Metronidazole-induced neurotoxicity: is iron a contributing factor?

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Moses W Bariweni, Vinood B Patel, Gulrez M Zariwala, Raymond I Ozolua This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5757381/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Metronidazole-induced neurotoxicity is a rising challenge in the management of susceptible infections. The mechanisms involved in metronidazole-induced neurotoxicity are not fully unraveled. This study was aimed at determining the effect of metronidazole on iron homeostasis in SH-SY-5Y neuroblastoma cells. Methods Confluent SH-SY-5Y neuroblastoma cells were treated with 1, 10, 25, 50, 100, 250 µM concentrations of metronidazole only or in combination with 20 µM iron. DMSO or culture media was used as control. Viability and ferritin assay were conducted on the treated cells. The treatments were for 24 hr, 48 hr and 72 hr respectively. Results In the viability assay, doses of metronidazole reduced viability of SH-SY-5Y neuroblastoma cells in a time and concentration dependent manner. After 24 hr treatment, 250 µM metronidazole reduced ( P < 0.001) cell viability while 50 µM, 100 µM and 250 µM metronidazole reduced ( p < 0.01, p < 0. 001) viability only after 48 and 72 hr compared with control. Doses of metronidazole 50 µM, 100 µM and 250 µM in 20 µM iron reduced viability in a time dependent manner in all the tests periods. Metronidazole also induced a time and concentration dependent increase ( P < 0.05) in cellular iron uptake in the 48 and 72 hr treated cells in concentrations above 25 µM metronidazole. Conclusion It is concluded that metronidazole induces a time and concentration dependent iron overload and consequent cell death in SH-SY5Y neuroblastoma cells and this may contribute to the mechanism of metronidazole-induced neurotoxicity. Iron Neurotoxicity Ferroptosis Ferritin Metronidazole Neuroblastoma cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background In vitro cell culture techniques have been successfully developed in the neurobiology field to address specific questions of cell biology and nervous system functioning. This has formed a basis to systematically study the nervous systems, revealing the complexity of cellular functions in the CNS. It has also provided a convenient experimental tool for studying defects and disease processes in the CNS [ 1 ]. Various techniques abound for the study of neurotoxicity using dedicated cell lines. Iron is an essential molecule with pro-oxidant characteristics, it catalyses synthesis of hydroxyl radicals in the intracellular environment owing to its reductive role in the Fenton reaction [ 2 , 3 ]. The brain consumes the largest amount of total oxygen in the body due to its high respiratory or oxidative metabolic ability, consequently it generates a large volume of reactive oxygen species [ 4 ]. Brain iron is mainly bound to ferritin and only unbound iron is physiologcally active in the brain. Hence ferritin is recognised as the protein responsible for brain iron homeastasis. The mechanism of metronidazole-induced neurotoxicity is still under research as the various proposed mechanisms have not answered all the questions arising from its occurrence. One of the proposed mechanisms of metronidazole-iduced neurotoxicity is interference with oxidative processes in the nervous system [ 5 – 8 ]. Metronidazole is a prodrug which undergoes reductive activation mediated by pyruvate:ferrodoxin oxidoreductase (PFOR) whose expression is positively regulated by iron [ 9 ]. Iron is ubiquitous and occurs in high abundance in the central nervous system. Iron overload is implicated as a major contributor to neurotoxicity and neurodegeneration as evidenced in several neurologic conditions [ 10 , 11 ]. This study is aimed at studying the effect of metronidazole on iron uptake in the human neuroblastoma SH-SY5Y cells. Materials Drugs and chemicals used include metronidazole (Sigma-Aldrich, Germany), Han’s F12 media (F12) (Sigma-Aldrich, Germany); Dulbecco’s minimum essential medium (DMEM) (Thermo-Fisher, USA); ferritin ELISA kit (Sigma-Aldrich, Germany); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kit (Sigma-Aldrich, Germany); Dulbecco phosphate-buffered saline (DPBS) (Sigma-Aldrich, Germany); serum free minimum essential medium (MEM) (Gibco, New York, USA); ferrous sulphate powder (Sigma-Aldrich, Germany); BCA assay kit (Sigma-Aldrich, Germany), GlutaMax (Gibco, USA). All reagents and chemicals used in the experiments were of analytical grade. Cell line Human neuroblastoma SH-SY5Y cells (CRL-2266, ATCC’ Rockville, USA) was used for the study and they were purchased from Gibco, USA. Their use required no institutional ethical approval. Methods Cell culture Unless stated otherwise, cells were grown in Dulbecco’s minimum essential medium (DMEM) in Han’s F12 media at pH 7.4, supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin solution and maintained at 37°C in 5% CO 2 at constant humidity, refreshing the medium every four days until the cells were confluent. Differentiation of the cells was initiated 48-hour after the initial plating by substituting the growth medium with serum free neurobasal medium (containing B27 supplement and GlutaMAX) and 10 µM all-trans-retinoic acid (ATRA). The cells were allowed to grow in ATRA-containing neurobasal medium for 5 days, refreshing the medium every 48-hour[ 12 ]. The confluent cells were subsequently passaged to maintain their viability and increase the quantity available for experiments. Treatment of cells with metronidazole Completely differentiated cells grown in 96-well plates were removed from the incubator, transferred to the hood, and the old media removed from the plates with a pipette and disposed into a bleach bottle. A quantity (200 µL) of each designated concentration (1, 10, 25, 50, 100, 250 µM) of the test media was added to each of the 6 replicate wells using a multichannel pipette. The plates were incubated for 24 h, 48 h and 72 h in the cell culture incubator. DMSO (200 µL) was used as negative control in the experiments. Treatment media was refreshed for the plates in the 48-hour and 72-hour groups. After the desired incubation period, the plates were removed from the incubator and the test media was aspirated and the cells harvested for endpoint assays to determine the effects of treatment on the cells. Protein quantification was done using the bicinchoninic acid assay method. Viability assay of SH-SY5Y following treatment with metronidazole The reagents for MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was prepared according to the manufacturer’s instructions. After incubating the plates for 24, 48 or 72 hours respectively in the test drug, they were removed from the cell culture incubator. The test substance was aspirated into a bleach bottle and the wells washed with DMSO. Each well was treated with MTT 20 µL /well and incubated for 4 hours at 37°C. After the incubation period, the plates were removed from the incubator, the media was gently aspirated into the bleach bottle without disrupting the cell monolayer. An aliquot (100 µL) of DMSO was added to each well and mixed properly with a pipette. The wells were covered with foil to avoid light and incubated in a rotary shaker for 15 minutes. The plates were then removed from the shaker and the absorbance measured at 570 nm in a spectrophotometer (Thermo-Fischer, UK) [ 13 ]. The effect of metronidazole on iron uptake A standard iron solution (1M) was prepared by dissolving 1.52 g ferrous sulphate powder in 10 mL 0.1 M HCl. Completely differentiated cells were incubated in serum free minimum essential medium (MEM) at pH 5.8 for 24-hour at 37 o C. After the incubation period, the cells were treated with filtered aliquots of metronidazole in MEM plus prepared