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Vibrational Spectroscopy 53 (2010) 39–44

Contents lists available at ScienceDirect

Vibrational Spectroscopy

journa l homepage: www.e lsev ier .com/ locate /v ibspec

ynchrotron radiation infrared microspectroscopy of arsenic-induced changes tontracellular biomolecules in live leukemia cells

ristie L. Munroa, Keith R. Bamberyb, Elizabeth A. Carterc, Ljiljana Puskard, Mark J. Tobind,ayden R. Woodb, Carolyn T. Dillona,∗

Centre for Medicinal Chemistry, School of Chemistry, Building 18, University of Wollongong, Wollongong, NSW 2522, AustraliaCentre for Biospectroscopy, School of Chemistry, Monash University, Vic 3800, AustraliaVibrational Spectroscopy Facility, School of Chemistry, The University of Sydney, NSW 2006, AustraliaAustralian Synchrotron, 800 Blackburn Road, Clayton, Vic 3168, Australia

r t i c l e i n f o

rticle history:eceived 3 September 2009eceived in revised form 10 February 2010ccepted 11 February 2010vailable online 19 February 2010

eywords:rseniceukemiaynchrotronTIR

a b s t r a c t

Arsenic trioxide, marketed as TrisenoxTM, successfully cures 60–85% of relapsed acute promyelocyticleukemia sufferers. However, the mechanisms of action remain unclear. In this work, SR-FTIR microspec-troscopy of live HL60 cells was used to monitor biomolecular changes that occur during exposure toarsenite (100 �M) over a period of 2 h. Importantly, the design of the sample holder enabled the collec-tion of high quality spectra that were not dominated by the ı(OH) mode of water. Significant spectraldifferences in the live treated cells were observed within 40 min after exposure to the drug. In particular,there was a decrease in the �(C O) band at 1742 cm−1 in the spectra of arsenite-treated cells. This isconsistent with initial damage to the membrane that leads to later loss of membrane integrity. In addi-tion, there was an initial (40–60 min) decrease in the intensities of the �s(PO2

−) and �as(PO2−) bands

which was attributed to IR “opaqueness” associated with chromatin condensation. In later stage apopto-

icrospectroscopy

ive cellssis (100–120 min) there was an increase in the intensity of these bands which was consistent with DNAfragmentation. The most significant differences (as determined by the second derivative spectra and PCAplots) were observed in the amide I band where the band was centred at 1639 cm−1 (�-sheet) in thecontrol cells and 1650 cm−1 (�-helix) in the arsenite-treated cells. The results of this study indicate thatthe mechanisms of action of arsenite-induced toxicity include alterations to the protein structure andinteractions with DNA. Further studies will be performed to verify whether these effects also occur at

entra

therapeutic arsenite conc

. Introduction

Arsenic possesses a complex toxicology with severe, acute,hronic and carcinogenic behaviour [1]. Ironically, these toxic prop-rties are pivotal for the success of As therapeutics. For instance,s2O3, marketed as TrisenoxTM, is an anti-leukemia agent for the

reatment of relapsed acute promyelocytic leukemia (APL) [2].

he successful cure rate of 60–85% of patients who had previ-usly relapsed after conventional therapy, has generated interestn As2O3 as a treatment for other forms of cancer [2]. New As com-ounds are also being explored as future therapeutics. To date As

Abbreviations: APL, acute promyelocytic leukemia; FBS, foetal bovine serum;MDM, Iscove’s modified Dulbecco’s media; PBS, phosphate buffered saline; PCA,rincipal component analysis; P/S, penicillin/streptomycin; SR-FTIR, synchrotronadiation-Fourier transform infrared.∗ Corresponding author. Tel.: +61 2 42214930; fax: +61 2 42214287.

E-mail address: carolynd@uow.edu.au (C.T. Dillon).

924-2031/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.vibspec.2010.02.004

tions (5–10 �M).© 2010 Elsevier B.V. All rights reserved.

has been described as a missile with multiple warheads since ithas been shown that As can interact with numerous biomoleculesincluding: DNA, tubulin and mitochondrial proteins [3]; however,determining the critical targets for As-induced toxicity towardscancer cells is a necessary precursor to the successful developmentof this class of drugs.

