Environmental Science and Pollution Research

, Volume 26, Issue 32, pp 33642–33653 | Cite as

Assessment of intermittent exposure of zinc oxide nanoparticle (ZNP)–mediated toxicity and biochemical alterations in the splenocytes of male Wistar rat

  • Neelu Singh
  • Monoj Kumar Das
  • Rohit Gautam
  • Anand Ramteke
  • Paulraj RajamaniEmail author
Research Article


Nanoparticles are being used extensively and found in applications to various fields ranging from agriculture to electronic devices, diagnosis to drug delivery, and cosmetics to food packaging. Increasing usage of engineered nanomaterials (ENM) raises potential concern for human health as well as to the environment. The present study aims to explore the effects of intermittent intraperitoneal exposure of ZNP on the spleen of male Wistar rat. Animals were divided into three groups, control and ZNP-treated groups (50 mg/kg and 250 mg/kg body weight), six in each group. Experimental animals were treated with different doses of ZNP once a week for 4 weeks, whereas control groups received water. After 28 days of exposure, animals were sacrificed, spleen tissue was excised, and various parameters such as hematological, genotoxicity, antioxidants, and histopathological were studied for changes in spleen if any. Results showed that ZNP exposure manages to induce alteration in various studied hematological parameters like neutrophils, platelets, and eosinophils which are found to increase significantly after the last treatment compared with the first treatment of ZNP. However, hemoglobin content, PCV, and MCV decrease with increasing dose of ZNP significantly in last treatment, when compared with the first treatment. DNA damage was observed in rats treated with a high dose of ZNPs compared with that in the control when analyzed through comet assay. Flow cytometric study was performed for better understanding of the underlying mechanism of the ZNP-mediated toxicity. From the present investigation, an increase in ROS production, a decrease in MMP, and increased apoptosis were exhibited. Further, altered antioxidant level (SOD, CAT, LDH, CYT P450, and CYT b5 r) has been observed in the studied splenic tissue, also histopathological changes observed in the rats exposed with high doses of ZNP. Therefore, ZNP may have the potential to induce a toxic effect even when exposed intermittently.


Nanotechnology Zinc oxide nanoparticles Oxidative stress Nanotoxicity Antioxidants Reactive oxygen species 


Compliance with ethical standards

Male Wistar rats were handled in accordance with the Institutional Animal Ethical Committee (Jawaharlal Nehru University, New Delhi).


