Brief communication
Towards hemerythrin-based blood substitutes: Comparative
performance to hemoglobin on human leukocytes...

Eva Fischer-Fodor et al.

suitable biological system is the in vitro evaluation of these
potential blood substitutes ...
Hemerythrin-based blood substitutes
requirements of the Ethical Committee of the I. Chiricuta
Cancer Institute in particul...

Eva Fischer-Fodor et al.

Figure 1. Effect of synthetic hemoglobin and hemerythrin on human lymphocytes after 24, 48 ...
Hemerythrin-based blood substitutes


Figure 2. Effect of Hb and Hr preparations on HUVEC cultures. Samples are as in ...

Eva Fischer-Fodor et al.

Hr preparations, the Trypan blue–stained cell populations
showed any significant decrease c...
Hemerythrin-based blood substitutes
stress in patients with rhabdomyolysis and subarachnoid
haemorrhage. Biochem. Soc. Tra...
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Towards hemerythrin based blood substitutes comparative performance to hemoglobin on human leukocytes and umbilical vein endothelial cells


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Towards hemerythrin based blood substitutes comparative performance to hemoglobin on human leukocytes and umbilical vein endothelial cells

  1. 1. Brief communication Towards hemerythrin-based blood substitutes: Comparative performance to hemoglobin on human leukocytes and umbilical vein endothelial cells EVA FISCHER-FODOR1 , AUGUSTIN MOT2 , FLORINA DEAC2 , MARIANN ARKOSI2 , and RADU SILAGHI-DUMITRESCU2,* Ion Chiricuta Cancer Institute – Comprehensive Cancer Center, 34–36 Republicii St, Cluj-Napoca 400015, Romania 2 Department of Chemistry, Babes-Bolyai University, 11 Arany Janos Street, Cluj-Napoca 400028, Romania 1 *Corresponding author (Fax, +40-26459-0818; Email, Hemerythrin is a dioxygen-carrying protein whose oxidative/nitrosative stress-related reactivity is lower than that of hemoglobin, which may warrant investigation of hemerythrin as raw material for artificial oxygen carriers (‘blood substitutes’). We report here the first biological tests for hemerythrin and its chemical derivatives, comparing their performance with that of a representative competitor, glutaraldehyde-polymerized bovine hemoglobin. Hemerythrin (native or derivatized) exhibits a proliferative effect on human umbilical vein endothelial cell (HUVEC) cultures, as opposed to a slight inhibitory effect of hemoglobin. A similar positive effect is displayed on human lymphocytes by glutaraldehyde-polymerized hemerythrin, but not by native or polyethylene glycol-derivatized hemerythrin. [Fischer-Fodor E, Mot A, Deac F, Arkosi M, Silaghi-Dumitrescu R 2011 Towards hemerythrin-based blood substitutes: Comparative performance to hemoglobin on human leukocytes and umbilical vein endothelial cells. J. Biosci. 36 215–221] DOI 10.1007/s12038-011-9066-5 1. Introduction The possibility of generating artificial oxygen carriers based on hemoglobins (Hbs) and chemical derivatives thereof has been a topic of intense research (Alayash 2004; Tsuchida et al. 2009). Several well-defined chemical side-reactions were found to correlate with undesired side-effects of such products (Alayash 2004). These side-reactions include autoxidation (Alayash 2004; Silaghi-Dumitrescu and Silaghi-Dumitrescu 2006) reaction with nitric oxide (Herold 1998; Herold et al. 2001; Blomberg et al. 2004; Olson et al. 2004) and freeradical-generating reactions with hydrogen peroxide (Reeder et al. 2002a, b, 2004, 2008; Reeder et al. 2002b; Vollaard et al. 2005a, b; Cooper et al. 2008). All of these reactions are facilitated by the slow but unavoidable heme release from the protein matrix as well as by Hb extravasation. Keywords. Hemerythrin (Hr) is an oxygen carrier first described in marine invertebrates that employs a non-heme diiron active site (Farmer et al. 2000; Jin et al. 2002; Kryatov et al. 2005). Hr was shown to avoid most of the stress-related sidereactions cited above for hemoglobin, showing much slower, if any, reactions with hydrogen peroxide, nitric oxide and nitrite (Kryatov et al. 2005; Mot et al. 2010). Furthermore, we described protocols for chemical modification of Hr with polyethylene glycol (PEG) and glutaraldehyde, pointing out that the effects of these chemical modifications on O2 affinity, autoxidation rate and molecular weight of Hr appear to be favourable for blood substitute applications. The use of synthetic hemoglobin and hemerythrin compounds in clinical practice is a great challenge, and to this end a rigorous evaluation of the biological effect of compounds is needed (Creteur and Vincent 2009). A Blood substitute; hemoglobin; hemerythrin; human umbilical vein endothelial cell (HUVEC); lymphocyte Abbreviations used: ANOVA, analysis of variance; FCS, fetal calf serum; Hb, hemoglobin; Hr, Hemerythrin; HUVEC, human umbilical vein endothelial cell; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium; MTT, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; PBS, phosphate buffer saline; PEG-Hr, polyethylene glycolderivatized Hr; PMS, phenazine methosulphate J. Biosci. 36(2), June 2011, 215–221, * Indian Academy of Sciences 215
