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Evidences of DMSO-Induced Protein Aggregation in Cells Alessandra Giugliarelli, Lorena Urbanelli, Maria Ricci, Marco Paolantoni, Carla Emiliani, Riccardo Saccardi, Benedetta Mazzanti, Letizia Lombardini, Assunta Morresi, and Paola Sassi J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.6b00178 • Publication Date (Web): 09 Feb 2016 Downloaded from http://pubs.acs.org on February 15, 2016

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The Journal of Physical Chemistry

Evidences of DMSO-Induced Protein Aggregation in Cells

A. Giugliarelli1, L. Urbanelli1, M. Ricci1,2, M. Paolantoni1 C. Emiliani1, R. Saccardi3, B. Mazzanti3, L. Lombardini3, A. Morresi1, P. Sassi1* 1. Dipartimento di Chimica, Biologia e Biotecnologie, Università di Perugia, Via Elce di sotto 8, 06123 Perugia, Italy 2. Istituto di Biofisica CNR (IBF-CNR), Unità di Trento, & FBK, Via Sommarive 18, 38123 Trento, Italy 3. Banca del Sangue Cordonale, Azienda Ospedaliera Universitaria Careggi, Firenze

Abstract We report on a study of protein aggregation induced on different cell samples by dimethylsulfoxide (DMSO) addition. DMSO is the most commonly used cryoprotectant because it is supposed to readily diffuse across lipid bilayers thus reducing water activity within cells; despite its large use, the mechanism of penetration and even the main interaction features with cell components are far from being understood. In the present work, infrared absorption spectroscopy is successfully applied to real time detection of chemical and structural changes occurring in cells during dehydration from water and water/DMSO suspensions. As a most interesting result, DMSO is observed to favor protein aggregation both in cellular model systems, as cultured lymphocytes and fibroblasts, and in human samples for clinic use, as hematopoietic stem cells from cord blood. This effect is evidenced at low water content, analogously to what is observed for protein solutions. Such tendency is not specific of the type of protein and suggests one possible origin of DMSO toxicity .

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*corresponding author: Paola Sassi Dipartimento di Chimica Biologia e Biotecnologie Università degli Studi di Perugia, via Elce di sotto 8 06123 Perugia, ITALY phone: +39 075 5855585 fax: +39 075 5855586


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1.1 Introduction Hydration is critical to living organism whose health is guaranteed by proper levels of tissue and cellular fluids and electrolytes. The role of water is crucial to the function of the cell in the intracellular environment, which is a complex, crowded mixture of many different species. In this mixture, macromolecules occupy a great part of the cell’s volume, at concentrations sometimes exceeding 300 g/L 1. For many years this crowded environment was thought to stabilize globular proteins and to guide their folding. The deep comprehension of causes and mechanisms of protein folding is probably one of the most fascinating challenges in molecular biology; it is the result of a millenary evolution route, with most aspects still unknown2. Recent studies suggest that crowding in living cells can also destabilize globular proteins, and, if a stress is induced on the cellular environment, protein misfolding can easily occur3-6. We can define misfolding as the process leading to a spatial organization of proteins different from the native configuration, due to not native-like interactions within and between the different local domains: the result can be the final formation of highly oriented and generally insoluble aggregates, usually defined as amyloid fibrils or plaques7. The intra or extra-cellular accumulation of such species can be dangerous to the function of different organs (i. e. brain skeletal muscle, liver); in addition, the presence of misfolded arrangements means that reduced quantities of native protein are available to their normal functional role. A wide literature refers to amyloid fibrils as connected to pathological conditions7-9; at the same time, it seems confirmed the idea that pre-fibrillar forms (protofibrils or protofilaments) or in general precursors of fibrillar species, are the real pathogenic species10-12. Also some genetic evidence seems to support this view13. In some cases, bigger aggregates as mature amyloid fibrils, have a lower toxicity and even seems to have a protective role in living organisms14,15.



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In the evolution of some fibril-connected diseases the role of dimension and structure of fibrils and of their precursors clearly emerges from experimental data. For example, fundamental differences have been recently found on the role of large and small aggregates of amyloid-β peptide, regarding autophagy and cell death in Alzheimer disease16. From the point of view of basic research, to monitor the very initial steps of protein aggregation is useful to individuate environmental conditions favoring precursors’ formation such as low pH, high temperature and possible presence of denaturant species17. In our laboratory we explored the thermal unfolding and aggregation processes of a simple globular protein as hen egg white lysozyme, dissolved in water, ethanol-water and DMSO-water mixtures by FTIR absorption spectroscopy17-22. We followed the characteristic FTIR absorption of the amide I stretching modes and particularly those at 1618 and 1690 cm-1 assigned to intermolecular β-sheet arrangements. Our studies confirmed the role of DMSO as a denaturant, likely due to the reduction of hydrophobic effect, reducing the thermal stability of the native state of the protein18,21. DMSO is a very important compound, due to its almost irreplaceable role as cryoprotectant in biobanking of cells and tissues, especially of human origin23,24. Despite its wide and consolidated use in most diffused cryopreservation protocols, its cytotoxicity is also well known, together with some dangerous adverse events on human beings treated with DMSO-cryopreserved biological samples25,26. In the present work we used FTIR spectroscopy to explore the aggregation of proteins inside some cellular systems, both cultured and clinical samples. In particular, we examined cord blood samples, following their dehydration, and compared the obtained results with those collected from cultured (fibroblast and lymphocytes) cellular lines, considered as models. We applied such alteration of the cellular environment to enhance the effects of sulfoxide-protein interaction; this should help clarifying the origin of DMSO cytotoxicity. Moreover, cell dehydration always occurs in cryopreservation procedures due to the formation of ice; this


