Plant Growth Regulation

, Volume 71, Issue 2, pp 157–170 | Cite as

Chromium (VI)-induced hormesis and genotoxicity are mediated through oxidative stress in root cells of Allium cepa L.

  • Anita R. Patnaik
  • V. Mohan M. Achary
  • Brahma B. Panda
Original paper


Chromium (VI) genotoxicity was evaluated in Allium bioassay by using different treatment protocols. Treatment of bulbs of Allium cepa L. with Cr(VI) at a range of concentrations for 5 days (120 h) exhibited low dose (12.5 μM) stimulation and high dose (25–200 μM) inhibition of root growth apparently indicating hormesis. Inhibition of root growth was correlated with the dose-dependent increase in generation of reactive oxygen species (ROS), cell death, lipid peroxidation, repression of antioxidative enzymes (catalase, superoxide dismutase, ascorbate peroxidase), induction of DNA damage, chromosome aberrations or micronuclei in root cells. The above effects were, however, reversed when the duration of Cr(VI) treatment was limited to 3–24 h followed by recovery in tap water for 4 days that resulted in the dose-dependent stimulation of root growth, mitosis and increased activity of the antioxidative enzymes that obliterated oxidative stress and genotoxicity. The above Cr(VI)-induced stimulation of root growth was effectively countered by pre- or post-treatments of dimethylthiourea, a ROS-scavenger. These findings underscored that Cr(VI), depending on the magnitude of the dose (concentration × time), could either be stimulatory or inhibitory for root growth that underlined the crucial role of ROS having obvious implications in agriculture, post harvest technology and human health.


Antioxidant enzymes Cell death Comet assay Oxidative stress Genotoxicity Hormesis 



Ascorbate peroxidase


Chromosome aberration






Ethylenediamine-tetra acetic acid


Fresh weight


Guaiacol peroxidase


Low melting point agarose




Mitotic index




Nitroblue tetrazolium


Olive tail moment


Reactive oxygen species


Superoxide dismutase


Thiobarbituric acid


Trichloroacetic acid



The present research was carried out through fellowships awarded to ARP and VMMA, respectively, from UGC and CSIR, New Delhi. The authors are thankful to the authorities of Berhampur University for providing administrative and infrastructural facilities to carry out the research and to Dr. B. B. Nayak, IMMT, Bhubaneswar for help with analysis of Cr(VI) in experimental solutions.


