Effect of Ingested Tungsten Oxide (WO x ) Nanofibers on Digestive Gland Tissue of Porcellio scaber...

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Article

Effect of ingested tungsten oxide (WOX) nanofiberson digestive gland tissue of Porcellio scaber (Isopoda,Crustacea): Fourier transform infrared (FTIR) imaging

Sara Novak, Damjana Drobne, Lisa Vaccari, Maya Kiskinova, PaoloFerraris, Giovanni Birarda, Maja Remskar, and Matej Ho#evar

Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es402364w • Publication Date (Web): 16 Aug 2013

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1

Effect of ingested tungsten oxide (WOX) nanofibers on digestive gland tissue of Porcellio

scaber (Isopoda, Crustacea): Fourier transform infrared (FTIR) imaging

Sara Novak‡, Damjana Drobne

‡,§,†,*, Lisa Vaccari

¶, Maya Kiskinova

¶, Paolo Ferraris

¶,

Giovanni Birarda‖, Maja Remškar

¥, Matej Hočevar

‖

‡Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia,

§Centre of Excellence, Advanced Materials and Technologies for the Future (CO

NAMASTE), Ljubljana, Slovenia,

†Centre of Excellence, Nanoscience and Nanotechnology (Nanocentre), Ljubljana, Slovenia,

¶Elettra-Sincrotrone Trieste, AREA Science Park, Basovizza, Trieste, Italy,

‖ Lawrence Berkeley National Laboratory, 1 Cyclotron Rd. Berkeley CA, USA,

¥Jožef Stefan Institute, Condensed Matter Physics Department, Jamova cesta 39, 1000

Ljubljana, Slovenia,

‖Institute of Metals and Technology, Lepi pot 11, 1000 Ljubljana, Slovenia

* Corresponding author: [email protected]

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Abstract 1

Tungsten nanofibers are recognised as biologically potent. We study deviations in molecular 2

composition between normal and digestive gland tissue of WOx nanofibers (nano-WOx) fed 3

invertebrate Porcellio scaber (Iosopda, Crustacea) and revealed mechanisms of nano-WOx 4

effect in vivo. Fourier transform infrared (FTIR) imaging performed on digestive gland 5

epithelium was supplemented by toxicity and cytotoxicity analyses as well as scanning 6

electron microscopy (SEM) of the surface of digestive gland epithelium. The difference in the 7

spectra of the WOx-treated and control cells showed up in the central region of the cells and 8

were related to a changed protein to lipid ratio, lipid peroxidation and structural changes of 9

nucleic acids. The conventional toxicity parameters failed to show toxic effects of nano-WOx, 10

whereas the cytotoxicity biomarkers and SEM investigation of digestive gland epithelium 11

indicated sporadic effects of nanofibers. Since toxicological and cytological measurements 12

did not highlight severe effects, the biochemical alterations evidenced by FTIR imaging have 13

been explained as the result of cell protection (acclimation) mechanisms to unfavourable 14

conditions and indication of non-homeostatic state, which can lead to toxic effects. 15

16

17

18

19

20

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Introduction 21

The amount of tungsten based products is increasing substantially.1 Tungsten oxides 22

(WO3, WO2, and WOx), which have attractive semiconductor properties, have been 23

considered for many important applications including optical devices, gas sensors, 24

electrochromic windows, and photocatalysts.2 Anthropogenic activities significantly increase 25

tungsten concentration in environmental systems.3 Tungsten powder mixed with soils at rates 26

higher than 1% on a mass basis, trigger changes in soil microbial communities resulting in the 27

death of a substantial portion of the bacterial component and an increase of the fungal 28

biomass. It also induces the death of red worms and plants.3 29

Synthesis of tungsten oxides can be also accompanied by release of fiber-like 30

nanoparticles which raises safety concerns reminiscent of those associated with asbestos 31

fibers, which were found to be highly toxic inducing irreversible health problems.4 WOx 32

nanofibers, whiskers or needles are recognised as being more biologically potent than non-33

fibrous WOx due to their ability to produce free radical damage in vitro.5 Tungsten carbide 34

particles (WCs) that can cause pneumoconiosisare also well known.6 35

Detection of biological effects can be gained from comparisons of healthy and 36

abnormal tissue, which can be carried out by a variety of physical, biological and biochemical 37

methods. The selection of methods is based on the expected alteration but, when it is 38

necessary to gain a better understanding on molecular and functional changes, methods which 39

can monitor a broad range of structural or functional alterations are required. Among these, 40

Fourier Transform InfraRed (FTIR) microscopy, which uses IR radiation to detect deviations 41

in molecular composition between normal and abnormal tissue is very promising.7 This 42

technique is based on absorption of infrared light that excites molecular vibrations, the 43

intensities of which provide quantitative information, while frequencies give qualitative 44

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knowledge about the nature of these bonds, their structure, and their molecular environment. 45

FTIR microspectroscopy is a label-free, non-destructive and objective tool for discriminating 46

between normal tissues and any alteration. In complex systems such as cells, the main spectral 47

features arise from the backbone vibrations of proteins, lipids, and nucleic acids. The infrared 48

spectrum of cells reflects all these contributions and provides information on the 49

concentration, organization and structure of the most fundamental macromolecules.8 50

Interactions between cells and nanoparticles lead to alteration in cell metabolism, 51

activation of mechanisms that protect against oxidative stress, toxic response and finally cell 52

death.9, 10

Many papers report effects of nano and microparticles on lipid and protein 53

oxidation, 11-13

changes in cell membrane fluidity,11

alterations of proteins12, 13

and of DNA.7 54

However, the assessment of nanomaterial toxicity is not always straightforward. The unique 55

chemo-physical properties of nanostructures compared with their bulk counterpart often tune 56

the system response to conventional bio-assays in unpredictable ways, raising many concerns 57

on the outcomes reliability.14

Since the vibrational features of the sample are the diagnostic 58

biomarkers in FTIR spectroscopy and microscopy, the technique is extremely promising in 59

this respect and it was indeed successfully exploited for highlighting the biomolecular 60

mechanisms of multiwalled carbon nanotube toxicity on both prokaryotic and eukaryotic cells 61

by combining synchrotron radiation FTIR imaging and multivariate analysis.15

Some authors 62

also demonstrated the technique sensitivity to cellular changes induced by environmentally-63

relevant concentrations of contaminants.15, 16

Overall, FTIR spectroscopic approaches allows 64

studying effects of different substances and stress agents in non-invasively, non-destructively 65

and in a high-throughput fashion. 15-17

66

In our study the capabilities of FTIR imaging has been complemented with 67

conventional bioassays to study deviations in molecular composition between normal 68

