Elsevier

Plant Physiology and Biochemistry

Volume 121, December 2017, Pages 206-215
Plant Physiology and Biochemistry

Research article
Engineered nickel oxide nanoparticles affect genome stability in Allium cepa (L.)

https://doi.org/10.1016/j.plaphy.2017.11.003Get rights and content

Highlights

  • First report on engineered NiO-NP induced genotoxicity in plants.

  • NiO-NP triggers indigenous ROS generation even at a low dose.

  • NiO-NP has detrimental effects on Genomic Template Stability and Band Sharing Indices in a dose dependent manner.

  • DNA damage signifies NiO-NP as an environmental hazard element.

Abstract

Indiscriminate uses of engineered nickel oxide nanoparticles (NiO-NPs) in heavy industries have ushered their introduction into the natural environment, ensuing novel interactions with biotic components of the ecosystem. Though much is known about the toxicity of NiO-NPs on animals, their phytotoxic potential is not well elucidated. NiO-NP hinders intra-cellular homeostasis by producing ROS in excess, having profound effect on the antioxidant profile of exposed animal and plant tissues. In the present study, bulbs of the model plant Allium cepa were treated with varying concentrations of NiO-NP (10 mg L−1 - 500 mg L−1) to study changes in ROS production and potential genotoxic effect. The data generated proved a concomitant upsurge in intracellular ROS accumulation with NiO-NP dosage that could be correlated with increased genotoxicity in A. cepa. Augmented in situ ROS production was revealed through DCFH-DA assay, with highest increase in fluorescence (70% over control) in bulbs exposed to 125 mg L−1 NiO-NP. Effect of NiO-NP on genomic DNA was studied through detailed analyses of RAPD profiles which allows detection of even slightest changes in DNA sequence of treated plants. Significant differences in band intensity, loss and appearance of bands as well as genomic template stability and band sharing indices of treated plants revealed increased vulnerability of genomic DNA to NiO-NP, at even lowest concentration (10 mg L−1). This is the first report of NiO-NP induced genotoxicity on A. cepa, which confirms the nanoparticle as a potent environmental hazard.

Introduction

Intensification in synthesis and application of engineered nanoparticles (ENPs) have augmented their large scale production and usage in various industrial and commercial projects in the last decade (Mueller and Nowack, 2008). From fast moving consumer goods (FMCG) sector to hard core semiconductor and electrical industry, ENPs now-a-days find use in myriad novel avenues (Zhang et al., 2012). The estimated worth of ENP industry worldwide might stood at 2.4 billion USD in 2016 (Global Markets for Nanocomposites, Nanoparticles, Nanoclays, and Nanotubes, 2012). Among these commercially exploited ENPs, metallic ENPs are most predominantly used and hence are extensively studied. Mostly oxides, these metal nanoparticles can occur simultaneously in nature, though wide-scale production in recent times, have raised concerns regarding their impact and association with the environment (Wiesner et al., 2006). Reports suggest that metallic ENPs can interact with and affect plants adversely. Genotoxic and cytotoxic outcomes consequent to exposure of metal ENPs like, Ag2O, ZnO, CeO2 and TiO2, are known in a variety of plants, including Arabidopsis thaliana, Allium cepa, Cucurbita pepo, Vicia faba, etc. (Kaveh et al., 2013, Ghosh et al., 2010, Zhang et al., 2012, López-Moreno et al., 2010). Nickel oxide nanoparticle (NiO-NP) has gained prevalence in the last few years, being used capaciously in the stainless steel and electrical industry, in making coin and artificial jewelleries etc. (Salimi et al., 2007, Rao and Sunandana, 2008). Large scale production and uses have raised apprehensions regarding their release into the environment and the consequences of their interaction with the biotic components in particular (Gong et al., 2011). Research on direct interaction of bulk nickel oxide with higher animals and humans helped identify it as a carcinogen, effective at even low concentrations (Oyabu et al., 2007). The interaction and effect of NiO-NP on plants, however, is relatively less studied. Since plants are the primary part and most critical component of the food chain in the ecosystem, understanding how NiO-NPs affect them becomes an urgent necessity.