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causes an increase of both protein and DMSO concentration inside the cell. High concentrations of DMSO are also used in cosmetics and to relief from chemotherapy and bones pain24,27. We observed the formation of pre-fibrillar aggregates during dehydration of cell samples only in the presence of DMSO. The experimental data evidenced the efficiency of this spectroscopic approach to reveal a protein self-aggregation phenomenon in cells. As a comparison, we also followed the effects of dehydration for two globular protein solutions and evidenced the formation of ordered aggregates only in the presence of DMSO, with a tendency that is not specific of the type of protein.

1.2 Materials and methods 1.2.1 Blood samples for clinical use Cord blood samples were prepared at the Florence Cord Blood Bank according to the standard procedure for cord blood volume reduction and cryopreservation28. Fresh cord blood units have been processed in an automated closed system (Sepax®, Biosafe, Eysin, Switzerland) to Red Blood Cells (RBC) removal by sedimentation in presence of HES (Hydroxyethyl starch). The sample was transferred in a cryovial with the cryopreservative agent (CPA) and stored in liquid nitrogen until further use. The product was cryopreserved by the slow injection of Cryosure solution –(WAK-Chemie Medical GmbH) as CPA, containing water for injectable use, Dextran and DMSO having a concentration corresponding to 10% v/v. Cells are preserved by slow-cooling using an automated programmed controlled rate freezer (slowfreezing). Before IR analysis, the sample was thawed in a thermal bath at 37 °C and a drop of cord blood was immediately analyzed in order to follow the behavior of the sample just after the thawing process. The dehydration of the sample was monitored for 3 hours. 5 ACS Paragon Plus Environment


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1.2.2 Cultured Cells HuDe (Human Dermal) adherent fibroblasts were purchased from the Istituto Zooprofilattico Sperimentale, Brescia (Italy) and cultured in DMEM (Dulbecco’s modified eagle medium) supplemented with 10% (v/v) heat inactivated fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, 100 U/ml streptomycin at 37°C in a humidified atmosphere containing 5% (v/v) CO2. Cell growth was determined by cell counting. Jurkat T lymphocytes were purchased from ATCC and cultured in RPMI medium (Roswell Park Memorial Institute) supplemented with 10% (v/v) heat inactivated fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% (v/v) CO2. Cell growth was determined by cell counting. Before each experiments, 10x106 HuDe cells (detached by trypsinization) and 70x106 (Jurkat) cells were pelleted and washed three times by centrifugation cycles at 800 g for 10 min in PBS (Phosphate Buffered Saline) solution. Then, the pellets were incubated with 500 µL of PBS or with 500 µL of PBS/DMSO 10% v/v solution for 10 minutes. The incubation time allows the equilibration between cells and solvents. After incubation, the solutions were centrifuged at 800 g for 5 minutes in order to obtain the fresh pellets for IR experiments. The pellets were immediately measured by deposing a drop on a ZnSe ATR crystal. 1.2.3 Protein solutions Hen egg white lysozyme and bovine serum albumin were purchased from Sigma Aldrich and used without further purification. 20 mg/ml solutions were prepared by dissolving the protein in water and DMSO/water (0.1 DMSO mole fraction) solutions at room temperature. 1.2.4 ATR-FTIR experiments One drop of the sample was distributed on the ATR29 crystal and let dry to air. ATR-FTIR spectra were acquired during the dehydration process by collecting spectra at different times. ATR-FTIR spectra were collected with a Bruker Tensor27 IR spectrometer equipped with the


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ATR stage (Specac Ins. Mod. Gateway) and the ZnSe crystal. Each spectrum is the average of 10 scans at a resolution of 2 cm-1. Spectra were acquired during the dehydration of the sample by collecting data every 2 minutes for the first 30 minutes and then every 10 minutes for about 3 hours. Reproducibility was evaluated by triplicate dehydration scans. Spectra of the dried samples are shown after normalization on the combination band of water at 2200 cm-1 ca. and subtraction of the solvent spectrum.