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digestive gland tissue and digestive gland tissue of WOx nanofibers (nano-WOx) fed animals. 69

The aim this work was to reveal the mechanism of in vivo effect of nano-WOx on digestive 70

gland cells of a model organism, terrestrial invertebrate Porcellio scaber (Isopoda, 71

Crustacea). The advantage of using this organism relies on the possibility to establish a direct 72

correlation between the actual exposure dose to nanofibers and the observed effects at 73

different levels of biological organization, since it is possible to estimate the exposure dose of 74

each test organisms. The feeding parameters are an integrated organism-level response, 75

appropriate evidence of the effects of different chemicals at organism level.18,

19

In addition, 76

cellular and biochemical analyses indicate cell level evidence after exposure to chemicals or 77

nanoparticles and to a certain degree their mode of action. Digestive gland cells 78

(hepotopancreas) of terrestrial isopods which combine the functions of pancreas and liver in 79

vertebrates are preferred tissue to study effects of substances with unknown and untargeted 80

action in digestive system. 81

82

Experimental 83

Model organisms 84

Terrestrial isopods (Porcellio scaber, Isopoda, Crustacea) were collected during July 85

2010 at an uncontaminated location near Ljubljana, Slovenia. The animals were kept in a 86

terrarium filled with a layer of moistened soil and a thick layer of partly decomposed hazelnut 87

tree leaves (Corylus avellana), at a temperature of 20 ± 2 °C and a 16:8-h light:dark 88

photoperiod. Only adult animals of both sexes and weighing more than 30 mg were used in 89

the experiments. If moulting or the presence of marsupia were observed, the animals were not 90

included in the experiment in order to keep the investigated population physiologically as 91

homogenous as possible. 92

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The digestive system of the terrestrial isopod P. scaber is composed of a stomach, four 93

blind-ending digestive gland tubes (hepatopancreas) and a gut. Food enters the digestive 94

glands directly via a short stomach or after the reflux from the gut and ingested material is 95

mixed with digestive fluids. 96

Synthesis and characterization of WOx nanofibers 97

The WOx nanofibers were synthesized by a chemical transport reaction20

at Jozef 98

Stefan Institute, Condensed Matter Physics Department, from tungsten powder (99.9%, 99

Sigma-Aldrich) and WO3 powder (99.9%, Sigma-Aldrich) in the stoichiometric ratio of 100

WO2,86. Iodine (99.8%) in a volume fraction of 3.2 mg/cm3

was added as a transport agent. 101

The material was transported from the source (hot zone at 1123 K) to the growth zone (1009 102

K), with a 5.7 K/cm temperature gradient. The produced nanofibers were studied using 103

electron transmission microscopy 200 keV Jeol 2010F, scanning electron microscopy (FE-104

SEM, Supra 35 VP, Carl Zeiss). XRD spectra were recorded with an AXS D4 Endeavor 105

diffractometer (Bruker Corporation, Karlsruhe,Germany), with Cu Ka1 radiation and a SOL-106

X energy dispersive detector with the angular range of 2θ from 5° to 75°, a step size of 0.04° 107

and a collection time of 3 to 4 s. Specific surface area of the sample was measured by single 108

point nitrogen absorption technique using a Micromeritics Gemini 2370 instrument. 109

Food preparation 110

In this study the animals consumed particles applied in a suspension to the leaf 111

surface. Hazelnut leaves were collected in an uncontaminated area and dried at room 112

temperature. Dried leaves were cut into pieces of approximately 100 mg. The WOx nanofibers 113

were suspended in distilled water before each experiment to obtain the final concentration of 114

5000 µg nano-WOx/ml. To diminish agglomeration of nanofibers in distilled H2O the 115

suspension was sonicated in an ultrasonic bath for 1h and mixed using a vortex mixer before 116

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brushing it on the leaves. In the control group, the leaves were treated only with distilled 117

water. The suspension of nanofibers or the distilled water was brushed onto the abaxial leaf 118

surface to give final nominal concentrations of nanoparticles on the leaves of 5000 µg nano-119

WOx per gram (dry wt) of leaf and left until dry. 120

Experimental setup 121

Two experiments were performed. The number of animals per group (control or nano-122

WOx exposed) was 5 in first experiment while 20 in the second one. The set up for both 123

experiments was same. Each individual animal was placed in a 9 cm Petri dish. A single 124

hazelnut leaf segment treated with distilled water or nano-WOx suspension (5000 µg/g of 125

WOx) and placed in each Petri dish was the animal's only food source. Humidity in the Petri 126

dish was maintained by spraying tap water on the internal side of the lid every day. All Petri 127

dishes were kept in a large glass container under controlled conditions in terms of air 128

humidity (≥80%), temperature (21±1°C) and light regime (16:8h light: dark photoperiod). 129

After seven days of exposure to nanofibers, the animals were anaesthetized at low 130

temperature and then decapitated and their digestive glands isolated and subsequently used for 131

different analyses. 132

Feeding parameters were observed in both experiments. Tissue from model organisms 133

from first experiment was used for scanning electron microscopy while digestive gland cell 134

membrane stability assay and FTIR experiments were carried out on the second group. 135

Feeding parameters, weight change and survival 136

After 7 days of exposure of the animals to treated leaves, the faecal pellets and leaves were 137

removed from the Petri dishes, dried at room temperature for 24 h and weighed separately. 138

The feeding rate of isopods was calculated as the mass of leaves consumed per animal’s wet 139

weight per day. The food assimilation efficiency was calculated as the difference between the 140

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mass of consumed leaves and mass of faecal pellets divided by the mass of consumed leaves. 141

The weight change of an animal was the difference in its mass from the beginning to the end 142

of the experiment. 143

Digestive gland cell membrane stability assay 144

The cell membrane stability was tested with the modified method previously described 145

by Valant et al.21

A single isolated hepatopancreatic tube was incubated for 5 min in a mixture 146

of the fluorescent dyes acridine orange (AO, Merck) and ethidium bromide (EB, Merck) and 147

then put on a microscope slide. Fresh samples were examined by an Axioimager.Z1 148

fluorescent microscope (Zeiss) and photographed with two different sets of filters. The 149

excitation filter 450 to 490 nm and the emission filter 515 nm (filter set 09) were used to 150

visualize AO and EB stained nuclei, while the excitation filter 365 nm and the emission filter 151