  1. Achary VMM, Panda BB (2010) Aluminium-induced DNA-damage and adaptive response to genotoxic stress in plant cells are mediated through reactive oxygen intermediates. Mutagenesis 25:201–209CrossRefGoogle Scholar
  2. Achary VMM, Jena S, Panda KK, Panda BB (2008) Aluminium induced oxidative stress and DNA damage in root cells of Allium cepa L. Ecotoxicol Environ Saf 70:300–310PubMedCrossRefGoogle Scholar
  3. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126PubMedCrossRefGoogle Scholar
  4. Agutter PS (2008) Elucidating the mechanism(s) of hormesis at the cellular level: the universal cell response. Am J Pharmacol Toxicol 3:100–110CrossRefGoogle Scholar
  5. Ahmad P, Sarwat M, Sharma S (2008) Reactive oxygen species, antioxidants and signalling in plants. J Plant Biol 51:167–173CrossRefGoogle Scholar
  6. Aiyar J, Buerkovits HJ, Floyd RA, Borges K (1991) Reaction of chromium (VI) with glutathione or with hydrogen peroxide: identification of reactive intermediates and their role in chromium (VI)-induced DNA damage. Environ Health Perspect 92:53–62PubMedCrossRefGoogle Scholar
  7. Alvarez ME, Pennell RI, Meijer PJ, Ishikawa A, Dixon RA, Lamb C (1998) Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity. Cell 9:773–784CrossRefGoogle Scholar
  8. Baker CJ, Mock NM (1994) An improved method for monitoring cell death in cell suspension and leaf disc assays using Evans blue. Plant Cell Tissue Organ Cult 39:7–12CrossRefGoogle Scholar
  9. Beauchamp C, Fridovich I (1971) Superoxide dismuatse: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44:276–287PubMedCrossRefGoogle Scholar
  10. Belz RG, Cedergreen N, Duke SO (2011) Herbicide hormesis—can it be useful in crop production? Weed Res 51:321–332CrossRefGoogle Scholar
  11. Bradford MM (1976) A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  12. Bray CM, West CE (2005) DNA repair mechanisms in plants: crucial sensors and effectors for the maintenance of genome integrity. New Phytol 168:511–528PubMedCrossRefGoogle Scholar
  13. Bryant HE, Ying S, Helleday T (2006) Homologous recombination is involved in repair of chromium-induced DNA damage in mammalian cells. Mutat Res 599:116–123PubMedCrossRefGoogle Scholar
  14. Calabrese EJ (2005) Paradigm lost, paradigm found: the re-emergence of hormesis as a fundamental dose response model in the toxicological sciences. Environ Pollut 138:378–411CrossRefGoogle Scholar
  15. Calabrese EJ (2009) Getting the dose–response wrong: why hormesis became marginalized and the threshold model accepted. Arch Toxicol 83:227–247PubMedCrossRefGoogle Scholar
  16. Calabrese EJ, Blain RB (2004) Metals and hormesis. J Environ Monit 6:14–19CrossRefGoogle Scholar
  17. Calabrese EJ, Blain RB (2009) Hormesis and plant biology. Environ Pollut 157:42–48PubMedCrossRefGoogle Scholar
  18. Casadevall M, da Cruz Fresco P, Kortenkamp A (1999) Chromium(VI)-mediated DNA damage: oxidative pathways resulting in the formation of DNA breaks and abasic sites. Chem-Biol Interact 123:117–132PubMedCrossRefGoogle Scholar
  19. Cedergreen N, Streibig JC, Kudsk P, Mathiassen SK, Duke SO (2007) The occurrence of hormesis in plants and algae. Dose-Response 5:150–162CrossRefGoogle Scholar
  20. Cervantes C, Campos-Garcia J, Devars S, Gutierrez-Corona F, Loza-Tavera H, Torres-Guzman JC (2001) Interactions of chromium with microorganisms and plants. FEMS Microbiol Rev 25:335–347PubMedCrossRefGoogle Scholar
  21. Chakraborty R, Mukherjee AK, Mukherjee A (2009) Evaluation of genotoxicity of coal fly ash in Allium cepa root cells by combining comet assay with the Allium test. Environ Monitor Assess 53:351–357CrossRefGoogle Scholar
  22. Chance B, Maehly AC (1955) Assay of catalase and peroxidases. Methods Enzymol 2:764–775CrossRefGoogle Scholar
  23. Chen GX, Asada K (1989) Ascorbate peroxidase in tea leaves: occurrence of two isozymes and the differences in enzymatic and molecular properties. Plant Cell Physiol 30:987–998Google Scholar
  24. Dhindsa RS, Plumb-Dhindsa P, Thorpe TA (1981) Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxided dismutase and catalase. J Exp Bot 32:93–101CrossRefGoogle Scholar
  25. Di Salvatore M, Carafa AM, Carratù G (2008) Assessment of heavy metals phytotoxicity using seed germination and root elongation tests: a comparison of two growth substrates. Chemosphere 73:1461–1464PubMedCrossRefGoogle Scholar
  26. Diwan H, Khan I, Ahmad A, Iqbal M (2010) Induction of phytochelatins and antioxidant defence system in Brassica juncea and Vigna radiata in response to chromium treatments. Plant Growth Regul 61:97–107CrossRefGoogle Scholar
