atw International Journal for Nuclear Power | 04.2020

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atw Vol. 65 (2020) | Issue 4 ı April RESEARCH AND INNOVATION 232 | Fig. 4. Luminescence spectra for the initial medium R red with 200 µM U(VI) as well as cells and supernatants after 24 h exposure to 200 µM U(VI). Spectra recorded after a delay of 0.1 µs. medium as well as plant cells and supernatants after exposure to U(VI). These samples were examined by means of TRLFS. Complexation with inorganic and organic ligands present in solution may change the speciation, i.e. the physico-chemical form in which the uranium is present in solution. These changes in speciation can be detected by TRLFS. Each U(VI) species has a spectrum with characteristic band positions and a typical luminescence lifetime, which can be determined by time-dependent measurements. These data can be used to determine how many and which species occur in the system. Reference substances can be used to identify them. For biological samples, cryo-TRLFS measurements are carried out in particular, in which the samples are cooled to -120 °C. This step is necessary because numerous quenching effects occur in the system due to medium and cell components that suppress the luminescence of U(VI). At low temperatures, quenching effects are suppressed as far as possible and spectra can be obtained. Figure 4 gives an overview of the luminescence spectra for the initial nutrient medium with 200 µM U(VI) and cells and supernatants after 24 h exposure to 200 µM U(VI). It can be seen that the cell species differs from the species in the medium or the supernatant. According to the literature [3] it can be assumed that this cell species is formed by the binding of UO 2 2+ to cell membranes or cell walls. Especially phosphate groups are of importance for the UO 2 2+ sorption processes. A closer look at the spectrum from the initial medium and from the supernatant after 24 h cell contact shows that speciation in the medium changes over time due to cell contact. From the experiments it can be concluded that the ((UO 2 ) 3 (OH) 5 ) + complex dominates in the initial medium according to the speciation calculations performed (not shown) and the band positions [2] of the luminescence spectrum. However, the species that dominates the luminescence in the supernatant is different. The measurements of the supernatants at different exposure times showed that various species occur in the supernatants over time (not shown). Starting from the medium species, the ((UO 2 ) 3 (OH) 5 ) + complex [2], two more species appear with increasing exposure time. Thus, the sub-process of biocomplexation could be detected spectroscopically for B. napus cells. Both species represent potential U(VI) complexes with plant cell metabolites. To identify these species it is necessary to identify released metabolites and to investigate their complexation behavior with U(VI). Plant cells release metabolites in response to exposure to uranium and europium For the experiments on the enrichment and identification of plant cell metabolites only very high concentrations of U(VI) and Eu(III) were used and the exposure time was fixed at 1 week in order to accumulate as many metabolites as possible in the nutrient medium. For the identification of metabolites in the supernatants after cell contact, sequentially solid phase extraction, HPLC (highperformance liquid chromatography) and MS (mass spectrometry) measurements were performed. For solid phase extraction columns were used that were suitable for enrichment of phenolic compounds and flavonoids. This procedure made it possible to obtain chromatograms of the enriched eluates from the extraction (Figure 5). | Fig. 5. Chromatograms of solid phase extraction eluates of cell culture media after plant cell exposure to 200 µM U(VI) or Eu(III) in comparison to those of control samples. Peaks that are new or exhibit higher intensities compared to the control sample are marked. Of particular interest are peaks in the chromatograms, which either show increased intensities or are new, compared to the control sample. A first peak assignment is possible by measuring reference compounds by HPLC and comparing their retention times with those found for the eluates from the solid phase extraction. In the literature, phenolic compounds and flavonoids in particular are mentioned as typical plant metabolites that are exuded in response to heavy metal stress. [21,26] In addition, however, smaller molecules such as organic acids or amino acids, peptides and amines are also mentioned. [21] A number of representatives from these mentioned substance classes have therefore been measured by HPLC. Peaks, which matched in their retention times for reference and solid phase extraction eluates were examined more closely. The cor responding peaks were fractionated and two metabolites could be identified using MS: p-coumaric acid and fumaric acid. However, the investi gation of the complexation behavior of these compounds with U(VI) showed that these substances are not involved in the formation of the two species identified in the TRLFS measurements of the supernatants. Therefore, further investigations on metabolite release are still necessary. However, a complexation constant could be obtained from the complexation experiments with fumaric acid, which will be used for calculations. Summary The investigation of the timedependent bioassociation behavior as well as the spectroscopic, spectrometric and chromatographic investigation of the formed cell metabolites and metal species led to molecular understanding of the interaction of Research and Innovation Studies on the Interaction of Plant Cells with U(VI) and Eu(III) and on Stress- induced Metabolite Release ı Jenny Jessat, Susanne Sachs, Robin Steudtner and Thorsten Stumpf