397 nm (filter set 01) were used to visualize nuclei stained with EB only. The cell membrane 152

integrity was assessed by examination of the micrographs. Photographs of intact digestive 153

glands were examined by the same observer twice at intervals of at least 24h. The integrity of 154

cell membrane was assessed visually and classified on the basis of a predefined scale from 0 155

to 9. From preliminary experiments, it was concluded that the non-treated (control) animals 156

show <5% of nuclei stained by EB, while severely stressed animals have up to 100% of EB-157

stained nuclei. The <5% of hepatopancreatic tubes stained with EB were classified as 0, and 158

those with the highest proportion (>95%) of EB stained nuclei as 9.21

Our previously 159

published results have demonstrated that in animals in good physiological condition from a 160

stock culture, the digestive gland cell membrane stability value was higher than 2 in only 5% 161

of animals and this was taken as a benchmark.21

The cell membranes are considered to be 162

destabilised when the value is higher than 2. 163

164

165

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FTIR imaging 166

For FTIR imaging, digestive glands were shock-frozen in liquid N2, using tissue-167

freezing medium (Jung Tissue Freezing Medium, Leica). 15 µm thick samples were sectioned 168

using a Leica CM3050 cryotome with the temperature of the microtome head and chamber 169

maintained between -25 °C and -20 °C. The sections were then placed onto CaF2 IR 170

transparent windows 2 mm thick, which were transferred to an freeze dryer (Alpha 2-4 Christ) 171

using a cryo-transfer assembly cooled with liquid nitrogen, and then freeze dried at 30°C and 172

0.4 mbar for 24 h. 173

FTIR measurements were carried out at the infrared beamline SISSI (Synchrotron 174

Infrared Source for Spectroscopy and Imaging) of Elettra Synchrotron laboratory22

using the 175

Vertex 70 interferometer coupled with Hyperion 3000 Vis-IR microscope. Both the 176

interferometer and microscope were purged with N2. The IR images were acquired in 177

transmission mode using the bidimensional Focal Plane Array (FPA, 64X64 pixel) detector, 178

averaging 256 scans per spectrum. Each FPA image is composed by 4096 spectra and by 179

using a 15X condenser/objective (NA=0.4), a ~170X170 µm2 sample area was imaged 180

achieving a pixel resolution of about 2.6 µm. A background spectrum for each image was 181

acquired on a portion of the CaF2 window devoid of cells. Each image was pre-processed by 182

running the atmospheric compensation routine of OPUS 6.5 (Bruker Optics GmbH, Ettlingen, 183

Germany) in order to minimize spectral contributions from water vapour and carbon dioxide. 184

The simpler FTIR image is generated by integrating a specific spectral band or a spectral 185

region for each image pixel, following univariate analysis. The integration results are then 186

plotted in 2D using a colour scale and providing information on the distribution of a 187

functional group within the sample, and consequently of the bio-macromolecules that contain 188

it (Figure S1A,B, Supporting Information). Figure S1B (Supporting Information) was 189

obtained following this procedure, integrating the 1720-1480 cm-1

spectral region, which 190

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contains the most intense bands of cellular proteins, Amide I and II (Table S1, Supporting 191

Information). The spectral bands relevant for this manuscript are reported in Table S1, 192

Supporting Information, and their assignment was done in accordance with the peer-review 193

articles listed within. Penetration of the tissue-freezing medium (TFM) into the sample is 194

usually minimal and dependent on the tissue type.23, 24

Typically, the TFM can be detected as 195

an additional thin layer surrounding the tissue. However, the TFM penetration within our 196

samples was presumed to be different between different specimen regions and among them, 197

depending also upon dragging effects during cutting. Figure 1 shows the spectra of the tissue 198

freezing medium (black dotted line), of a peripheral and central point of the sample control 199

K2, black and grey continuous lines, respectively. The Jung TFM has vibrational features that 200

overlap with some characteristic spectral bands and this may impose some limitation to the 201

diagnostic potentials of the technique. In particular, a strong and sharp tissue freezing medium 202

peak centred at 1116 cm-1

could affect the spectral profile below 1200 cm-1

: its contribution is 203

clearly visible in the peripheral region spectrum (black continuous line) while it is not 204

detectable in the central part of the same sample. Consequently, a procedure was developed 205

for subtracting the TFM contribution from the sample vibrational pattern. 206

The TFM was subtracted from each pixel (pixel size: ~2.6X2.6 μm) of the map 207

accordingly to its concentration and its penetration within the tissue. TMF removal was done 208

by using a script written in R environment and using the HyperSpecJSS package 209

(http://hyperspec.r-forge.r-project.org). 25,

26

The routine steps, independently applied for each 210

IR image, are presented schematically in Figure S2, Supporting Information for the control 211

sample K1 (see optical image S2A) and listed as follows: 212

i) Identification of the TFM cluster centroid. The FTIR image of the protein 213

distribution, obtained by peak integration in the spectral region 1720-1480 cm-1

, is shown in 214

Fig.S2, Supporting Information. By both Figure S2A and 1 it can be seen that the cellular 215

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http://hyperspec.r-forge.r-project.org).25/

11

proteins have spectral features not overlapping with the ones of the TFM. Therefore, I order to 216

obtain the TFM average spectrum, each image was processed by Hierarchical Cluster 217

Analysis (HCA), based on Euclidean distances and Wards’ algorithm on absorbance spectra 218

in the 1720-1480 cm-1

spectral region. The cluster of the freezing medium was then identified: 219

it corresponded to the sampled region outside the specimen, the cluster number 1, black 220

coloured in Figure S2C, Supporting Information. The cluster centroid, that is the average 221

spectrum of the ones belonging to the cluster, was then set as the mean TFM spectrum for the 222

analyzed sample 223

ii) Distribution of the TFM and determination of the scaling factor, α. The distribution 224

of the tissue freezing medium over the sampled region was evaluated by the peak height value 225

of the characteristic TFM band centred at 1116 cm-1

(Figure S2D, Supporting Information). 226

By this procedure we obtained a matrix of values representing the TMF distribution within the 227

map, and by dividing this matrix by its maximum value we got a normalized scaling factor α 228

(that goes from 0 to 1), that, applied to the TMF spectrum, gave the hyperspectral image of 229

the freezing medium within the map that could be called “TMF map”. A new hyperspectral 230

image was then obtained by subtracting pixel by pixel from the raw image the “TMF map”. 231

The procedure was repeated twice per sample (see Figures S2E, F, Supporting Information). 232

Actually, the obtained scaling factors accounts for both TFM and non-TFM contribution in 233

the specimen regions. However, the spectral shape of the TFM spectrum suggests that the 234

specimen contribution to the overall spectral shape should be minimal when considering the 235

spectral region around at 1116 cm-1

. For supporting our hypothesis, the integrated intensity of 236

the spectral region 1300-900 cm-1

before and after the correction was plotted (see Figures 237

S2G and H, Supporting Information). This region comprehends the bands of nucleic acids and 238

carbohydrates and it is the one mostly affected by TFM. Before running the subtraction 239

routine, the chemical image of the K1 sample showed areas of medium intensity also outside 240

