Estimation of genomic instability and mutation induction by graphene oxide nanoparticles in mice liver and brain tissues

  • Hanan R. H. MohamedEmail author
  • Mary Welson
  • Ahmed Essa Yaseen
  • Akmal A. EL-Ghor
Research Article


The rapidly growing interest in using graphene-based nanoparticles in a wide range of applications increases human exposure and risk. However, very few studies have investigated the genotoxicity and mutagenicity of the widely used graphene oxide (GO) nanoparticles in vivo. Consequently, this study estimated the possible genotoxicity and mutagenicity of GO nanoparticles as well as possible oxidative stress induction in the mice liver and brain tissues. Nano-GO particles administration at the dose levels of 10, 20, or 40 mg/kg for one or five consecutive days significantly increased the DNA breakages in a dose-dependent manner that disrupts the genetic material and causes genomic instability. GO nanoparticles also induced mutations in the p53 (exons 6&7) and presenilin (exon 5) genes as well as increasing the expression of p53 protein. Positive p53 reaction in the liver (hepatic parenchyma) and brain (cerebrum, cerebellum, and hippocampus) sections showed significant increase of p53 immunostaining. Additionally, induction of oxidative stress was proven by the significant dose-dependent increases in the malondialdehyde level and reductions in both the level of reduced glutathione and activity of glutathione peroxidase observed in GO nanoparticles administered groups. Acute and subacute oral administration of GO nanoparticles induced genomic instability and mutagenicity by induction of oxidative stress in the mice liver and brain tissues.


Graphene oxide nanoparticles Genotoxicity Mutagenicity p53 Presenilin Oxidative stress and mice 



