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Environmental and Experimental Botany 91 (2013) 1– 11

Contents lists available at SciVerse ScienceDirect

Environmental and Experimental Botany

jou rn al h om epa ge: www.elsev ier .com/ locate /envexpbot

oxicity of aluminium oxide nanoparticles demonstrated using a BY-2lant cell suspension culture model

uzana Poborilovaa, Radka Opatrilovab, Petr Babulaa,∗

Department of Natural Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1/3, CZ-61242 Brno, CzechepublicDepartment of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1/3, CZ-61242 Brno, Czechepublic

r t i c l e i n f o

rticle history:eceived 11 December 2012eceived in revised form 2 February 2013ccepted 7 March 2013

eywords:luminium oxide nanoparticleshytotoxicityxidative stressrogrammed cell deathnvironmental toxicology

a b s t r a c t

Aluminium oxide nanoparticles have been applied in many branches of industry. They are also used inpersonal care products, such as cosmetics. Because of these uses, their impact on the environment must beconsidered and investigated. Almost nothing is known about the effects of aluminium oxide nanoparticleson plants at the cellular level; the objective of this work was thus to study the effects of nanoparticleson the plant cell model tobacco BY-2 cell suspension culture, which serves as a model comparable withthe HeLa cells used for animal cell studies. We observed the impact of these nanoparticles at differentlevels. The inhibitory effect on growth was observed in both time- and concentration-dependent studies.In addition, the ability of the nanoparticles to generate reactive oxygen (hydrogen peroxide, superoxideanion radical) and nitrogen species (nitric oxide) has been established. The principal part of the workwas focused on the ability of aluminium oxide nanoparticles to induce the processes of programmed cell

death. Changes observed in the permeability of the plasma membrane are connected with the effectsof the reactive oxygen species and lipid peroxidation. In addition, the loss of mitochondrial potential,the enhancement of the caspase-like activity and the fragmentation of DNA determined in both time-and concentration dependent studies are closely connected with the execution of the programmed celldeath. Our results indicate the ability of aluminium oxide nanoparticles to induce programmed cell deathin plant cells and may explain the toxic effect of these nanoparticles on plants.

. Introduction

Aluminium is the third most abundant element in the Earth’srust and the most abundant metallic element. It has a very wideange of uses, especially in the form of different types of alloys,here it improves the mechanical properties (Johnson and Sanders,

012; Ruan and Schuh, 2012). However, elemental aluminium isot the only chemical form used. Because aluminium compoundsemonstrate relatively low toxicity, they are found in significant

nd sometimes large-scale applications. Aluminium oxide, Al2O3,s usually used for the reduction to aluminium, but it has also beenpplied in industry, especially as a catalyst or catalyst support,

Abbreviations: Ac-DEVD-pNA, acetyl-Asp-Glu-Val-Asp-p-nitroanilide; DHE,ihydroethidium; DW, dry weight; FDA, fluorescein diacetate; FW, fresh weight;2DCFDA, 2′ ,7′-dichlorodihydrofluorescin diacetate; MDA, malondialdehyde; MS,urashige and Skoog; NPs, nanoparticles; PI, propidium iodide; RNS, reactive nitro-

en species; ROS, reactive oxygen species.∗ Corresponding author. Tel.: +420 776 206027; fax: +420 541 562835.

E-mail address: petr-babula@email.cz (P. Babula).

098-8472/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.envexpbot.2013.03.002

© 2013 Elsevier B.V. All rights reserved.

in gas purification, and as an abrasive (Buzdugan and Beckman,2007; Platonov et al., 2007). Aluminium oxide plays an importantpart in cosmetics. Ceramics based on Al2O3 have high mechanicalstrength, hardness, wear resistance, and chemical inertness withgood biocompatibility, so, they are attractive for use in dental andbone implants (Lukin et al., 2001). Carbon-doped aluminium oxidefilm can be used to measure luminiscence signals optically stimu-lated by irradiation, so a possible application in the film dosimetryused in radiotherapy may be expected (Schembri and Heijmen,2007). Aluminium oxide is among the chemicals most abundantlyproduced in nano-sized particles. These are used by the militaryand by commercial industries in many applications includingcoatings, thermites, and propellants. Generally, nanoparticles havevery high surface reactivity; this means that they may have a neg-ative impacts on health or the environment (Handy et al., 2008).Whereas the toxicity of aluminium is relatively well known andhas been discussed in many publications, the toxicity of aluminium

oxide nanoparticles remains almost unknown and is still an objectof experimental work. The possibility of an impact of thesenanoparticles on the environment has been demonstrated by theirbioaccumulation in various organisms, including Tubifex tubifex

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üller, Hyalella azteca Saussure, Lumbriculus variegates Müller,nd Corbicula fluminea Müller (Stanley et al., 2010). The resultsf a study by Coleman et al. indicate that nano-sized Al2O3 canccumulate and impact the reproduction and behaviour of Eiseniaetida Savigny, although at high levels that are unlikely to be foundn the natural environment (Coleman et al., 2010). This fact meanshat aluminium oxide nanoparticles may possibly enter the foodhain and be responsible for toxicity in animals. In light of theseacts the further impact of nanoparticles on various organisms

ust be considered. The oral exposure of rats to aluminium oxideanomaterial has demonstrated the potential to cause genotoxicamage (Balasubramanyam et al., 2009). Work by Chen et al. usinghe model of the microvascular endothelial cells of the human brainndicates that the nano-alumina affects the cerebral vasculaturen time- and concentration-dependent ways (Chen et al., 2008).prague-Dawley rats injected intraperitoneally with nano-sizedluminium oxide showed an effect on the innate immune system ofhe brain (Li et al., 2009). A cytotoxic effect of these nanoparticlesn a model of UMR 106 cells has been revealed by Di Virgiliot al. (2010), and their transport and accumulation in L929 andJ cells (Radziun et al., 2011) and L5178Y and BEAS-2B cells (Kimt al., 2009) have been described. Possible mechanisms for theytotoxicity of nano-sized Al2O3 particles are still being discussed,ut oxidative stress (Prabhakar et al., 2012) and DNA damageKim et al., 2009) may be responsible for their cytotoxic effect.n conclusion, some work focused on the possible cytotoxicity ofluminium oxide nanoparticles on cell models and animals haseen published, but, their toxic effects on plants, e.g. phytotoxicity,emain almost entirely unknown. Possible interactions betweenhese nanoparticles and the cell walls of algae (Scenedesmus sp.,hlorella sp.) have been shown in the work of Sadiq et al. (2011).hese authors observed a decrease in the chlorophyll content. Inight of the above-mentioned facts, further studies focused on theossible toxic effects of aluminium oxide nanoparticles on plants,specially as the particles accumulate, are highly necessary. Weought to determine the toxicity of aluminium oxide nanoparticlesn the plant cell model, cell suspension culture BY-2, focusing onrowth parameters, stress markers, and polyphenols. In addition,he content of aluminium in the form of soluble Al was determinedsing simple but sensitive spectrophotometric methods and visu-lized by the method of fluorescence microscopy. The principaloal of the work was to determine the ability of nanoparticles tonduce the processes of programmed cell death.