Fourier transform infrared (FTIR) microspectroscopy is anexcellent technique for studying changes in the biomoleculesassociated with biological tissue and cells. Typical spectral featuresinclude: amide I (�(C O) ∼1650 cm−1) and amide II (�(CN)/�(NH)∼1549 cm−1) bands representative of proteins; symmetric(∼1080 cm−1) and anti-symmetric PO2

− (∼1232 cm−1) bandsassociated with DNA; C–H stretches of �as(CH3) (∼2955 cm−1)and �as(CH2) groups (∼2850 cm−1), the ester band �(C O)ester

(1740 cm−1) and the C–O–P (∼1080 cm−1) band associatedwith lipids/phospholipids; and C–O stretches (1050 cm−1) ofcarbohydrates [4–7]. One of the most important features ofIR spectroscopy is the ability to detect changes in the averageprotein secondary structure by analysing the components of the

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mide I band; specific secondary structural assignments include:-helices (1662–1645 cm−1), �-sheets (1637–1613 cm−1), turns

1682–1662 cm−1) and random coils (1645–1637 cm−1) [4,5,8–10].onsequently, the technique is gaining prominence as a tool capa-le of diagnosing diseased tissue and monitoring apoptosis5,7,10,11]. Of particular interest to the area of drug developmentnd design is the implementation of IR spectroscopy to monitornd assess the biomolecular responses to chemotherapeutic agents12]. Sule-Suso et al. [12] recently reported the use of SR-FTIR

icrospectroscopy to assess the response of human lung cancerells to the anti-cancer agent, gemcitabine. Significant changesere observed in the IR spectral region between 1150 cm−1 and

50 cm−1 following treatment with gemcitabine. In particular,here was an increase in the peak at 1080 cm−1 and an increase inhe ratio of the peaks at 1080:1050 cm−1 which were attributedo the symmetric vibrations of PO2 groups and the C–O stretchesf carbohydrates, respectively [12]. IR spectroscopy has alsoeen used to successfully monitor apoptosis in leukemia cellshereby increases in the lipid content and decreases in cellularNA were detected. In addition, contributions to the amide I bands

howed secondary structure shifts from a predominantly �-sheeto unordered protein [13].

Synchrotron radiation (SR) used in combination with FTIRicrospectroscopy provides an intense light source (up to 1000×

righter than a globar source) that improves the signal-to-noise atigh spatial resolution. This is particularly advantageous in record-

ng IR spectra of single cells where high spatial resolution and,herefore, smaller apertures are essential. Since the size of theocused synchrotron beam is of the order of the cell size it sig-ificantly improves the quality of the spectra that can be acquirednd reduces the collection time [7,14]. Although an experimentalhallenge, live cell analysis under aqueous media is desirable ast removes the potential for spectral artefacts resulting from fixa-ion procedures and provides the added advantage of allowing realime analyses. As such SR-FTIR microspectroscopy was used for therst time to analyse live leukemia cells following their exposure torsenite, the physiological (bioavailable) form of TrisenoxTM. Theurpose of the study was to distinguish and identify differences

n the intracellular biomolecules (DNA, proteins and membraneipids) associated with arsenite treatment of leukemia cells in ordero identify the targets of As-induced toxicity.

. Experimental

Human APL cells (HL60) were obtained from the ATCC and wererown in 75 cm2 cell culture flasks (Interpath Services) containingrowth medium which consisted of Iscove’s modified Dulbecco’sedia (IMDM, Invitrogen) supplemented with foetal bovine serum

FBS, 20%, In Vitro Corporation) and penicillin/streptomycin (P/S,00 U/mL, 100 �g/mL, respectively, In Vitro Corporation) in a 37 ◦C,% CO2, humidified incubator. After reaching confluency, the cellsere transferred to 15 mL centrifuge tubes (Interpath Services) and

entrifuged at 1000 r.p.m. for 5 min. The cells were then washedn sterile phosphate buffered saline (PBS) and IMDM+P/S (2 mL,

ithout FBS) was added to the centrifuge tubes. Freshly preparedrsenite solution (sodium arsenite, Sigma–Aldrich, 99%) was thendded to produce a final As concentration of 100 �M. This concen-ration was chosen since it caused notable toxicity (IC20) in the MTTssessment of arsenite following a 4-h exposure period (Supportingata, Fig. S1).