  2. 2. 216 Eva Fischer-Fodor et al. suitable biological system is the in vitro evaluation of these potential blood substitutes on human peripheral blood mononuclear cells. Indeed, once transfused into the blood vessels, blood substitutes will encounter lymphocytes, monocytes and other cells, implicated in the cellular immune response (Malkevich et al. 2008). Another useful test system has previously been shown to be the culture of human umbilical vein cord cells. Such results are reported here, alongside with preliminary results on the in vivo interaction of hemerythrin with mice, all of which point towards hemerythrin derivatives as non-toxic materials for blood substitute preparation. 2. Materials and methods 2.1 Materials Native bovine hemoglobin (Hb) (Antonini and Brunori 1971) and recombinant hemerythrin (Hr) (Mot et al. 2010) were obtained as previously described and manipulated in phosphate buffer saline (PBS) unless otherwise mentioned. Hemoglobin and hemerythrin concentrations in text are given per heme rather than per tetramer. Protocols for obtaining glutaraldehyde-polymerized-Hr (poly-Hr), polyethylene glycol-derivatized Hr (PEG-Hr) and glutaraldehydederivatized Hb were previously described in detail (Deac et al. 2009; Mot et al. 2010). PBS, RPMI-1640, Hank’s media, FCS, glutamine, penicillin-streptomycin solution, Histopaque 1077 and Trypan blue solution were acquired from Sigma. Cell Titer 96 AQueus Non-Radioactive Cell Proliferation Assay Kit was obtained from Promega Corporation. Nunclon Delta surface dishes, centrifuge tubes and other plastic disposables from Nunc Company were employed. 2.2 Instrumentation UV-vis spectra were recorded on Agilent 8453 (Agilent, Inc.) and Cary 50 (Varian, Inc) instruments. Cell culture tests were performed under sterile conditions in a cell culture laboratory equipped with LaminAir Class II laminary hood, Hettich Universal 32R centrifuge, Uniequip incubator, Olympus BX40 optical microscope, Olympus CKX41 inverted phase fluorescence microscope and Heidolph Titramax 1000 shaker coupled with Heidolph Incubator 1000. Spectrometric measurements were made using BioTek Synergy universal microplate plate reader with monochromator for absorbance measurements. Nechifor from the University of Bucharest, Faculty of Biology. Cells were defrosted carefully, cultivated in special culture flasks (Nunclon) with RPMI-1640 (Sigma) culture medium, supplemented with fetal calf serum (FCS, Sigma), penicillin-streptomycin (Sigma) and glutamine in humidified Heto Holten Cellhouse 154 incubator at 37°C and 5% CO2 level. Cells were adherent, and several cell passages were performed using enzymatic procedures. Experiments were performed when confluence of the cells achieved 80% on the flask surface. Cells were then plated on 96-well flat-bottomed microtitre plates (Nunclon), and were kept 24 h in the incubator. To obtain a suitable density of cells on each well on the day of the measurement, and considering the HUVEC cells proliferation rate, we used different cell densities per well: 1×105 per well cell for the 24 h experiment, 7.5×103 cells for 48 h incubation, and 6×103 cells for the 72 h experiment. Wells were then treated with compounds to be tested (10 μl in each well), with untreated cells left as reference; blank cell-free wells contained cell culture media; as coloration reference, cell-free wells were treated with culture media and compound. Each compound was tested in triplicate, and three different experiments were completed. To assess cytotoxicity we performed the widely used quantitative colorimetric MTT assay (Denizot and Lang 1986; Furukawa et al. 1991) for determination of cell viability changes. The method’s advantages are its sensitivity, simplicity and relatively short duration. The assay is based on the ability of viable cells mitochondria to reduce 3-(4,5-dimethylthiazolyl-2)-2,5diphenyltetrazolium bromide (MTT) to a purple formazan product. The formazan product was analysed with