Thanks and great appreciation to the Department of Zoology, Faculty of Science, Cairo University, for providing us with the necessary equipment to conduct experiments of this study.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Afzali M, Parivar K, Roodbari Nand Badiei A (2015) Study of Nano-Graphene oxide effects on the number of Kupffer cells and megakaryocytes in liver of NMRI strain mouse embryo in vivo. Current World Environment 10(1):713–718CrossRefGoogle Scholar
  2. Athan ES, Williamson J, Ciappa A, Santana V, Romas SN, Lee JH, Rondon H, Lantigua RA, Medrano M, Torres M, Arawaka S, Rogaeva E, Song YQ, Sato C, Kawarai T, Fafel KC, Boss MA, Seltzer WK, Stern Y, St George-Hyslop P, Tycko B, Mayeux R (2001) A founder mutation in presenilin 1 causing early-onset Alzheimer disease in unrelated Caribbean Hispanic families. JAMA. 286(18):2257–2263. CrossRefGoogle Scholar
  3. Beutler E, Duron O, Kelly BM (1963) Improved method for the determination of blood glutathione. J Lab Clin Med 61:882–888Google Scholar
  4. Bjelland, Seeberg E (2003) Mutagenicity, toxicity and repair of DNA base damage induced by oxidation. MutatRes-Fundam Mol Mech Mutagen 531:37–80CrossRefGoogle Scholar
  5. Chaenyung C, Ryon SS, Xiguang G, Nasim A, Dokmeci MR, Xiaowu Shirley T et al (2014) Controlling mechanical properties of cell-laden hydrogels by covalent incorporation of graphene oxide. Small 10(3):514–523. CrossRefGoogle Scholar
  6. Chen M, Yin J, Liang Y, Yuan S, Wang F, Song M, Wang H (2016) Oxidative stress and immunotoxicity induced by graphene oxide in zebrafish. Aquat Toxicol 174(1879–1514 (electronic)):54–60CrossRefGoogle Scholar
  7. Crews L, Masliah E (2010) Molecular mechanisms of neurodegeneration in Alzheimer’s disease. Hum Mol Genet 19(1):R12–R20CrossRefGoogle Scholar
  8. De Marzi L, Ottaviano L, Perrozzi F, Nardone M, Santucci S, De Lapuente J, Borras M, Treossi E, Palermo V, Poma A (2014) Flake size-dependent cyto and genotoxic evaluation of graphene oxide on in vitro A549, CaCo2 and vero cell lines. J Biol Regul Homeost Agents 28(2):281–289Google Scholar
  9. Ding Z, Zhang Z, Ma H, Chen Y (2014) In vitro hemocompatibility and toxic mechanism of graphene oxide on human peripheral blood T lymphocytes and serum albumin. ACS Appl Mater Interfaces 6(22):19797–19807 18CrossRefGoogle Scholar
  10. Dorszewska J, Oczkowska A, Suwalska M, Rozycka A, Florczak-Wyspianska J, Dezor M, Lianeri M, Jagodzinski P, Kowalczyk MJ, Prendecki M, Kozubski W (2014) Mutations in the exon 7 of Trp53 gene and the level of p53 protein in double transgenic mouse model of Alzheimer’s disease. Folia Neuropathol 52(1):30–40CrossRefGoogle Scholar
  11. El-Yamanya AN, Mohamed FF, Salaheldin TA, Tohamya AA, Abd El-Mohsena WN, Amine AS (2017) Graphene oxide nanosheets induced genotoxicity and pulmonary injury in mice. Exp Toxicol Pathol 69:383–392CrossRefGoogle Scholar
  12. Ferraro D, Anselmi-Tamburini U, Tredici IG, Ricci V, Sommi P (2016) Overestimation of nanoparticles-induced DNA damage determined by the comet assay. Nanotoxicology 10(7):861–870CrossRefGoogle Scholar
  13. Gautheron V, Auffret A, Mattson MP, Mariani J, Garabedian B V-d (2009) A new and simple approach forgenotyping Alzheimer’s disease presenilin-1 mutant knock-inmice. J Neurosci Methods 181(2):235–240CrossRefGoogle Scholar
  14. Guo X, Mei N (2014) Review Article:Assessment of the toxic potential of grapheme family nanomaterials. J Food Drug Anal 2 2:1 0 5–1 1 5CrossRefGoogle Scholar
  15. Gurunathan S, Han JW, Dayem AA, Eppakayala V, Kim JH (2012) Oxidative stress-mediated antibacterial activity of graphene oxide and reduced graphene oxide in Pseudomonas aeruginosa. Int J Nanomedicine 7:5901–5914. CrossRefGoogle Scholar
  16. Gutierrez MI, Bhatia K, Siwarski D, Wolff L, Magrath IT, Mushinski JF, Huppi K (1992) Infrequent p53 Mutation in Mouse Tumors with Deregulated myc. Cancer Res 52:1032–1035Google Scholar
  17. Hsu SM, Raine L, Fanger H (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J HistochemCytochem 29(4):577–580Google Scholar
  18. Jarosz A, Skoda M, Dudek I, Szukiewicz D (2016) Oxidative stress and mitochondrial activation as the main mechanisms underlying graphene toxicity against human cancer cells. Oxidative Med Cell Longev 2016:5851035CrossRefGoogle Scholar
  19. Jasim DA, M’enard-Moyon C, B’egin D, Bianco A, Kostarelos K (2015) Tissue distribution and urinary excretion of intravenously administered chemically functionalized graphene oxide sheets. Chem Sci 6:3952–3964CrossRefGoogle Scholar
  20. Jaworski S, Sawosz E, Grodzik M, Winnicka A, Prasek M, Wierzbicki M, Chwalibog A (2013) In vitro evaluation of the effects of graphene platelets on glioblastoma multiforme cells. Int J Nanomedicine 8:413–420Google Scholar