IR spectra of live cells were collected using a demountable liquidell comprising a 1-mm thick CaF2 window onto which a spacer hadeen patterned by polymer spin coating and UV photolithography,nd an upper 0.5-mm thick CaF2 window [15]. The two windowsere held tightly together in a Thermo micro compression cell. To

ctroscopy 53 (2010) 39–44

load the sample into the liquid cell, the resultant cell suspension(1 �L) was immediately deposited onto the lower spacer-bearingcalcium fluoride disk, and the upper window was placed on topof the sample before closing the compression cell [15]. Excess liq-uid was expressed through a gap in the microfabricated spacer [15].Spectra were obtained for ∼20 individual cells after treatment peri-ods of 40 min, 60 min, 100 min and 120 min with arsenite (100 �M).Separate calcium fluoride disks were used for HL60 control cells andarsenite-treated cells.

The samples were analysed using a Bruker Vertex V80 v Fouriertransform infrared (FTIR) spectrometer coupled with a Hype-rion 2000 microscope equipped with a liquid nitrogen coolednarrow-band MCT detector at the Australian Synchrotron infraredbeamline. Samples were analysed in transmission mode using a7 �m × 7 �m aperture with 128 co-added sample scans per cell and256 co-added background scans (after every 6 cells) using OPUS 6.5software. Background spectra were obtained from an area (contain-ing IMDM) inside the sample holder which was free from cells andcellular material.

Principal component analysis (PCA) was performed usingUnscrambler (Version 9.8, Camo, Norway), on all raw spectra thatwere firstly derivatized (second) (Savitzky–Golay algorithm with13 smoothing points) and then unit vector normalised. The PCAresidual X-variance versus leverage plot for all the spectra wasobtained (Supporting Data, Fig. S2) and revealed some outlierswhich were found to have significant dispersion and Mie scat-tering contributions. These were removed from the resultant datasets. The resultant spectra for each treatment were averaged usingOPUS 6.5 and were presented as raw (no baseline correction) spec-tra. The spectra were plotted between 3000 cm−1 and 1000 cm−1

due to the presence of water in the aqueous media resulting in alarge OH band above 3000 cm−1 which overshadowed many of theother important and relevant peaks. Below 1000 cm−1 the spectralfeatures were difficult to interpret due to the noise. PCA was alsoperformed on spectral regions to identify any significant variationbetween the data sets.

2.1. Trypan blue exclusion assay

HL60 cells were seeded (5 × 105 cells) into 60 mm Petri dishes(Bacto Laboratories) containing growth medium (5 mL) and incu-bated for 24 h at 37 ◦C, 5% CO2 in a humidified incubator(Revco Ultima, USA). Following the incubation period the cellswere centrifuged at 1000 r.p.m. for 5 min, the supernatant wasremoved, and the cells were resuspended in IMDM+P/S (controlcells) or IMDM+P/S containing 100 �M sodium arsenite (treatedcells).

Following the exposure period (0 min, 40 min, 60 min, 100 min,or 120 min) the cells were washed twice in sterile PBS (1 mL) andwere then resuspended in fresh growth media (1 mL) to maintaincell integrity. The cell solution (50 �L) was added to trypan bluesolution (50 �L, 0.5% in PBS, Thermo Electron Corporation) and thecells were scored after 5 min using a haemocytometer. Each assaywas performed in triplicate. The number of normal cells (those withintact membranes that contained no stain) and membrane com-promised cells (those that did not exclude trypan blue) was plottedas the mean and standard deviation for each time point. Statisticalanalysis of the data was performed using the ANOVA Tukey–Kramermethod whereby a value of p < 0.05 (p = probability) was consideredsignificant [16].