. Material and methods

.1. Chemicals

All of the chemicals used were obtained from Sigma–Aldrich,SA, unless otherwise noted. They were stored in accordance with

he manufacturer’s recommendations. Working solutions wererepared immediately before use.

.2. BY-2 cell suspension culture and determination of growth

Nicotina tabacum L. cv. Bright Yellow-2 suspension-culturedells (BY-2) were grown in liquid MS medium (Murashige andkoog, 1962) as modified by Nagata et al. (1992) under constanthaking (130 rpm) at 27 ◦C in the dark in 250 ml Erlenmeyer flasks.he pH of the cultivation media was adjusted to 5.6. Cells in thexponential growth phase were transferred into fresh cultivation

edia and aluminium oxide nanoparticles (<50 nm particle size,

haracterized by TEM, 20 wt.% in H2O, Sigma–Aldrich, USA) weredded to create concentrations of 0, 10, 20, 50 and 100 �g mL−1.o investigate possible impact of size of Al2O3 nanoparticles,

Experimental Botany 91 (2013) 1– 11

aluminium oxide microparticles (5 �m mean particle size,Sigma–Aldrich, USA) were applied at he same concentrations.Cells were subsequently cultivated for 96 h, with samples beingcollected at strictly defined time intervals of 12, 24, 36, 48, 72, and96 h. All experiments were carried out in triplicate. The cell density(growth of the cell suspension culture) was determined using aFuchs-Rosenthal haemocytometer (Fisher Scientific, Czech Repub-lic). The cells were counted at the above-defined time intervalsas viewed in white light under an Axioscop 40 microscope (Zeiss,Germany). Ten random fields were evaluated for each series, andthe cell numbers were determined in triplicate.

2.3. Observations with the microscope

2.3.1. Cell viability and visualization of soluble AlThe cell viability was measured using fluorescein diacetate (FDA,

Sigma–Aldrich, USA) and the propidium iodide (PI, Sigma–Aldrich,USA) as described by Babula et al. (2012). A 20 �L sample of thecell suspension culture was diluted to 50 �l by fresh MS culti-vation medium and incubated for 5 min at 25 ◦C with FDA (finalconcentration 2.4 �mol L−1) and PI (30 �mol L−1). PI, a nucleic acidstain, penetrates through damaged cell membranes and interca-lates the DNA of the cell, so PI positive cells are dead or dying.Living cells metabolize FDA to fluorescein which emits green lightupon excitation. The percentages of viable and dead cells wereevaluated by counting using a fluorescent microscope (Axioscop40, Zeiss, Germany) equipped with broad spectrum UV excitation.Ten random fields (minimally 100 cells per field, totally minimally1000 cells) from each series were evaluated in the microscopeand the viability was determined in triplicate. Soluble aluminiumwas visualized using fluorescence microscopy (Axioscop 40, Zeiss,Germany) in accordance with the work of Vitorello and Haug, whichwas based on morin staining and the formation of highly fluores-cent Al–morin complexes (Vitorello and Haug, 1997). NIS elementssoftware (Nikon, Japan) was used to process of images and to eval-uate the resultant pictures.

2.3.2. Visualization of ROSDihydroethidium (DHE, Invitrogen, USA) was used for the fluo-

rescent visualization of the reactive oxygen species, respectivelyoxidative stress (Garnczarska, 2005; Goto et al., 1993). Thrice-washed BY-2 cells were incubated for 1 h in 10 �M DHE in fresh MScultivation medium in darkness to avoid possible light-acceleratedDHE oxidation. They were then washed three times with MSmedium and observed with the microscope using the appropriateexcitation filter (Axioscop 40, Zeiss, Germany). In a similar manner,2′,7′-dichlorodihydrofluorescin diacetate (H2DCFDA, Invitrogen,USA) was used to determine the oxidative stress (Garnczarska,2005). Thrice-washed BY-2 cells were incubated for 1 h in 10 �MH2DCFDA in fresh MS cultivation medium in darkness to avoidpossible light-accelerated H2DCFDA oxidation. They were thenwashed three times with MS medium and observed with the micro-scope using the appropriate excitation filter (Axioscop 40, Zeiss,Germany).

2.3.3. Nuclear architecture and programmed cell deathIn order to observe nuclei and detect signs of programmed

death, a 20 �L sample of cells was treated with 20 �L of PEM-buffer(100 mM PIPES, 10 mM EGTA, 10 mM MgCl2, pH 6.9, all chemi-cals obtained from Sigma–Aldrich, USA) containing formaldehyde(4%, w/w; Sigma–Aldrich, USA). A Hoechst 33258 fluorescent probe

(Sigma–Aldrich, USA) was used for fluorescence microscopy. Onethousand nuclei in each preparation were observed using a fluo-rescence microscope (Olympus AX 70, Germany) equipped withbroad-spectrum UV excitation. Ten random fields (minimally 1000

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ells) from each series were evaluated in triplicate and each mor-hological change was expressed as a percentage of the total cells.

.3.4. Changes in mitochondrial potential and caspase-likectivity

Changes in mitochondrial potential were investigated using JC-1taining (Sigma–Aldrich, USA) according to the method publishedy Simeonova et al. (2004). Stock solution in DMSO (1 mg mL−1)as used to prepare the working solution. BY-2 cells were collected

t strictly defined time intervals, washed with fresh cultivationedium, and used to isolate the protoplasts according to theethod of Bandmann and Homann (2012). Two millilitre of the

Y-2 cell culture was mixed with 2 mL of digestion medium for3% cellulase Onzuka R10 obtained from Duchefa, The Netherlands,.2% macerozyme, 0.1% pectolyase, w/v, both Sigma–Aldrich, USA),

mM CaCl2, 25 mM MES-KOH, final pH 5.6, adjusted with sucrose)nd incubated for 3 h. Following this digestion, the cells were col-ected, carefully washed (30 mM CaCl2, 30 mM KCl, 20 mM MES,H 6.5, adjusted using sorbitol) and immediately stained using

C-1 at a final concentration of 2.5 �g mL−1 at 25 ◦C for 30 minn darkness. The intensity of the fluorescent emissions of the JC-

monomers (emission at 535 nm) and the aggregates (emissiont 595 nm) were determined using a spectrofluorimetric detectorRF-551, Shimadzu, Duisburg, Germany). Fluorescence measure-

ents were repeated four times for each concentration and timeeriod. Caspase-like activity was determined using a Caspase 3olorimetric Kit (Sigma–Aldrich, USA) with acetyl-Asp-Glu-Val-sp-p-nitroanilide (Ac-DEVD-pNA) as the substrate. Cells wererepared in accordance with the manufacturer’s instructions, andhe absorbance was measured at 405 nm with a Helios Epsilonnicam spectrophotometer (ThermoFisher Scientific, USA). Theaspase-like activity at time 0 was considered to be 100%, otheralues were referenced to this value.

