Biochemical effects of Eu3+ and Cm3+ on eukaryotic cell metabolism – a case study

Trivalent lanthanides and actinides (Ln3+/Ac3+) are mostly non-essential metal ions and, in case of trivalent actinides, also not naturally occurring elements. Nonetheless, both lanthanides and actinides can be found in significant quantities in our environment. Trivalent lanthanides have a geogenic origin and get further into the environment during mining, processing and subsequent disposal caused by their intensive use for modern high-tech products. In contrast, trivalent actinides can be unintentionally released by for example the failure of nuclear power plants or during their long-term disposal. Even though they are typically non-essential elements their chemical similarity to calcium (Ca) justifies a detailed investigation of their physiological relevance to organisms and in particular regarding to their interference with the Ca metabolism. In literature an accumulation of lanthanides and actinides in organisms is reported as a function of concentration. Thus, specific or nonspecific transport of these elements in cells can be expected. Possible pathways are via the Na-Ca antiporter, bio-ligand mediated endocytosis or via ionophores. Furthermore, a metal induced permeabilization is described in literature. At least some of these processes can lead to accumulation of these lanthanides or actinides in the food chain.
To study the interaction of lanthanides with Brassica napus on a cellular level, callus and suspension cells were exposed to Eu3+. Besides the kinetics of the bio-association, the amount of associated Eu3+ and its effect on cell growth and viability was determined. Time-resolved laser-induced fluorescence spectroscopy (TRLFS) was used as direct speciation technique to determine the Eu3+ species on callus cells and cell compartments after the cell fractionation. The latter showed that 95 % of the Eu3+ can be found on large cell fragments, e.g. the cell membrane, whereas 5 % can be found in the cytoplasm and the residual plasma and intracellular membranes.
Not only the uptake routes for these elements are unknown but also the molecular mechanism of toxicity is not yet fully understood. An interference with metal ion channels, transporters and/or an interaction with other proteins is possible. In this study calmodulin (CaM) was used as Ca-binding protein relevant for all eukaryotes and as protein with a well-established crystal structure. As such, CaM plays a key role in the Ca metabolism and interacts with more than 350 cellular targets. Combining enzymology, calorimetry, spectroscopy and computational modeling allowed a structural and mechanistic understanding of the effects of Eu3+ and Cm3+ cations on the CaM protein structure and function. The analyses proved that Eu3+ binds CaM with high affinity and displaces Ca2+. Additional experiments demonstrated that the latter applies also for Cm3+. In addition, the stronger binding and different chemical environment of Eu3+/Cm3+ leads to structural distortion and disorder of CaM, which is associated with a reduced enzymatic activity of CaM.