3. Results

Fig. 1 shows the averaged raw spectra obtained from live HL60cells that were untreated (40 min) or treated with arsenite (100 �M,

K.L. Munro et al. / Vibrational Spe

Fig. 1. SR-FTIR averaged raw spectra from live HL60 control cells 40 min after intro-duction into the sample holder, n = 19 (–); and HL60 cells treated with sodiumarsenite (100 �M) for 40 min, n = 20 ( ); 60 min, n = 20 ( );100 min, n = 18 ( ); and 120 min, n = 15 ( ). The inset shows thenov

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between the lipid bands and the protein/phosphodiester bands inarsenite-treated HL60 cells. PCA revealed no significant differencebetween the spectra obtained from HL60 cells treated with arsenitefollowing different treatment periods.

Fig. 2. Second derivative SR-FTIR spectra (1800–1000 cm−1, 13 smoothing points)

ormalisation of the spectra over the region (1750–1450 cm−1). (For interpretationf the references to color in this figure legend, the reader is referred to the webersion of the article.)

0–120 min). There is evidence of an undulating baseline in all thepectra obtained from the HL60 live cells which is attributed toie scattering and a dispersion artefact [17,18]. However, common

iomolecular markers, such as the amide I (∼1650–1639 cm−1) andmide II (∼1545–1539 cm−1) bands (representative of proteins),he anti-symmetric (∼1225 cm−1) and symmetric (∼1084 cm−1)O2

− bands (associated with DNA) and the carbonyl ester band1742 cm−1, associated with lipids) are clearly discernable in thepectra [4–7]. The most notable feature of the spectra is the similarntensities of the amide I and amide II bands (Fig. 1, inset) whichontrasts that seen in fixed cells whereby the amide I band is com-only more intense (approximately 1.5–2×) than the amide II band

7,14,19]. Close examination of the spectrum from the control sam-les versus the spectra obtained from the arsenite exposed cellsver the time course shows differences in the relative intensitiesf a number of peaks. For instance, the intensity of the �as(PO2

−)nd �s(PO2

−) bands at 1225 cm−1 and 1084 cm−1, respectively,ecreased in HL60 cells treated with 100 �M sodium arsenite solu-ion (compared to HL60 control cells). In arsenite-treated HL60ells the intensity of the �as(PO2

−) and �s(PO2−) increases in cells

reated for 100–120 min (compared to cells treated for 40–60 min).ig. 1(inset) reveals a decrease in the intensity of the �(C O)ester

1742 cm−1) band (often associated with phospholipids) in HL60ells treated with sodium arsenite (40–120 min). This suggests thathere is a decrease in the amount of observable lipids in arsenite-reated HL60 cells.

Fig. 1(inset) also shows a decrease in the intensity of the amideI band with respect to the amide I band following treatment withodium arsenite (100 �M). In addition, the position of the amide IIand shifts from 1539 cm−1 in HL60 control cells to 1545 cm−1 inrsenite-treated HL60 cells (Fig. 1) over the 2-h period.

The second derivative spectra (Fig. 2) confirm that the amideband has shifted in the spectra of the HL60 cells treated with00 �M sodium arsenite (40–120 min) compared to control HL60ells. The amide I band in HL60 control cells is centred around639 cm−1, whereas the amide I band for HL60 cells treated withodium arsenite (40–120 min) is centred around 1650 cm−1. Themide I band for HL60 control cells is far broader than what is

bserved for arsenite-treated HL60 cells indicating that it is poten-ially comprised of more than one band. This would indicate thathe predominant protein structure in HL60 control cells is �-sheet,hereas the predominant protein structure in HL60 cells treated

ctroscopy 53 (2010) 39–44 41

with sodium arsenite is �-helix and/or random coil. This change isevident in spectra obtained from HL60 cells after the 40 min treat-ment period indicating that sodium arsenite has a rapid affect onthe secondary structure of proteins within HL60 cells. Similarly, theposition of the amide II band shifts from 1545 cm−1 to 1547 cm−1

and there is a loss of the shoulder at the lower wavenumber(∼1517 cm−1) in arsenite-treated cells.

