Memantine Is Protective against Cytotoxicity Caused by Lead and

Subscriber access provided by University of Victoria Libraries

Article

Memantine is protective against cytotoxicity caused by lead and quinolinic acid in cultured rat embryonic hippocampal cells Abdur Rahman, Sara Al-Qenaie, Muddanna S Rao, Khalid M Khan, and Gilles J. Guillemin Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00421 • Publication Date (Web): 05 Apr 2019 Downloaded from http://pubs.acs.org on April 7, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

Memantine is protective against cytotoxicity caused by lead and quinolinic acid in cultured rat embryonic hippocampal cells

Abdur Rahman*1, Sara Al-Qenaie1,2, Muddanna S. Rao3, Khalid M. Khan3, Gilles J. Guillemin4 1Department

of Food Science and Nutrition, College of Life Sciences, Kuwait University Oil Company Hospital, Ahmadi, Kuwait 3Department of Anatomy, Faculty of Medicine, Kuwait University 4Neuroinflammation group, Faculty of Medicine and Health Sciences, Macquarie University, NSW, Australia 1,2Kuwait

*Corresponding Author: Abdur Rahman Department of Food Science and Nutrition College of Life Sciences Kuwait University P.O. Box 5969 Safat 13060, Kuwait Phone: +965 2463 3055 Fax: +965 2251 3929 Email: [email protected]

1

ACS Paragon Plus Environment

Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Content Graphic:

2


Page 2 of 36

Page 3 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Abstract Quinolinic acid (QA) is an excitotoxic metabolite of the kynurenine pathway of tryptophan metabolism produced in response to inflammation and oxidative stress. Lead (Pb) causes oxidative stress and thus may produce neurotoxicity by increasing QA production. We investigated the in vitro cytotoxic effects of Pb and QA and the protective effects of the NMDA receptor antagonist memantine. Primary cultures of embryonic hippocampal cells from Wistar rats were treated with different concentrations of Pb, QA and Pb+QA with and without memantine. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT). Apoptosis was analysed by flow cytometry after AnnexinV/propidium iodide staining. The numbers of immunostained neurons (with β3-Tubulin; Tuj1) and astrocytes (with glial fibrillary acidic protein) were counted. Pb at 20µg/dL (0.97μM) and QA at 500nM concentrations showed significant cytotoxic effects, as evidenced by decreased cell viability, increased apoptosis, and a decrease in the number of both astrocytes and neurons. The combination of Pb and QA showed significant synergistic apoptotic effects at lower doses. Memantine (500nM) was largely protective against the cytotoxic effects of both Pb and QA, suggesting that Pb’s and QA’s cytotoxicity involves NMDA receptor activation. Whereas the neuroprotection by memantine from QA-induced toxicity has been previously reported, this is the first study reporting the protection by memantine against Pb-induced cytotoxicity in cultured hippocampal cells. Protection by memantine against these neurotoxicants in vivo needs to be investigated.

Keywords: Quinolinic Acid, Lead, Memantine, Cell cycle, Viability, Apoptosis, Cultured Hippocampal Cells, Neurons, Astrocytes

3



Introduction: Lead (Pb) is a neurotoxic heavy metal1. Despite tremendous efforts, particularly in developed countries, to minimize the burden of Pb in the environment, Pb exposure is still a public health problem; particularly in children2-4. The central nervous system (CNS) is most sensitive to Pb toxicity, particularly during early development, and is affected by low doses5-8. Lower cognitive function during adolescence or childhood is associated with various adverse health outcomes in adulthood, with severe public health consequences9-12. The current blood Pb level of concern in children has been set at 5µg/dL13. The mechanism by which Pb affects CNS, in particular learning and memory, remains speculative despite being thoroughly investigated1. Quinolinic acid (QA) is a metabolite of the kynurenine pathway (KP) of tryptophan metabolism. In physiological conditions, approximately 95% of the brain’s tryptophan is metabolized by the KP, resulting in generation of the essential co-factor nicotinamide adenine dinucleotide (NAD+)14. However, during neuroinflammations, this pathway is diverted to produce several other metabolites with neuroactive properties15. Of these, QA is an N-methyl-D-aspartic acid receptor (NMDAR) agonist and is known to cause excitotoxicity in many pathological conditions15. Increased levels of QA are implicated in several neurodegenerative diseases such as Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis, AIDS-dementia, depression and schizophrenia15-22. QA is toxic to astrocytes23, oligodendrocytes24, and neurons25, 26,

particularly in the hippocampus, striatum and neocortex15. QA-induced apoptosis has also

been reported in these cells27-29.

