Experiments were carried out to investigate the effects of root citric acid on uptake and initial distribution of cadmium (Cd) in tomato plants (Lycopersicon esculentum, cv. Tiny Tim). Cd was measured by "y-spectrometry, using 115Cd spikes. Citric acid was measured by UV-detection, and, after spiking with lac-citric acid, by ~-spectrometry. Cd was applied for 48 h periods, in control experiments, in the presence of citric acid, and after 24 h plant pre-incubation with citric acid.Pre-incubation resulted in two-fold increases in fast-exchangeable amounts of root citric acid, as measured by the presence of citric acid in xylem exudates of decapitated and pressurized roots.Simultaneous application of Cd and citric acid did not change Cd accumulation in total plants and in the roots, nor did any significant change occur with respect to Cd root-to-shoot transport, and Cd concentrations in shoot tissues and xylem fluid. After citric acid pre-incubation, total plant uptake of Cd increased twofold, without any significant change in Cd accumulation in the roots. Cd root-to-shoot transport was increased 5-6 fold, and Cd concentrations in shoot tissues and xylem fluid were increased 6-8 fold. Speciation calculations indicated that, under the conditions applied, xylem Cd may be, at least partly, complexed in citric acid.

Alpha emitters have great potential in targeted tumour therapy, especially in destroying micrometastases, due to their high linear energy transfer (LET). To prevent toxicity caused by recoiled daughter atoms in healthy tissue, alpha emitters like Ac can be encapsulated in polymeric nanocarriers (polymersomes), which are capable of retaining the daughter atoms to a large degree. In the translation to a (pre-)clinical setting, it is essential to evaluate their therapeutic potential. As multicellular tumour spheroids mimic a tumour microenvironment more closely than a two-dimensional cellular monolayer, this study has focussed on the interaction of the polymersomes with U87 human glioma spheroids. We have found that polymersomes distribute themselves throughout the spheroid after 4 days which, considering the long half-life ofAc (9.9 d) (Vaidyanathan and Zalutsky, 1996), allows for irradiation of the entire spheroid. A decrease in spheroidal growth has been observed upon the addition of only 0.1 kBq Ac, an effect which was more pronounced for theAc in polymersomes than when only coupled to DTPA. At higher activities (5 kBq), the spheroids have been found to be destroyed completely after two days. We have thus demonstrated that Ac containing polymersomes effectively inhibit tumour spheroid growth, making them very promising candidates for future in vivo testing.

Abstract:The lactate dehydrogenase (LDH) assay was addressed for its sensitivity, disturbances by foaming, and cell number and size. Cells were from a U-251 MG grade IV human glioblastoma brain tumor cell line used in 100-l well volumes. Cells were counted by microscopy and Coulter counting; assays were LDH or trypan blue. The results indicate increased 490 nm signals (level, variance) by using phenol red or by increasing fetal bovine serum from 5% to 10%. The data also indicate that defoaming results in reduced variances ranging from a factor of 2 at 1-3 units of absorption, up to a factor of 4-5 at Ͻ1 units of absorption. Coulter counting indicated a decrease in cell volume with increasing end-point cell density, attributed to general shrinking at increasing density. In comparisons, total LDH was considered relative to both cell total volume and cell numbers. The result suggests that total LDH should be regarded as reflecting cell total volume rather than cell numbers. In a comparative Cu exposure test, signals of both LDH and a sodium salt of 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) decreased with increasing Cu supply, while bromodeoxyuridine signals remained largely unaffected. The data show the differences in responses in cell viability and proliferation, but, above all, indicate that LDH should be expressed on a per cell volume basis rather than per cell, to avoid the problem that mere density effects contribute to signals on compound or metal toxicity. 675

Mutual interactions between cadmium ions, citric acid and xylem cell walls were examined. Cadmium and citric acid were measured as "^Cd and [1,5-^*'C] citric acid, respectively. Xylem cell walls were obtained by bacterial degradation of tomato stem sections {Lycopersicon esculentum Mill, cv. Tiny Tim), and applied as ion-exchange columns. The xylem column material carried 2 4dm3 HzO kg "^ dry weight, and was temporarily capable of buffering perfusates at pH 5 7, Sorbed cadmium and citric acid were determined from H2O and HCI rinses after perfusion periods. In all experiments, total cadmium and/or citric acid recoveries were better than 98%, indicating both the effectiveness of the rinses applied and the possibility of full regeneration of the xylem column. The results indicate that the presence of 2 45 mol m"^ citric acid causes an approximately 50% reduction of adsorbed cadmium levels, irrespective of the applied total cadmium concentrations (004-04 mol m"Ĉ d(NO3)2.4H2O). This reduction is probably related to a corresponding reduction to approximately 2% of the control applied free Cd^^ concentration, the latter also independent of the total cadmium concentrations. Furthermore, without inducing positively charged citrate complexes in the applied solution, the presence of cadmium resulted in increased levels of citric acid absorbed in the xylem column. The Donnan Free Space accumulation of citric acid in the presence of Cd(NO3)2.4H2O, observed in the experiments described, could be expressed by its distribution coefficient, as approximately 15 times the control accumulation. These data indicate that the xylem column may operate as a ligand exchanger, suggesting the importance of metal ions for the longitudinal and lateral movement of organic complexing compounds in the xylem.

Major amino acids and organic acids in xylem exudates of tomato plants were separated by reversed phase high performance liquid chromatography (RP-HPLC) and quantified by UV detection. Before separation, amino acids were converted into their phenylisothiocyanate (PITC) derivatives. In a single run, Asp, Glu, Ser, Gin, His, Thr, Ala, Tyr, Val, Met, Cys, lie, Leu, Phe, and Lys could be separated and detected down to the pmol level. Unresolved peaks were obtained for Asn and Gly and for Arg and Pro. For organic acid analysis, exudates were pre-treated by perfusion over a prepacked Adsorbex SCX cation exchange column, to eliminate exudate amino acids. Elution recoveries for organic acids were close to 100%. The exudate organic acids were separated by ion suppression RP-HPLC chromatography, and peaks could be resolved for L-malic acid, malonic acid, maleic acid, citric acid and fumaric acid, down to the pmol level. UV signals for exudate ascorbic acid, and succinic acid were below the limits of detection. Determination of oxalic acid and tartaric acid was impossible, due to the presence of the exudate salt peak in the chromatogram. The results indicate the potential of the methods applied, and show the applicability of RP-HPLC analysis for the determination of both amino acids and organic acids in xylem exudates.