Significant changes are observed in the lineshape, intensity andposition of the �as(PO2

−) band. A significant reduction in intensityis observed in the second derivative spectra of the arsenite-treatedcells compared to the control cells and there is a shift in the bandto ∼1220 cm−1 from 1215 cm−1. The shoulder at 1237 cm−1 in thecontrol cells is more resolved and distinct in the second derivativespectra of the treated cells.

PCA (Fig. 3(a)–(c)) revealed that significant variance existsbetween the spectra obtained from HL60 control cells and thosetreated with sodium arsenite (100 �M, 40–120 min) for a numberof important biomolecules including proteins, lipids, and nucleicacids. The variance was attributed spectral regions containing theamide I and II bands, as well as the �(C O)ester, �s(PO2

−) and�as(PO2

−) bands. PCA of the �(C O)ester (1758–1671 cm−1) region(Fig. 3(a)) shows strong separation along PC1 for HL60 control cellsversus arsenite-treated cells. The spectra obtained from arsenite-treated HL60 cells are associated with positive scores in the scoresplot. This indicates that they are associated with negative loadingsas the PCA was performed on second derivative spectra. The nega-tive loadings correspond to the ester carbonyl band (1742 cm−1)and the band associated with DNA base pairing (1715 cm−1).Fig. 3(b) shows the results obtained from the PCA performed onthe phosphodiester region of the spectrum (1293–1000 cm−1). Theresults show a strong separation along PC1 but unlike Fig. 3(a) thespectra obtained from arsenite-treated cells are associated withnegative scores and hence positive loadings. The positive load-ings for Fig. 3(b) are associated with the �as(PO2

−) and �s(PO2−)

bands. The same trends are seen in Fig. 3(c) whereby the spec-tra obtained from arsenite-treated HL60 cells also associated withpositive scores and hence negative loadings. The negative loadingsare associated with the amide I, amide II, �as(PO2

−) and �s(PO2−)

bands and the positive loadings are associated with the ester car-bonyl band. This would indicate that there is an opposite correlation

obtained from HL60 live control cells 40 min after introduction into the sampleholder, n = 19 (–) and HL60 live cells treated with sodium arsenite (100 �M) for40 min, n = 20 ( ); 60 min, n = 20 ( ); 100 min, n = 18 (); and 120 min, n = 15 ( ). (For interpretation of the references to color inthis figure legend, the reader is referred to the web version of the article.)

42 K.L. Munro et al. / Vibrational Spectroscopy 53 (2010) 39–44

F b) anti-symmetric and symmetric phosphate stretches (1293–1000 cm−1) and (c) amider ith sodium arsenite (100 �M, 40–120 min) (B). PCA was performed on second derivative(

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Fig. 4. Trypan blue exclusion assay of HL60 cells treated with sodium arsenite

ig. 3. PCA scores plot and PC1 X-loadings for (a) �(C O)ester (1758–1671 cm−1), (egion (1671–1471 cm−1) of HL60 control cells (40 min) (A) and HL60 cells treated wSavitzky Golay, 13 smoothing points), unit vector normalised spectra.

.1. Trypan blue exclusion assay

The results obtained using the trypan blue exclusion assay tossess the condition of the arsenite-treated HL60 cells for the dura-ion (40–120 min) of the IR assay are shown in Fig. 4. The populationf cells drops off substantially from that of the control following the0-min arsenite exposure. However, due to the variability in theell populations, significant differences in cell populations are notbserved until 100- and 120-min exposure periods. While there isore than a doubling in the number of cells that fail to exclude

rypan blue for the treated cells (40–120 min) compared with theontrol cells, this value is not significant (p > 0.05) due to the vari-tion in the cell populations.