iron solution to give a final concentration of the different test conditions in 20 µM elemental iron and all the wells buffered to pH 5.8. A positive control (iron solution in MEM) and negative control (MEM only) were also included in the plates. The plates were incubated on a rotary shaker (6 g ) at 37 o C for 24, 48 and 72 hours and the media refreshed for the 48-hour and 72-hour groups respectively. On completion of incubation period, the plates were removed, and the media aspirated into the bleach bottle. The plates were washed twice with wash solution and the media replaced with serum-free MEM and incubated for 24 hours. After incubation, the cells were washed, lysed and the supernatant collected in aliquots for ferritin ELISA and bicinchoninic acid (BCA) assay [ 13 ]. The plates for MTT were not lysed after washing rather, they were treated with MTT reagent and assayed as previously described. Ferritin assay The manufacturer’s instructions were followed in the preparation of reagents and standards for the ferritin assay. Ferritin standards (50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 and 0.00 ng / mL) were prepared by serial dilution method starting from 50 ng/mL stock. An aliquot (50 µL) of ferritin standard or sample were added in duplicates per well and the plate tapped gently to thoroughly coat the wells and incubated for two hours at room temperature. The plate was washed five times with 200 µL of the wash buffer and gently tapped on an absorbent paper towel to completely remove the liquid after the last wash. The biotinylated ferritin antibody (50 µL) was added to each well and incubated for one hour at room temperature. The plates were washed five times as previously done followed by the addition of 50 µL SP-conjugate to each well. All bubbles were removed by tapping and the plates incubated at room temperature for 30 minutes. After the incubation each plate was washed as above. Some chromogen substrate (50 µL) was added to each well and spread well by tapping to remove bubbles, then each plate was incubated at room temperature for 12 minutes. At the end of the incubation, 50 µL of the stop Solution was added to each well and the plates read immediately at 450 nm (MicroPlate Reader 550, Bio-Rad Laboratories, Denmark) [ 14 , 15 ]. Statistical analysis Results are presented as mean ± standard error of mean (SEM). Statistical analysis was done using one-way ANOVA followed by Dunnet’s post hoc test for multiple comparisons (GraphPad Prism 6 Software, San Diego California USA). Correlations for protein analysis were calculated by Pearson’s test. Statistical differences between compared data were considered significant at p < 0.05. Results Metronidazole reduces viability of SH-SY5Y cells dependent of time and concentration In the cell viability experiments, metronidazole exhibited time and concentration dependent effects on the treated cells, the viability of the treated cells reduced with increase in metronidazole concentration, the 250 µM treated cells were the least viable (< 10% viability across all treatment periods). The effect of treatment duration on cell viability of identical metronidazole concentration was marginal at concentrations of 25 µM and below. After 24 h treatment, 250 µM metronidazole reduced ( P < 0.001) cell viability compared with the growth media-treated cells (Fig. 1 ). Cell viability ranged between 8.9 to 83.2% after 48 h treatment (Fig. 2 ) with a significant reduction in viability for the 50 µM, 100 µM and 250 µM ( p < 0.01, p < 0. 001) treated cells. These effects were similar in the 72 h treated cells with viability ranging between 7.5 to 78.5% (Fig. 3 ). Metronidazole increases SH-SY5Y cell iron uptake. In determining the effect of metronidazole on cellular iron uptake, cytotoxicity experiments were conducted to determine the viability of SH-5YSY cells when concomitantly treated with the various concentrations of metronidazole and 20 µM ferrous sulphate. Treatment with metronidazole and ferrous sulphate yielded a concentration and time dependent effect on the treated cells with cell viability reducing with increase in dose and time of treatment. After 24 h incubation, cell viability reduced ( P < 0.05, 0.001) in the 50, 100 and 250 µM metronidazole treated wells (Fig. 4 ). The effect of co-culturing the cells in metronidazole and ferrous sulphate was more pronounced after 48 h as shown in Fig. 5 . Metronidazole concentrations of 50 µM and above caused a reduction ( P < 0.01, 0.001) in cell viability. The reduction in cell viability seen after 72 h incubation with metronidazole and ferrous sulphate was more intense with 100 and 250 µM concentrations (Fig. 6 ). The total iron absorption data is shown in Figs. 7 , 8 and 9 . Metronidazole induced slight increase in iron uptake into the cells after 24 h treatment although the changes were not statistically different from the iron only treated cells (Fig. 7 ). However, metronidazole induced a time and concentration dependent increase ( P < 0.05) in cellular iron uptake in the 48 (Fig. 8 ) and 72-h treated cells and in concentrations above 25 µM metronidazole. In the 72-h treated group (Fig. 9 ), 1 µM metronidazole treated cells had reduced iron absorption compared with the iron only treated cells. Discussion Although, the various effects, targets and modes of action of metronidazole are well characterized in bacteria, the consequences and how metronidazole interacts with mammalian cells is still not fully deciphered. In this study, human neuroblastoma (SH-SY5Y) cells were employed to elucidate the effect of metronidazole on cell viability and iron uptake. SH-SY5Y neuroblastoma cells are a replicated offspring of SK-N-SH cells originally isolated from bone marrow biopsy of a neuroblastoma patient in the early 1970s [ 16 ]. This cell line has been extensively used as a neuronal model since the early 1980s, as they possess many biochemical and functional properties of human neurons [ 17 , 18 ]. They are useful in assessing morphological and many differentiated neuronal functions hence their usefulness in neurotoxicity screening of parent drug molecules [ 19 ]. Cytotoxicity testing is an essential procedure in cell-based neurotoxicity testing. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay is a high throughput screening model for determination of cell viability in toxicity testing [ 20 ]. Healthy growing cells with intact metabolic capacity convert MTT to an insoluble purple-colored formazan product which accumulates inside cells. Dead cells lose the capability to convert MTT to formazan, hence formation of the insoluble color serves as an accessible and convenient marker for healthy growing cells [ 21 ]. The quantum of formazan formed is directly proportional to the number of healthy growing cells as determined by the optical density at 570 nm using a plate reading spectrophotometer [ 22 ]. In this study, 50 µM metronidazole and concentrations above it exhibited a time and concentration-dependent effect on the treated cells. There is paucity of reports on the outcome of metronidazole treatment on SH-SY5Y cells. Xiao et al. , (2018) reports that metronidazole induced apoptosis of neuronal cell cultures (PC12, human primary neurons and CAD) by increasing the expression of annexin-v at a concentration of 40 µg/mL [ 23 ]. This may be a possible effect of metronidazole on the SH-SY5Y cells, however more research into the genotoxicity of metronidazole on SH-SY5Y needs to be done to ascertain its effects on the cellular componenets of SH-SY5Y cells. Iron is vitally important for most life forms and is broadly utilized by different proteins to execute a number of functions including respiration, metabolism and synthesis of new proteins, in essence iron is essential for survival of living systems [ 24 ]. Dysfunction in iron metabolism