Optimization of ZnO-NPs to Investigate Their Safe Application by Assessing Their Effect on Soil Nematode Caenorhabditis elegans
© Gupta et al. 2015
Received: 2 January 2015
Accepted: 12 July 2015
Published: 28 July 2015
Zinc oxide nanoparticles (ZnO-NPs) are increasingly receiving attention due to their widespread application in cosmetics, pigments and coatings. This has raised concerns in the public and scientific communities regarding their unexpected health effects. Toxicity effect of ZnO-NPs on the environment was assessed in the present study using Caenorhabditis elegans. Multiple toxicity end points including their mortality, behaviour, reproduction, in vitro distribution and expression of stress response mtl-1 and sod-1 genes were observed to evaluate safe application of ZnO-NPs. C. elegans were exposed to 10, 50, and 100 nm ZnO-NPs (0.1 to 2.0 g/l). Application of 10 nm ≥0.7g/l adversely affects the survivability of worms and was significantly not affected with exposure of 50 and 100 nm ≤1.0 g/l. However, reproduction was affected at much low concentration as compared to their survivability. LC50 was recorded 1.0 ± 0.06 (g/l) for 100 nm, 0.90 ± 0.60 for 50 nm and 0.620 ± 0.08 for 10 nm. Expression of mtl-1 and sod-1 was significantly increased with application of 10 nm ≥0.7g/l and significantly unaffected with exposure of 50 and 100 nm at the same concentration. ZnO-NPs (10 nm) had shown even distribution extended nearly the entire length of the body. The distribution pattern of ZnO-NPs indicates that the intestine is the major target tissues for NP toxicity. Study demonstrates that small-sized (10 nm) ZnO-NPs ≥0.7g/l is more toxic than larger-sized particles. This may be suggested on the basis of available data; application of 50 and 100 nm ≤1.0 g/l ZnO-NPs may be used to the environment as this shows no significant toxicity. However, further calibration is warranted to explore safe dose on soil compartments prior to their field application.
Nanotechnology is rapidly expanding the field of science with continuous development of nanomaterials and their industrial application. Nanoforms of metals, metal oxides, carbon-based materials and biopolymers are being used in several industries including diagnosis, drug delivery, cosmetics, sunscreens, food, paints, electronics, sports, imaging, etc. Among them, zinc oxide nanoparticles (ZnO-NPs) are most exploited at nano-dimension level. They are abundantly used nanomaterials in cosmetics and sunscreens as they exhibit high catalytic efficiency, as well as strong absorption ability for UV light. This makes them transparent and more aesthetically acceptable compared to their bulk counterpart as suggested by Schilling et al. . ZnO-NPs have vast area of application including biosensor, gas sensor, cosmetics, storage, optical devices, window materials and drug delivery. Colloidal solutions of ZnO-NPs as nano-fertilizer (500–1000 ppm) have potential to boost yield and growth of crops. Prasad et al.  recorded application of ZnO-NPs (25 nm @ 1000 ppm) enhanced seed germination, seedling vigor, plant and root growth in peanuts. They are also being used in the food industry as additives and packaging due to their antimicrobial properties [3, 4]. Hu et al.  explored their potential use as fungicides in agriculture. Toxicological studies with NPs have mainly performed on aquatic organisms as recorded by Heinlaan et al. and Zhu et al. [6, 7]. However, Peralta-Videa et al. , Roh et al. , Hu et al. , Van der Ploeg et al.  and Gupta et al.  studied effects of short-term NP exposure to the isopod Porcellio scaber, Caenorhabditis elegans and earthworms Eisenia fetida and Lumbricus rubellus, respectively. Furthermore, researches are needed to provide insight into the toxicological effect of exposure to NPs on organisms especially living in the soil. Auffan et al.  reported metal-based NPs like ZnO, TiO2, Ag and CeO2; toxicity is at least partly due to the specific properties related to the small size and consequent high surface activity of NPs, while their effects may be further enhanced by the release of the free metal ions. If the free ions are more toxic than the original particles, this process of dissolution is likely to lead an increase of the overall toxicity. Speciation of metal NPs is soils is not yet clearly understood. Some works talk about the distribution of Zn, from ZnO-NPs in soil is the most liable fractions (those accessible to inhabiting soil organism) and differences between ZnO-NPs and derivate ion Zn2+ . However, assessment of toxicity of the ionic forms of metal oxide nanoparticles may require particular attention on their solubility. ZnO-NPs can impose serious toxicity to bacteria, Daphnia magna, freshwater microalgae, mice and even human cell [14–17]. These nanoparticles have the ability to penetrate to the skin and to exert toxicity to viable cells. Jeng and Swanson  observed dramatic changes in cell morphology and apoptosis in mammalian cell after exposure of 50 μg/ml for 24 h. At exposure of 100 μg/ml, the recorded death of the cells was 15 to 50 %; Jeng and Swanson  also observed reduced mitochondrial activity (>80%).. Mitochondrial function showed that ZnO exhibits more toxicity than other metal oxide nanoparticles . Jeng and Swanson  also observed reduced mitochondrial activity (>80 %).
The increased production and use of ZnO-NPs enhances the probability of their exposure in occupational and environmental settings. Although this NP has great commercial importance and present in various commercial products, so therefore is growing concern in public and scientific communities regarding their unanticipated and adverse health effects. A significant portion of ZnO-NPs may be expected to be released in sewage sludge via waste water. Varying portions of sewage sludge were disposed in landfills and incinerated or applied to agricultural land . Therefore, terrestrial ecosystems are expected to be an ultimate sink for a large portion of NPs. This raises concerns about potential effects, entry in food webs and ultimately human exposure from consumption of contaminated agricultural products. In the current study, the toxicity of different-sized ZnO-NPs were assessed on the soil nematodes C. elegans using multiple toxicity end points to record optimal dose and size of these NPs for their safe application.