. Discussion

Live leukemia (HL60) cells were studied in an effort to anal-se cells in an environment which was identical to the conditionsn which they were grown. The main advantage of this is that itvoids the incorporation of spectral artefacts that may be associated

(100 �M) for 0 min, 40 min, 60 min, 100 min and 120 min. The columns representthe mean of triplicate measurements and the error bars represent the standarddeviation from the mean. Cells with intact membranes are represented by whitecolumns (red error bars) and those with compromised membranes are representedby blue columns (blue error bars). (For interpretation of the references to color inthis figure legend, the reader is referred to the web version of the article.)

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ith fixation or other sample preparation procedures. In addition,ive cell spectroscopic analysis enables a real time study of theiomolecular changes associated with As exposure. Importantly,owever, cell hydration and the surrounding solution can markedlyffect the resultant spectra [6]. For instance, the ı(OH) mode ofater at 1645 cm−1 is found within the same spectral region as the

mide bands and contributes to the intensities of these bands [6].n addition, the amide I carbonyl group possesses two lone pairs oflectrons, and is a likely candidate for H-bonding. This can causeshift in the position of the amide band. Consequently, a sampleolder that enables collection of spectra that are a true representa-ion of the cell but at the same time prevents spectral dominationrom the ı(OH) water band, is important. The quality of the spectran Fig. 1 shows that this has been achieved to some extent usinghe sample holder described [15].

An important difference in these spectra versus those obtainedrom fixed HL60 cells is the similar intensities of the amide I andmide II bands in the live cells; the amide I band is normally approx-mately double the amide II band in fixed cells. Moss et al. [20] alsoeported near-equal intensities of the amide I and II bands in liveell spectra. This has been attributed to the fact that the cell freeegion used contains more water than the region within a cell, lead-ng to overcompensation of the water band at 1645 cm−1 [20,21].

hile mechanisms of correcting for this have been reported (i.e.he qualitative addition of a pure water spectrum to achieve a flataseline in the spectral region between 3100 cm−1 and 3000 cm−1),

t is apparent that this is unreliable in microscopic samples [21]. Itas concluded by Moss et al. [20] that as long as the same methodas used throughout the analysis, this would not void the data

omparison.The undulating baseline observed for the HL60 control spectra

s characteristic of Mie scattering which results in the observedinusoidal oscillation in the baseline of the spectrum and is dueo dielectric spheres which are known to scatter electromagneticadiation if the wavelength of the light is comparable to the sizef the sphere [18,22]. In this instance the cells are approximately0 �m diameter and contain within them an even smaller nucleus6–8 �m) which makes them an ideal (and unavoidable) candidateor this phenomenon. An additional artefact that can contribute tohe baseline is the ‘dispersion artefact’ described by Gardner and co-orkers [18]. This is believed to result from scattering by the edges

f the cells; although in order to reduce this effect, the aperture sizeas confined to 7 �m in these studies.

Nonetheless, careful inspection of the raw spectra (Fig. 1) andecond derivative spectra (Fig. 2) reveals important spectral fea-ures that can be used to identify the biomolecules in the cellsnd discern differences between the sample types. The spectralifferences observed for the control and arsenite-treated cells areonsistent with the fact that there is also a significant decreaseFig. 4) in the number of viable cells over the exposure period.

Firstly, the spectral band observed at 1715 cm−1, is characteris-ic of leukemia cells and is believed to be a base-pairing marker ofNA [5]. The decrease in the intensity of this band in the spectraf arsenite-treated HL60 cells indicates a decrease in the amountf detectable DNA. This is consistent with DNA cleavage follow-ng the initiation of apoptosis. There is some debate regardinghe effects of apoptosis on the intensity of the bands associatedith nucleic acids. Studies by Zhou et al. [11] have shown that the

ntensity of the IR bands assigned to nucleic acids increases dur-ng apoptosis while studies by Liu et al. [13] have shown that thentensity of the IR bands assigned to nucleic acids decreases. The

hange in the intensity of the bands associated with nucleic acidsbserved in this study may be representative of the progressionf apoptosis whereby cells exhibiting nuclear condensation showands of lower intensity due to low transmission in the infraredf highly condensed areas [22,23]. Non-linear infrared absorption

