atw International Journal for Nuclear Power | 04.2020

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atw Vol. 65 (2020) | Issue 4 ı April RESEARCH AND INNOVATION 230 WiN Germany Price 2019 Studies on the Interaction of Plant Cells with U(VI) and Eu(III) and on Stress- induced Metabolite Release Jenny Jessat, Susanne Sachs, Robin Steudtner and Thorsten Stumpf The interaction of plant cells with U(VI) and Eu(III) was investigated as a function of exposure time and metal concentration. Brassica napus (canola) cells were used in the experiments. For europium and uranium, an immobilization by the plant cells could be observed in first instance, which can be attributed to sorption processes. During the experiments an equilibrium state was reached, i.e. for none of the investigated concentrations a complete bio association occurred. Especially for the examined concentration of 200 µM U(VI) a multi-stage bioassociation process was observed, i.e. after the described bioassociation a new release of uranium by the cells occurred. By means of time-resolved laserinduced fluorescence spectroscopy it could be shown that the U(VI) is complexed by cell metabolites in the course of exposure. Several newly formed species could be detected. It can be assumed that metabolites released by plant cells in response to heavy metal stress complex U(VI) and keep it in solution. Using a combination of solid phase extraction, high-performance liquid chromatography and mass spectrometry, p-coumaric acid and fumaric acid were identified as released metabolites and their complexation behavior with U(VI) was investigated. | Fig. 1. Schematic illustration of possible interaction processes of heavy metals with a plant cell. (adapted from Francis 1998 [13]) Introduction In Germany, the Ethics Committee on Secure Energy Supply proposed the phasing out of the use of nuclear energy in 2011 and found its realization possible within the next ten years. [1] As a result, it was decided to shut down all German nuclear power plants by 2022. The decision for a suitable repository site for high radioactive nuclear waste is a major challenge for politicians, energy supply companies and society. It is the task of science to understand and predict the behavior of radionuclides in the environment to create the basis for a safety assessment and thus for repository site selection. For a reliable safety assessment of nuclear waste reposi tories, it is necessary to consider possible scenarios in which radionuclides are released into the environment. In this context, the transfer behavior of the radionuclides from the groundwater zone via the soil into the plant is of importance. Radionuclides can be accumulated by plants and thus, they can enter the food chain. This creates a health risk for humans and animals. However, there is little knowledge about the mechanisms of interaction between radionuclides and plants. Of course, there are some studies on this topic [2–5], but in many cases only transfer factors are determined which do not provide any information on the underlying processes taking place. [5–7] It is therefore a major goal to generate a molecular process understanding in this field. Thus, one major objective of the present work was to investigate the time dependence of the bioassociation of uranium (U(VI)) and europium (Eu(III)) with Brassica napus (canola) cells. These two elements were chosen because uranium is a major component of spent nuclear fuel rods and europium can be used as an analogue for trivalent actinides, like plutonium (Pu(III)), americium (Am(III)) and curium (Cm(III)). [8–10] Americium and curium, in turn, are significantly responsible for the long-lasting radiotoxicity of the nuclear waste. [8] Canola was selected because it is a typical feed and crop plant in Germany. It is also known for tolerating high amounts of heavy metals and is therefore a suitable model organism for these studies. [7,11] The suspension cell cultures used are obtained from callus cultures. Callus cells have the advantage that they are able to synthesize typical secondary metabolites that are also produced in whole tissues. [12] It is also of interest to investigate the formation of metabolites that are released by the cells as a response to the heavy metal stress as well as to study the complexation behavior of these metabolites with uranium. These data are required for an improved process understanding and are the basis for the modeling of the radionuclide transfer in the environment up to the food chain. Therefore it is necessary to know possible interaction processes of plant cells with heavy metals including radio nuclides, as illustrated in Figure 1. On the one hand there is the sorption [14] of heavy metals on cell walls and membranes, which takes place passively and fast, on the other hand there is the accumulation [15] as an active process through which heavy metals are taken up into the cell. As a result of the release of bioligands by the cell, heavy metals can be complexed. This process influences their solubility (biocomplexation). [16,17] Due to biotransformation [18] (usually that is a reduction) the oxidation state of the heavy metal is changed by the cell metabolism. For uranium this could be a reduction from U(VI) to U(IV). This reduction is closely related to biomineralization, which can lead to the formation of insoluble metal precipitates. [11,19] To put it simply, all processes with the exception of complexation lead to an immobilization of the heavy metals and are therefore summarized under the term bioassociation. Nevertheless, it is possible to obtain information on the individual processes, as will be 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 