.3.5. TUNEL assayA terminal deoxynucleotidyl transferase (Tdt)-mediated deoxy-

ridinetriphosphate (dUTP)-nick labelling (TUNEL) assay waserformed using a TMR-red in situ cell death detection kit (Roche,ermany) in accordance with the manufacturer’s instructions. Con-

rols of untreated BY-2 cells, BY-2 cells treated by Al2O3 NPs,ositive BY-2 cells treated with DNAse I, and negative controlsBY-2 cells treated only with the labelling solution without termi-al transferase) were included in the experiments. Minimally 1000ells per sampling time per treatment were counted in triplicate.

.4. Spectrophotometric measurements

.4.1. Measurement of soluble AlSoluble Al was determined by adding aluminium oxide NPs to

amples of MS medium and using the BY-2 cells thus exposed toluminium oxide nanoparticles for further analysis. To determinehe soluble Al in the MS medium, samples of medium wereentrifuged (15,000 rpm for 30 min, centrifuge 5415R, Eppendorf,ermany), and the supernatant liquid was then used for both

he preconcentration step and the analysis itself. The BY-2 cellsere washed carefully three times with fresh MS medium andsed for analysis. The BY-2 cells were frozen using liquid nitrogen,omogenized and divided into two halves. The first half was usedo determine the soluble Al in the BY-2 cells; the second half of theomogenized BY-2 cells was mineralized overnight in a mixturef HNO3 and H2O2 (1:1, v/v, both Sigma–Aldrich, USA). The cellsere then dried at 90 ◦C. The dry residue was dissolved in 1 mL of

7% HCl of ACS purity (Sigma–Aldrich, USA) and diluted to a finalolume of 5 mL with miliQ water. After mineralization, the contentf Al3+ ions was determined according to the procedure describedy Liu for the rapid and sensitive determination of Al3+ using morin

Experimental Botany 91 (2013) 1– 11 3

(Liu, 1999). To compare and verify the determination of soluble Al,an ultrasensitive method for the determination of trace aluminiumusing eriochrome cyanine R was used (Shokrollahi et al., 2008).This method suppresses interferences caused by a wide range ofcations and anions.

2.4.2. MTT and TTC assaysThe dehydrogenase and oxidoreductase activities were

evaluated by measuring the mitochondrial-dependent reduc-tion of 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazoliumbromide (MTT, Sigma–Aldrich Chem. Corp., USA) and 1,3,5-triphenyltetrazolium chloride (TTC, Sigma–Aldrich Chem. Corp.,USA) to a formazan in separate assays. For both assays the BY-2cells were washed twice with 0.05 M Na2HPO4–KH2PO4 buffer(pH 7.4) and mixed with 0.6% (w/v) MTT or TTC dissolved in0.05 M Na2HPO4–KH2PO4 buffer (pH 7.4) and 0.05% (v/v) Tween80 and incubated at 28 ◦C for 60 min. The cells were then lysedby addition of an equal volume of 2-propanol and 0.1 mM HCl.The amount of the formazan produced was proportional to theabsorbance measured at � = 570 nm (MTT) or 490 nm (TTC) nm(Babula et al., 2009). The dehydrogenase and oxidoreductaseactivities were each expressed as a percentage of the activitydetermined in a control sample of untreated tobacco BY-2 cells atthe time 0 (= 100%). A Helios Epsilon Unicam spectrophotometer(ThermoFisher Scientific, USA) was used for all spectrophotometricmeasurements.

2.4.3. Reactive oxygen species (ROS) and reactive nitrogen species(RNS), and lipid peroxidation

The amount of hydrogen peroxide was determined using theTiCl4 method based on the reaction between hydrogen perox-ide and TiCl4 that forms coloured Ti complexes. The absorbancewas measured at 410 nm (Kovacik et al., 2009b). Superoxide anionradical (O2

−•) was determined by a method that measures thereduction of yellow nitro blue tetrazolium (NBT) to blue NBT form-azan measured at 620 nm (Choi et al., 2006). The NO content wasdetermined using Griess reagent. In this case, homogenates wereprepared in sodium acetate buffer (50 mM, pH 3.6) enriched withzinc acetate (4%, w/w). The absorbance was measured at 530 nm(Kovacik et al., 2009a). For the determination of lipid peroxida-tion, the Thiobarbituric Acid Reactive Substances (TBARS) Assaydescribed by Stewart and Bewley (1980) was used. A sample offrozen BY-2 cells was washed with fresh liquid cultivation mediumand immediately crushed in liquid nitrogen using a pre-chilledmortar and pestle. The homogenized sample was then resuspendedin cultivation medium (50 mg mL−1) and sonicated for 15 s overice (K5 sonicator, Kraintek, Czech Republic). The product was thenused to determine MDA. The sample was mixed with an equalvolume of 0.5% (w/v) thiobarbituric acid solution containing 20%(w/v) trichloroacetic acid. This mixture was heated (95 ◦C, 30 min)and the reaction was terminated by abrupt placement in an ice-bath. The cooled mixture was centrifuged at 10,000 rpm for 10 min(centrifuge 5415R, Eppendorf, Germany) and subsequently, theabsorbance of the supernatant liquid was measured at 532 nm. TheTBARS concentration was estimated as a malondialdehyde equiva-lent using the standard curve.

2.4.4. Loss of the integrity of the cell membraneThe integrity of the plasma membrane was measured using the

method described by Baker and Mock (1994) and Ikegawa et al.(1998). BY-2 cells were washed and resuspended in 2 mL of 0.05%(w/v) solution of Evans blue in the MS medium and incubated for

15 min at 25 ◦C. After incubation, the BY-2 cells were harvestedby centrifugation and washed three times with MS medium. TheEvans blue was released by resuspending the BY-2 cells in a 1%(w/v) aqueous solution of sodium dodecyl sulphate and rupturing

4 l and Experimental Botany 91 (2013) 1– 11

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he BY-2 cells at room temperature in a sonicator (K5, Krain-ek, Czech Republic). Finally, the homogenate was centrifuged at5,000 rpm for 15 min (centrifuge 5415R, Eppendorf, Germany) andhe absorbance was measured at 600 nm.

.5. Quantification of phenolics

Soluble phenolics were extracted with 80% methanol (v/v, 30 mgW per 3 mL) and quantified using the Folin-Ciocalteu method withallic acid as the standard (Sochor et al., 2011).

.6. Statistical analyses

Microsoft® Office Excel (Microsoft®, USA) was used for statis-ical analyses. Student’s t test was used to evaluate the data. Theesults are expressed as the mean ± S.D. unless noted otherwise.alues of P < 0.05 were considered significant.