RAPD (Random Amplified Polymorphic DNA) is a PCR (Polymerase Chain Reaction) based technique, and relatively quick, easy and efficient for studying subtle differences in genomic DNA bands (Williams et al., 1990). The technique was originally extensively used for species classification, genetic mapping and phylogeny analyses, etc. (Powell et al., 1996, Bartish et al., 2000, Ranade et al., 2001). When used in surveying genomic DNA for evidence of DNA damage and mutation, RAPD has the potential to form the bases of novel biomarker assays for the detection of DNA damage and mutational events (e.g. rearrangements, point mutation, small insertions or deletions of DNA and ploidy changes) in cells of bacteria, plants, invertebrate and vertebrate animals (Savva, 1996, Savva, 2000, Atienzar et al., 2000), thus qualifying as the technique of choice for most genotoxicity researchers. Detection of genotoxic effect using RAPD involves the comparison of profiles generated from control (unexposed) and treated (exposed) DNA. The resultant amplified DNA amplification profiles may differ due to band shifts, missing bands or the appearance of new bands.

Allium cepa is the model system for studying ecotoxicity potential of any chemical or xenobiotic agent, and as a test system has been used since the 1940s to evaluate genotoxicity. Its ease of growth, wide availability, genomic plasticity, low cost and easy handling makes A. cepa the ideal organism to study genotoxicity, mutagenecity and cytotoxicity (Leme and Marin-Morales, 2009). It was also adopted by the International program on plant bioassays (IPPB) for evaluation of environmental pollutants (Ma, 1999). In the present study, RAPD technique was used to investigate genotoxicity of various concentrations of NiO-NP suspensions (10 mg L−1 to 500 mg L−1) in Allium cepa. To the authors’ knowledge, this is the first report of utilization of RAPD profiling for toxicity assessment after NiO-NP on either plants or animal systems.

Section snippets

Plant materials and growth condition

Seeds of Allium cepa (var. Nasik Red), were procured from Suttons Seeds Pvt. Ltd., Kolkata, germinated and grown into healthy plants in the Experimental Garden, Department of Botany, University of Calcutta. Fresh, healthy bulbs, grown under controlled condition, were obtained which were put on wet bed made of moist, sterilised sand and kept in a growth chamber at 23 ± 2 °C in dark for rooting. Healthy uniformly rooted bulbs were selected for treatment with various concentrations of NiO-NP

Characterisation of NiO-NP

TEM and AFM analyses were performed to elucidate and evaluate morphology, size and ultrastructure of NiO-NPs in solid state. TEM analysis revealed that most particles tended to form agglomerates with a non-uniform morphology and a single particle appeared as a crystallite with polyhedral morphology [Fig. 1a]. The average size calculated from measuring 100 particles in random fields of TEM view, was found approximately 33.65 nm [Fig. 1b]. AFM images indicated the average size to be around

Discussion

Present evidence indicates that engineered nanoparticle exposure can induce genotoxicity in plants (López-Moreno et al., 2010). Many findings suggest potential long-term risk to ecosystems and both long term (epigenetic changes) (Wang et al., 2012) through changes in methylation status (Ghosh et al., 2015) and short term damages to plants (Morales et al., 2013). The effects might be detrimental or positive, totally depending on the internal physiology of the plant (Ma et al., 2010a). Many

Conflicts of interest

The authors would like to declare that there is no conflict of interest, and no commercial or financial aid has been involved for the study.

Author's contribution

Both the authors contributed equally for the work.

Acknowledgement

The authors would like to acknowledge the facilities provided by the Department of Botany (CAS Phase VI, VII), University of Calcutta, DBT-IPLS facility, University of Calcutta for providing access to confocal microscopy and HOD, Department of Biochemistry, University of Calcutta for letting use of General Instrument Facility for spectroflourometry, and DST-FIST for infrastructural support and UGC under Government of India for general financial support.

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