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the specimen (see in particular upper right corner in Figure S2G, Supporting Information), 241

while after subtraction the intensity of the 1300-900 cm-1

spectral band is at the noise level in 242

the region external to the specimen while it well follows the profile of the specimen both 243

before and after the subtraction. This result is an indirect proves of the goodness of the 244

approach. 245

Images corrected for the TFM contribution were analysed independently in order to 246

highlight the biochemical diversity between central and peripheral sample regions. The 247

aforementioned regions have been discriminated against by applying HCA in the spectral 248

region 3050-2800 cm-1

on vector normalized absorbance spectra (Euclidean distances & 249

Wards’ algorithm). Comparing optical and FTIR images, it was deduced that apical and basal 250

parts of the epithelium represent the peripheral (black coloured cluster 1 in Figure S2I, 251

Supporting Information), while the central part of epithelium, located around nuclei formed 252

the central region (red colored cluster 2 in Figure S2I, Supporting Information). For the FTIR 253

microspectroscopic imaging, two control and three WOx-fed animals where considered. For 254

each sample, two to three different locations have been imaged: more than 6000 spectra, 255

divided between peripheral and central regions, have been collected and further analysed 256

following the procedure described above. 257

Scanning electron microscopy (SEM) and Energy dispersive X-ray analysis (EDX) 258

After the feeding experiment, animals were decapitated and the hepatopancreas was 259

isolated and immediately transferred with tweezers to the fixative containing 2.5% 260

glutaraldehyde (Spi Supplies), 0.4% paraformaldehyde (Sigma-Aldrich) and 0.1M sodium 261

phosphate buffer (pH 7.2). After primary aldehyde fixing, digestive glands were put in 1% 262

osmium tetroxide and stained with TOTO (thiocarbohydrazide 263

/osmiumtetroxide/thiocarbohydrazide/osmiumtetroxide) conductive, a method previously 264

described by Leser et al.21

The fixed hepatopancreas glands were dehydrated in absolute 265

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alcohol and dried with hexamethyldisilizane (HMDS, Merck). The dry samples were mounted 266

on holders and sputter coated with gold-palladium (Sputter coater SCD 050, BAL-TEC). 267

Samples were investigated by field emission scanning electron microscopy (SEM; Jeol 268

JSM-6500F, at the Institute of Metals and Technology in Ljubljana). Energy dispersive X-ray 269

analysis (EDX) was used to analyse the chemical composition of selected parts of the 270

epithelial surface (EDX/WDX Oxford Instruments INCA, Jeol JSM-6500F, at the Institute of 271

Metals and Technology). 272

Data analysis 273

Data were analyzed by standard statistical methods. The difference in the median 274

measured parameters in exposed and unexposed groups was tested with the non-parametric 275

Mann-Whitney U test. All calculations were performed with Statgraphics Plus 4.0. 276

277

Results 278

Characteristics of WOx nanofibers 279

The WOx fibers were grown as single crystalline fibers with high aspect ratio (Figure 280

S4A). While their diameter typically did not exceed 100 nm, their length was on the 281

millimetre scale (Figure S3A, Supporting Information) and reduced after sonication (Figure 282

S3B, Supporting Information). An electron diffraction pattern taken on a single fiber Figure 283

S4B, Supporting Information) corresponds to the W18O49 phase. The [ ] zone axis is 284

shown. X-ray diffraction confirms the monoclinic W18O49 phase (JCPDS-71-2450). 285

Impurities of other WOx phases are possible with quantities below the detection limit of 2-3 286

%. Specific surface area of the samples is 3.2 m2/g. 287

Feeding parameters, weight change and survival 288

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Weight, survival and feeding parameters were not affected when animals were exposed 289

to leaves contaminated with WOx nanofiber suspension, providing nominal concentrations of 290

5000 µg nano-WOx/g of leaf. 291

Digestive gland cell membrane stability assay 292

In the control group, 90% of animals had a cell membrane stability of, nominally 2 293

(Figure 2). In 10% of control animals, it was slightly higher and assigned the value 3. We 294

ascribe this to suboptimal experimental conditions. 295

In animals fed on food dosed with nano-WOx the stability of the digestive gland cell 296

membrane was affected in almost 20% of animals, while 10% of them had severely affected 297

cell membranes (Figure 2). 298

FTIR imaging 299

The average spectra of peripheral and central regions for each sample as obtained from 300

HCA have been compared by verifying their spectral similarities. In particular, we analysed 301

differences between control and exposed samples in the peripheral region, the apical part of 302

the digestive gland epithelia, and in the central part of the epithelia, which is the region 303

between the apical and the basal layer of the cells. The analysis did not highlight any 304

significant differences between the control and nano-WOx treated peripheral regions that are 305

also those mostly affected by tissue freezing medium penetration. In contrast, following 306

nanoparticle ingestion noticeable biochemical alterations were found in the central sectors. 307

Figures 3A, B show the average spectra of central parts of the WOx and control 308

samples, as obtained upon standard vector normalization on the 3000-2800 cm-1

range. In 309

order to highlight compositional modification affecting the samples following ingestion of 310

nano-WOx, several spectral regions have been taken into consideration. Since inhomogeneity 311

in the sample thickness, both within and between samples, influences the values of integrated 312

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band areas and peak heights, accordingly with Lambert-Beer law, area or height ratios have 313

been considered. Second derivatives of average spectra are shown in Figures 3C, D, and have 314

been analysed for highlighting structural differences among the samples. 315

The ratio of area integrals between 1720-1480 cm-1

and 3000-2800 cm-1

decreased 316

significantly upon ingestion of nanofibers, from 3.536±0.033 to 2.1563±0.221, revealing a 317

remarkable decrease in the protein to lipid content (Table S1, Supporting Information). 318

However, no significant differences in either relative intensity or energy were detected for 319

methyl and methylene stretching bands. Asymmetric and symmetric -CH3 and -CH2 stretching 320

bands were found at 2960±2, 2873±2, 2925±2 and 2852±2 cm-1

, respectively, in both control 321

and treated samples. Moreover, both symmetric and asymmetric CH3-to-CH2 peak height 322

ratios did not change upon treatment: for the control samples (CH3/CH2)asym = 0.620±0.006 323

and (CH3/CH2)sym = 0.691±0.065, while for the WOx treated samples (CH3/CH2)asym = 324

0.619±0.021 and (CH3/CH2)sym = 0.734±0.012. Conversely, an upshift of both vinyl (3007-325