  27. Duan P, Zhai T, Xu C, Ding J, Chen Y (2013) A simple and effective method for detecting toxicity of chromium trioxide on Vicia faba. Eur Food Res Technol 236:517–521CrossRefGoogle Scholar
  28. Eleftheriou EP, Adamakis I-DS, Melissa P (2012) Effects of hexavalent chromium on microtubule organization, ER distribution and callose deposition in root tip cells of Allium cepa L. Protoplasma 249:401–416PubMedCrossRefGoogle Scholar
  29. Eleftheriou EP, Adamakis I-DS, Fatsiou M, Panteris E (2013) Hexavalent chromium disrupts mitosis by stabilizing microtubules in Lens culinaris Moench. root tip cells. Physiol Plant 147:169–180PubMedCrossRefGoogle Scholar
  30. Fiskesjo G (1988) The Allium test—an alternative in environmental studies: the relative toxicity of metal ions. Mutat Res 197:243–260PubMedCrossRefGoogle Scholar
  31. Foyer CH, Noctor G (2005) Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. Plant Cell 17:1866–1875PubMedCrossRefGoogle Scholar
  32. Gomez KA, Gomez AA (1984) Statistical procedures for agricultural research. Wiley, New YorkGoogle Scholar
  33. Gratao PL, Polle A, Lea P, Azevedo RA (2005) Making life of heavy metal stressed plants a little easier. Funct Plant Biol 32:481–494CrossRefGoogle Scholar
  34. Ha L, Ceryak S, Patierno SR (2003) Chromium (VI) activates ataxia telangiectasia mutated (ATM) protein: requirement of ATM for both apoptosis and recovery from terminal growth arrest. J Chem Biol 278:17885–17894CrossRefGoogle Scholar
  35. Halliwell B, Gutteridge JMC, Aruoma O (1987) The deoxyribose method: a simple ‘test tube’ assay for determination of rate constants for reactions of hydroxyl radicals. Anal Biochem 165:215–219PubMedCrossRefGoogle Scholar
  36. Hayashi M, Dearfield K, Kasper P, Lovell D, Martus HJ, Thybaud V (2011) Compilation and use of genetic toxicity historical control data. Mutat Res 723:87–90PubMedCrossRefGoogle Scholar
  37. Kiba A, Miyake C, Toyoda K, Ichinose Y, Yamada T, Shiraishi T (1997) Superoxide generation in extracts from isolated plant cell walls is regulated by fungal signal molecules. Phytopathology 87:846–852PubMedCrossRefGoogle Scholar
  38. Kumaravel TS, Vilhar B, Faux SP, Jha AN (2009) Comet assay measurements: a perspective. Cell Biol Toxicol 25:53–64PubMedCrossRefGoogle Scholar
  39. Leme DM, Marin-Morales MA (2009) Allium cepa test in environmental monitoring: a review on its application. Mutat Res 682:71–81PubMedCrossRefGoogle Scholar
  40. Leonard SS, Harris GK, Shi X (2004) Metal-induced oxidative stress and signal transduction. Free Rad Biol Med 37:1921–1942PubMedCrossRefGoogle Scholar
  41. Liszkay A, van der Zalm AE, Schopfer P (2004) Production of reactive oxygen intermediates (O2·, H2O2 and ·OH) by maize roots and their role in wall loosening and elongation growth. Plant Physiol 136:3114–3123PubMedCrossRefGoogle Scholar
  42. Liu DH, Jiang WS, Li W (1992) Effects of trivalent and hexavalent chromium on root growth and cell division of Allium cepa. Hereditas 117:23–29CrossRefGoogle Scholar
  43. Loreto F, Velikova V (2001) Isoprene produced by leaves protects the photosynthetic apparatus against ozone damage, quenches ozone products, and reduces lipid peroxidation of cellular membranes. Plant Physiol 127:1781–1787PubMedCrossRefGoogle Scholar
  44. Micera G, Dessi A (1988) Chromium adsorption by plant roots and formation of long-lived Cr(V) species: an ecological hazard? J Inorg Biochem 34:157–166CrossRefGoogle Scholar
  45. Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410PubMedCrossRefGoogle Scholar
  46. Nickens KP, Patierno SR, Ceryak S (2010) Chromium genotoxicity: a double-edged sword. Chem Biol Interact 188:276–288PubMedCrossRefGoogle Scholar
  47. Nriagu JO (1988) Production and uses of chromium. In: Nriagu JO, Nieboer E (eds) Chromium in the natural and human environments. Wiley, New York, pp 81–103Google Scholar
  48. Oliveira H (2012) Chromium as an environmental pollutant: insights on induced plant toxicity. J Bot. doi: 10.1155/2012/375843 (in press)
  49. Panda SK (2007) Chromium-mediated oxidative stress and ultrastructural changes in root cells of developing rice seedlings. J Plant Physiol 164:1419–1428PubMedCrossRefGoogle Scholar
  50. Panda KK, Achary VMM, Krishnaveni R, Padhi BK, Sarangi SN, Sahu SN, Panda BB (2011) In vitro biosynthesis and genotoxicity bioassay of silver nanoparticles using plants. Toxicol In Vitro 25:1097–1105PubMedCrossRefGoogle Scholar
  51. Pandey V, Dixit V, Shyam R (2005) Antioxidative responses in relation to growth of mustard (Brassica juncea cv. Pusa Jaikisan) plants exposed to hexavalent chromium. Chemosphere 61:40–47PubMedCrossRefGoogle Scholar