. Results

We focused on the toxicity of Al2O3 nanoparticles in this work.n the first step, we studied the effect of size of used Al2O3 formsnanoparticles versus microparticles) on toxicity to the tobaccoY-2 cells. Whereas application of Al2O3 nanoparticles caused sig-ificant changes in all monitored parameters, Al2O3 microparticlesaused only nonsignificant changes that were comparable withontrol, untreated BY-2 cells. These results were confirmed alsoicroscopically. With respect to these facts, we further focused on

anoparticulated form of Al2O3.

.1. Effect of aluminium oxide nanoparticles on cell viability androwth

The application of aluminium oxide nanoparticles led to dis-inctive changes in cell viability, which were determined usingouble FDA/PI fluorescence staining. Whereas the viability of theontrol, untreated BY-2 cells, was about 98.5% during the wholexperiment, the application of Al2O3 nanoparticles at the low-st concentration (10 �g mL−1) significantly diminished the celliability, which was 77% after a 96-h treatment. At the highestoncentration (100 �g mL−1), the application of the Al2O3 nanopar-icles significantly diminished the cell viability in as little as 24 hviability about 84%). At the end of the experiment, the viability ofhe BY-2 cells in this particular experimental variant was only 22%see Fig. 1A for a graphical presentation of these results). Exceptor shrinkage of the cytoplasm, the Al2O3 nanoparticles evidentlyaused no significant morphological changes in the BY-2 cells.owever, this symptom is connected with the processes of pro-rammed cell death and will be further discussed. The viability ofY-2 cells was not the only change caused by treatment with Al2O3anoparticles treatment; growth parameters were also changed.anoparticles were introduced at the beginning of the phase ofxponential growth of the BY-2 cell culture, so, it was assumedhat they had an effect on the dynamics of cell growth. In the casef the lowest concentration of Al2O3 nanoparticles (10 �g mL−1),he increase in cell density compared to the control (untreated)Y-2 cells was well evident. In the first day (after 24 h of treat-ent), the cell density in this variant reached 107% compared to

he control; by the end of the experiment (96 h), the cell densityad reached almost 129% of that of the control. On the other hand,

rowth was obviously supressed at the higher concentrations. Inhe case of the highest applied concentration of Al2O3 nanoparti-les (100 �g mL−1), the final cell density was only 54% compared tohe control (see Fig. 1B).

BY-2 cells. (For interpretation of the references to color in this figure legend, thereader is referred to the web version of the article.)

3.2. Uptake of aluminium oxide nanoparticles and release ofaluminium

In the next step, we focused on the release of aluminium andthe possible uptake of Al2O3 nanoparticles by BY-2 cells. Despitethe fact that there are many methods used to determine alu-minium, which differ in sensitivity and limit of detection, weused two different methods to verify the results of the analyses.Modern spectrophotometric methods enable the easy and inexpen-sive determination of aluminium in real samples, whereas atomicabsorption spectrometry (AAS) and inductively coupled plasmaatomic emission spectrometry (ICP-AES) are more expensive andnot without drawbacks. We used a method described and usedby Vitorello et al. that provides a high correlation (r2 = 0.94) ofresults with those obtained using graphite furnace atomic absorp-tion spectrometry (Vitorello and Haug, 1997). The second method,the eriochrome cyanine R assay, provides high sensitivity and lowlimits of determination (0.05 ng mL−1) (Shokrollahi et al., 2008).Both methods revealed the presence of soluble Al in the treatedmedia. In the control variant, we determined only a weak responseprobably caused by interfering ions. However, due to the generalabsence of aluminium ions, respectively soluble Al forms, in thecultivation media of the control variant, we assumed that no sol-uble Al was detected and that any values determined representother sources. In the other experimental variants, aluminium oxidenanoparticles were added (with no other changes in the cultivationmedia), and an increase in the values determined must correspond

directly to soluble Al. The concentrations of soluble Al in the treatedmedia, determined at the end of the experiment using the morinmethod, are summarized in Table 1. The presence of soluble Al

Z. Poborilova et al. / Environmental and Experimental Botany 91 (2013) 1– 11 5

Table 1Content of soluble aluminium in cultivation media, BY-2 cells, and mineralized BY-2 cells as determined by morin assay. Note values determined for the control untreatedvariant caused by interfering ions * indicates a significant difference between the BY-2 cells treated with Al2O3 nanoparticles and the corresponding control (untreated) cellsat P < 0.05, ** indicates a significant difference between the BY-2 cells treated with Al2O3 nanoparticles and the corresponding control (untreated) cells at P < 0.01.

Time of treatment (h) Experimental variant(�g mL−1)

Cultivation medium(�g mL−1)

BY-2 cells(ng g−1 FW)

BY-2 cells after mineralization(ng g−1 FW)

0 0 0.137 ± 0.052 0.085 ± 0.007 0.119 ± 0.02124 0 0.141 ± 0.026 0.114 ± 0.021 0.136 ± 0.018

10 0.258 ± 0.025* 0.359 ± 0.037** 1.583 ± 0.042**20 0.374 ± 0.036** 0.379 ± 0.032** 1.755 ± 0.091**50 0.349 ± 0.073* 0.472 ± 0.019** 1.792 ± 0.054**

100 0.576 ± 0.073** 0.670 ± 0.042** 3.126 ± 0.105**48 0 0.180 ± 0.062 0.129 ± 0.025 0.232 ± 0.151

10 0.285 ± 0.023 0.396 ± 0.020** 2.271 ± 0.158**20 0.349 ± 0.039* 0.407 ± 0.065* 2.437 ± 0.269**50 0.437 ± 0.035* 0.503 ± 0.014** 3.340 ± 0.229**

100 0.612 ± 0.028** 1.003 ± 0.106** 3.801 ± 0.227**72 0 0.265 ± 0.044 0.123 ± 0.024 0.126 ± 0.021

10 0.348 ± 0.030 0.424 ± 0.022** 2.389 ± 0.350*20 0.413 ± 0.028* 0.647 ± 0.047** 2.697 ± 0.263**50 0.536 ± 0.046** 0.749 ± 0.053** 3.524 ± 0.402**

100 0.866 ± 0.067** 1.045 ± 0.087** 4.266 ± 0.172**96 0 0.260 ± 0.029 0.147 ± 0.028 0.131 ± 0.025

10 0.386 ± 0.014* 0.587 ± 0.044** 2.513 ± 0.139**20 0.452 ± 0.032** 0.700 ± 0.026** 3.488 ± 0.333**

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n the treated media was evident as little as 24 h after the intro-uction of Al2O3 nanoparticles. The uptake of Al2O3 nanoparticlesnd the uptake and accumulation of soluble Al released from Al2O3anoparticles in the BY-2 cells were considered next. Soluble Alas detected in all of the treated variants; the concentration of

oluble Al in each variant was determined with respect to bothime and original concentration of Al2O3 nanoparticles. The highestoncentration of total aluminium was determined for the highestpplied concentration of Al2O3 nanoparticles (100 �g mL−1). Theifference between the concentrations of aluminium determinedefore and after mineralization was considered to be the amountf Al2O3 nanoparticles taken up by the BY-2 cells, or Al2O3 nanopar-icles adhering closely to the BY-2 cells, respectively their cell walls.he highest concentration of Al2O3 nanoparticles was determinedor the highest applied concentration (100 �g mL−1). Both methodsevealed the presence of soluble Al in the treated media and the BY-

cells and the possibility that NPs are taken up by the BY-2 cellsnd interact with them. Moreover, the results of the morin assaynd eriochrome cyanine R assay are comparable.