a scanning multiple spectrophotometer and its quantity as measured by the amount of 492 nm absorbance values was directly proportional to the number of living cells in cell culture. After 24, 48 or 72 h incubations with the Hb molecules, MTT solution was added to each well at a final concentration of 1 mg/mL per well and the plates were incubated at 37°C for another hour. Dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan and spectrophotometric absorbance measurements were made using a BioTek Synergy 2 multimodal fluorescence microplate plate reader. Statistical analysis employed the GraphPad Prism 5 biostatistics software. We established the survival curves for the potential blood substitutes. One-way analysis of variance (ANOVA) and Dunnett Multiple Comparison test (P<0,05, r2 =0.94–0.86) were completed for all individually inhibitory effects. 2.4 2.3 Lymphocyte preparation and testing HUVEC cultures The human umbilical vein endothelial cell line (HUVEC) was a generous gift from Associate Professor Marina J. Biosci. 36(2), June 2011 Human whole blood was collected by phlebotomy from a 44-year-old healthy male volunteer. We obtained the patient’s written informed consent, according to the
  3. 3. Hemerythrin-based blood substitutes requirements of the Ethical Committee of the I. Chiricuta Cancer Institute in particular and respecting the general E u r op e a n e t h i ca l g u i d e l i n e s ( R e c om m e nd a t i o n Concerning Medical Research on Human Beings, 1990). Whole blood was collected in Li-Heparin coated tubes, and lymphocyte separation was completed using the gradient density method. Thus, blood was mixed in 1:1 ratio with PBS. Lymphocyte separation solution Histopaque with density 1077 was added in centrifuge tubes, and the diluted blood was carefully transferred onto the Histopaque column, without disturbing the surface. After centrifugation at 2000 rpm, in the centrifuge tubes several layers were separated: lymphocytes were in an opaque peripheral blood mononuclear cell layer between the Histopaque and the blood plasma. Lymphocytes were extracted from this layer, washed twice in Hank’s medium in centrifuge tubes at 1200 rpm, and resuspended in cell culture medium. For lymphocyte primary cultures, RPMI-1640 medium was employed, supplemented with fetal calf serum (FCS), glutamine and penicillin-streptomycin solution. Cells were plated on 96-well cell culture dishes, and they were kept in an incubator for 1 h at 37°C and 5% CO2 level. Human normal lymphocytes do not adhere to the culture plate surface. We obtained a primary lymphocyte culture, in which cells do not proliferate, and their number decreases in time. In order to obtain an appropriate number of lymphocytes during measurements, the density of lymphocytes seeded on 96-well plates was 2×105 cells/mL for the 24 h experiment, 3.75×105 cells/mL for the 48 h experiment and 5×105 cells/mL for the 72 h experiment . Wells contained 200 μl cell suspension and were treated with 10 μl of synthetic compounds. For every experiment, untreated cells were used as reference; wells without lymphocytes, with media only, were used as blanks. For every compound analysed, wells containing media and protein sample were used for colour control. Measurements were made in quadruplicate, and three individual experiments were accomplished. Evaluation of cell growth was made after 24, 48 and 72 h. Lymphocytes chemosensitivity was evaluated with a spectrometric method, using the MTS assay. The assay (Arafat et al. 2000) uses the novel tetrazolium compound MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, and an electron coupling agent, the phenazine methosulphate (PMS). MTS is bio-reduced by viable cells to a water-soluble formazan product, and samples can be measured directly from the plate, without further processing (Wang et al. 2010). The absorbance of the MTS–formazan product was measured at 490 nm. The quantity of formazan product is directly proportional to the number of living cells in culture. Absorbance values were recorded and data were implemented in biostatistics. 217 Simultaneously, cell counting using the Trypan blue dye were also performed. Cells were re-suspend by pipetting up and down, and they were removed from the wells of the culture plate. The number of cells from the well was counted using a Bürker-Türk hemocytometer chamber. In the next step, an aliquot of cell suspension was mixed in 1:1 proportion with Trypan blue 1% solution, and another count was completed in order to monitor the number of viable cells in the population. The calculation of survival rate was based on proportion between plated cells and the number of harvested cells after 24, 48 and 72 h. Statistical processing of data provided by spectrometric measurements was accomplished using the GraphPad Prism 5 software program. Statistical comparison between groups was made by one-way ANOVA Test with Bonferroni Multiple Comparison Posttest. 