3012 cm-1

) and methine (2893-2896 cm-1

) moieties was evident upon treatment of nanofibers, 326

followed by the downshift of the carbonyl ester stretching band from 1743 to 1739 cm-1

. No 327

protein structural variations were detected upon nano-WOx ingestion. Amide I and Amide II 328

components remained similar and contribution from α-helix (AmI, 1660 cm-1

; AmII, 1545 cm-329

1), random coil (AmI, 1640 cm

-1) and β-sheet (AmI, 1688 and1613 cm

-1) structures were 330

found for both control and nano-WOx treated samples. 331

The overlapping of the asymmetric bending mode of methyl groups and the 332

deformation of methylene moieties originates a band between 1479 and 1427 cm-1

. This band 333

is centred at 1456 cm-1

for both control and treated samples, but the two contributions could 334

be clearly distinguished in WOx treated samples from second derivatives υasymCH3 at 1464 335

cm-1

and υCH2, at1453cm-1

. The symmetric bending mode of methyl groups reflected the 336

former trend, splitting from 1394 to 1382 and 1399 cm-1

upon ingestion of NPs. The height 337

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ratio of the bands centred at ~1740 and ~1456 cm-1

is much higher for treated samples 338

(0.692±0.105) than for the controls (0.378±0.069). Similarly, the height ratio between the 339

band centred at 1456 to the component centred at 1400 cm-1

of the band extending from 1427 340

to 1357 cm-1

changed from 0.600±0.062 to 1.187±0.057 upon nanofibers ingestion. Finally, 341

asymmetric phosphate band had 2 major components, centred at 1217 and 1232 cm-1

in both 342

control and treated samples, but the former became much weaker upon treatment. 343

In has to be stated that any of the detected differences between control animals and 344

nano-WOx-fed ones cannot be attribute to the spectral features of nano-fibers themselves. 345

Actually, the most relevant bands related to WOx compounds (W=O and O-W-O) are in the 346

region of 1000-500 cm-1

.27, 28

347

Scanning electron microscopy (SEM) and Energy dispersive X-ray analysis (EDX) 348

Digestive glands of animals fed with food contaminated with 5000 µg nano-WOx/g of 349

food were prepared for scanning electron microscopy. Fiber-like structures were observed on 350

the surface of some cells of digestive glands (Figure 4A). We analysed these structures with 351

EDX and the chemical composition reveals the presence of tungsten (Figure 4B, C). 352

In 25% of animals these irregularly shaped structures were found to be thrust into the 353

cells. Such structures have never been found in control animals or observed during our 354

previous research of morphological characteristics of digestive gland cells. The EDX analyses 355

indicate elevated amount of tungsten approximately 2.4 wt% in these structures as shown in 356

Table 1. 357

358

Discussion 359

The application of FTIR microspectroscopic to studies of molecular alterations in the 360

digestive gland cells due to ingestion of WOx nanofibers by model invertebrate organism 361

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17

P.scaber has provided new insight into cellular response to nanofibers in vivo. Spectroscopic 362

information has been complemented with the toxicological and cytological data, scanning 363

electron microscopy and energy dispersive X-ray spectrometry. 364

In the presented study classical toxicological parameters (weight change, survival and 365

feeding rate) were not affected when animals were exposed to leaves contaminated with WOx 366

nanofiber suspension, providing nominal concentrations of 5000 µg nano-WOx/g of leaf. 367

However, after seven days of feeding with nano-WOx contaminated food, the stability of the 368

digestive gland cell membrane was affected in almost 20% of animals. Moreover, by SEM 369

inspection of the surface of digestive gland epithelium, rod like structures containing tungsten 370

have been found thrust into the apical part of a few epithelial cells in 2 of 8 investigated 371

animals. This indicates that the ingested WOx nanofibers have the potential to interact with 372

individual cells and may lead to deleterious effect. This phenomenon has been well studied in 373

the case of asbestos. Wang et al.29

report that asbestos fibres insert into pleural mesothelia 374

cells, inducing chromosomal changes by direct biological or mechanical damage. Similarly, 375

some well knew toxic effect of asbestos nanofibers have also been found.4 376

The FTIR spectral imaging revealed several significant differences in molecular 377

composition of digestive gland epithelium between control animals and animals exposed to 378

nano-WOx. Hierarchical cluster analysis of all analysed sections of gland epithelium indicated 379

that the epithelium could be divided into two distinct regions: the central one around the 380

nuclei, and the remainder, which is termed a peripheral region. Since distinct differences 381

between the control and nano-WOx treated digestive gland epithelium were observed only in 382

the central region, only this one will be considered here. 383

The most evident compositional difference between control and WOx treated samples was 384

found in the protein to lipid ratio. Compared to control cells, the central region of treated cells 385

is enriched in lipids with respect to proteins. This could be interpreted either as accumulation 386

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18

of fatty acids and phospholipids or a decreased cellular protein concentration due to nano-387

WOx ingestion. The Amide I to Amide II ratio is only slightly lower in treated samples, 388

2.847±0.291 versus 3.111±0.023, nevertheless comparable within standard deviation. This 389

ratio is usually regarded as indicative of variations in structural versus metabolic (new 390

synthesis) proteins that seems to be almost unaffected by NPs’ ingestion.30

Moreover, no 391

differences in the Amides’ shape, possibly indicating detectable variations in cellular protein 392

global folding, were highlighted. Therefore we speculate that the difference in the protein to 393

lipid ratio may be attributed to an altered lipid metabolism rather than to a decreased protein 394

synthesis activity. Similar conclusions were drawn out by Gaigneaux and co-workers.8 395

It is noteworthy that that our spectroscopic evidences support the hypothesis that 396

neither the cellular membrane composition nor structure changed as a consequence of nano-397

WOx consumption. As a matter of fact, the methyl to methylene ratio constancy reveals that 398

the average length and ramification of acyl chains of lipids, and phospholipids in particular, 399

was unaffected by the ingestion of nanofibers as well as the energies of the methyl and 400

methylene stretching bands. The positions of the signals corresponding to the –CH2 and –CH3 401

moieties provide information regarding the packing characteristics of the acyl chains, which 402

in turn may be related to the fluidity-rigidity of membrane.31

In particular, we did not observe 403

any measurable variations in saturation level of lipids but the detected upshift on both vinyl 404

and methine stretching indicates that the unsaturated acyl chains are to a certain extent 405

affected by the ingestion of nanofibers. Indeed, the FTIR data also show that the ratio of 406

carbonyl stretching to methyl&methylene deformations (1740/1456 cm-1

) increased upon 407

nanofibers ingestion. Levine et al.,32

Palaniappan and Pramod,33

and Vileno et al.34

408

interpreted similar observations as a sign of lipid peroxidation. In our study, this hypothesis is 409

supported by the downshift of the carbonyl band at ~1740 cm-1

and the decrease of the 410

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19

methyl&methylene to carbonyl ratio (1456/1400 cm-1

) that indicates that oxidative processes 411

are taking place.35

412

Overall, our results suggest that the accumulation of end products of lipid peroxidation 413

is not combined with a detectable and massive degradation of the plasma membrane order and 414

composition. This is also confirmed by the results on digestive gland membrane stability that 415

showed only 10% of the WOx-fed animals had cell membrane integrity severely affected. 416