1.3 Results and Discussion 1.3.1 Cord Blood analysis

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As described in the experimental section, fresh cord blood units were added to CPA solution, stored in liquid nitrogen and then thawed in a thermal bath at 37 °C before IR measurements. Fig. 1 shows the low and high frequency regions of IR spectra of cord blood registered at different times during dehydration. These spectral regions contain signals associated to the different components of the cell and of DMSO present in the Cryosure solution; for further



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details on vibrational assignment of IR absorptions see Table 1S of the Supporting Information. In particular, the absorption at about 1650 cm-1 is assigned to amide I band of protein species. Amide I band is a sensitive probe of protein denaturation processes17-22; it is partially overlapped to the profile of HOH bending signal of water at 1640 cm-1 ca. The loss of water content in the sample can be monitored through the analysis of OH stretching signal at 3000 – 3800 cm-1 (see Fig.1b); a strong intensity decrease can be detected in the first hour. On the contrary, an intensification can be observed for all the other bands: they are associated to vibrations of the cellular components and the CPA (Figs.1a and 1b). During dehydration, significant changes occur on amide I bandshape. The band shifts to higher frequency (see inset of Fig.1a) due to the unfolding of protein units19; moreover, beyond 90 minutes desiccation the water contribution to this spectral region is almost negligible and two new spectral components at about 1620 and 1690 cm-1 progressively gains intensity. These spectral features, and particularly the most intense at 1620 cm-1 (aggregate band) are commonly assigned to the formation of intermolecular β-sheets structures of protein aggregates17,18,20,30. As recently reported for lysozyme, high DMSO concentration can induce protein aggregation even at room temperature21,22 and this process is related to the formation of amyloid structures involved in many neurodegenerative diseases7,8. It is well known that IR spectroscopy is sensitive to the first steps of the aggregation process30,31: this allows monitoring the formation of oligomers that possibly evolve to fibrillar structure. These oligomeric species seem to be more toxic than amyloid fibrils and play a key role in the related diseases32. According to our results, IR studies can give an important contribution to the characterization of this aggregation process in cord blood samples. These latter mainly contain white blood and stem cells suspended in the serum; as a consequence, different protein sources are present which cannot be distinguished spectroscopically. In order to understand the possible origin of aggregates, we analyzed the effect of DMSO addition to two


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different cell cultures often taken as models of adherent and non-adherent cells: respectively HuDe (Human Dermal fibroblasts) and Jurkat T cells. This allowed avoiding the presence of any residual, serum and medium thus focusing the analysis on DMSO interactions occurring within the cells, due to the permeability of plasma membranes to DMSO. Recently, we observed that a higher amount of Jurkat and HuDe cells retain their membrane integrities in the presence of DMSO, thus confirming this additive to permeate the plasmatic membrane and to be a valid CPA for both types of cells33.

1.3.2 Cell cultures ATR-FTIR spectra of Jurkat and HuDe cells were measured during water evaporation at ambient conditions. Both types of cells were dried from a PBS and a PBS/DMSO solution and the water loss was monitored through the decrease of intensity ratio of OH stretching mode at 3400 cm-1 respect to lipid absorption at 2852 cm-1. In Fig. 2 this intensity variation is shown as a function of desiccation time for the different samples.

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Figure 2 Intensity ratio of OH stretching mode at 3400 cm-1 to the CH2 symmetric stretching mode of lipid chains at 2852 cm-1, as a function of desiccation time: this quantity is indicative of water content in the sample. Intensity is referred to the band height; normalization is referred to the value obtained at t=0. 9 ACS Paragon Plus Environment


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We revealed a strong intensity variation due to water loss in the first 26-28 minutes treatment; at longer times only minor changes were observed (see Fig. 1SI of the Supporting Information). To notice that in case of blood unit much longer times were needed to dry the sample due to the lower cell concentration. Data of Fig. 2 show that for both HuDe and Jurkat samples the water loss is much reduced in the presence of DMSO indicating for these samples a higher water retention. A similar water retention was also observed for suspensions of artificial lipid vesicles (see Fig 2SI of the Supporting Information). This effect can be probably related to the strong interactions between water and DMSO34-36. In a recent study we used FTIR spectroscopy to monitor the freezing of HuDe and Jurkat suspended in PBS and DMSO/PBS solution at 0.1 DMSO mole fraction33. At the temperature of -80°C we monitored the fraction of crystallized water and observed a great reduction due to the presence of DMSO. The formation of ice causes a concentration increase of solutes dispersed in the extracellular compartment and promotes the cell dehydration33,37,38; accordingly, a reduced fraction of crystallized water is related to a reduced cell dehydration. From this point of view, the effects on the composition of the intracellular compartment (increase of protein concentration) are similar on water evaporation in air and on freezing of cellular samples: the water leakage is lower in the presence of DMSO. Together with the change of water OH stretching profile, we also followed the effects of dehydration on protein signals of both HuDe and Jurkat cells. In Fig.3 a comparison between spectra of dried samples obtained from PBS and PBS/DMSO suspensions, shows that the signal at 1620 cm-1, characteristic of pre-fibrillar aggregates18,20,28,29, can be detected only for DMSO-containing samples. In PBS samples, despite the reduced hydration can certainly alter the cell structure, the profile of amide I band is almost unchanged: this suggests that self-