atw Vol. 65 (2020) | Issue 4 ı April the repository-relevant elements uranium and europium (as analogue for tri valent actinides) with B. napus. When these elements are released from a repository site into the environment, they can initially be immobilized via bioassociation with plants, mainly by binding to the cell surface. As a result, these elements may enter the food chain and create a health risk for the population. Furthermore, in case of U(VI) exposure a release and thus mobilization of uranium with the involvement of metabolites is observed. This can contribute to a higher bioavailability for, e.g. soil microorganisms and to the transfer of uranium in the environment. It can be assumed that, in addition to uranium, other actinides are also mobilized by interactions with plants. Results of this study contribute to an enhanced understanding of the actinide uptake into plants on a molecular level. Such data are necessary to improve biogeochemical models to predict the transfer of these elements in the environment, which enables the assessment of more reliable radiation doses with lower uncertainties. [13] A. J. Francis, J. Alloys Compd. 1998, 271–273, 78–84. [14] M. Vogel, A. Günther, A. Rossberg, B. Li, G. Bernhard, J. Raff, Sci. Total Environ. 2010, 49, 384–395. [15] S. Singh, R. Malhotra, B. S. Bajwa, Radiat. Meas. 2005, 40, 666–669. [16] A. Günther, G. Geipel, G. Bernhard, Radiochim. Acta 2006, 94, 845–851. [17] A. Günther, R. Steudtner, K. Schmeide, G. Bernhard, Radiochim. Acta 2011, 99, 535–542. [18] K. Viehweger, G. Geipel, G. Bernhard, Biometals 2011, 24, 1197–1204. [19] J. Misson, P. Henner, M. Morello, M. Floriani, T. Wu, J.-L. Guerquin-Kern, L. Février, Environ. Exp. Bot. 2009, 67, 353–362. [20] E. Weiler, L. Nover, Allgemeine Und Molekulare Botanik, Georg Thieme Verlag, Stuttgart, 2008. [21] O. Sytar, A. Kumar, D. Latowski, P. Kuczynska, K. Strzałka, M. N. V. Prasad, Acta Physiol. Plant. 2013, 35, 985–999. [22] A. Emamverdian, Y. Ding, F. Mokhberdoran, Y. Xie, Sci. World J. 2015, 2015, 1–19. [23] https://www.dsmz.de/fileadmin/Bereiche/PlantCellLines/ Dateien/R.pdf, zuletzt aufgerufen am 20.08.2018. [24] T. Mosmann, J. Immunol. Methods 1983, 65, 55–63. [25] M. Bader, K. Müller, H. Foerstendorf, B. Drobot, M. Schmidt, N. Musat, J. S. Swanson, D. T. Reed, T. Stumpf, A. Cherkouk, J. Hazard. Mater. 2017, 327, 225–232. [26] K. Viehweger, Bot. Stud. 2014, 55, 1–12. Authors M. Sc. Jenny Jessat, Dr. Susanne Sachs, Dr. Robin Steudtner, Prof. Dr. Thorsten Stumpf Helmholtz-Zentrum Dresden-Rossendorf Institute of Resource Ecology Bautzner Landstraße 400 01328 Dresden Germany RESEARCH AND INNOVATION 233 Acknowledgement The authors thank S. Beutner and S. Bachmann for performing the ICP-MS measurements as well as M. Raiwa from the Institute of Radioecology and Radiation Protection, Leibniz University Hannover for performing the MS measurements. The work is part of the project “TRANS-LARA”, which is funded by the Federal Ministry of Education and Research under contract number 02NUK051B. References [1] K. Töpfer, M. Kleiner, Deutschlands Energiewende – Ein Gemeinschaftswerk Für Die Zukunft, Berlin, 2011. [2] S. Sachs, G. Geipel, F. Bok, J. Oertel, K. Fahmy, Environ. Sci. Technol. 2017, 51, 10843–10849. [3] A. Günther, G. Bernhard, G. Geipel, T. Reich, A. Roßberg, H. Nitsche, Radiochim. Acta 2003, 91, 319–328. [4] S. D. Ebbs, D. J. Brady, L. V Kochian, J. Exp. Bot. 1998, 49, 1183–1190. [5] L. Laroche, P. Henner, V. Camilleri, M. Morello, J. Garnier-Laplace, Radioprotection 2005, 40, 33–39. [6] F. V. Tomé, P. B. Rodríguez, J. C. Lozano, Chemosphere 2009, 74, 293–300. [7] P. Chang, K. W. Kim, S. Yoshida, S. Y. Kim, Environ. Geochem. Health 2005, 27, 529–538. [8] M. Salvatores, Physics and Safety of Transmutation Systems – A Status Report, Nuclear Energy Agency, Paris, 2006. [9] D. Westlén, Prog. Nucl. Energy 2007, 49, 597–605. [10] Z. Zha, D. Wang, W. Hong, L. Liu, S. Zhou, X. Feng, B. Qin, J. Wang, Y. Yang, L. Du, et al., J. Radioanal. Nucl. Chem. 2014, 301, 257–262. [11] J. Laurette, C. Larue, I. Llorens, D. Jaillard, P.-H. Jouneau, J. Bourguignon, M. Carrière, Environ. Exp. Bot. 2012, 77, 87–95. [12] N. V. Zagoskina, E. A. Goncharuk, A. K. Alyavina, Russ. J. Plant Physiol. 2007, 54, 237–243. Research and Innovation Studies on the Interaction of Plant Cells with U(VI) and Eu(III) and on Stress- induced Metabolite Release ı Jenny Jessat, Susanne Sachs, Robin Steudtner and Thorsten Stumpf
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