Soluble Al was observed by using morin staining and fluores-ence microscopy. Only minimal emission was detected from theontrol (untreated) variant, whereas all of the treated variantsemonstrated a gradual concentration-dependent increase in the

ntensity of emission. Soluble Al was detected in the cytoplasmnd the cell wall, but they were localised in the nuclei distinc-ively (Fig. 2A), so nuclei represented the site of the accumulatedoluble Al (Fig. 2B). The pictures obtained were used for the evalu-tion using NIS element software that enables image processingnd the calculation of selected parameters, in this case the rel-tive intensity of emission. The results are shown in Fig. 2C andre in agreement with the spectrophotometric results in that theyhow that the intensity of emission increases with the concentra-ion of aluminium oxide NPs and the duration of treatment. Pictureshat show the distribution of soluble Al within the BY-2 cells after6 h treatment converted into intensity maps (see Fig. 2A, last col-mn) are also introduced, and the highest applied concentration

s converted into a surface intensity graph showing the three-

imensional distribution of soluble Al (Fig. 2B). The presence oflusters of NPs was clearly evident and is also marked in these fig-res. Processing of the pictures resulted in the calculation of theelative intensity of emission, which determines the concentration

1.045 ± 0.070** 4.179 ± 0.244**1.203 ± 0.056** 5.048 ± 0.048**

of soluble Al. These data are shown in Fig. 2C for all of the experi-mental variants, including the untreated control, at the end of theexperiment, i.e. after 96-h treatment. On the other hand, there isonly a comparison of the relative values of emission intensity thatcorrespond to soluble Al.

3.3. Reactive oxygen (ROS) and reactive nitrogen (RNS) species,lipid peroxidation, and loss of the integrity of the cell membrane

Reactive oxygen (hydrogen peroxide and superoxide anion rad-ical) and nitrogen species belong to the important markers ofoxidative stress. Nitrogen reactive species were determined usingthe Griess reagent and expressed as �g g−1 of NO and related tothe fresh weight (Fig. 3A-1). The highest RNS levels were foundfor the two highest applied concentrations of Al2O3 nanoparticlesafter 96 h of treatment. A concentration of 50 �g mL−1 produced3.61 �g g−1 FW, 100 �g mL−1 yielded 3.66 �g g−1 FW, and con-trol with no NPS showed 2.11 �g g−1 FW. However, increases inRNS as a response to treatment with Al2O3 nanoparticles wereobserved in all of the experimental variants. Levels of hydrogenperoxide increased with both time and concentration in all of thevariants treated by Al2O3 NPs except for the highest concentrationof Al2O3 nanoparticles (100 �g mL−1) at 72 and 96 h, where mod-erate declines in the hydrogen peroxide level as compared to thelower concentration (50 �g mL−1) were determined (see Fig. 3A-2). A similar course was observed for superoxide anion radical(Fig. 3A-3). The greatest increase in the amount of superoxide anionradical (63.21 ng g−1 FW) was observed for the highest applied con-centration of Al2O3 nanoparticles (100 �g mL−1) as compared to35.11 ng g−1 FW for the control (untreated) BY-2 cells after 24 hof treatment). ROS were observed visually using two fluorescentprobes, dihydroethidium and 2′,7′-dichlorodihydrofluorescin diac-etate, after 96 h of treatment. The intensity of emission increased ina concentration-dependent manner (Fig. 3C). Both probes showedthat the greatest concentrations of ROS were localized in the nucleiand the adjacent cytoplasm. On the other hand, the cell walls, andplasma membranes were also stained. This fact is closely connected

with lipid peroxidation. Lipid peroxidation damages membranesby forming lipid radicals that destroy characteristics of the plasmamembrane characteristics and cause it to lose integrity. In addi-tion, the final product of the lipid peroxidation, malondialdehyde

6 Z. Poborilova et al. / Environmental and Experimental Botany 91 (2013) 1– 11

Fig. 2. Visualization and distribution of soluble Al in BY-2 cells using fluorescence microscopy and morin (A) after 96 h of treatment. Distribution of soluble Al in BY-2cells demonstrated on a three-dimensional model after image processing (B). Intensity of emission in BY-2 cells treated with Al2O3 nanoparticles, evaluated using imagep 2 cell.

t emitb end, t

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rocessing (C). The white arrows indicate the localization of the nucleus in the BY-he emission of these clusters stained with morin. On the other hand, the cell wallsar represents 50 �m. (For interpretation of the references to color in this figure leg

MDA), can subsequently react with DNA to form DNA adducts.

alondialdehyde was determined spectrophotometrically in ourork. Whereas control (untreated) BY-2 cells had produced only a

mall amount of malondialdehyde (5.02 nmol g−1 FW) by the endf the experiment (96 h), the content of MDA in BY-2 cells that had

ig. 3. Generation of reactive oxygen and nitrogen species caused by treatment with Al2Oitric oxide (1), hydrogen peroxide (2), and superoxide (3). Changes in the integrity of thevaluated by using the Evans blue method (1) and the production of malondialdehydihydroethidium (DHE) and 2′ ,7′-dichlorodihydrofluorescin diacetate (H2DCFDA) in BY-

ocalized nuclei in both cases (white arrows) and weak emission corresponding to the plahe reader is referred to the web version of the article.)

Note the strands of cytoplasm. The red arrows indicate clusters of Al2O3 NPs. Note only moderately. N indicates a nucleus, CW indicates a cell wall. The length of thehe reader is referred to the web version of the article.)

been treated with Al2O3 nanoparticles gradually increased (Fig. 3B-

2). For the highest Al2O3 NPs concentration, the MDA content wasalmost 4.55-times as much as that of the control (22.84 nmol g−1

FW). In conclusion, the MDA content increased with both increasesin concentration and the passage of time. The integrity of the

3 nanoparticles (A). Changes in the levels of reactive nitrogen species expressed as plasma membrane (B). Changes in the semipermeability of the plasma membrane

e estimated by TBARS assay (2). Localization of reactive hydrogen species using2 cells after 96 h of treatment (C). Note the intensive emission in the area of thesma membrane. (For interpretation of the references to color in this figure legend,

Z. Poborilova et al. / Environmental and Experimental Botany 91 (2013) 1– 11 7

Fig. 4. Changes in the activities of dehydrogenase and oxidoreductase in BY-2 cells treated with Al2O3 nanoparticles (A 1, 2). Parameters of programmed cell death (B) –changes in mitochondrial dysfunction (mitochondrial potential) (1), caspase-like activity (2) and changes in the nuclear architecture expressed as the rates of productionof normal, pre-apoptic, and apoptic-like nuclei and micronuclei (3). TUNEL assay–Hoechst, TUNEL and merge in BY-2 cells treated with NPs for 96 h with evaluation of theT age ot