  1. Aebi H (1984) [13] Catalase in vitro. Methods Enzymol 105:121–126Google Scholar
  2. Aitken R, Chaudhry MQ, Boxall ABA, Hull M (2006) Manufacture and use of nanomaterials: current status in the UK and global trends. Occup Med 56(5):300–306CrossRefGoogle Scholar
  3. Ansar S, Alshehri SM, Abudawood M, Hamed SS, Ahamad T (2017) Antioxidant and hepatoprotective role of selenium against silver nanoparticles. Int J Nanomedicine 12:7789–7797CrossRefGoogle Scholar
  4. Badria FA, Ibrahim AS, Badria AF, Elmarakby AA (2015) Curcumin attenuates iron accumulation and oxidative stress in the liver and spleen of chronic iron-overloaded rats. PLoS One 10(7):e0134156CrossRefGoogle Scholar
  5. Bandyopadhyay S, Plascencia-Villa G, Mukherjee A, Rico CM, José-Yacamán M, Peralta-Videa JR, Gardea-Torresdey JL (2015) Comparative phytotoxicity of ZnO NPs, bulk ZnO, and ionic zinc onto the alfalfa plants symbiotically associated with Sinorhizobium meliloti in soil. Sci Total Environ 515:60–69CrossRefGoogle Scholar
  6. Becheri A, Dürr M, Nostro PL, Baglioni P (2008) Synthesis and characterization of zinc oxide nanoparticles: application to textiles as UV-absorbers. J Nanopart Res 10(4):679–689CrossRefGoogle Scholar
  7. Ben-Slama I, Mrad I, Rihane N, Mir LE, Sakly M, Amara S (2015) Sub-acute oral toxicity of zinc oxide nanoparticles in male rats. J Nanomed Nanotechnol 6(3):1Google Scholar
  8. Bergmeyer HU, Bernt E (1971) In: Methods of Enzymatic Analysis, Vol IIGoogle Scholar
  9. Bogutska KI, Sklyarov YP, Prylutskyy YI (2013) Zinc and zinc nanoparticles: biological role and application in biomedicine. Ukr Bioorg Acata 1:9–16Google Scholar
  10. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1-2):248–254CrossRefGoogle Scholar
  11. Cash KJ, Clark HA (2010) Nanosensors and nanomaterials for monitoring glucose in diabetes. Trends Mol Med 16(12):584–593CrossRefGoogle Scholar
  12. Chung MK, Baek SS, Lee SH, Kim H, Choi K, Kim JC (2009) Combined repeated dose and reproductive/developmental toxicities of copper monochloride in rats. Environ Toxicol 24(4):315–326CrossRefGoogle Scholar
  13. Dahdouh F, Attalah S, Djabar MR, Kechrid Z (2016) Effect of the joint supplementation of vitamin c and vitamin e on nickel heamatotoxicity and nephrotoxicity in male Swiss albino mice. Int J Pharm Pharm Sci 8(6):234–239Google Scholar
  14. Dowling A, Clift R, Grobert N, Hutton D, Oliver R, O’Neill O, Pethica J, Pidgeon N, Porritt J, Ryan Seaton A (2004) Nanoscience and nanotechnologies: opportunities and uncertainties. The Royal Society, The Royal Academy of Engineering, LondonGoogle Scholar
  15. EI-Morshedi N, AlZahrani I, Kizibash NA, Al-Fayez HAA (2014) Toxic effect of zinc oxide nanoparticles on some organs in experimental male Wistar rats. Int J Adv Res 2:907–915Google Scholar
  16. Guo D, BiH LB, Wu Q, Wang D, Cui Y (2013) Reactive oxygen species-induced cytotoxic effects of zinc oxide nanoparticles in rat retinal ganglion cells. Toxicol in Vitro 27(2):731–738CrossRefGoogle Scholar
  17. Hackenberg S, Zimmermann FZ, Scherzed A, Friehs G, Froelich K, Ginzkey C, Burghartz M, Hagen R, Kleinsasser N (2011) Repetitive exposure to zinc oxide nanoparticles induces DNA damage in human nasal mucosa mini organ cultures. Environ Mol Mutagen 52(7):582–589CrossRefGoogle Scholar
  18. Heinlein C, Deppert W, Braithwaite A, Speidel D (2010) A rapid and optimization-free procedure allows the in vivo detection of subtle cell cycle and ploidy alterations in tissues by flow cytometry. Cell Cycle 9(17):3584–3590CrossRefGoogle Scholar
  19. Horie M, Nishio K, Fujita K, Endoh S, Miyauchi A, Saito Y, Iwahashi H, Yamamoto K, Murayama H, Nakano H, Nanashima N (2009) Protein adsorption of ultrafine metal oxide and its influence on cytotoxicity toward cultured cells. Chem Res Toxicol 22(3):543–553CrossRefGoogle Scholar
  20. Koeneman BA, Zhang Y, Westerhoff P, Chen Y, Crittenden JC, Capco DG (2010) Toxicity and cellular responses of intestinal cells exposed to titanium dioxide. Cell Biol Toxicol 26(3):225–238CrossRefGoogle Scholar
  21. Marklund S, Marklund G (1974) Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. FEBS J 47(3):469–474Google Scholar
  22. Negahdary M, Chelongar R, Zadeh SK, Ajdary M (2015) The antioxidant effects of silver, gold, and zinc oxide nanoparticles on male mice in in vivo condition. Adv Biomed Res 4Google Scholar
  23. Paulraj R, Behari J (2006) Single strand DNA breaks in rat brain cells exposed to microwave radiation. Mutat Res Fund Mol Mech Mutagen 596(1):76–80CrossRefGoogle Scholar
  24. Prasad AS (1991) Discovery of human zinc deficiency and studies in an experimental human model. Am J Clin Nutr 53(2):403–412CrossRefGoogle Scholar