shown later. Due to these interactions, stress reactions can be induced in the plant cells. The heavy metals can act directly as chemical stressors and displace essential metal ions from the active centers of enzymes or damage proteins, especially those with thiol groups. [20] They can also induce a secondary form of stress – the oxidative stress – by disturbing the cellular redox status. This leads to the formation of reactive oxygen species, which in turn can damage cell organelles. [20–22] Plants react differently to stress. One possible reaction is the formation of protective metabolites that can affect the bioavailability of heavy metals. [20,22] To investigate the time-dependent bioassociation behavior of U(VI) und Eu(III) with canola, B. napus callus cells (PC-1113 from DSZM, Braunschweig, Germany) were transferred to suspension cell cultures and exposed to a phosphate-reduced cell culture medium R [23] (R red ) with different concentrations of U(VI) (20 and 200 µM) or Eu(III) (30 and 200 µM). Exposure times were thereby varied between 1 and 72 h. At the end of the exposure time the cells were separated from the supernatants. The metabolic activity of the cells ( vitality) was determined by the MTT test [24] (MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The supernatants were analyzed concerning their heavy metal content by ICP-MS (inductively coupled plasma-mass spectrometry) as well as regarding the speciation (physico-chemical form) of U(VI) by TRLFS (time-resolved laser-induced fluorescence spectroscopy). How plant cells react to radionuclides and their analogues As mentioned before, all cell-related processes that can immobilize heavy metals are called bioassociation. This immobilization of U(VI) and Eu(III) by B. napus cells can be compared for different heavy metal concentrations. Taking into account the metabolic activity of the plant cells at the considered exposure times, conclusions can be drawn about the physiological state of the cell and the reactions taking place. For 20 µM U(VI) as well as for 30 and 200 µM Eu(III) a comparable behavior was observed: up to about 24 h of exposure a rapid increase of bioassociation occurs, followed by an equilibrium adjustment. This behavior is exemplary shown in Figure 2 for the experiments carried out with 30 µM Eu(III). Two conclusions can be drawn from this. On the one hand, the rapid increase in bioassociation within the first few hours suggests that biosorption as a passive, fast process plays an important role in the immobilization of metals. However, it can be assumed that with increasing exposure time, other processes such as bioaccumulation, biocomplexation and bioprecipitation are also involved in the overall process. Furthermore, an equilibrium is reached after 24 h and it is noticeable that for none of the investigated concentrations 100 % of the metal was bioassociated at equilibrium. This means that there is no complete immobilization of the metals by the plant cells. This suggests that bioprecipitated U(VI) remains unnoticed as colloids in the supernatants and/or that processes are actively maintained by the cells to keep the heavy metals out of or away from the cells. For the lower U(VI) and Eu(III) concentrations, cell vitality (metabolic activity) remains at the level of the control samples (100 %) over the entire exposure period. It also turned out, as expected, that the cell vitality is increased for higher heavy metal concentrations than for lower concentrations. This difference can be explained by the fact that plant cells are exposed to increased stress due to the higher heavy metal load and react metabolically to it. The bioassociation behavior of B. napus cells exposed to 200 µM U(VI) differs from the behavior described so far (shown as an example in Figure 3). Here, the initially rapid increase in bioassociation can also be seen, which is probably again mainly attributable to biosorption. This is followed by a decrease of the bioassociated amount of uranium, i.e. as the exposure time increases, less immobilized uranium is present at or in the cells. Therefore, it can be concluded that B. napus cells show a multistage bioassociation process in the presence of 200 µM U(VI). At the same time, cell vitality shows clear fluctuations during this period, indicating metabolic processes induced in the cell as a result of radionuclide exposure. In the literature such a multistage bioassociation process could be observed for halophilic archaea. [25] The release of U(VI) indicates a biocomplexation. In the literature it is mentioned that plant cells react to heavy metal stress, among other things, by releasing protective metabolites, especially flavonoids and phenols. [22,26] It can therefore be assumed that, as a result of the exposure of the cells to U(VI), the release of protective metabolites occurs, which complex U(VI) and thus convert it into a more mobile physicochemical form. Spectroscopic investigations with cells and supernatants were carried out in order to verify this. Luminescence spectroscopy shows that plant cells react to uranium The investigated system includes the initial U(VI) containing nutrient RESEARCH AND INNOVATION 231 | Fig. 2. Bioassociation behavior and cell vitality of B. napus cells exposed to 30 µM Eu(III) (mean values of 3 experiments). Cell vitalities are expressed as a percentage of the cell vitality of the control samples. | Fig. 3. Bioassociation behavior and cell vitality of B. napus cells exposed to 200 µM U(VI) (1 experiment). Cell vitalities are expressed as a percentage of the cell vitality of the control samples. 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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