has been implicated in several disease states [ 25 , 2 ]. Normally, iron is absorbed into cells by means of the divalent metal transporter 1 (DMT1), which is a steadfastly modulated process. Additionally, a new iron transporter ferropotin (IREG1) has been identified recently. It is reported that both DMT1 and IREG1 exist in the central nervous system and is expressed on neurons, glia cells and astrocytes are thought to be responsible for maintaining iron homeostasis in the CNS [ 26 , 27 ]. These transporters have been characterized also in SH-SY5Y cells, and IREG1 is thought to mediate iron efflux while DMT1 is thought to mediate iron influx in SH-SY5Y cells [ 28 ]. Excessive iron aggregation can activate oxidative stress and cell damage as it reacts with hydrogen peroxide to produce the hydroxyl radical and other ROS in the Fenton reaction [ 2 , 3 ]. Iron accumulation and reduced viability of SH-SY5Y cells have been reported recently following treatment with iron (1.5–80 µM). Also, DMT1 expression decreased but persisted even at 80 µM while IREG1 expression increased with increased cellular iron content [ 28 ]. This implies that iron influx did not stop completely even at very high cellular iron concentration though an increased efflux process seemed to have been activated. In this study, the presence of metronidazole caused a drastic decline in cell viability compared to the cells treated with only iron. It is possible to then suggest that metronidazole favors an increase in DMT1 expression as opposed to IREG1 expression in the presence of iron and subsequent oxidative stress and cell death arising from iron accumulation. Iron is stored in the cytosol or in mitochondria as ferritin. Ferritin has recently been shown to play a role in iron homeostasis, immunomodulation, inflammation and antioxidation [ 3 ]. Ferritin structurally consists of an outer shell known as apoferritin and a hollow inner-core which serves as a store for iron. The apoferritin shell also contains ferroxidase which converts iron from its active ferrous (Fe 2+ ) form into the inactive ferric (Fe 3+ ) form for storage [ 29 ]. Experimental evidence implicates ferritin as the protein responsible for iron delivery to the brain [ 30 ]. In low iron conditions, the ferritin molecule is degraded by activation of the lysosomal- autophagy pathway where nuclear receptor coactivator-4 binds to ferritin and causes the release of iron into the cytosol [ 29 , 30 ]. Elevated ferritin levels have been implicated in various neuropsychiatric and neurodegenerative diseases in recent times [ 10 , 11 ]. In such cases, the storage capacity of ferritin is thought to be overwhelmed resulting in an increased concentration of unsequestered iron in the cytosol with resultant production of free radicals and oxidative stress [ 10 , 30 , 29 ]. Aguirre et al (2005), reports a four-fold increase in ferritin levels in SH-SY5Y cells treated with iron (1.5-5.0 µM) and a ten-fold increase for iron concentrations higher than that resulting in a commensurate elevation of the labile iron pool [ 28 ]. Metronidazole undergoes reductive activation to form reactive intermediates which are toxic to susceptible organisms in anerobic conditions. Several enzymes suggested to be involved in the reductive activation of MTZ include the pyruvate: ferredoxin oxidoreductase (PFOR) which catalyzes electron transfer via its iron-sulphur clusters resulting in the generation of nitro-radical anion as metronidazole serves as a receptacle of electrons released by the action of PFOR [ 31 , 9 ]. PFOR is a key enzyme in the metabolic pathway of many anaerobic microorganisms and some parasites. It plays a critical role in the conversion of pyruvate to acetyl-CoA for energy production in anaerobes. PFOR expression is thought to increase in the presence of iron, this is corroborated by the increased conversion of metronidazole to its active nitro-radical forms with consequent increase in its antibacterial activity [ 9 ] and toxic radical production [ 32 ]. In this study, there was a concentration and time-dependent elevation of ferritin levels in the metronidazole-plus-iron treated cells compared to the iron only treated cells. It is hence suggested that metronidazole induced iron overload as evidenced by the increased ferritin concentration in the treated cells with a consequent decrease in cell viability. Conclusion SH-SY5Y cells are known to possess an active system responsible for regulation of iron homeostasis. Iron overload is implicated in several disease conditions affecting the brain and neurons in particular. Ferritin is the main storage protein for iron in brain and neuronal structures and correlates with the amount of iron in circulation in these cells. In this study, metronidazole induced a time and concentration dependent decrease in SH-SY5Ycell viability and an increase in ferritin concentration in the treated cells. Thus, we conclude that metronidazole induces iron overload and consequent cell death in SH-SY5Y cells and this may contribute to the mechanism of metronidazole-induced neurotoxicity. Abbreviations ATRA- all-trans-retinoic acid; MEM -Minimum essential medium; BCA- Bicinchoninic acid; DMEM- Dulbecco’s minimum essential medium; ELISA- enzyme linked immunoassay; ROS- reactive oxygen species; PFOR- pyruvate-ferredoxin oxidoreductase; MTT- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; DMSO- dimethylsulfoxide; DMT1- divalent metal transporter 1; IREG1- iron transporter ferropotin; FBS- foetal bovine serum; CNS- central nervous system. Declarations Author Contribution MWB and RIO designed the study, MWB, VBP and GMZ participated in the experiments and data collection, RIO, GMZ and VBP analyzed the data, MWB and RIO wrote the manuscript, all authors read and approved the final manuscript. Data Availability All data generated or analyzed during this study are included in this manuscript References Barbosa DJ, Capela JP, Bastosa M, Cavarlho F. In vitro models for neurotoxicology research. Toxicol Res. 2015; 4(4):801-42. Rouault T. Biogenesis of iron–sulfur clusters in mammalian cells: new insights and relevance to human disease. Dis Models Mech.2012;5(2):155–64. Bresgen N, Eckl PM. Oxidative stress and the homeodynamics of iron metabolism. Biomolec. 2015;5(2): 808–47. Singh N, Haldar S, Tripathi A, Horback K, Wong J, Sharma D, et al . Brain iron homeostasis: from molecular mechanisms to clinical significance and therapeutic opportunities. Antioxid Redox Signal. 2014; 20:1324–63. 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Chapman A, Cammack R, Linstead R, Lloyd D. The generation of metronidazole radicals in hydrogenosomes isolated from Trichomonas vaginalis. J Gen Microbiol. 1985;131(9): 2141–44. Moreno S, Mason R, Docampo R. Nitroimidazole cellular activities. J Biol Chem. 1984;256: 6298-6305. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5757381","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":402077980,"identity":"3a9977ca-7f1e-41b7-afd0-4ef171d18456","order_by":0,"name":"Moses W Bariweni","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYDACCSBOAGI29gYwm7GBeC08B0jRAmEkEKlFd3bzsw8P22zy+CTfHrzNw2Aju+EA7+EX+LSY3TlmPCOxLa2YTTov2ZqHIc14wwG+NAu8Wm4kGDMkbjuc2CadYybNw3A4ccMBHjMD/FrSPwO1/E9skzwD0vKfGC05IFsOJLZJ8IC0HABpMX6A3y9nihkS/yUntvHkGFvOMUg2nnmYxwyfDgaz2+2bGX+csUuc337G8MabCjvZvuM9xh/w6kEFIE8wM7BJEFSIDphJsWUUjIJRMAqGPwAA2yRJy1cgtbUAAAAASUVORK5CYII=","orcid":"","institution":"Niger Delta University","correspondingAuthor":true,"prefix":"","firstName":"Moses","middleName":"W","lastName":"Bariweni","suffix":""},{"id":402077981,"identity":"056216f9-ce88-4ffa-b12e-2c9ee3190fcf","order_by":1,"name":"Vinood B Patel","email":"","orcid":"","institution":"University of Westminster","correspondingAuthor":false,"prefix":"","firstName":"Vinood","middleName":"B","lastName":"Patel","suffix":""},{"id":402077982,"identity":"1bfd44d5-b0be-4f43-82f5-c6fda37c4e8c","order_by":2,"name":"Gulrez M Zariwala","email":"","orcid":"","institution":"University of Westminster","correspondingAuthor":false,"prefix":"","firstName":"Gulrez","middleName":"M","lastName":"Zariwala","suffix":""},{"id":402077983,"identity":"387b7cae-c53a-4e00-b241-884dd2e04835","order_by":3,"name":"Raymond I Ozolua","email":"","orcid":"","institution":"University of Benin","correspondingAuthor":false,"prefix":"","firstName":"Raymond","middleName":"I","lastName":"Ozolua","suffix":""}],"badges":[],"createdAt":"2025-01-03 10:08:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5757381/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5757381/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73861328,"identity":"c6f3eef1-263a-4161-8b06-5d614934a4d5","added_by":"auto","created_at":"2025-01-15 11:02:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":112047,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability after 24 hours treatment with concentrations of metronidazole. \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u0026lt;\u003c/em\u003e0.001 compared with minimum essential media (MEM). 4 x 10\u003csup\u003e3\u003c/sup\u003e cells/well. Met: metronidazole.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5757381/v1/4d100bcab4249341229c62ff.png"},{"id":73861325,"identity":"913a0053-3d1c-47b2-bc2f-f636042361ee","added_by":"auto","created_at":"2025-01-15 11:02:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":105197,"visible":true,"origin":"","legend":"\u003cp\u003eViability data of SH-SY5Y cells treated with various concentrations of metronidazole for 48 hours. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u0026lt;\u003c/em\u003e0.01\u003cem\u003e, \u003c/em\u003e\u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u0026lt;\u003c/em\u003e0.001 compared with minimum essential media (MEM). 4 x 10\u003csup\u003e3\u003c/sup\u003e cells/well.\u0026nbsp; Met: metronidazole.\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5757381/v1/3252f2e7b30387eb690e6a12.png"},{"id":73861327,"identity":"0cd6f602-0862-43a7-ae5d-26888acf0d6f","added_by":"auto","created_at":"2025-01-15 11:02:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":103376,"visible":true,"origin":"","legend":"\u003cp\u003eViability data of SH-SY5Y cells treated with metronidazole for 72 hours. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01\u003cem\u003e, \u003c/em\u003e\u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u0026lt;\u003c/em\u003e0.001 compared with minimum essential media (MEM), 4 x 10\u003csup\u003e3\u003c/sup\u003e cells/well. Met: metronidazole.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5757381/v1/b763bcdc7f3dca022d986198.png"},{"id":73861329,"identity":"87ba86e9-01a2-41af-9d2c-dd3c0b98f97b","added_by":"auto","created_at":"2025-01-15 11:02:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":105076,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability after 24 h incubation in ferrous sulphate and metronidazole. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u0026lt;\u003c/em\u003e0.05, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u0026lt;\u003c/em\u003e0.001 compared with minimum essential media (MEM) only. 4 x 10\u003csup\u003e3\u003c/sup\u003e cells/well. Met: metronidazole, Fe: ferrous sulphate\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5757381/v1/1e164d98716d764d81d35bb6.png"},{"id":73861332,"identity":"998f76d0-5b13-4be0-81a4-2dca3143f794","added_by":"auto","created_at":"2025-01-15 11:02:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":102850,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of metronidazole at various concentrations and ferrous sulphate after 48 hours co-treatment on viability of SH-SY5Y cells. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01\u003cem\u003e, \u003c/em\u003e\u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 compared with minimum essential media (MEM). 4 x 10\u003csup\u003e3\u003c/sup\u003e cells/well. Met: metronidazole, Fe: ferrous sulphate.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5757381/v1/5f2795f3c3f6e9a301573a3c.png"},{"id":73862608,"identity":"d5d77720-3d51-449e-b6ce-f0c534a2226e","added_by":"auto","created_at":"2025-01-15 11:10:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":102895,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of metronidazole and ferrous sulphate 72 hours co-treatment on viability of SH-SY5Y cells. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01\u003cem\u003e, \u003c/em\u003e\u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u0026lt;\u003c/em\u003e0.001 compared with minimum essential media (MEM). 4 x 10\u003csup\u003e3\u003c/sup\u003e cells/well. Met: metronidazole, Fe: ferrous sulphate.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5757381/v1/377d6e26d8b1d74190677143.png"},{"id":73862625,"identity":"23ca4553-88b6-4a40-b8c8-25e9b2d6ebba","added_by":"auto","created_at":"2025-01-15 11:10:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":118231,"visible":true,"origin":"","legend":"\u003cp\u003eIron absorption of SH-SY5Y cells following treatment with concentrations of metronidazole, and ferrous sulphate for 24 hours. Each data set is the mean of 6 replicates. \u003cem\u003eP\u003c/em\u003e\u0026gt;0.05 compared with the 20 µM iron treated cells. 4 x 10\u003csup\u003e3\u003c/sup\u003e cells/well. Met: metronidazole, Fe: ferrous sulphate.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5757381/v1/0d12b8cf74b205aa4c21e8b7.png"},{"id":73861334,"identity":"5706ad9c-58c5-438b-a55e-d193045bfbd3","added_by":"auto","created_at":"2025-01-15 11:02:26","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":111781,"visible":true,"origin":"","legend":"\u003cp\u003eCellular iron absorption data following 48 hours treatment with ferrous sulphate and various concentrations of metronidazole. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 compared with the ferrous sulphate 20 µM only treated cells. 4 x 10\u003csup\u003e3\u003c/sup\u003e cells/well. Met: metronidazole, Fe: ferrous sulphate.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5757381/v1/d14a1c47f1df89b1d0713fa3.png"},{"id":73861354,"identity":"8499cc5b-3ff4-40e4-bb1a-ec64ded1d049","added_by":"auto","created_at":"2025-01-15 11:02:27","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":116093,"visible":true,"origin":"","legend":"\u003cp\u003eCellular iron absorption data after 72 hours treatment with ferrous sulphate and various concentrations of metronidazole. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 compared with the ferrous sulphate 20 µM only treated cells. 4 x 10\u003csup\u003e3\u003c/sup\u003e cells/well. Met: metronidazole, Fe: ferrous sulphate.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5757381/v1/b7c68832dadce972e6dfee5e.png"},{"id":80623695,"identity":"8db23432-ba20-4d4d-9a88-c98ef70383da","added_by":"auto","created_at":"2025-04-15 10:16:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1507376,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5757381/v1/a5723429-0234-4541-9617-b514e05d9d3a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Metronidazole-induced neurotoxicity: is iron a contributing factor? ","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e cell culture techniques have been successfully developed in the neurobiology field to address specific questions of cell biology and nervous system functioning. This has formed a basis to systematically study the nervous systems, revealing the complexity of cellular functions in the CNS. It has also provided a convenient experimental tool for studying defects and disease processes in the CNS [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Various techniques abound for the study of neurotoxicity using dedicated cell lines.