Conversely, an accumulation of lipids have been highlighted. It is indeed known that 417

oxidative stress can increase lipid biosynthesis and accumulation, which in turn promotes the 418

generation of reactive oxygen species (ROS).36,

37

Reactive oxygen species concentration at 419

cellular level upon NPs ingestion probably have not exceed the antioxidant cellular capacity, 420

since features of extensive oxidative damage have not been detected.38

Indeed we have no 421

FTIR spectra indication for detectable changes in the secondary structure of the proteins, 422

which would occur in case of an extensive oxidative damage.39,

40

423

Comparing the results for control and nano-WOx–treated samples we also noticed 424

some alterations in the spectral range between 1300 and 1000 cm-1

, characteristic for nucleic 425

acids and carbohydrates. The asymmetric stretching band of the PO2- in the backbone of 426

nucleic acids shifts to higher wave numbers upon nano-WOx ingestion (from 1226 to 1232 427

cm-1

). This trend can be a consequence of a partial reorganization of the nucleic acid structure. 428

Similar FTIR results, were reported also by Whelan et al.41

and Vaccari et al.42

and have been 429

explained as an indication of transitions from the B form toward the A form of DNA in 430

response to dehydration. However, since both control and treated samples have been 431

subjected to the same preparation procedure, stored in the dried environment and measured 432

during the same days, dehydration is unlikely to be the cause of the experimental conditions 433

or sample preparation artifacts. Nevertheless, Dovbeshko et al.43

interpreted changes in 434

position of both asymmetric and symmetric stretching vibrations of phosphate moieties to be a 435

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20

consequence of damage caused to cells by irradiation, possibly related to spatial changes in 436

the positions of the phosphate groups in the RNA helix. In addition, there are also literature 437

reports suggesting that the B toward A transition of DNA might play role in the resistance of 438

DNA to potential damage caused by heat, desiccation and toxic damage.44

439

The FTIR results obtained in our study are in agreement with toxicological and 440

cytological measurements, which indicate that ingestion of nano-WOx does not affect severely 441

the cell membrane stability and feeding behaviour. However, FTIR analyses clearly reveal 442

that the cells are disturbed and respond to the ingested nano-WOx. 443

We succeeded to show that ingested WOx nanofibers activate some cellular 444

mechanisms that may act as a protection against unfavourable conditions. Changed protein to 445

lipid ratio, lipid peroxidation and structural changes of nucleic acids were interpreted as 446

responses indicative of non-homeostatic state before oxidative stress and toxic responses are 447

evidenced. We speculate that both, partial structural reorganisation of nucleic acids as well as 448

slight lipid peroxidation indicate adaptation strategy of cells exposed to irritating nanofibres. 449

Whether is this nanofibre-specific response or a response to variety of unfavourable 450

conditions is a matter of further research. 451

452

Author information 453

Corresponding author 454

Phone: + 386 1 423 33. Fax: +386 1 257 33 90. E-mail: [email protected] 455

Notes 456

The authors declare no competing financial interest. 457

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mailto:[email protected]

21

458

Tables 459

Table 1: EDX elemental composition 460

Spectrum C O Ag W Os

Spectrum 1 43.32 15.03 5.34 2.40 33.91

Table 21: EDX elemental composition of spectrum 1 observed in area showed in Figure 4B., 461

results in wt.% with some other elements present in cell. 462

463

Figures 464

465

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22

Figure 1. Spectra of tissue freezing medium used in samples for FTIR analyses. FTIR 466

spectra of pure tissue freezing medium (dotted spectrum), of a sample point containing a 467

negligible amount of tissue freezing medium (gray spectrum) and a sample point where the 468

tissue freezing medium contribution is clearly visible from the sharp band centred at 1116 cm-

469

1 (black spectrum). 470

471

Figure 2. Digestive gland cell membrane stability of control and nano-WOx exposed 472

animals. Percentage of animals in each exposed group, with different degrees of destabilised 473

cell membrane, assessed visually and classified from 0 to 9 according to the predefined scale 474

as described in Materials and Methods. A digestive gland cell membrane stability value of 2 475

or less denotes animals which did not have destabilized cell membranes and digestive gland 476

cell membrane stability values from 3 to 5 animals with destabilized cell membranes. The 477

value of 5 corresponds to the most highly destabilized cell membranes. 478

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23

479

Figure 3. Averge spectras of the central regions of digestive gland tissue. A,B Average 480

spectra of the central regions of WOx treated (grey) and control (black) samples. C,D Second 481

derivative of the average spectra of treated (grey) and control (black) samples, respectively; 482

Savitzky-Golay algorithm, (17 smoothing points). 483

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24

484

Figure 4. Scanning electron microscopy (SEM) and Energy dispersive x-Ray (EDX) 485

composition of fiber-like structures in digestive gland. Surface of digestive gland 486

epithelium of animal fed with tungsten nanofibers for 7 days with fiber like structures found 487

in one of the cells (A). Digestive gland epithelium cell with thrusted fiber like structures 488

where EDX spectrum was taken (B). EDX spectra of observed area (pointed in fFigure B) 489

(C). 490

491

Acknowledgements 492

Work of PhD student Sara Novak was supported by Slovenian Research Agency 493

within the framework of young researchers. Part of work was conducted within research 494

projects financed by Slovenian Research Agency (J1-4109) and within the 7th FP EU Project 495

‘‘NANOVALID’’ (contract: 263147). We thank G.W.A Milne for editorial assistance. Lisa 496

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25

Vaccari and M. Kiskinova acknowledge the grant from Friuli Venezia Giulia Region: 497

Nanotox 0060 -2009. We thank Janez Jelenc for synthesis of nanomaterial used in the study. 498

499

Supporting Informations 500

Spectral band assignment where the relevant positions and assignments of the spectral bands 501

are reported and listed in Table S1. In Figure S1 is optical and Fourier transform infrared 502