4


Page 4 of 36

Page 5 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Both Pb and QA are neurotoxic and share several features of neurotoxicity. For example, both Pb and QA impair learning and memory 30-33, destabilize the cytoskeleton21, 34, trigger oxidative stress14, 35, 36, and increase synaptic glutamate levels15, 37. Pb, a pro-oxidant metal, may increase QA levels by microglial and astroglial activation and increase the expression of proinflammatory cytokines like interlukin-1 (IL-1), interlukin-6 (IL-6), tumor necrosis factor- (TNF-) and interferon gamma IFN-γ38-41. These cytokines are known to activate the KP and increase production of QA14, 35, 42, 43. We have recently shown increased production of QA in the blood and brain of rat pups exposed to Pb during the early postnatal period44. As QA is an NMDAR agonist, we hypothesized that NMDAR antagonists will mitigate the neurotoxic effects of Pb. The objectives of this study were (1) to investigate the effect of memantine, an NMDAR antagonist, on neurotoxicity and gliotoxicity caused by Pb and QA (used alone and in combination) in cultured rat embryonic hippocampal cells and (2) to investigate if Pb and QA in combination have synergistic cytotoxic effects on cultured hippocampal cells. Methods Animals: Adult (3 months old) male and female Wistar rats, provided by the Animal Resources Center, Faculty of Medicine, Kuwait University, were used for getting embryonic day-18 (E18) embryos. The rats were housed at constant temperature (21 ± 2°C) and relative humidity (50 ± 10%) with a 12-hour light/dark cycle (0700–1900 h). Animal maintenance and exposure were according to the protocol approved by the Animal Care and Use Committee of Kuwait University and the experimental protocol followed the ARRIVE guidelines for the care and use of laboratory animals. Rats were mated (two males and two females) and, after pregnancy was confirmed by vaginal smear method, pregnant rats were transferred to individual cages.

5



Primary cell culture: Primary cultures of the embryonic hippocampi were prepared by dissecting the fetuses at E18. Pregnant rats were euthanized with CO2; the anterior abdominal walls were sterilized with 70% ethanol and the fetuses were removed by laparotomy. The fetuses were collected and washed in ice cold sterile saline; the hippocampi were dissected in ice-cold Dulbecco's Modified Eagle's media-F12 (DMEM-F12) (Gibco Life Technologies, USA) under a stereomicroscope. The fetal hippocampal tissue (20 hippocampi) was gently triturated mechanically with a fire-polished pasture pipette in 5mL ice-cold media. Cell suspension was centrifuged, and the cell pellet was re-suspended in the DMEM media. Viable cell density was estimated by trypan blue staining and counting the dead and live cells in a Neubauer hemocytometer. Final cell seeding density was 5000 cell/mL media. The fetal hippocampal cells were cultured in DMEM-F12 medium, supplemented with 10% heat inactivated fetal bovine serum and 100U/ml of penicillin/streptomycin (all from Gibco Life Technologies, USA). For viability assays, cells were cultured in 24 well-collagenase-II coated culture plates (in 500µL media/well). For apoptosis and cell counting, cells were cultured in 60mm culture dishes in 2mL media/dish (Nunc™ Cell-culture treated, Thermo Fisher Scientific MA, USA). The cultures were maintained in a humidified 5% CO2 incubator at 37oC. The culture media was replaced 24 hours after culturing and every 72 hours thereafter. After 10 days, the cultures were exposed to Pb [10μg/dL (Pb10) and 20μg/dL (Pb20)], QA [250nM (QA250) and 500nM (QA500] or different combinations of Pb+QA (Pb10+QA250, Pb10+QA500, Pb20+QA250 and Pb20+QA500) for 7 days. Appropriate control cultures were maintained for comparison. In a separate experiment, 10-day-old mixed cell cultures were exposed to Pb, QA or Pb+QA as in the above experiment but were treated along with memantine (500nM). Stock solutions of Pb (10µg/mL; Sigma-Aldrich, St. Louis MO, USA), and QA