\u003c/p\u003e \u003cp\u003eIron is an essential molecule with pro-oxidant characteristics, it catalyses synthesis of hydroxyl radicals in the intracellular environment owing to its reductive role in the Fenton reaction [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The brain consumes the largest amount of total oxygen in the body due to its high respiratory or oxidative metabolic ability, consequently it generates a large volume of reactive oxygen species [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Brain iron is mainly bound to ferritin and only unbound iron is physiologcally active in the brain. Hence ferritin is recognised as the protein responsible for brain iron homeastasis.\u003c/p\u003e \u003cp\u003eThe mechanism of metronidazole-induced neurotoxicity is still under research as the various proposed mechanisms have not answered all the questions arising from its occurrence. One of the proposed mechanisms of metronidazole-iduced neurotoxicity is interference with oxidative processes in the nervous system [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Metronidazole is a prodrug which undergoes reductive activation mediated by pyruvate:ferrodoxin oxidoreductase (PFOR) whose expression is positively regulated by iron [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Iron is ubiquitous and occurs in high abundance in the central nervous system. Iron overload is implicated as a major contributor to neurotoxicity and neurodegeneration as evidenced in several neurologic conditions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This study is aimed at studying the effect of metronidazole on iron uptake in the human neuroblastoma SH-SY5Y cells.\u003c/p\u003e"},{"header":"Materials","content":"\u003cp\u003eDrugs and chemicals used include metronidazole (Sigma-Aldrich, Germany), Han\u0026rsquo;s F12 media (F12) (Sigma-Aldrich, Germany); Dulbecco\u0026rsquo;s minimum essential medium (DMEM) (Thermo-Fisher, USA); ferritin ELISA kit (Sigma-Aldrich, Germany); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kit (Sigma-Aldrich, Germany); Dulbecco phosphate-buffered saline (DPBS) (Sigma-Aldrich, Germany); serum free minimum essential medium (MEM) (Gibco, New York, USA); ferrous sulphate powder (Sigma-Aldrich, Germany); BCA assay kit (Sigma-Aldrich, Germany), GlutaMax (Gibco, USA). All reagents and chemicals used in the experiments were of analytical grade.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCell line\u003c/strong\u003e \u003cp\u003eHuman neuroblastoma SH-SY5Y cells (CRL-2266, ATCC\u0026rsquo; Rockville, USA) was used for the study and they were purchased from Gibco, USA. Their use required no institutional ethical approval.\u003c/p\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMethods\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eUnless stated otherwise, cells were grown in Dulbecco\u0026rsquo;s minimum essential medium (DMEM) in Han\u0026rsquo;s F12 media at pH 7.4, supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin solution and maintained at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e at constant humidity, refreshing the medium every four days until the cells were confluent. Differentiation of the cells was initiated 48-hour after the initial plating by substituting the growth medium with serum free neurobasal medium (containing B27 supplement and GlutaMAX) and 10 \u0026micro;M all-trans-retinoic acid (ATRA). The cells were allowed to grow in ATRA-containing neurobasal medium for 5 days, refreshing the medium every 48-hour[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The confluent cells were subsequently passaged to maintain their viability and increase the quantity available for experiments.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eTreatment of cells with metronidazole\u003c/h3\u003e\n\u003cp\u003eCompletely differentiated cells grown in 96-well plates were removed from the incubator, transferred to the hood, and the old media removed from the plates with a pipette and disposed into a bleach bottle. A quantity (200 \u0026micro;L) of each designated concentration (1, 10, 25, 50, 100, 250 \u0026micro;M) of the test media was added to each of the 6 replicate wells using a multichannel pipette. The plates were incubated for 24 h, 48 h and 72 h in the cell culture incubator. DMSO (200 \u0026micro;L) was used as negative control in the experiments. Treatment media was refreshed for the plates in the 48-hour and 72-hour groups. After the desired incubation period, the plates were removed from the incubator and the test media was aspirated and the cells harvested for endpoint assays to determine the effects of treatment on the cells. Protein quantification was done using the bicinchoninic acid assay method.\u003c/p\u003e\n\u003ch3\u003eViability assay of SH-SY5Y following treatment with metronidazole\u003c/h3\u003e\n\u003cp\u003eThe reagents for MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was prepared according to the manufacturer\u0026rsquo;s instructions. After incubating the plates for 24, 48 or 72 hours respectively in the test drug, they were removed from the cell culture incubator. The test substance was aspirated into a bleach bottle and the wells washed with DMSO. Each well was treated with MTT 20 \u0026micro;L /well and incubated for 4 hours at 37\u0026deg;C. After the incubation period, the plates were removed from the incubator, the media was gently aspirated into the bleach bottle without disrupting the cell monolayer. An aliquot (100 \u0026micro;L) of DMSO was added to each well and mixed properly with a pipette. The wells were covered with foil to avoid light and incubated in a rotary shaker for 15 minutes. The plates were then removed from the shaker and the absorbance measured at 570 nm in a spectrophotometer (Thermo-Fischer, UK) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eThe effect of metronidazole on iron uptake\u003c/h3\u003e\n\u003cp\u003eA standard iron solution (1M) was prepared by dissolving 1.52 g ferrous sulphate powder in 10 mL 0.1 M HCl. Completely differentiated cells were incubated in serum free minimum essential medium (MEM) at pH 5.8 for 24-hour at 37\u003csup\u003eo\u003c/sup\u003eC. After the incubation period, the cells were treated with filtered aliquots of metronidazole in MEM plus prepared iron solution to give a final concentration of the different test conditions in 20 \u0026micro;M elemental iron and all the wells buffered to pH 5.8. A positive control (iron solution in MEM) and negative control (MEM only) were also included in the plates. The plates were incubated on a rotary shaker (6 \u003cem\u003eg\u003c/em\u003e) at 37\u003csup\u003eo\u003c/sup\u003eC for 24, 48 and 72 hours and the media refreshed for the 48-hour and 72-hour groups respectively. On completion of incubation period, the plates were removed, and the media aspirated into the bleach bottle. The plates were washed twice with wash solution and the media replaced with serum-free MEM and incubated for 24 hours. After incubation, the cells were washed, lysed and the supernatant collected in aliquots for ferritin ELISA and bicinchoninic acid (BCA) assay [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The plates for MTT were not lysed after washing rather, they were treated with MTT reagent and assayed as previously described.