(FTIR) image of control sample. Figure S2 is showing the routine steps of chemometric 503

approach in control sample after FTIR analyses. In Figure S3 are Scanning electron 504

microscopy (SEM) images of WOx nanofibers and on Figure S4 Transmission electron 505

microscopy (TEM) images of WOx nanofibers. 506

507

References 508

(1) Koutsospyros, A.; Braida, W.; Christodoulatos, C.; Dermatas, D.; Strigul, N. A review of 509

tungsten: From environmental obscurity to scrutiny. J. Hazard. Mate. 2006, 136, (1), 1-19. 510

(2) Lu, X. F.; Liu, X. C.; Zhang, W. J.; Wang, C.; Wei, Y. Large-scale synthesis of tungsten oxide 511

nanofibers by electrospinning. J. Colloid. Interf. Sci. 2006, 298, (2), 996-999. 512

(3) Strigul, N.; Koutsospyros, A.; Arienti, P.; Christodoulatos, C.; Dermatas, D.; Braida, W. 513

Effects of tungsten on environmental systems. Chemosphere. 2005, 61, (2), 248-258. 514

(4) Jaurand, M. C. F.; Renier, A.; Daubriac, J. Mesothelioma: Do asbestos and carbon nanotubes 515

pose the same health risk? Part. Fibre. Toxicol. 2009, 6. 516

(5) Leanderson, P.; Sahle, W. Formation of Hydroxyl Radicals and Toxicity of Tungsten-Oxide 517

Fibers. Toxicol. in Vitro. 1995, 9, (2), 175-183. 518

Page 25 of 30



26

(6) Enriquez, L. S.; Mohammed, T. L. H.; Johnson, G. L.; Lefor, M. J.; Beasley, M. B. Hard metal 519

pneumoconiosis: A case of giant-cell interstitial pneumonitis in a machinist. Respir. Care. 520

2007, 52, (2), 196-199. 521

(7) Lasch, P.; Pacifico, A.; Diem, M. Spatially resolved IR microspectroscopy of single cells. 522

Biopolymers 2002, 67, (4-5), 335-338. 523

(8) Gaigneaux, A.; Ruysschaert, J. M.; Goormaghtigh, E. Infrared spectroscopy as a tool for 524

discrimination between sensitive and multiresistant K562 cells. Eur. J. Biochem. 2002, 269, 525

(7), 1968-1973. 526

(9) Karlsson, H. L.; Cronholm, P.; Gustafsson, J.; Moller, L. Copper oxide nanoparticles are 527

highly toxic: A comparison between metal oxide nanoparticles and carbon nanotubes. Chem. 528

Res. Toxicol. 2008, 21, (9), 1726-1732. 529

(10) Lin, W. S.; Xu, Y.; Huang, C. C.; Ma, Y. F.; Shannon, K. B.; Chen, D. R.; Huang, Y. W. 530

Toxicity of nano- and micro-sized ZnO particles in human lung epithelial cells. J. Nanopart. 531

Res. 2009, 11, (1), 25-39. 532

(11) Peng, L.; Wenli, D.; Qisui, W.; Xi, L. The envelope damage of Tetrahymena in the presence 533

of TiO2 combined with UV light. Photochem. Photobiol. 2010, 86, (3), 633-8. 534

(12) Riding, M. J.; Martin, F. L.; Trevisan, J.; Llabjani, V.; Patel, I. I.; Jones, K. C.; Semple, K. T. 535

Concentration-dependent effects of carbon nanoparticles in gram-negative bacteria determined 536

by infrared spectroscopy with multivariate analysis. Environ. Pollut. 2012, 163, 226-234. 537

(13) Saulou, C.; Jamme, F.; Maranges, C.; Fourquaux, I.; Despax, B.; Raynaud, P.; Dumas, P.; 538

Mercier-Bonin, M. Synchrotron FTIR microspectroscopy of the yeast Saccharomyces 539

cerevisiae after exposure to plasma-deposited nanosilver-containing coating. Anal. Bioanal. 540

Chem. 2010, 396, (4), 1441-1450. 541

(14) Nel, A.; Xia, T.; Meng, H.; Wang, X.; Lin, S. J.; Ji, Z. X.; Zhang, H. Y. Nanomaterial Toxicity 542

Testing in the 21st Century: Use of a Predictive Toxicological Approach and High-543

Throughput Screening. Accounts. Chem. Res. 2013, 46, (3), 607-621. 544

Page 26 of 30



27

(15) Riding, M. J.; Trevisan, J.; Hirschmugl, C. J.; Jones, K. C.; Semple, K. T.; Martin, F. L. 545

Mechanistic insights into nanotoxicity determined by synchrotron radiation-based Fourier-546

transform infrared imaging and multivariate analysis. Environ. Int. 2012, 50, 56-65. 547

(16) Llabjani, V.; Malik, R. N.; Trevisan, J.; Hoti, V.; Ukpebor, J.; Shinwari, Z. K.; Moeckel, C.; 548

Jones, K. C.; Shore, R. F.; Martin, F. L. Alterations in the infrared spectral signature of avian 549

feathers reflect potential chemical exposure: A pilot study comparing two sites in Pakistan. 550

Environ. Int. 2012, 48, 39-46. 551

(17) Llabjani, V.; Trevisan, J.; Jones, K. C.; Shore, R. F.; Martin, F. L. Derivation by Infrared 552

Spectroscopy with Multivariate Analysis of Bimodal Contaminant-Induced Dose-Response 553

Effects in MCF-7 Cells. Environ. Sci. Technol. 2011, 45, (14), 6129-6135. 554

(18) Drobne, D.; Hopkin, S. P. The Toxicity of Zinc to Terrestrial Isopods in a Standard Laboratory 555

Test. Ecotox. Environ. Safe. 1995, 31, (1), 1-6. 556

(19) Drobne, D.; Blazic, M.; Van Gestel, C. A. M.; Leser, V.; Zidar, P.; Jemec, A.; Trebse, P. 557

Toxicity of imidacloprid to the terrestrial isopod Porcellio scaber (Isopoda, Crustacea). 558

Chemosphere 2008, 71, (7), 1326-1334. 559

(20) Remskar, M.; Kovac, J.; Virsek, M.; Mrak, M.; Jesih, A.; Seabaugh, A. W5O14 nanowires. 560

Adv. Funct. Mater. 2007, 17, (12), 1974-1978. 561

(21) Valant, J.; Drobne, D.; Sepcic, K.; Jemec, A.; Kogej, K.; Kostanjsek, R. Hazardous potential 562

of manufactured nanoparticles identified by in vivo assay. J. Hazard .Mater. 2009, 171, (1-3), 563

160-165. 564

(22) Lupi, S.; Nucara, A.; Perucchi, A.; Calvani, P.; Ortolani, M.; Quaroni, L.; Kiskinova, M. 565