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UNEL-positive nuclei relative to time and concentration, expressed as the percenthe concentration 100 �g mL−1 (arrows).

embrane was evaluated by using the Evans blue method, wheren cells with disrupted or damaged plasma membranes is accu-

ulated Evans blue stain, which can subsequently be extractednd determined spectrophotometrically. The values obtained wereecalculated to the value at the beginning of the experiment (time). The accumulation of Evans blue stain was greatest in the BY-2ells exposed to the highest concentration of Al2O3 nanoparticles100 �g mL−1) (Fig. 3B-1). However, the Evans blue accumulationncreased with both time and concentration. At the end of thexperiment (96 h), the relative values were as follows: control (nol2O3 NPs) 1.55; 10 �g mL−1 Al2O3 NPs 1.98; 20 �g mL−1 Al2O3 NPs.57; 50 �g mL−1 Al2O3 NPs 3.24 and 100 �g mL−1 Al2O3 NPs 5.69.he value of the Evans blue accumulation in the control cells at theeginning of the experiment was set at 1. These results indicateamage of the integrity of the plasma membrane and are furtheriscussed in connection with the production of ROS, the formationf malondialdehyde, and the processes of programmed cell death.

.4. Changes in dehydrogenase and oxidoreductase activity,itochondrial potential and the changes in nuclear architecture

MTT assay and TTC assay are generally accepted and usedo determine dehydrogenase and oxidoreductase activities. Ourxperiments showed a gradual increase in the activity of bothnzymes in the control (untreated) variant, and in all treatedariants affecting of their enzyme activity; the results are shownn Fig. 4A-1 and A-2. At lower applied concentrations (10 and0 �g mL−1) of Al2O3 nanoparticles, the activities of both enzymesere increased as compared to the control. By the end of the

xperiment (96 h), the activity of dehydrogenase had 133.5% ofhe level of activity found in the control for the variant with a

oncentration of 10 �g mL−1 of Al2O3 nanoparticles and 140.1%or that with 20 �g mL−1. For oxidoreductase, the correspondingalues were 108.9% for 10 �g mL−1 and 111.3% for 20 �g mL−1. Onhe other hand, the application of Al2O3 nanoparticles at higher

f TUNEL-positive nuclei (C). Note the high percentage of TUNEL-positive nuclei for

concentrations led to a significant reduction in the activities of bothdehydrogenase and oxidoreductase. The activity of dehydrogenaseactivity was reduced to 65.4% compared to the control and that ofoxidoreductase to 73.5% compared to the control for the highestapplied concentration of Al2O3 nanoparticles (100 �g mL−1). Mito-chondrial dysfunction is reflected in changes in the mitochondrialpotential. In our case, the mitochondrial potential was determinedusing a JC-1 fluorescent probe, where the rate between JC-1aggregates (JC-1A) and JC-1 monomers (JC-1M) was determined.For control (untreated) BY-2 cells, this rate varied between 91.36and 93.11%. The application of Al2O3 nanoparticles significantlyreduced the presence of JC-1 aggregates. For the lowest concentra-tion (10 �g mL−1), the rate was 87.01% at the end of the experiment(96 h) and for the highest concentration (100 �g mL−1), it was only41.23%. The results are depicted in Fig. 4B-1. The mitochondrialpotential is closely connected with the release of cytochrome cand programmed cell death. Programmed cell death is related tothe activity of executive proteins called caspases, but these havebeen found only in animal cells. In plants, a group of proteins withcaspase-like activity have been identified. In our study, we detectedcaspase-like activity using acetyl-Asp-Glu-Val-Asp-p-nitroanilidesubstrate. All results were referenced to the caspase-like activitydetected at time 0 (= 100%). The highest caspase-like activity wasfound for the highest applied concentration of Al2O3 nanoparticles(100 �g mL−1). It was 305% relative to the control at time 0. Incomparison with this result, by the end of the experiment (96 h)the control (untreated) BY-2 cells showed 186% as much activityas they had shown at the beginning of the experiment (time 0). Forthe details, see Fig. 4B-2. Programmed cell death was determinedmicroscopically using Hoechst 33258 staining. Changes in cytologyand in nuclear architecture were determined for all variants at

the end of the experiment (96 h). The results are summarized inFig. 4B-3. The following parameters were evaluated: the presenceof normal nuclei, nuclei with condensed chromatin (pre-apopticnuclei), and apoptic-like nuclei. The presence of micronuclei was

8 l and Experimental Botany 91 (2013) 1– 11

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Table 2Content of phenolics in BY-2 cells treated by Al2O3 nanoparticles. * indicates a sig-nificant difference between the BY-2 cells treated with Al2O3 nanoparticles and thecorresponding control (untreated) cells at P < 0.05, ** indicates a significant differ-ence between the BY-2 cells treated with Al2O3 nanoparticles and the correspondingcontrol (untreated) cells at P < 0.01.

Time oftreatment (h)

Experimentalvariant (�g mL−1)

Phenolics(�g g−1 FW)

S.D. Significance

0 0 24.50 2.2224 0 20.08 1.34

10 22.01 2.3820 29.12 1.54 *50 28.11 3.45

100 32.09 3.4048 0 25.68 2.16

10 29.92 2.56 *20 37.27 2.8650 70.51 4.36 **

100 89.44 7.96 **72 0 41.93 4.36

10 49.53 6.0920 75.29 9.4050 98.81 8.06 *

100 181.40 2.10 **96 0 57.20 3.94

10 77.70 7.29 *20 109.60 5.95 **

Z. Poborilova et al. / Environmenta

lso monitored. It was clearly evident that the most significanthanges took place in the highest applied concentration of Al2O3anoparticles (100 �g mL−1). Apoptic-like nuclei were found toe present in 1.12% of the BY-2 cells (compared with the controluntreated) at 0%), pre-apoptic nuclei were observed in 24.5% ofhe BY-2 cells (compared to1.34% in the control BY-2 cells) and theormation of micronuclei was detected in 0.85% of the BY-2 cells0% in the control) in this case. Changes in the nuclear architectureere not so evident for the lowest applied concentrations of Al2O3anoparticles, see Fig. 4B-3. That aluminium oxide nanoparticlesriggered the process of programmed cell death was confirmed byhe TUNEL test, a method of detecting the fragmentation of DNAy the labelling the terminal ends of the DNA. The percentageositive cells found by the TUNEL test increased with both timend concentration (Fig. 4C). At the end of the experiment (96 h),he percentages of TUNEL positive cells were as follows: 1.14% inhe control, 3.24% in the 10 �g mL−1 Al2O3 variant, 6.12% in the0 �g mL−1 Al2O3 variant, 14.96% in the 50 �g mL−1 Al2O3 variantnd 26.84% in the 100 �g mL−1 Al2O3 variant. All of these resultsive complex information about the ability of aluminium oxideanoparticles to induce the process of programmed cell deathelative to time and concentration.