6


Page 6 of 36

Page 7 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(500µM; Sigma-Aldrich, St. Louis MO, USA) were prepared in sterile distilled water and further diluted appropriately in culture media to get the above final concentration. The cultures were terminated after 7 days of treatment for various end-point assays. Three independent experiments were done. For each group triplicate samples were analysed. Cell Viability: Cell viability was quantitated by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay45. At termination of the experiments, the control and treated cultures were washed three times with sterile phosphate buffered saline (PBS), and incubated with 250µL (0.5mg/ml) of MTT solution (Sigma-Aldrich, St. Louis MO, USA) for 30 min. After washing with PBS, the formazan crystals formed were solubilized with 100µL dimethyl sulphoxide (DMSO) (Sigma-Aldrich, St. Louis MO, USA) and transferred into 96 well plates. Absorbance was read at 570 nm by an automated ELISA reader (Sunrise-Basic Tecan with Magellan software, Tecan Austria GmbH, Qrodig, Austria). For each group triplicate cultures were used for quantification. Apoptosis: At termination of the experiment, the control and treated cultures were stained with an Annexin-V antibody and propidium iodide (PI) using an Annexin-V/FITC staining kit (APOAF-20TST, Sigma-Aldrich, St. Louis MO, USA). Media from the culture dishes were removed and collected in labelled centrifuge vials to collect the floating cells. For harvesting the adherent cells, culture dishes were washed two times with PBS, trypsinised by incubating in 250µL Triple-Express solution (Gibco Life Technologies, USA) at 37oC for 10 minutes, neutralized with fresh media and centrifuged at 2500 rpm for 5 minutes at 4oC. Adherent and floating cells were combined and were pelleted by centrifugation at 2500 rpm for 5 minutes. The annexin staining solution (250µL of 1X binding buffer + 2.5µL of Annexin-V/FITC + 5µL of PI) was added to each culture vial and incubated for 10 mins at room temperature. The cells were

7



transferred into centrifuge tubes and analysed by flow cytometry. For each group triplicate cultures were used for quantification. Quantitation of neurons and astrocytes: The control and treated cultures were fixed with 2% paraformaldehyde and were immunostained for neurons, with mouse anti-β-3 tubulin (Tuj1, Abcam, Cambridge, United Kingdom), and astrocytes with rabbit anti-GFAP (Abcam, Cambridge, United Kingdom). The culture dishes were washed with PBS. Nonspecific antibody binding was blocked with blocking solution containing 0.01% triton-x 100, 5% normal goat serum (For Tuj1) or 5% normal horse serum (For GFAP) for 30 minutes at room temperature. The dishes were incubated with mouse anti-β-3 tubulin (Tuj1, 1:1000) or rabbit anti-GFAP (1:500) antibody overnight at 4oC. Dishes were washed three times with PBS and incubated with biotinylated horse anti-mouse (1:200) or goat anti rabbit (1:200) secondary antibodies, respectively. After washing with PBS, cultures were incubated with Avidin Biotin Complex (ABC kit, Vector Laboratories, Burlingame, CA). Diaminobenzidine was used as a chromogen. The number of immunostained neurons and astrocytes in the culture dishes were quantified under a Zeiss inverted microscope at 40x magnification. In each dish, the number of immunostained neurons or astrocytes in 10 non-overlapping microscopic fields was counted. Finally, the total number of stained cells for the entire dish area (21.5mm2) were calculated. For each group duplicate cultures were used for quantification. Evaluation of synergistic effects of Pb and QA: To evaluate the synergistic effect of Pb and QA combinations, the coefficient of drug interaction (CDI) was calculated according to the equation CDI= AB/(A× B), where AB is the relative effect of the combination effect on various parameters; and A or B are the relative effects of the single treatment, either Pb or QA. A CDI of

Memantine Is Protective against Cytotoxicity Caused by Lead and

Recommend Documents