxicity in neurodegenerative diseases like Parkinson and Alzheimer, mediated by release of calcium, which can initiate a cascade of intracellular signals that result in oxidative stress [4,5]. As N-methyl-d-aspartate (NMDA) receptors are mediators in glutamate-induced neurodegeneration, one line of therapeutic research has been focused on the development of non-competitive NMDA receptor antagonists that would block the undesired elevations of Ca+2 caused by the prolonged action of glutamate in the extracellular space, without interfering with glutamate physiological actions required for learning and memory [6–9].
Aminoadamantanes represent a class of drugs that block NMDA glutamate receptors and have already been used clinically as antiviral and anti-parkinsonian agents. Some of these drugs are known to have a neuroprotective effect in animal models [10–13]. Memantine is a neuroprotective aminoadamantane, which blocks NMDA receptor in a non-competitive fashion. It has been reported that memantine increases brain-derived neurotrophic factor levels [14,15] and synaptic plasticity in the aged rat , protects neurons from glutamate-induced neurotoxicity [17,18] and reduces β-amyloid-induced apoptotic death and neuroinflammation in the hippocampus .
There are few studies on the effects of aminoadamantane drugs on genomic stability. A recent study has shown genotoxic effects of amantadine assessed by comet assay in blood and brain tissues of mice . In this study, we evaluated the possible genotoxic/antigenotoxic and mutagenic effects of memantine. DNA damage was assessed using the alkaline comet assay, while mutagenicity was evaluated by measuring micronucleus frequency in bone marrow of treated mice. In addition, a survival assay on Saccharomyces cerevisiae yeast strains lacking superoxide dismutase was performed to detect antioxidant effects.
Materials and Methods
Animals. Thirty-three CF-1 male mice (3 months old; 35–40 g) were obtained from the Lutheran University of Brazil (ULBRA). The mice were housed in plastic cages with ad libitum access to water and food, under a 12-hr light/dark cycle and at a constant temperature of 23 ± 2°C. All experimental procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and with the consent of the Ethics Committee of the Lutheran University of Brazil (CEP-ULBRA number: 2008-030A).
Drug. Memantine (1,3-dimethyl-5-aminoadamantane hydrochloride) (CAS Number 41100-52-1) was purchased from Acros Organics (Belgium). All solutions were prepared immediately prior to administration.
Animal treatment and samples. The animals were divided into four groups of seven animals, which were given an intraperitoneal (i.p.) injection (10 mL/kg b.w.) of saline solution (NaCl 0.9%) or memantine (7.5, 15 or 30 mg/kg), once a day for 3 days, reaching the maximum tolerated dose, as recommended by the revised guideline protocol . Peripheral blood samples were collected in four periods (5 and 24 hr after the first and third injections) for the comet assay. All animals were killed by decapitation on the fourth day. Brain tissue was dissected for the comet assay, and bone marrow was sampled from femurs for the micronucleus test. A positive control group of five animals (treated with cyclophosphamide 25 mg/kg, i.p) was included for the micronucleus test, and ex vivo treatment with hydrogen peroxide (H2O2) 0.20 mM in cells of the saline group was considered as a positive control for the comet assay.