Performance of SISSI, the infrared beamline of the ELETTRA storage ring. J. Opt. Soc. Am. B 566

2007, 24, (4), 959-964. 567

(23) Liao, C.; Piercey-Normore, M. D.; Sorensen, J. L.; Gough, K. In situ imaging of usnic acid in 568

selected Cladonia spp. by vibrational spectroscopy. Analyst. 2010, 135, (12), 3242-3248. 569

(24) Stitt, D. M.; Kastyak-Ibrahim, M. Z.; Liao, C. R.; Morrison, J.; Albensi, B. C.; Gough, K. M. 570

Tissue acquisition and storage associated oxidation considerations for FTIR 571

microspectroscopic imaging of polyunsaturated fatty acids. Vib. Spectrosc. 2012, 60, 16-22. 572

Page 27 of 30



28

(25) Beleites, C.; Neugebauer, U.; Bocklitz, T.; Krafft, C.; Popp, J. Sample size planning for 573

classification models. Anal. Chim. Acta. 2013, 760, 25-33. 574

(26) Beleites, C.; Sergo, V. hyperSpec: A Package to Handle Hyperspectral Data Sets in R. 2012. 575

(27) Tocchetto, A.; Glisenti, A. Study of the interaction between simple molecules and W-Sn-based 576

oxide catalysts. 1. The case of WO3 powders. Langmuir 2000, 16, (15), 6173-6182. 577

(28) Nguyen, T. A.; Jun, T. S.; Rashid, M.; Kim, Y. S. Synthesis of mesoporous tungsten oxide 578

nanofibers using the electrospinning method. Mate.r Lett. 2011, 65, (17-18), 2823-2825. 579

(29) Wang, N. S.; Jaurand, M. C.; Magne, L.; Kheuang, L.; Pinchon, M. C.; Bignon, J. The 580

Interactions between Asbestos Fibers and Metaphase Chromosomes of Rat Pleural Mesothelial 581

Cells in Culture - a Scanning and Transmission Electron-Microscopic Study. Am. J. Pathol. 582

1987, 126, (2), 343-349. 583

(30) Diem, M.; Chiriboga, L.; Yee, H. Infrared spectroscopy of human cells and tissue. VIII. 584

Strategies for analysis of infrared tissue mapping data and applications to liver tissue. 585

Biopolymers 2000, 57, (5), 282-290. 586

(31) Jackson, M.; Mantsch, H. H. The Use and Misuse of Ftir Spectroscopy in the Determination of 587

Protein-Structure. Crit. Rev. Biochem. Mol. 1995, 30, (2), 95-120. 588

(32) LeVine, S. M.; Wetzel, D. L. Chemical analysis of multiple sclerosis lesions by FT-IR 589

microspectroscopy. Free Radical. Bio. Med. 1998, 25, (1), 33-41. 590

(33) Palaniappan, P. R.; Pramod, K. S. FTIR study of the effect of nTiO(2) on the biochemical 591

constituents of gill tissues of Zebrafish (Danio rerio). Food. Chem. Toxicol. 2010, 48, (8-9), 592

2337-2343. 593

(34) Vileno, B.; Jeney, S.; Sienkiewicz, A.; Marcoux, P. R.; Miller, L. M.; Forro, L. Evidence of 594

lipid peroxidation and protein phosphorylation in cells upon oxidative stress photo-generated 595

by fullerols. Biophys. Chem. 2010, 152, (1-3), 164-169. 596

(35) Di Giambattista, L.; Pozzi, D.; Grimaldi, P.; Gaudenzi, S.; Morrone, S.; Castellano, A. C. New 597

marker of tumor cell death revealed by ATR-FTIR spectroscopy. Anal. Bioanal. Chem. 2011, 598

399, (8), 2771-2778. 599

Page 28 of 30



29

(36) Liu, J. F.; Ma, Y.; Wang, Y.; Du, Z. Y.; Shen, J. K.; Peng, H. L. Reduction of Lipid 600

Accumulation in HepG2 Cells by Luteolin is associated with Activation of AMPK and 601

Mitigation of Oxidative Stress. Phytother. Res. 2011, 25, (4), 588-596. 602

(37) Sekiya, M.; Hiraishi, A.; Touyama, M.; Sakamoto, K. Oxidative stress induced lipid 603

accumulation via SREBP1c activation in HepG2 cells. Biochem. Bioph. Res. Co 2008, 375, 604

(4), 602-607. 605

(38) Thannickal, V. J.; Fanburg, B. L. Reactive oxygen species in cell signaling. Am. J. Physiol-606

Lung. C. 2000, 279, (6), L1005-L1028. 607

(39) Dudala, J.; Janeczko, K.; Setkowicz, Z.; Eichert, D.; Chwiej, J. The use of SR-FTIR 608

microspectroscopy for a preliminary biochemical study of the rat hippocampal formation 609

tissue in case of pilocarpine induced epilepsy and neutroprotection with FK-506. Nukleonika 610

2012, 57, (4), 615-619. 611

(40) Berlett, B. S.; Stadtman, E. R. Protein oxidation in aging, disease, and oxidative stress. J. Biol. 612

Chem. 1997, 272, (33), 20313-20316. 613

(41) Whelan, D. R.; Bambery, K. R.; Heraud, P.; Tobin, M. J.; Diem, M.; McNaughton, D.; Wood, 614

B. R. Monitoring the reversible B to A-like transition of DNA in eukaryotic cells using 615

Fourier transform infrared spectroscopy. Nucleic. Acids. Res. 2011, 39, (13), 5439-5448. 616

(42) Vaccari, L.; Birarda, G.; Businaro, L.; Pacor, S.; Grenci, G. Infrared Microspectroscopy of 617

Live Cells in Microfluidic Devices (MD-IRMS): Toward a Powerful Label-Free Cell-Based 618

Assay. Anal. Chem. 2012, 84, (11), 4768-4775. 619

(43) Dovbeshko, G. I.; Gridina, N. Y.; Kruglova, E. B. Pashchuk, O. P., FTIR spectroscopy studies 620

of nucleic acid damage. Talanta. 2000, 53, (1), 233-246. 621

(44) Lee, K. S.; Bumbaca, D.; Kosman, J.; Setlow, P.; Jedrzejas, M. J. Structure of a protein-DNA 622

complex essential for DNA protection in spores of Bacillus species. P .Natl. Acad. Sci. USA 623

2008, 105, (8), 2806-2811. 624

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Effect of Ingested Tungsten Oxide (WO x ) Nanofibers on Digestive Gland Tissue of Porcellio scaber...

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