  25. Ren Z, Wang Y, Deng H, Deng Y, Deng J, Zuo Z, Wang Y, Peng X, Cui H, Shen L (2015) Deoxynivalenol induces apoptosis in chicken splenic lymphocytes via the reactive oxygen species-mediated mitochondrial pathway. Environ Toxicol Pharmacol 39(1):339–346CrossRefGoogle Scholar
  26. Riccardi C, Nicoletti I (2006) Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat Protoc 1(3):1458–1461CrossRefGoogle Scholar
  27. Saliani M, Jalal R, Goharshadi EK (2016) Mechanism of oxidative stress involved in the toxicity of ZnO nanoparticles against eukaryotic cells. Nanomed J 3(1):1–14Google Scholar
  28. Satapathy SR, Mohapatra P, Preet R, Das D, Sarkar B, Choudhuri T, Wyatt MD, Kundu CN (2013) Silver-based nanoparticles induce apoptosis in human colon cancer cells mediated through p53. Nanomedicine 8(8):1307–1322CrossRefGoogle Scholar
  29. Sharma V, Anderson D, Dhawan A (2012a) Zinc oxide nanoparticles induce oxidative DNA damage and ROS-triggered mitochondria mediated apoptosis in human liver cells (HepG2). Apoptosis 17(8):852–870CrossRefGoogle Scholar
  30. Sharma V, Singh P, Pandey AK, Dhawan A (2012b) Induction of oxidative stress, DNA damage and apoptosis in mouse liver after sub-acute oral exposure to zinc oxide nanoparticles. Mutat Res Genet Toxicol Environ Mutagen 745(1-2):84–91CrossRefGoogle Scholar
  31. Sheweita SA (2000) Drug-metabolizing enzymes mechanisms and functions. Curr Drug Metab 1(2):107–132CrossRefGoogle Scholar
  32. Shukla RK, Kumar A, Pandey AK, Singh SS, Dhawan A (2011) Titanium dioxide nanoparticles induce oxidative stress-mediated apoptosis in human keratinocyte cells. J Biomed Nanotechnol 7(1):100–101CrossRefGoogle Scholar
  33. Singh N, Manshian B, Jenkins GJ, Griffiths SM, Williams PM, Maffeis TG, Wright CJ, Doak SH (2009) NanoGenotoxicology: the DNA damaging potential of engineered nanomaterials. Biomaterials 30(23-24):3891–3914CrossRefGoogle Scholar
  34. Srivastav AK, Kumar M, Ansari NG, Jain AK, Shankar J, Arjaria N, Jagdale P, Singh D (2016) A comprehensive toxicity study of zinc oxide nanoparticles versus their bulk in Wistar rats: toxicity study of zinc oxide nanoparticles. Hum Exp Toxicol 35(12):1286–1304CrossRefGoogle Scholar
  35. Strunk J, Kähler K, Xia X, Muhler M (2009) The surface chemistry of ZnO nanoparticles applied as heterogeneous catalysts in methanol synthesis. Surf Sci 603(10):1776–1783CrossRefGoogle Scholar
  36. Swain PS, Rao SB, Rajendran D, Dominic G, Selvaraju S (2016) Nano zinc, an alternative to conventional zinc as animal feed supplement: a review. Anim Nutr 2(3):134–141CrossRefGoogle Scholar
  37. Teow Y, Asharani PV, Hande MP, Valiyaveettil S (2011) Health impact and safety of engineered nanomaterials. Chem Commun 47(25):7025–7038CrossRefGoogle Scholar
  38. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J (2007) Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39(1):44–84CrossRefGoogle Scholar
  39. Wang J, Deng X, Zhang F, Chen D, Ding W (2014) ZnO nanoparticle-induced oxidative stress triggers apoptosis by activating JNK signaling pathway in cultured primary astrocytes. Nanoscale Res Lett 9(1):117CrossRefGoogle Scholar
  40. Wang X, Yang X, Chen S, Li Q, Wang W, Hou C, Gao X, Wang L, Wang S (2016) Zinc oxide nanoparticles affect biomass accumulation and photosynthesis in Arabidopsis. Front Plant Sci 6:1243Google Scholar
  41. Xue Y, Chen Q, Ding T, Sun J (2014) SiO2 nanoparticle-induced impairment of mitochondrial energy metabolism in hepatocytes directly and through a Kupffer cell-mediated pathway in vitro. Int J Nanomedicine 9:2891Google Scholar
  42. Yang K, Xing B (2009) Sorption of phenanthrene by humic acid-coated nanosized TiO2 and ZnO. Environ Sci Technol 43(6):1845–1851CrossRefGoogle Scholar
  43. Yang Z, Chen J, Dou R, Gao X, Mao C, Wang L (2015) Assessment of the phytotoxicity of metal oxide nanoparticles on two crop plants, maize (Zea mays L.) and rice (Oryza sativa L.). Int J Environ Res Public Health 12(12):15100–15109CrossRefGoogle Scholar
  44. Zhao X, Wang S, Wu Y, You H, Lv L (2013) Acute ZnO nanoparticles exposure induces developmental toxicity, oxidative stress and DNA damage in embryolarval zebrafish. Aquat Toxicol 136:49–59CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Neelu Singh
    • 1
  • Monoj Kumar Das
    • 2
  • Rohit Gautam
    • 1
  • Anand Ramteke
    • 2
  • Paulraj Rajamani
    • 1
    Email author
  1. 1.School of Environmental SciencesJNUNew DelhiIndia
  2. 2.Cancer Genetics and Chemoprevention Research Group, Department of Molecular Biology and BiotechnologyTezpur UniversityTezpurIndia

Personalised recommendations