ZnO-NPs (100, 50 and 10 nm) were purchased from Sigma Aldrich Chemical (St Louis, MO, USA). Particles were labelled as suggested by Tachikawa et al.  with fluorescent polymer. The size of the particles was measured in 20-μl particle suspension from the test medium on 400 mesh carbon-coated copper grid and observed using a transmission electron microscope (40-100KV) at Sophisticated Analytical Instrumentation Facility of Electron Microscopy, Department of Anatomy, All India Institute of Medical Sciences, New Delhi, India.
Test Organism and Method of Exposure
The wild-type C. elegans Bristol strain N2 was obtained from Caenorhabditis Genetic Centre (CGC), USA, and culture was maintained on nematode growth medium (NGM) plates seeded with Escherichia coli strain OP50 at 20 °C, using the standard method . Young adult (3 days old) synchronized culture were used in all the experiments. Worms were incubated at 20 °C for 24 h without a food source and were then subjected to the analysis . Nematodes were exposed to three different-sized ZnO-NPs (10, 50 and 100nm). The test consisted series of seven ZnO-NP concentrations (0.1–2.0 g/l). NPs were diluted in K-medium (32 mM KCl, 51 mM NaCl) following Williams and Dusenbery  and buffered in 140 mM sodium acetate (pH 6.0) to avoid aggregation. Each treatment was replicated for three times, and control (K-medium + buffer) was maintained for the entire test.
Assessment of Mortality and Behaviour
Toxicity end points including LC50 (probit method), mortality and mobility were observed after 24-h incubation with exposure of NPs, and juveniles were recorded at 48 h. Visually dead worms were sorted out with a platinum wire under stereo microscope.
Analysis Expression of Stress Response Gene mtl-1 and sod-1
After 24-h incubation with exposure of NPs, nematodes were harvested for the analysis of mtl-1 (metal response proteins) and sod-1(anti oxidant enzyme) gene expression. Standard procedures were followed as suggested by Roh et al. . Reverse transcription (two steps) polymerase chain reaction (RT-PCR) method was used, and PCR products were separated on 1.5 % agarose gel through electrophoresis and observed by using ethidium bromide. Relative density of each band was observed in BioRed gel documentation system.
Assessment of In Vivo Distribution of NPs
After exposure of ZnO-NPs, fluorescence distribution images were observed by using fluorescence microscope equipped with a peltier cooled charge-coupled camera. Both differential interference contrast (DIC) and epi-fluorescence images were taken. Filter set with maxima of 460 nm was used for visualization of fluorescence.
Results are the means of three replicates. Two-way analysis of variance (ANOVA) was performed by using the SPSS 10.5 software. The objective of statistical analysis was to determine any significant differences among the parameters analysed in different treatments to record optimal dose and size of ZnO NPs for their safe application. p values used to decide statistical significance based on the somewhat arbitrary choice of level were often set at 0.05 except for mortality and behaviour (0.01).
Results and Discussion
Mortality and Behaviour of ZnO-NP-exposed C. elegans
Expression of mtl-1 and sod-1 Gene
Gene expression may consider as high sensitivity and mechanistic values to diagnose environmental contamination. Nowadays, environmental stress response in toxicology was increasingly recorded by using gene profiling by many workers including Snell et al. , Lee and Choi , Roh et al.  and Poynton et al. . In the present investigation, the effect of ZnO-NPs on alteration of gene expression after exposure of ZnO-NPs was analysed in two genes, namely, mtl-1 and sod-1 by using stress response gene expression profiling analysis . Low-molecular weight cystine-rich protein, metallothionein (mtl), helps in metal detoxification and homeostasis. It is ubiquitous in most of the animals. A cell-specific factor, as well as developmentally modulated and metal response pathways controls their transcription. Due to their inducibility to transition metals, they are considered as an important specific biomarker to detect organism response to heavy metals. They have been involved in the defence against reactive oxygen species and also in the homeostatic regulation of heavy metals. Otuska  assumed them as multifunctional proteins with additional unidentified physiological roles. Therefore, mtl-1 gene may serve as significant model for investigating the toxicity of ZnO-NPs and mechanism to cellular response of uptake of heavy metals.
Biodistribution of ZnO-NPs
The present study was focussed to optimize the safe application of ZnO-NPs by assessing multiple toxicity points and in vitro distribution in nanoparticle-exposed C. elegans. We conclude that the activity of worms was not adversely affected with exposure of 50 and 100 nm ZnO-NPs ≤1.0 g/l, while small-sized particles 10 nm ≥0.7g/l affects them at large as shown by an increase in the expression of mtl-1 and sod-1 gene. Distributional pattern of ZnO-NPs reveals that the intestine is the major target tissues for NP toxicity. The application of 50 and 100 nm ZnO-NPs may be safe to the environment in comparison to 10 nm. However, further calibration and validation of identified dose are warranted to explore more dose-response toxicity assessment and their kinetics of in vivo distribution.
Authors acknowledge the financial support of the Department of Biotechnology, Ministry of Science and Technology, Govt. of India, New Delhi and to Dr K. Subramaniam, Indian Institute of Technology, Kanpur, India for culture techniques of C. elegans in the present study.
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