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assembling of protein units is not induced after desiccation. On the contrary, when the cells are exposed to high percentage of DMSO due to the water loss, proteins undergo aggregation with the formation of intermolecular β-sheets. Actually, with respect to the absorption at 1650 cm-1, the relative intensity of the aggregate band is similar for the Jurkat and HuDe samples. The intensity of aggregate band is proportional to the concentration of aggregate species, and this is in turn related to the concentration of both protein and DMSO18.

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Our results suggest that, despite the different types of cell and possibly of protein molecules, a similar tendency to protein aggregation can be observed for the two systems here studied. The analysis with optical microscopy did not show the formation of aggregated species or plaques in DMSO samples (data not shown). This means that the protein clusters detected by FTIR are of sub-micrometric dimensions. The possibility to reveal the presence of such small aggregates is important due to their high toxicity as previously mentioned10-12. 11 ACS Paragon Plus Environment


1.3.3 Protein solutions

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wavenumber / cm-1 Figure 4 ATR-FTIR spectra in the region of amide I band of lysozyme (Lys) and albumin (BSA) at 3 hours desiccation from 20 mg/ml PBS and PBS/DMSO solutions, after subtraction of the solvent spectrum. Asterisk denotes the aggregate band.

The ATR-FTIR monitoring of water evaporation was also performed for lysozyme (Lys) and albumin (BSA) solutions in PBS and PBS/DMSO. As shown in Fig. 4, we observed once again that protein aggregation is DMSO-driven; moreover, we obtained similar aggregate concentrations for Lys and BSA samples. These evidences suggest that the tendency to form ordered clusters having β-sheet structure is not specific of certain kinds of globular proteins and is not induced by the cell crowding. The aggregates appear as a consequence of dehydration because of the augment of DMSO concentration, and the only increase of protein concentration in the absence of DMSO is not sufficient to induce the formation of pre-fibrillar species in the cell as well as in protein solution. 12 ACS Paragon Plus Environment

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1.4 Conclusions The protein aggregation process can be successfully detected in real time with the ATR-FTIR absorption technique, following the modifications of the band assigned as Amide I. This procedure is able to reveal the presence of small aggregates in simple molecular systems, as protein solutions, in cellular model systems, like fibroblasts and lymphocytes of human origin, and in more complex cellular samples, as cord blood stored for clinical use. It is the first time this procedure has been applied to such cellular and blood systems in order to detect DMSO-induced aggregation processes. Our data evidence that dehydration is much stronger in the absence of sulfoxide: nevertheless it doesn’t cause protein aggregation even in the crowded cellular environment. On the contrary, DMSO addition favors protein assembling in blood samples, cultured cellular samples as well as in protein solutions. Similarities between the cord blood sample and cultured cells suggest that this effect in blood is not limited to serum but also affects the protein content of the cells. Moreover, it is specific nor of the type of protein nor of the type of cell. We observed that this aggregation is revealed at very low water content and high DMSO concentration; from this point of view we suggest that similar effects could be also induced during freezing of cell samples due to the formation of ice in the extracellular compartment. This phenomenon could be one of the causes of the well-known cytotoxicity of DMSO, despite it is widely used as a cryoprotectant. In fact, we evidenced the formation of small protein clusters and literature data suggest that these aggregated species have a toxic effect on cells.



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Acknowledgments This research project was supported by 2007-2013 ESF “Competitiveness and Employment objective” Umbrian Regional Operational Programme (ROP), Avviso pubblico aiuti individuali per la realizzazione di progetti di ricerca. M.R. acknowledge support from MIURPRIN (Project 2012J8X57P) and PAT (Autonomous Province of Trento) “Grandi Progetti 2012” Project “MaDEleNA”. Authors also aknowledge the Centro Nazionale Trapianti for financial support by the project “Crioconservazione di cellule per uso clinico umano, con particolare riferimento ad alcune tipologie come le cellule staminali emopoietiche, mesenchimali e cellule riproduttive”.

Supporting Information description Table with vibrational assignment of cell IR absorptions. ATR-FTIR spectra of HuDe cells in PBS solution as a function of dehydration. ATR-FTIR spectra of phospholipid samples (DPPC liposomes) dried from aqueous suspension and DMSO/water suspension. This information is available free of charge via the Internet at http://pubs.acs.org


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