Comet assay. The alkaline comet assay was performed as described in the comet assay guidelines [21,22]. Briefly, the brains were dissected and placed in 0.5 mL cold phosphate-buffered saline (PBS) and finely minced to obtain a cellular suspension; blood samples were placed in 15 μL anticoagulant (heparin sodium 25.000 IU-liquemine®). These suspension cells from brain and from the peripheral blood (5.0 μL) were embedded in 95 μL 0.75% low melting point agarose (Gibco BRL, Gaithersburg, MD, USA). The mixture (cell/agarose) was spread on a fully frosted microscope slide coated with a layer of 300 μL normal melting agarose (1%) (Gibco BRL). After solidification, slides were transferred to either PBS or 0.20 mM freshly prepared H2O2 solution (ex vivo treatment) for 5 min., at 4°C as described by Pereira et al. . Slides were washed three times with PBS and then placed in lysis buffer [2.5 M NaCl, 100 mM EDTA and 10 mM Tris, freshly added 1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) and 10% DMSO, pH 10.0] for 48 hr at 4°C. Subsequently, the slides were incubated in freshly made alkaline buffer (300 mM NaOH and 1 mM EDTA, pH > 13) for 20 min., at 4°C. An electric current of 300 mA and 25 V (0.90 V/cm) was applied for 15 min. to induce DNA electrophoresis. The slides were then neutralized (0.4 M Tris, pH 7.5), stained with silver  and analysed using a microscope. Images of 100 randomly selected cells (50 cells from each of two replicate slides) were analysed from each animal. The cells were also scored visually according to tail size into five classes, ranging from undamaged (0) to maximally damaged (4), resulting in a single DNA damage score for each animal, and consequently, each group was studied. The damage index (DI) can range from 0 (completely undamaged, 100 cells × 0) to 400 (with maximum damage, 100 × 4). The damage frequency (DF) was calculated based on number of cells with tail versus those with no tails . The percentage of reduction in DI was calculated for: R% = [DI saline with H2O2 − DI memantine with H2O2]/[DI saline with H2O2 − DI saline without H2O2] × 100.
Micronucleus test. The micronucleus test was performed according to the US Environmental Protection Agency Gene-Tox Program . Bone marrow from both femurs was suspended in foetal calf serum, and smears on clean glass slides were prepared as in a previous report . Slides were air-dried, fixed in methanol, stained in 10% Giemsa and coded for a blind analysis. To avoid false-negative results and as to obtain a measure of toxicity on bone marrow, the polychromatic erythrocytes/normochromatic erythrocytes (PCE/NCE) ratio was scored in 1000 cells. Micronucleus frequency (MN) was observed in 2000 PCE for each animal.
Survival assay of Saccharomyces cerevisiae yeast strains. Four strains kindly provided by E. B. Gralla (Los Angeles, CA, USA) were used in this study: wild-type (WT) strain, EG103: MATα leu2-3 112 his3Δ1 trp1-289 ura3-52 GAL+; mutant lacking cytosolic superoxide dismutase, EG118 (sod1Δ): MATα leu2-3 112 his3Δ1 trp1-289 ura3-52 GAL+sod1ΔA::URA3; mutant lacking mitochondrial superoxide dismutase, EG110 (sod2Δ): MATα leu2-3 112 his3Δ1 trp1-289 ura3-52 GAL+sod2Δ::TRP1; and mutant lacking cytosolic and mitochondrial superoxide dismutase, EG133 (sod1Δsod2Δ): MATα leu2-3 112 his3Δ1 trp1-289 ura3-52 GAL+sod1ΔA::URA3 sod2Δ::TRP1.
The survival assay was performed as described by Picada et al. . Briefly, complete YPD medium (containing 0.5% yeast extract, 2% bacto-peptone and 2% glucose) was used for routine growth of yeast cells. For plates, the medium was solidified with 2% bactoagar. Stationary-phase cultures were obtained by inoculation of an isolated colony into liquid YPD, and after a 48-hr incubation period at 30°C, the cultures were YPD grown to 1–2 × 108 cells/mL. To assess the antioxidant or pro-oxidant effects, the cells were cotreated for 1 hr with non-cytotoxic concentrations of memantine (50, 100 and 500 μg/mL) and a lethal concentration of H2O2 (0.4 mM). The cells were appropriately diluted and plated in triplicate on solid YPD. After 3 days, colony-forming units were counted. All tests were repeated at least twice, and plating was carried out in triplicate for each concentration.
Data analysis. Results are expressed as mean ± S.D, and statistical significance was determined by one-way analysis of variance (anova) complemented by the Dunnett’s test. In all comparisons, p < 0.05 was considered as indicating statistical significance.