ctroscopy 53 (2010) 39–44 43

behaviour of the nucleus in a pyknotic (contracted) state associatedwith increased condensation of DNA has previously been reported[22]. The very high local concentration of DNA is believed to impartan opaqueness of the chromatin, and consequently the absence ofDNA in very small nuclei [22]. The small size and the density ofthe chromatin particles have been implicated in explaining thisphenomenon [22]. Cells in later stages of apoptosis after fragmen-tation of the DNA have been shown to exhibit phosphate bandsof higher intensity [23]. These reports [22,23] are consistent withthe results of this study, whereby the intensity of the phosphatebands decreased after the 40–60 min treatment period and thensubsequently increased after a 100–120 min treatment period. Asis expected following arsenite exposure, the spectral results arenot consistent with necrosis (versus apoptosis) which is associatedwith a unique absorption band at 1621 cm−1 [10] and an increasein the intensity of the band at 1740 cm−1 [23].

The decrease in the intensity of the �(C O)ester (1741 cm−1)band which is often attributed to phospholipids (Fig. 1(inset)) maybe indicative of initiation in loss of membrane integrity which isapparent by the increased number of cells that took up trypan blue(Fig. 4) following treatment with sodium arsenite (40–120 min).

Finally, the most convincing spectral differences were observedin the contributions to the amide I band providing clear evidenceof protein secondary structural changes. These results are also con-sistent with FTIR microspectroscopy studies of the time dependentexposure of HepG2 cells to arsenite and suggest the probability ofdirect interaction of As with intracellular proteins [24]. The resultis also consistent with our earlier work [25] whereby XAS stud-ies indicated that intracellular As was predominantly Tris–sulphurbound and that this was likely to be protein-bound following treat-ment with 100 �M arsenite (not oxygen- or nitrogen-bound aswould be expected following predominance of As–DNA interac-tions). The results in Figs. 1 and 2 suggest that arsenite treatmentresults in the loss of the predominance of the �-sheet structure[5,8]. The dominance of the contribution at 1650 cm−1 could beattributed to an increase in the relative amount of �-helical proteinconformation [5,8]. Liu and Mantsch [26] reported that apoptoticleukemia cells also exhibit a change in the protein secondary struc-ture from predominantly �-sheet in CEM (T-lymphoblastic cells)control cells to more random coils proteins structures in CEM cellstreated with etoposide [13,26]. Studies by Jamin et al. [27] foundthat in both early and late stage apoptosis the percentage of helicalprotein increases in relation to the percentage of �-sheet protein.

5. Conclusions

In this work, the restriction of aqueous solution in the sam-ple holder facilitated a reduction of the dominance of the OHbands in the resultant SR-FTIR spectra. While baseline phenomena,were observed in the control spectra reasonable quality spectrawere obtained that showed distinct differences between the con-trol and time dependent arsenite-treated cells. Of major note werethe differences in the position of the characteristic amide I bandthat shifted from 1639 cm−1, indicative of proteins possessing apredominate �-sheet structure (control cells), to 1650 cm−1 (inarsenite-treated cells) which reflects a decrease in the relative �-sheet structure and dominance of the �-helical structure. Theseresults show a clear impact on the protein structure followingarsenite treatment, and are consistent with a number of studiesthat show that As interacts readily with proteins [3,25,28,29]. In

addition, there was evidence of changes to DNA (condensation andfragmentation) which is consistent with reports that show thatarsenite causes DNA strand breaks at higher concentrations [30].The successful collection of spectra from live cells is highly valu-able for monitoring the effectiveness of anti-leukemia agents such

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s TrisenoxTM, and the ability to distinguish differences betweenontrol cells and arsenite-treated cells highlights the validity of per-orming further studies at therapeutic doses of arsenite (5–10 �M).

cknowledgements

This research was undertaken on the infrared beamline at theustralian Synchrotron, Victoria, Australia. K.L.M. acknowledges

an and Jean Simpson and Australian Rotary Health for her Ph.D.cholarship. C.T.D. thanks the University of Wollongong Small Grantcheme for financial support.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.vibspec.2010.02.004.

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