  21. Kelleher RJ, Shen J (2010) γ -Secretase and human disease. Science. 33(6007):1055–1056CrossRefGoogle Scholar
  22. Kurantowicz N, Strojny B, Sawosz E, Jaworski S, Kutwin M, Grodzik M, Chwalibog A (2015) Biodistribution of a high dose of diamond, graphite, and Graphene oxide nanoparticles after multiple Intraperitoneal injections in rats. Nanoscale Res Lett 10(1):398. CrossRefGoogle Scholar
  23. Lanoiselée HM, Nicolas G, Wallon D, Rovelet-Lecrux A, Lacour M, Rousseau S (2017) APP, PSEN1, and PSEN2 mutations in early-onset Alzheimer disease: a genetic screening study of familial and sporadic cases. PLoS Med 14(3):e1002270. CrossRefGoogle Scholar
  24. Liu S, Zeng TH, Hofmann M, Burcombe E, Wei J, Jiang R, Chen Y (2011) Antibacterial activity of graphite, graphite oxide, grapheme oxide, and reduced graphene oxide: membrane and oxidative stress. ACS Nano 5:6971–6980CrossRefGoogle Scholar
  25. Liu Y, Luo Y, Wu J, Wang Y, Yang X, Yang R, Wang B, Yang J, Zhang N (2013) Graphene oxide can induce in vitro and in vivo mutagenesis. Sci Rep 3:3469CrossRefGoogle Scholar
  26. Liu Z, Choi SW, Crott JW, Smith DE, Mason JB (2008) Multiple B-vitamin inadequacy amplifies alterations induced by folate depletion in p53 expression and its downstream effector MDM2. Int J Cancer 123(3):519–525CrossRefGoogle Scholar
  27. Lockman PR, Koziara JM, Mumper RJ, Allen DD (2004) Nanoparticle surface charges alter blood-brain barrier integrity and permeability. J Drug Target 12(9–10):635–641CrossRefGoogle Scholar
  28. Mendonça MC, Soares ES, de Jesus MB, Ceragioli HJ, Ferreira MS, Catharino RR, da Cruz-Höfling MA (2015) Reduced graphene oxide induces transient blood-brain barrier opening: an in vivo study. Journal of nanobiotechnology 13:78. CrossRefGoogle Scholar
  29. Nezakati T, Cousins BG, Seifalian AM (2014) REVIEW ARTICLE: Toxicology of chemically modified graphene-based materials for medical application. Arch Toxicol 88:1987–2012CrossRefGoogle Scholar
  30. Ohkawa H, Ohishi W, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reactionAnal. Biochem 95:351–358Google Scholar
  31. Paglia DE, Valentine WN (1967) Studies on the quantitativeand qualitative characterization of erythrocyte glutathioneperoxidase. J Lab Clin Med 70:158–169Google Scholar
  32. Pan Y, Sahoo NG, Li L (2012) The application of graphene oxide in drug delivery. Expert Opin Drug Deliv 9(11):1365–1376. CrossRefGoogle Scholar
  33. Recio L, Hobbs C, Caspary W, Witt KL (2010) Dose-response assessment of four genotoxic chemicals in a combined mouse and rat micronucleus (MN) and comet assay protocol. J Toxicol Sci 35(2):149–162CrossRefGoogle Scholar
  34. Ren H, Wang C, Zhang J, Zhou X, Xu D, Zheng J, Guo S, Zhang J (2010) DNA cleavage system of nanosized graphene oxide sheets and copper ions. ACS Nano 4(12):7169–7174CrossRefGoogle Scholar
  35. Sanchez VC, Jachak A, Hurt RH, Kane AB (2012) Biological interactions of graphene-family nanomaterials: an interdisciplinary review. Chem Res Toxicol 25:15–34CrossRefGoogle Scholar
  36. Seabra AB, Paula AJ, de Lima R, Alves OL, Duran N (2014) Nanotoxicity of graphene and graphene oxide. Chem Res Toxicol 27(2):159–168CrossRefGoogle Scholar
  37. Shen H, Zhang L, Liu M, Zhang Z (2012) Biomedical applications of Graphene. Theranostics 2(3):283–294CrossRefGoogle Scholar
  38. Tice RR, Agurell E, Anderson V, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–221CrossRefGoogle Scholar
  39. Valko M, Morris H, Cronin MT (2005) Metals, toxicity and oxidative stress. Curr Med Chem 12(10):1161–1208CrossRefGoogle Scholar
  40. Waiwijit U, Kandhavivorn W, Oonkhanond B, Lomas T, Phokaratkul D, Wisitsoraat A, Tuantranont A (2014) Cytotoxicity assessment of MDA-MB-231 breast cancer cells on screen-printed graphene-carbon paste substrate. Colloids Surf B: Biointerfaces 113:190–197CrossRefGoogle Scholar
  41. Wang A, Pu K, Dong B, Liu Y, Zhang L, Zhang Z, Duan W, Zhu Y (2013) Role of surface charge and oxidative stress in cytotoxicity and genotoxicity of graphene oxide towards human lung fibroblast cells. J Appl Toxicol 33:1156–1164CrossRefGoogle Scholar
  42. Yang K, Wan J, Zhang S, Zhang Y, Lee S-T, Liu Z (2011) In vivo pharmacokinetics, long-term biodistribution, and toxicology of PEGylated Graphene in mice. ACS Nano 5(1):516–522. CrossRefGoogle Scholar
  43. Zhang S, Yang K, Feng L, Liu Z (2011) In vitro and in vivo behaviors of dextran functionalized graphene. Carbon 49:4040–4049CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Zoology Department, Faculty of ScienceCairo UniversityGiza GovernorateEgypt
  2. 2.Zoology Department, Faculty of ScienceSuez UniversitySuez GovernorateEgypt

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