No genotoxic effect was observed in blood samples collected 5 and 24 hr after the first administration of memantine (table 1). To evaluate the antigenotoxic activity, the blood cells were challenged ex vivo with H2O2. A decrease in DI and DF at 15 mg/kg (24 hr) and 30 mg/kg (5 and 24 hr) was observed in comparison with the saline group. The R% reached 88 in the group treated with memantine 30 mg/kg.
Comet assay in peripheral blood of mice treated with a single dose of saline or memantine (7.5, 15 or 30 mg/kg).
|Saline||12.4 ± 7.8||4.2 ± 2.7||140.0 ± 17.0||48.9 ± 6.0|
|7.5 mg/kg||7.9 ± 5.6||4.6 ± 3.5||157.4 ± 78.2||54.0 ± 25.2|
|15 mg/kg||12.1 ± 7.0||7.4 ± 5.5||93.0 ± 40.5||43.7 ± 13.9|
|30 mg/kg||13.9 ± 6.7||7.2 ± 3.9||88.1 ± 55.1* (41)||37.2 ± 20.8*|
|Saline||8.8 ± 4.8||8.8 ± 4.8||96.8 ± 18.1||60.6 ± 10.8|
|7.5 mg/kg||5.4 ± 3.4||6.2 ± 5.7||95.6 ± 68.0||49.9 ± 25.7|
|15 mg/kg||11.6 ± 9.2||9.6 ± 5.2||58.2 ± 32.0* (44)||32.5 ± 16.7*|
|30 mg/kg||14.5 ± 11.6||7.2 ± 4.1||19.8 ± 11.1** (88)||18.2 ± 9.0**|
After repetitive doses administered for 3 days, memantine did not induce DNA damage in blood or brain tissues (table 2). In the ex vivo treatments with H2O2, the blood samples from the group treated with the highest dose of memantine showed significantly lower DI in blood, as compared with the saline group. In brain tissue, a reduction in DI and DF was observed after treatment with the highest doses, reaching R% above 70.
Comet assay in mice treated with saline or memantine (7.5, 15 or 30 mg/kg) on three consecutive days.
|Saline||8.7 ± 5.3||5.0 ± 2.5||101.0 ± 27.0||44.7 ± 6.8|
|3 × 7.5 mg/kg||6.2 ± 3.8||3.5 ± 2.1||110.6 ± 29.6||47.3 ± 7.3|
|3 × 15 mg/kg||12.3 ± 6.3||6.1 ± 2.5||118.6 ± 41.3||46.8 ± 14.1|
|3 × 30 mg/kg||7.3 ± 5.6||4.9 ± 3.9||87.7 ± 19.6* (14)||43.7 ± 7.2|
|Saline||7.7 ± 5.8||6.2 ± 4.4||95.2 ± 34.2||48.8 ± 12.1|
|3 × 7.5 mg/kg||12.9 ± 9.2||7.3 ± 4.3||117.2 ± 54.5||44.7 ± 15.8|
|3 × 15 mg/kg||13.8 ± 10.6||8.3 ± 7.4||107.7 ± 45.2||49.4 ± 15.6|
|3 × 30 mg/kg||14.4 ± 11.3||9.1 ± 6.9||69.2 ± 17.6* (30)||41.7 ± 11.3|
|Saline||13.5 ± 8.7||8.3.9 ± 5.2||151.4 ± 29.1||73.1 ± 15.9|
|3 × 7.5 mg/kg||18.7 ± 12.6||17.8 ± 13.2||151.0 ± 33.7||78.1 ± 16.4|
|3 × 15 mg/kg||10.8 ± 5.3||10.0 ± 5.3||52.0 ± 20.5**(72)||42.2 ± 22.1**|
|3 × 30 mg/kg||9.9 ± 4.8||6.8 ± 4.4||38.2 ± 14.0**(82)||21.3 ± 11.1**|
As shown in table 3, there was a decrease in the PCE/NCE ratio in groups treated with higher doses of memantine. However, there was no significant increase in the micronucleus frequency in the treated groups in comparison with the saline group. The positive control group (cyclophosphamide) showed a significant increase in micronucleus frequency in PCE, agreeing with the historical values reported in our laboratory.