3. Results and discussion The effect of the 24 h contact of human lymphocytes with the artificial hemoglobin and hemerythrin compounds was quantified by the absorbance values obtained following the colorimetric measurements in the MTS assay. The Hb and Hr compounds in the cell growth media did not inhibit significantly the lymphocyte viability (figure 1). In fact, PEG-Hr and polymerized Hr exhibit a cytoprotective effect, increasing the lymphocyte survival after 24 h; this effect is statistically very significant for polymerized Hr (two-way ANOVA test, Bonferroni post-test, 95% confidence interval, P<0.05). For the 48 h contact with human lymphocytes, the tendency is the same: hemoglobin derivatives show a slightly inhibitory effect, but not statistically significant, while hemerythrins, particularly Poli-Hr, protect the cells (figure 1, middle panel). The same tendency is observed after 72 h (figure 1, bottom panel), with poly-Hr faring distinctly better than any other preparation. At this point, slight differences also begin to develop between native and polymerized Hb, in favour of the polymerized protein. Figure 1 also shows data where poly-Hb was added in the form of a mixture with antioxidants. Components of these mixtures included catalase (1000 U), ascorbate (100 μM), urate (100 μM), cysteine (10 μM) and BSA (100 μM) for ‘+a1’ and ascorbate (100 μM), catalase (1000 U) for ‘+a2’. Neither of the two antioxidant mixtures had a positive effect in these tests. Simultaneously, cell viability count was performed using the Trypan blue method. We evaluated the number of the living cells found in each well treated with the new compounds. The number of untreated lymphocytes decreased in time, as expected since under in vitro conditions in growth media part of the normal human lymphocytes loose their viability. For none of the Hb and J. Biosci. 36(2), June 2011
  4. 4. 218 Eva Fischer-Fodor et al. Figure 1. Effect of synthetic hemoglobin and hemerythrin on human lymphocytes after 24, 48 and 72 h treatments. J. Biosci. 36(2), June 2011
  5. 5. Hemerythrin-based blood substitutes 219 Figure 2. Effect of Hb and Hr preparations on HUVEC cultures. Samples are as in figure 1; details are provided in the section on materials and methods. J. Biosci. 36(2), June 2011
  6. 6. 220 Eva Fischer-Fodor et al. Hr preparations, the Trypan blue–stained cell populations showed any significant decrease compared with the untreated control populations. The capacity of Poli-Hr and PEG-Hr to slow down this tendency was confirmed by the viability test. Figure 2 shows results obtained upon treating human umbilical vein endothelial cell (HUVEC) cultures with the same Hb and Hr samples as in figure 1. In agreement with previous results according to which hemoglobin and its derivatives engage in free-radical generating reactions affecting such cells (McLeod and Alayash 1999; D'Agnillo and Alayash 2001; Alayash 2004), native hemoglobin as well as poly-Hb display a slight inhibitory effect on HUVEC cultures. As in the case of the lymphocytes, addition of antioxidants to poly-Hb (samples ‘+a1’ and ‘+a2’ in figure 2) does not appear to improve performance to any significant extent. On the other hand, all three Hr samples are seen to fare distinctly better than any of the Hb preparations, with indication of a slight proliferative effect especially at 48 h. To conclude, hemerythrin and its chemical derivatives were found to be less toxic than native and glutaraldehydepolymerized hemoglobin towards human lymphocytes and endothelial cells. Glutaraldehyde crosslinking did not improve the performance of hemoglobin in these tests, nor did the addition of antioxidants at concentrations that would otherwise protect against autoxidation or oxidation by hydrogen peroxide. Acknowledgements This work was supported by the Romanian Ministry for Education and Research (grants PNII ID565/2007 and PCCE 140/2008) and by PhD scholarships to FD and AM (Contract POSDRU/88/1.5/S/60185 – ‘Innovative doctoral studies in a knowledge based society’). References Alayash AI 2004 Oxygen therapeutics: can we tame haemoglobin? Nat. Rev. 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