.5. Changes in the contents of phenolics in BY-2 cells in responseo treatment with Al2O3 nanoparticles

The application of Al2O3 nanoparticles to BY-2 cells causedlearly evident changes in their contents of phenolics. The con-ent of phenolics in the BY-2 cells was very low. The total levelf phenolics in the control (untreated) BY-2 cells was determinedo be 24.5 �g g−1 FW at the beginning of the experiment (time 0).he application of Al2O3 nanoparticles led to a significant increasen phenolics in the BY-2 cells. At the end of the experiment (after6 h of treatment), the detected concentrations were as follows:7.2 �g g−1 FW for the control (untreated) variant, 77.7 �g g−1 FWor the Al2O3 NPs concentration 10 �g mL−1, 109.6 �g g−1 FW forhe Al2O3 NPs concentration of 20 �g mL−1, and 174.4 �g g−1 FWor the Al2O3 NPs concentration of 50 �g mL−1 and 211.7 �g g−1 FWor the highest applied concentration of Al2O3 NPs (100 �g mL−1).or details, see Table 2.

. Discussion

Nanopollution represents a serious problem. The toxicity ofanoparticles arises from their chemical composition and espe-ially their large surface/volume ratio, which gives them uniqueroperties (especially chemical reactivity) not seen in larger parti-les. The contribution of large surface/volume ratio of nanoparticleso toxicity was also confirmed in our experiments, where Al2O3anoparticles showed significant toxicity, and microparticles, onhe contrary, almost none. Once released in the environment, NPs,epending on their size, can be taken up by microorganisms, fungi,r plants (Dimkpa et al., 2011; Navarro et al., 2008). They mayubsequently enter the food chain and cause serious alterationsn humans and other animals. NPs can also modify the uptakend accumulation of other pollutants in organisms (Dubchak et al.,010). Aluminium oxide nanoparticles have been applied in manyranches of human life. However, their possible negative impactn living organisms must be studied (Griffitt et al., 2011; Stanleyt al., 2010). Whereas the effects of aluminium oxide nanoparti-les on animal cell models have been described in some works (Di

irgilio et al., 2010; Dong et al., 2011; Kim et al., 2009), knowl-dge of their effects on autotrophic organisms, including plants,s almost totally missing. The work of Sadiq et al. has broughtnowledge of their toxicity for algal species (Sadiq et al., 2011).

50 174.40 8.05 **100 211.70 9.93 **

The authors described diminished content of chlorophyll and pos-sible interactions with the cell walls of algae. Few studies havefocused directly on the effects of aluminium oxide nanoparticleson plants. However, problems with the stability of Al2O3 nanopar-ticles in cultivation media must be considered carefully. They havea tendency to form aggregates, as has been shown by the workof Garcia-Saucedo et al. (2011). We too observed the formationof aggregates of Al2O3 nanoparticles too (see Fig. 2, red arrows).We also observed the formation of aggregates of NPs on the sur-face of the cell wall and in cultivation medium. The formation ofaggregates is connected with changes in the uptake of NPs and thepossible release of aluminium. In general, the number of publica-tions focused on the phytotoxicity of Al2O3 nanoparticles is stillvery limited and provides space for further research. Burklew et al.studied the effects of relatively high concentrations (0.1–1.0%) ofAl2O3 nanoparticles on in vitro cultivated tobacco plants. A sig-nificant reduction in the length of the roots and changes in theexpression of miRNA expression were found (Burklew et al., 2012).Because the authors focused only on nanoparticles, they did notinvestigate the possibility that aluminium might be released intothe cultivation media during cultivation and subsequently havetoxic effects on plants. The phytotoxicity of soluble Al, respec-tively Al3+ ions has been described in many works and includes,especially, inhibiting the growth of the roots. The inhibition of thegrowth of roots is based on the accumulation of aluminium ions inthe root cap, the subapical meristem, and the zone of elongation(Matsumoto and Motoda, 2012). These changes were observablewithin 60 min after the application of aluminium ions. It has beendemonstrated that the cytoskeleton is affected by aluminium ions,as has been proved in a study by Swarzerova et al., who used thetobacco BY-2 cell line as a model to elucidate cytoskeleton-basedaluminium toxicity (Schwarzerova et al., 2002). The cytoskeletonalone is involved in all cellular processes, including cell division,and the most harmful effects may be expected in the rapidly divid-ing and proliferating cells. We found significant concentration-and time-dependent phytotoxicity of Al O nanoparticles in the

2 3tobacco BY-2 plant cell model. These results are in agreement withthe work of Yoon et al. (2011), and Dong et al. (2011), who deter-mined the size-, concentration- and time-dependent cytotoxicity

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Z. Poborilova et al. / Environmenta

f Al2O3 nanoparticles in different cell models. In addition, webserved reduced growth of the BY-2 cell suspension at higherpplied concentrations and stimulated growth of the BY-2 celluspension at the lowest applied concentration (10 �g mL−1) com-ared to the control. This phenomenon must be studied furtherspecially in view of the fact that controversial studies have beenublished that focused only on the effect of aluminium ions, not alu-inium oxide nanoparticles. Whereas Pan et al. (2002) observed

nhibited cell growth in a barley suspension culture treated withluminium, Dominguez et al. (2002) found increased biosynthesisf DNA in human fibroblasts treated with aluminium ions in vitro.inally, Perreault et al. observed aluminium-induced cell divisionn the unicellular green alga Chlamydomonas acidophila NegoroPerreault et al., 2010). The foregoing facts are connected with theffects of Al3+ ions. For this reason, we determined the amount ofoluble Al in both the treated cultivation media and the BY-2 cellssing two methods, morin and eriochrome cyanine R. Both meth-ds revealed the presence of soluble Al. In addition, we visualisedhe soluble Al in the BY-2 cells by the morin method (Vitorellond Haug, 1997) as used for the rapid determination of Al3+ ionsLiu, 1999; Yuan and Fu, 2011). Soluble Al was determined in bothhe treated cultivation media and the BY-2 cells as confirmed by

icroscopical observation with subsequent image analysis thatevealed the highest concentrations of soluble Al in the cell wallsnd nuclei of the cells. Image analysis alone represents an importantool in the quantification of the emission intensity, the quantifi-ation of the monitored ions. The presence of soluble Al in theuclei may explain the effect on cell division. The presence ofoluble Al (and/or nanoparticles) in the cell walls or on their sur-ace is in the agreement with the work of Sadiq et al., who foundnteractions between the algal cell wall and Al2O3 nanoparticlesSadiq et al., 2011). We observed the formation of aggregates ofPs associated with the BY-2 cell walls. However, the exact mech-nism of interactions between aluminium oxide nanoparticles andhe cell walls remains unknown. The toxicity of the nanoparticles

ay be connected not only with the formation and presence oflusters of NPs on the cell surface, but also with the release of alu-inium into the cultivation medium. However, it is very difficult

o predict the release of aluminium from Al2O3 nanoparticles andts subsequent behaviour in the cultivation medium. Aluminiumn the solutions generally hydrolyzes. Under strongly acidic con-itions, Al3+ predominantly exists as Al(H2O)6