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFerritin assay\u003c/h2\u003e \u003cp\u003eThe manufacturer\u0026rsquo;s instructions were followed in the preparation of reagents and standards for the ferritin assay. Ferritin standards (50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 and 0.00 ng\u003cem\u003e/\u003c/em\u003emL) were prepared by serial dilution method starting from 50 ng/mL stock. An aliquot (50 \u0026micro;L) of ferritin standard or sample were added in duplicates per well and the plate tapped gently to thoroughly coat the wells and incubated for two hours at room temperature. The plate was washed five times with 200 \u0026micro;L of the wash buffer and gently tapped on an absorbent paper towel to completely remove the liquid after the last wash. The biotinylated ferritin antibody (50 \u0026micro;L) was added to each well and incubated for one hour at room temperature. The plates were washed five times as previously done followed by the addition of 50 \u0026micro;L SP-conjugate to each well. All bubbles were removed by tapping and the plates incubated at room temperature for 30 minutes. After the incubation each plate was washed as above. Some chromogen substrate (50 \u0026micro;L) was added to each well and spread well by tapping to remove bubbles, then each plate was incubated at room temperature for 12 minutes. At the end of the incubation, 50 \u0026micro;L of the stop Solution was added to each well and the plates read immediately at 450 nm (MicroPlate Reader 550, Bio-Rad Laboratories, Denmark) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eResults are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean (SEM). Statistical analysis was done using one-way ANOVA followed by Dunnet\u0026rsquo;s post hoc test for multiple comparisons (GraphPad Prism 6 Software, San Diego California USA). Correlations for protein analysis were calculated by Pearson\u0026rsquo;s test. Statistical differences between compared data were considered significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMetronidazole reduces viability of SH-SY5Y cells dependent of time and concentration\u003c/h2\u003e \u003cp\u003eIn the cell viability experiments, metronidazole exhibited time and concentration dependent effects on the treated cells, the viability of the treated cells reduced with increase in metronidazole concentration, the 250 \u0026micro;M treated cells were the least viable (\u0026lt;\u0026thinsp;10% viability across all treatment periods). The effect of treatment duration on cell viability of identical metronidazole concentration was marginal at concentrations of 25 \u0026micro;M and below. After 24 h treatment, 250 \u0026micro;M metronidazole reduced (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) cell viability compared with the growth media-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Cell viability ranged between 8.9 to 83.2% after 48 h treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) with a significant reduction in viability for the 50 \u0026micro;M, 100 \u0026micro;M and 250 \u0026micro;M (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.\u003c/em\u003e001) treated cells. These effects were similar in the 72 h treated cells with viability ranging between 7.5 to 78.5% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMetronidazole increases SH-SY5Y cell iron uptake.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn determining the effect of metronidazole on cellular iron uptake, cytotoxicity experiments were conducted to determine the viability of SH-5YSY cells when concomitantly treated with the various concentrations of metronidazole and 20 \u0026micro;M ferrous sulphate. Treatment with metronidazole and ferrous sulphate yielded a concentration and time dependent effect on the treated cells with cell viability reducing with increase in dose and time of treatment. After 24 h incubation, cell viability reduced (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05, 0.001) in the 50, 100 and 250 \u0026micro;M metronidazole treated wells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The effect of co-culturing the cells in metronidazole and ferrous sulphate was more pronounced after 48 h as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Metronidazole concentrations of 50 \u0026micro;M and above caused a reduction (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, 0.001) in cell viability. The reduction in cell viability seen after 72 h incubation with metronidazole and ferrous sulphate was more intense with 100 and 250 \u0026micro;M concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe total iron absorption data is shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. Metronidazole induced slight increase in iron uptake into the cells after 24 h treatment although the changes were not statistically different from the iron only treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). However, metronidazole induced a time and concentration dependent increase (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in cellular iron uptake in the 48 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) and 72-h treated cells and in concentrations above 25 \u0026micro;M metronidazole. In the 72-h treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e), 1 \u0026micro;M metronidazole treated cells had reduced iron absorption compared with the iron only treated cells.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough, the various effects, targets and modes of action of metronidazole are well characterized in bacteria, the consequences and how metronidazole interacts with mammalian cells is still not fully deciphered. In this study, human neuroblastoma (SH-SY5Y) cells were employed to elucidate the effect of metronidazole on cell viability and iron uptake. SH-SY5Y neuroblastoma cells are a replicated offspring of SK-N-SH cells originally isolated from bone marrow biopsy of a neuroblastoma patient in the early 1970s [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This cell line has been extensively used as a neuronal model since the early 1980s, as they possess many biochemical and functional properties of human neurons [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. They are useful in assessing morphological and many differentiated neuronal functions hence their usefulness in neurotoxicity screening of parent drug molecules [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCytotoxicity testing is an essential procedure in cell-based neurotoxicity testing. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay is a high throughput screening model for determination of cell viability in toxicity testing [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Healthy growing cells with intact metabolic capacity convert MTT to an insoluble purple-colored formazan product which accumulates inside cells. Dead cells lose the capability to convert MTT to formazan, hence formation of the insoluble color serves as an accessible and convenient marker for healthy growing cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The quantum of formazan formed is directly proportional to the number of healthy growing cells as determined by the optical density at 570 nm using a plate reading spectrophotometer [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In this study, 50 \u0026micro;M metronidazole and concentrations above it exhibited a time and concentration-dependent effect on the treated cells. There is paucity of reports on the outcome of metronidazole treatment on SH-SY5Y cells. Xiao \u003cem\u003eet al.\u003c/em\u003e, (2018) reports that metronidazole induced apoptosis of neuronal cell cultures (PC12, human primary neurons and CAD) by increasing the expression of annexin-v at a concentration of 40 \u0026micro;g/mL [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This may be a possible effect of metronidazole on the SH-SY5Y cells, however more research into the genotoxicity of metronidazole on SH-SY5Y needs to be done to ascertain its effects on the cellular componenets of SH-SY5Y cells.