Micronucleus test in bone marrow of mice treated with saline or memantine (7.5, 15 or 30 mg/kg) on three consecutive days and killed 24 hr after the last administration.
|Saline||2.8 ± 0.5||3.3 ± 1.8|
|3 × 7.5 mg/kg||2.6 ± 0.8||3.1 ± 2.3|
|3 × 15 mg/kg||2.0 ± 0.9*||4.1 ± 2.3|
|3 × 30 mg/kg||1.7 ± 0.2**||4.9 ± 1.9|
|Positive control1||1.0 ± 0.1**||9.8 ± 3.1**|
Memantine enhanced the survival of the WT S. cerevisiae cells against the challenger H2O2 in a dose-dependent way, but it was not able to protect the isogenic mutant strains lacking cytosolic (sod 1Δ), mitochondrial (sod 2Δ) and both (sod1Δsod2Δ) superoxide dismutase (table 4).
Survival percentage of wild-type (WT) and lacking superoxide dismutase Saccharomyces cerevisiae isogenic yeast strains, after cotreatment of memantine (50, 100 or 500 μg/mL) with hydrogen peroxide (H2O2 0.4 mM).
|H2O2 0.4 mM||45.6 ± 17.5||61.75 ± 14.2||78.5 ± 5.2||61.7 ± 14.5|
|50 μg/mL + H2O2||89.7 ± 7.1*||66.2 ± 13.5||85.7 ± 20.1||59.3 ± 11.8|
|100 μg/mL + H2O2||96.0 ± 17.8*||93.7 ± 11.1||71.6 ± 11.1||46.6 ± 6.7|
|500 μg/mL + H2O2||112.3 ± 19.3**||82.6 ± 10.5||76.2 ± 20.6||49.4 ± 7.5|
Evidence has accumulated that lack of protection against reactive oxygen species (ROS) and lack of repair of DNA oxidative damage play a significant role in the progression of neurodegenerative diseases [1,2]. Extensive research has linked neurodegenerative diseases with a hyperactive glutamatergic system, which results in the overactivation of NMDA receptors, excessive Ca2+ influx and eventual cell death [5,28,29]. Memantine is a neuroprotective agent able to inhibit the pathological features of NMDA receptor activation, and at the same time, it allows the necessary physiological and cognitive functions to remain intact [7,17]. This drug has been shown to normalize the impairments in synaptic plasticity and cognition that typically follow excitotoxic neuronal injury [30,31]. Neuroprotective drugs as memantine with clinical application in neurological diseases could decrease genomic instability in brain tissue in addition to improvement in cognitive functions.
In this study, genotoxic/antigenotoxic effects of memantine were evaluated in mice, using the comet assay to assess DNA damage in several tissues. Peripheral blood samples were collected 5 and 24 hr after a single injection of memantine to detect possible DNA damage and its repair; however, no DNA damage was observed in any of the samples collected at different treatment times (tables 1 and 2), suggesting no genotoxic effect. To evaluate genotoxic/antigenotoxic effects in target tissue of neuroprotective actions by memantine, brain tissue was dissected 24 hr after the last administration. Similarly as in blood, there was no genotoxic effect (table 2).
The animals receiving the highest dose showed stereo-typed behaviour for about 1.5 hr after each injection. The toxicity was confirmed by decreasing the PCE/NCE ratio (table 3). In addition, in relation to the highest dose, a decrease in DNA damage was observed in blood collected 5 and 24 hr after single or repetitive treatments using comet assay with H2O2 (tables 1 and 2). This effect was more evident at 24 hr than at 5 hr, suggesting that memantine might induce adaptive responses resulting in protection against H2O2. Also, a decreased DI and DF in brain tissue was observed (table 2), most likely for the same reason.