3+. An increase inhe pH causes deprotonation of the hexahydrate and produces

ore Al(OH)2+ and Al(OH)2+. These forms differ not only in their

ioavailability to plants, but also in their ability to interact withther ions and compounds, including organic acids, proteins, andipids. The pH of the cultivation media was adjusted to 5.6, which

eant that the aluminium was present in the forms Al(OH)2+ andl(OH)2

+. All positively charged forms of aluminium are phytotoxic.he foregoing facts may possibly explain why Al2O3 nanoparticleselease aluminium and are toxic. Exposing tobacco BY-2 cells tol2O3 nanoparticles led to significant cellular changes that wereecognized as the signs of programmed cell death (PCD). Thesehanges included shrinkage of cytoplasm, changes in the nuclearrchitecture, condensation of the chromatin, formation of apoptic-ike bodies, and fragmentation of DNA that are direct symptomsf PCD. Changes in the integrity of the plasma membrane identi-ed using the Evans blue method, were among the further signsf PCD. BY-2 cells treated with hydrogen peroxide (Houot et al.,001), plant secondary metabolite naphthoquinones (Babula et al.,009), or heavy metals, such as cadmium (Ma et al., 2010) formedpoptic-like bodies. Both hydrogen peroxide and nitric oxide are

mportant signal molecules in plants exposed to biotic or abiotictress (De Pinto et al., 2012; Gechev and Hille, 2005). Nitric oxidean also modulate the influx and accumulation of heavy metals inlant cells (Ma et al., 2010). We recorded that in BY-2 cells exposed

Experimental Botany 91 (2013) 1– 11 9

to Al2O3 nanoparticles levels of RNS (nitric oxide) and ROS (hydro-gen peroxide and superoxide anion radical) increased with timeand concentration. The largest amounts of ROS were found in thenuclei and the adjacent cytoplasm, but these were found also inthe plasma membrane. We also determined a marker of oxidativestress and membrane damage, malondialdehyde, a product of lipidperoxidation. The formation of lipid peroxides was accompaniedby changes in the permeability of the plasma membrane and theaccumulation of Evans blue stain. On the other hand, the relation-ship between the ROS and RNS during abiotic or biotic response tostress must be studied further. Not only oxidative stress, but alsomitochondrial dysfunction belongs to the early signs of PCD. Theproduction of reactive oxygen species and mitochondrial dysfunc-tion with subsequent signs of PCD were observed in tobacco BY-2cells treated with the alkaloid narciclasin (Lu et al., 2012). The lossof membrane potential can be measured using rhodamine-basedfluorescent probes. Rhodamine 123 belongs to the stains com-monly used to determine the loss of membrane potential in bothanimal and plant cells. However, quantification of the observedchanges is relatively difficult and requires image analysis. A JC-1 probe was used in the work of Diaz-Tielas et al. (2012), whoinvestigated trans-chalcone-induced PCD in the roots of Arabidopsisthaliana (L.) Heynh., and in the work of Simeonova et al. (2004), whomonitored the mitochondrial membrane potential during senes-cence of leaf mesophyll in Pisum sativum L. The application of aJC-1 probe enables relatively easy quantification of any changes inthe mitochondrial potential and thus the mitochondrial dysfunc-tion. Our work revealed changes, even loss of membrane potentialas a consequence of treatment with Al2O3 nanoparticles. Finally,we were focused on the caspase-like activity. Caspases, cysteine-aspartic proteases, are enzymes that play a crucial role in apoptosis.Whereas caspases have been intensely studied in animal cells, therole and function of enzymes that resemble caspases (“caspase-like” enzymes) in plant cells remain almost unknown. Vacuolarprocessing enzymes, saspases, metacaspases, and phytaspases areassociated with the execution of programmed cell death in plantcells. Vacuolar processing enzymes, saspases, and phytaspaseshave demonstrated caspase activity (Siczek and Mostowska, 2012).Caspase-3-like activity was monitored in plants and connectedwith oxidative stress and programmed cell death. It was detectedin real time in single living plant cells undergoing PCD by Zhanget al. (2009). Garcia-Heredia successfully used caspase-3 substrateto quantify the caspase-like activity in Arabidopsis thaliana cell cul-tures exposed to acetylsalicylic acid (Garcia-Heredia et al., 2008). Inour case, we used Ac-DEVD-pNA substrate, which is a specific cleav-age substrate for caspase-3. Compared to the control (untreated)BY-2 cells, tobacco BY-2 cells treated with Al2O3 nanoparticlesshowed significantly increased level of caspase-like activity. Thisfact gives evidence of the induction of the PCD machinery in BY-2cells treated with Al2O3 nanoparticles and completes the knowl-edge about their phytotoxic effect. Finally, we observed changesin the secondary metabolism of BY-2 cells as a result of treatmentwith Al2O3 nanoparticles. The level of phenolics was significantlyincreased, especially at the two highest applied concentrations ofAl2O3 nanoparticles (50 and 100 �g mL−1). Kovacik et al. showedchanges in secondary metabolism of Matricaria chamomilla plantsexposed to aluminium (Kovacik et al., 2010, 2012). Phenolics alonerepresent an important protective mechanism against the action ofROS. However, the BY-2 plant cell model is not commonly used asa model culture for studying the phenolic secondary metabolismand further study is quite necessary. We can conclude that thetreatment of BY-2 cells, which serve as the model cells in plant biol-

ogy, with Al2O3 nanoparticles leads to the generation of reactiveoxygen and nitrogen species, which are involved in lipid peroxi-dation, change the integrity of the plasma membrane and initiatethe processes of programmed cell death executed by enzymes with

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aspase-like activity. Our work presents a comprehensive view ofhe mechanisms involved in the phytotoxicity of Al2O3 nanoparti-les.

. Conclusion

This work presents new knowledge about the systematic effectsf Al2O3 nanoparticles on a model plant cell culture, a suspensionulture of tobacco BY-2 cells. Al2O3 nanoparticles exhibited toxicityhat was closely connected with the generation of reactive oxygennd nitrogen species in both concentration- and time-dependentays. The generation of ROS and RNS is involved in the processes

f programmed cell death. We observed not only cytological signsf PCD (shrinkage of cytoplasm, changes in the nuclear architecture,ragmentation of the DNA and the formation of apoptic-like bodies),ut also changes in the integrity of plasma membrane, changes inhe mitochondrial potential and the initiation of PCD mechanismsxecuted by caspase-like proteins. Our work represents the firsttudy of the toxicity of Al2O3 nanoparticles and demonstrates thenduction of PCD in a plant cell model.

cknowledgement

Grants GACR P304/11/2246 and 42/2011/FaF are highlycknowledged.

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