\u003c/p\u003e \u003cp\u003eIron is vitally important for most life forms and is broadly utilized by different proteins to execute a number of functions including respiration, metabolism and synthesis of new proteins, in essence iron is essential for survival of living systems [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Dysfunction in iron metabolism has been implicated in several disease states [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Normally, iron is absorbed into cells by means of the divalent metal transporter 1 (DMT1), which is a steadfastly modulated process. Additionally, a new iron transporter ferropotin (IREG1) has been identified recently. It is reported that both DMT1 and IREG1 exist in the central nervous system and is expressed on neurons, glia cells and astrocytes are thought to be responsible for maintaining iron homeostasis in the CNS [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These transporters have been characterized also in SH-SY5Y cells, and IREG1 is thought to mediate iron efflux while DMT1 is thought to mediate iron influx in SH-SY5Y cells [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExcessive iron aggregation can activate oxidative stress and cell damage as it reacts with hydrogen peroxide to produce the hydroxyl radical and other ROS in the Fenton reaction [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Iron accumulation and reduced viability of SH-SY5Y cells have been reported recently following treatment with iron (1.5\u0026ndash;80 \u0026micro;M). Also, DMT1 expression decreased but persisted even at 80 \u0026micro;M while IREG1 expression increased with increased cellular iron content [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This implies that iron influx did not stop completely even at very high cellular iron concentration though an increased efflux process seemed to have been activated. In this study, the presence of metronidazole caused a drastic decline in cell viability compared to the cells treated with only iron. It is possible to then suggest that metronidazole favors an increase in DMT1 expression as opposed to IREG1 expression in the presence of iron and subsequent oxidative stress and cell death arising from iron accumulation.\u003c/p\u003e \u003cp\u003eIron is stored in the cytosol or in mitochondria as ferritin. Ferritin has recently been shown to play a role in iron homeostasis, immunomodulation, inflammation and antioxidation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Ferritin structurally consists of an outer shell known as apoferritin and a hollow inner-core which serves as a store for iron. The apoferritin shell also contains ferroxidase which converts iron from its active ferrous (Fe\u003csup\u003e2+\u003c/sup\u003e) form into the inactive ferric (Fe\u003csup\u003e3+\u003c/sup\u003e) form for storage [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Experimental evidence implicates ferritin as the protein responsible for iron delivery to the brain [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In low iron conditions, the ferritin molecule is degraded by activation of the lysosomal- autophagy pathway where nuclear receptor coactivator-4 binds to ferritin and causes the release of iron into the cytosol [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Elevated ferritin levels have been implicated in various neuropsychiatric and neurodegenerative diseases in recent times [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In such cases, the storage capacity of ferritin is thought to be overwhelmed resulting in an increased concentration of unsequestered iron in the cytosol with resultant production of free radicals and oxidative stress [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Aguirre \u003cem\u003eet al\u003c/em\u003e (2005), reports a four-fold increase in ferritin levels in SH-SY5Y cells treated with iron (1.5-5.0 \u0026micro;M) and a ten-fold increase for iron concentrations higher than that resulting in a commensurate elevation of the labile iron pool [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMetronidazole undergoes reductive activation to form reactive intermediates which are toxic to susceptible organisms in anerobic conditions. Several enzymes suggested to be involved in the reductive activation of MTZ include the pyruvate: ferredoxin oxidoreductase (PFOR) which catalyzes electron transfer via its iron-sulphur clusters resulting in the generation of nitro-radical anion as metronidazole serves as a receptacle of electrons released by the action of PFOR [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. PFOR is a key enzyme in the metabolic pathway of many anaerobic microorganisms and some parasites. It plays a critical role in the conversion of pyruvate to acetyl-CoA for energy production in anaerobes. PFOR expression is thought to increase in the presence of iron, this is corroborated by the increased conversion of metronidazole to its active nitro-radical forms with consequent increase in its antibacterial activity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and toxic radical production [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In this study, there was a concentration and time-dependent elevation of ferritin levels in the metronidazole-plus-iron treated cells compared to the iron only treated cells. It is hence suggested that metronidazole induced iron overload as evidenced by the increased ferritin concentration in the treated cells with a consequent decrease in cell viability.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eSH-SY5Y cells are known to possess an active system responsible for regulation of iron homeostasis. Iron overload is implicated in several disease conditions affecting the brain and neurons in particular. Ferritin is the main storage protein for iron in brain and neuronal structures and correlates with the amount of iron in circulation in these cells. In this study, metronidazole induced a time and concentration dependent decrease in SH-SY5Ycell viability and an increase in ferritin concentration in the treated cells. Thus, we conclude that metronidazole induces iron overload and consequent cell death in SH-SY5Y cells and this may contribute to the mechanism of metronidazole-induced neurotoxicity.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eATRA- all-trans-retinoic acid; MEM\u0026nbsp;-Minimum essential medium; BCA- Bicinchoninic acid; DMEM- Dulbecco\u0026rsquo;s minimum essential medium; ELISA- enzyme linked immunoassay; ROS- reactive oxygen species; PFOR- pyruvate-ferredoxin oxidoreductase; MTT- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; DMSO- dimethylsulfoxide; DMT1- divalent metal transporter 1; IREG1- iron transporter ferropotin; FBS- foetal bovine serum; CNS- central nervous system.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMWB and RIO designed the study, MWB, VBP and GMZ participated in the experiments and data collection, RIO, GMZ and VBP analyzed the data, MWB and RIO wrote the manuscript, all authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this manuscript\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBarbosa DJ, Capela JP, Bastosa M, Cavarlho F. In vitro models for neurotoxicology research. Toxicol Res. 2015; 4(4):801-42.\u003c/li\u003e\n\u003cli\u003eRouault T. Biogenesis of iron\u0026ndash;sulfur clusters in mammalian cells: new insights and relevance to human disease. Dis Models Mech.2012;5(2):155\u0026ndash;64.\u003c/li\u003e\n\u003cli\u003eBresgen N, Eckl PM. Oxidative stress and the homeodynamics of iron metabolism. Biomolec. 2015;5(2): 808\u0026ndash;47.\u003c/li\u003e\n\u003cli\u003eSingh N, Haldar S, Tripathi A, Horback K, Wong J, Sharma D, \u003cem\u003eet al\u003c/em\u003e. Brain iron homeostasis: from molecular mechanisms to clinical significance and therapeutic opportunities. Antioxid Redox Signal. 2014; 20:1324\u0026ndash;63.