  52. Pandey V, Dixit V, Shyam R (2009) Chromium (VI) induced changes in growth and root plasma membrane redox activities in pea plants. Protoplasma 235:49–55PubMedCrossRefGoogle Scholar
  53. Patnaik AR, Achary VMM, Panda BB (2011) Comet assay to assess DNA damage and genotoxic stress in plants. In: Roy BK, Chaudhary BR, Sinha RP (eds) Plant Genome: Biodiversity, Conservation and Manipulation. Narosa Publications, New Delhi, pp 17–29Google Scholar
  54. Patra J, Sahoo MK, Panda BB (2003) Persistence and prevention of aluminium- and paraquat-induced adaptive response to methyl mercuric chloride in plant cells in vivo. Mutat Res 538:51–61PubMedCrossRefGoogle Scholar
  55. Radak Z, Chung HY, Kotai E, Taylor AW, Goto S (2008) Exercise, oxidative stress and hormesis. Age Res Rev 7:34–42CrossRefGoogle Scholar
  56. Rank J, Nielsen MN (1994) Evaluation of the Allium anaphase-telophase test in relation to genotoxicity screening of industrial wastewater. Mutat Res 312:17–24PubMedCrossRefGoogle Scholar
  57. Reichheld J-F, Vernoux T, Lardon F, Mantagu MV, Inze D (1999) Specific checkpoints regulate plant cell cycle progression in response to oxidative stress. Plant J 17:647–656CrossRefGoogle Scholar
  58. Rodriguez E, Azevedo R, Fernandes P, Santos C (2011) Cr(VI) induces DNA damage, cell cycle arrest and polyploidization: a flow cytometric and comet assay study in Pisum sativum. Chem Res Toxicol 24:1040–1047PubMedCrossRefGoogle Scholar
  59. Rozman KK, Doull J, Hayes WJ Jr (2010) Dose and time determining, and other factors influencing, toxicity. In: Krieger R (ed) Hayes’ handbook of pesticide toxicology. Elsevier, New York, pp 3–101CrossRefGoogle Scholar
  60. Shama G, Alderson P (2005) UV hormesis in fruits: a concept ripe for commercialisation. Trends Food Sci Tech 16:28–136CrossRefGoogle Scholar
  61. Shanker AK, Cervantes C, Loza-Tavera H, Avudainayagam S (2005) Chromium toxicity in plants. Environ Intern 31:739–753CrossRefGoogle Scholar
  62. Sharma SS, Dietz KJ (2008) The relationship between metal toxicity and cellular redox imbalance. Trends Plant Sci 14:43–50PubMedCrossRefGoogle Scholar
  63. Shi X, Dalal NS (1990) Evidence for a fenton-type mechanism for the generation of ·OH radicals in the reduction of Cr(VI) in cellular media. Arch Biochem Biophys 281:90–95PubMedCrossRefGoogle Scholar
  64. Shulaev V, Oliver DJ (2006) Metabolic and proteomic markers for oxidative stress. New tools for reactive oxygen species research. Plant Physiol 141:367–372PubMedCrossRefGoogle Scholar
  65. Stearns DM, Courtney KD, Giangrande PH, Phieffer LS, Wetterhahn KE (1994) Chromium(VI) reduction by ascorbate: role of reactive intermediates in DNA damage in vitro. Environ Health Perspect 102:21–25PubMedGoogle Scholar
  66. Stebbing ARD (1982) Hormesis: the stimulation of growth by low levels of inhibitors. Sci Total Environ 22:213–234PubMedCrossRefGoogle Scholar
  67. Sugiyama M (1992) Role of physiological antioxidants in chromium (VI)-induced cellular injury. Free Rad Biol Med 12:397–407PubMedCrossRefGoogle Scholar
  68. Ueno S, Sugiyama M, Susa N, Furukawa Y (1995) Effect of dimethylthiourea on chromium (VI)-induced DNA single-strand breaks in Chinese hamster V-79 cells. Mutat Res Lett 346:247–253CrossRefGoogle Scholar
  69. Ünyayar S, Çelik A, Çekiç FO, Gözel A (2006) Cadmium-induced genotoxicity, cytotoxicity and lipid peroxidation in Allium sativum and Vicia faba. Mutagenesis 21:77–81PubMedCrossRefGoogle Scholar
  70. Wakeman TP, Xu B (2006) ATR regulates hexavalent chromium-induced S-phase checkpoint through phosphorylation of SMC1. Mutat Res 610:14–20PubMedCrossRefGoogle Scholar
  71. Wang W (1991) Literature review on higher plants for toxicity testing. Water Air Soil Pollut 59:381–400CrossRefGoogle Scholar
  72. Wang X, Sun C, Gao S, Wang L, Shuokui H (2001) Validation of germination rate and root elongation as indicator to assess phytotoxicity with Cucumis sativus. Chemosphere 44:1711–1721PubMedCrossRefGoogle Scholar
  73. Zayed AM, Terry N (2003) Chromium in the environment: factors affecting biological remediation. Plant Soil 249:139–156CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Anita R. Patnaik
    • 1
  • V. Mohan M. Achary
    • 2
  • Brahma B. Panda
    • 1
  1. 1.Molecular Biology and Genomics Laboratory, Department of BotanyBerhampur UniversityBerhampurIndia
  2. 2.Plant Molecular Biology GroupInternational Centre for Genetic Engineering and BiotechnologyNew DelhiIndia

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