Previous studies using memantine doses reaching up to 20 mg/kg have shown neuroprotective effects of this drug . Memantine improves the survival of rat neurons exposed to glutamate ; protects rats from carbofuran-induced hypercholinergic behavioural activity, including seizures, by blocking pathways associated with oxidative damage in neurons ; prevents the increase in biomarkers of ROS (F2-isoprostanes and F4-neuroprostanes) induced by the organophosphate diisopropylphosphorofluoridate in rat brain  and reduces protein carbonylation in the cerebral cortex and the hippocampus of aged rats . Recent studies have demonstrated that memantine diminishes neuronal cell damage evoked by staurosporine and doxorubicin [34,35]. Corroborating those studies, memantine at 3 × 15 mg/kg decreased DNA damage in brain tissue (table 2). In this dose, no stereo-typed behaviour or signals of toxicity were observed, suggesting that the decrease in the brain DNA damage might be associated with neuroprotective effects of memantine.
Micronucleus frequency increased, though without statistical significance, suggesting that memantine was unable to induce mutagenicity (table 3). The lowest dose used in this study (7.5 mg/kg) is equivalent to 25 times the therapeutic dose, because the usual dose in human beings is 20 mg/day (0.3 mg/kg for human beings of 70 kg). This dose leads to neither toxicity nor mutagenic effects in mice. Additionally, there was no antigenotoxic effect in blood and brain tissues in the lowest dose using comet assay (table 2).
To evaluate protective antioxidant effect of memantine, we used the model eukaryote S. cerevisiae, which has been shown to be ideal to investigate oxidative stress responses, not only because it has been genetically well defined but also because its defence systems against ROS are well characterized, including both enzymatic and non-enzymatic antioxidants [27,36]. Here, WT S. cerevisiae yeast strain and defective in superoxide dismutase enzymes were treated with memantine and H2O2 simultaneously. As shown in table 4, there was a higher sensitivity of WT to H2O2 in comparison with mutants. Some antioxidant defences in these mutants have been higher in comparison with WT, like total glutathione and glutathione peroxidase, which could increase the efficiency to detoxify H2O2 .
Memantine was shown to protect WT S. cerevisiae yeast strain (table 4). In single and double mutant strains lacking superoxide dismutase, memantine was unable to protect against hydrogen peroxide-induced damage. Results using in vitro systems to evaluate antioxidant properties of aminoadamantanes have suggested that this class of drugs may not act as free radical scavengers or as singlet oxygen quenchers in protecting brain cells from the deleterious influence of ROS produced at glutamate-activated receptor sites . Apomorphine, a dopamine receptor agonist, enhanced the survival of WT and its isogenic strains lacking superoxide dismutase in cotreatment with H2O2, and those results were attributed to its scavenger ability . In the present study, only WT was protected. Thus, an increase in antioxidant defence in WT elicited by memantine leading to protection against H2O2 appears to be more likely than an antioxidant direct action.
An interaction with intracellular signal transduction seems to be indicated, which could lead to protective actions against ROS . In a study using lipopolysaccharide infusion to induce neuroinflammatory damage, Rosi et al.  showed that memantine decreases the probability of microglial activation, preventing the release of arachidonic acid and pro-inflammatory factors from neurons. Memantine increased the levels of brain-derived neurotrophic factor mRNA, which may mediate its neuroprotective effects , reversed loss of cell viability and decreased caspase-3/7 activities and ROS level induced by catechol (a component from cigarette smoke) in Müller cells (MIO-M1) .
In conclusion, memantine did not induce mutagenicity or DNA damage in blood or brain tissues, and it was able to protect against hydrogen peroxide-induced oxidative damage. These results corroborate the findings obtained in other studies, which have shown neuroprotective activities of memantine, besides its actions on memory and cognitive functions.
This work was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FAPERGS (Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul), Brazil.