\u003c/li\u003e\n\u003cli\u003eTalapatra S, Dasgupta S, Guha G, Auddy M and Mukhopadhyay A. Therapeutic efficacies of Coriandrum sativum aqueous extract against metronidazole induced genotoxicity in Channa punctatus peripheral erythrocytes. Food Chem Toxicol. 2010;48(12): 3458-61.\u003c/li\u003e\n\u003cli\u003eBahn Y, Kim E, Park C, Park HC. Metronidazole induced encephalopathy in a patient with brain abscess. J Korean Neurosurg Soc. 2010;48: 301-4.\u003c/li\u003e\n\u003cli\u003eWard F, Crowley P and Cotter P. Acute cerebellar syndrome associated with metronidazole. Pract Neurol. 2015;15(4): 298-99.\u003c/li\u003e\n\u003cli\u003eCeruelos HA, Romero-Quezada L, Ledezma J, Contreras L. Therapeutic uses of metronidazole and its uses: an update. Eur Rev Med Pharmacol Sci. 2019;23: 397-401.\u003c/li\u003e\n\u003cli\u003eElwakil H, Tawfik R, Alam-Eldin Y, Nassar D. The effect of iron on metronidazole activity against Trichomonas vaginalis in vitro. Expt parasitol. 2017;182: 34\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003ePetzold A, Worthington V, Appleby I, Kerr M, Kitchen N, Smith M, \u003cem\u003eet al\u003c/em\u003e. Cerebrospinal fluid ferritin level, a sensitive diagnostic test in late-presenting subarachnoid hemorrhage. J Stroke Cerebrovasc Dis. 2011;20(6): 489\u0026ndash;93.\u003c/li\u003e\n\u003cli\u003eDa Costa R, Szyper-Kravitz M, Szekanecz Z, Csepany T, Danko K, Shapira Y, \u003cem\u003eet al\u003c/em\u003e. Ferritin and prolactin levels in multiple sclerosis. Israel Med Assoc J: IMAJ. 2011;13(2), 91\u0026ndash;5.\u003c/li\u003e\n\u003cli\u003eKovalevich J, Langford D. Considerations for the use of SH-SY5Y neuroblastoma cells in neurobiology. Methods Molec Biol. 2013; 1078: 9\u0026ndash;21.\u003c/li\u003e\n\u003cli\u003eZariwala M, Somavarapu S, Farnaud S, Renshaw D. Comparison Study of Oral Iron Preparations Using a Human Intestinal Model. Scientia Pharmaceutica8. 2013;1(4), 1123\u0026ndash;39\u003c/li\u003e\n\u003cli\u003ePark E and Chung S (2019). ROS-mediated autophagy increases intracellular iron levels and ferroptosis by ferritin and transferrin receptor regulation. Cell Death Dis. 2019;10(11):822.\u003c/li\u003e\n\u003cli\u003eElhassanny A, Soliman E, Marie M, McGuire P, Gul W, ElSohly M, \u003cem\u003eet al\u003c/em\u003e. Heme-dependent ER stress apoptosis: a mechanism for the selective toxicity of the dihydroartemisinin, NSC735847, in colorectal cancer cells. Front Oncol. 2020;10: 965.\u003c/li\u003e\n\u003cli\u003eBiedler JL, Helson L, Spengler BA. Morphology and growth, tumorigenicity, and cytogenetics of human neuroblastoma cells in continuous culture. Cancer Res. 1973;33(11): 2643\u0026ndash;52.\u003c/li\u003e\n\u003cli\u003eFerreira PS, Nogueira TB, Costa VM, Branco PS, Ferreira LM, Fernandes E, \u003cem\u003eet al\u003c/em\u003e. Neurotoxicity of \u0026quot;ecstasy\u0026quot; and its metabolites in human dopaminergic differentiated SH-SY5Y cells. Toxicol Lett. 2013;216(2-3):159-70.\u003c/li\u003e\n\u003cli\u003eGandhi D, Tarale P, Naoghare P. Integrative genomic and proteomic profiling of human neuroblastoma SH-SY5Y cells reveals signatures of endosulfan exposure. Env Toxicol Pharmacol. 2016;41:187\u0026ndash;94.\u003c/li\u003e\n\u003cli\u003eBell M, Zempel H. SH-SY5Y-derived neurons: a human neuronal model system for investigating TAU sorting and neuronal subtype-specific TAU vulnerability. Rev Neurosci. 2022;33(1): 1\u0026ndash;15.\u003c/li\u003e\n\u003cli\u003eMosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1-2): 55\u0026ndash;63.\u003c/li\u003e\n\u003cli\u003eBerridge MV, Tan AS, McCoy KD, Wang R. The biochemical and cellular basis of cell proliferation assays that use tetrazolium salts. Biochemica. 1996;4:14\u0026ndash;19.\u003c/li\u003e\n\u003cli\u003eMarshall NJ, Goodwin CJ, Holt SJ. A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function. 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Interleukin-1beta up-regulates iron efflux in rat C6 glioma cells through modulation of ceruloplasmin and ferroportin-1 synthesis. Neurosci Lett. 2004;363:182-6.\u003c/li\u003e\n\u003cli\u003eAguirre P, Mena N, Tapia V, Arredondo M, N\u0026uacute;\u0026ntilde;ez M. Iron homeostasis in neuronal cells: a role for IREG1. BMC Neurosci. 2005;https://doi.org/:10.1186/1471-2202-6-3.\u003c/li\u003e\n\u003cli\u003ePanther E, Zelmanovich R, Hernandez J, Dioso E, Foster D, Lucke-Wold B. Ferritin and Neurotoxicity: A contributor to deleterious outcomes for subarachnoid hemorrhage. Eur Neurol. 2022;85(6): 415\u0026ndash;23.\u003c/li\u003e\n\u003cli\u003eZhang N, Yu X, Xie J, Xu H. New insights into the role of ferritin in iron homeostasis and neurodegenerative diseases. Molec Neurobiol. 2021;58(6): 2812\u0026ndash;23.\u003c/li\u003e\n\u003cli\u003eChapman A, Cammack R, Linstead R, Lloyd D. The generation of metronidazole radicals in hydrogenosomes isolated from Trichomonas vaginalis. J Gen Microbiol. 1985;131(9): 2141\u0026ndash;44.\u003c/li\u003e\n\u003cli\u003eMoreno S, Mason R, Docampo R. Nitroimidazole cellular activities. J Biol Chem. 1984;256: 6298-6305.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Iron, Neurotoxicity, Ferroptosis, Ferritin, Metronidazole, Neuroblastoma cells","lastPublishedDoi":"10.21203/rs.3.rs-5757381/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5757381/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMetronidazole-induced neurotoxicity is a rising challenge in the management of susceptible infections. The mechanisms involved in metronidazole-induced neurotoxicity are not fully unraveled. This study was aimed at determining the effect of metronidazole on iron homeostasis in SH-SY-5Y neuroblastoma cells.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eConfluent SH-SY-5Y neuroblastoma cells were treated with 1, 10, 25, 50, 100, 250 \u0026micro;M concentrations of metronidazole only or in combination with 20 \u0026micro;M iron. DMSO or culture media was used as control. Viability and ferritin assay were conducted on the treated cells. The treatments were for 24 hr, 48 hr and 72 hr respectively.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn the viability assay, doses of metronidazole reduced viability of SH-SY-5Y neuroblastoma cells in a time and concentration dependent manner. After 24 hr treatment, 250 \u0026micro;M metronidazole reduced (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) cell viability while 50 \u0026micro;M, 100 \u0026micro;M and 250 \u0026micro;M metronidazole reduced (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.\u003c/em\u003e001) viability only after 48 and 72 hr compared with control. Doses of metronidazole 50 \u0026micro;M, 100 \u0026micro;M and 250 \u0026micro;M in 20 \u0026micro;M iron reduced viability in a time dependent manner in all the tests periods. Metronidazole also induced a time and concentration dependent increase (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in cellular iron uptake in the 48 and 72 hr treated cells in concentrations above 25 \u0026micro;M metronidazole.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIt is concluded that metronidazole induces a time and concentration dependent iron overload and consequent cell death in SH-SY5Y neuroblastoma cells and this may contribute to the mechanism of metronidazole-induced neurotoxicity.\u003c/p\u003e","manuscriptTitle":"Metronidazole-induced neurotoxicity: is iron a contributing factor?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-15 11:02:21","doi":"10.21203/rs.3.rs-5757381/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"940e1142-1378-4982-97d6-d377e749a2df","owner":[],"postedDate":"January 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-15T10:08:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-15 11:02:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5757381","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5757381","identity":"rs-5757381","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}