Imaging of Vanadium in Microfossils: A New Potential Biosignature.

The inability to unambiguously distinguish the biogenicity of microfossil-like structures in the ancient rock record is a fundamental predicament facing Archean paleobiologists and astrobiologists. Therefore, novel methods for discriminating biological from nonbiological chemistries of microfossil-like structures are of the utmost importance in the search for evidence of early life on Earth. This, too, is important for the search for life on Mars by in situ analyses via rovers or sample return missions for future analysis here on Earth. Here, we report the application of synchrotron X-ray fluorescence imaging of vanadium, within thermally altered organic-walled microfossils of bona fide biological origin. From our data, we demonstrate that vanadium is present within microfossils of undisputable biological origin. It is well known in the organic geochemistry literature that elements such as vanadium are enriched and contained within crude oils, asphalts, and black shales that have been formed by diagenesis of biological organic material. It has been demonstrated that the origin of vanadium is due to the diagenetic alteration of precursor chlorophyll and heme porphyrin pigment compounds from living organisms. We propose that, taken together, microfossil-like morphology, carbonaceous composition, and the presence of vanadium could be used in tandem as a biosignature to ascertain the biogenicity of putative microfossil-like structures. Key Words: Microfossils-Synchrotron micro-X-ray fluorescence-Vanadium-Tetrapyrrole-Biosignature. Astrobiology 17, 1069-1076.

Glutathione transferase P1-1 as an arsenic drug-sequestering enzyme.

Arsenic-based compounds are paradoxically both poisons and drugs. Glutathione transferase (GSTP1-1) is a major factor in resistance to such drugs. Here we describe using crystallography, X-ray absorption spectroscopy, mutagenesis, mass spectrometry, and kinetic studies how GSTP1-1 recognizes the drug phenylarsine oxide (PAO). In conditions of cellular stress where glutathione (GSH) levels are low, PAO crosslinks C47 to C101 of the opposing monomer, a distance of 19.9 Å, and causes a dramatic widening of the dimer interface by approximately 10 Å. The GSH conjugate of PAO, which forms rapidly in cancerous cells, is a potent inhibitor (Ki = 90 nM) and binds as a di-GSH complex in the active site forming part of a continuous network of interactions from one active site to the other. In summary, GSTP1-1 can detoxify arsenic-based drugs by sequestration at the active site and at the dimer interface, in situations where there is a plentiful supply of GSH, and at the reactive cysteines in conditions of low GSH.

Reactivity and Speciation of Anti-Diabetic Vanadium Complexes in Whole Blood and Its Components: The Important Role of Red Blood Cells.

Reactions with blood components are crucial for controlling the antidiabetic, anticancer, and other biological activities of V(V) and V(IV) complexes. Despite extensive studies of V(V) and V(IV) reactions with the major blood proteins (albumin and transferrin), reactions with whole blood and red blood cells (RBC) have been studied rarely. A detailed speciation study of Na3[V(V)O4] (A), K4[V(IV)2O2(citr)2]·6H2O (B; citr = citrato(4-)); [V(IV)O(ma)2] (C; ma = maltolato(-)), and (NH4)[V(V)(O)2(dipic)] (D; dipic = pyridine-2,6-dicarboxylato(2-)) in whole rat blood, freshly isolated rat plasma, and commercial bovine serum using X-ray absorption near-edge structure (XANES) spectroscopy is reported. The latter two compounds are potential oral antidiabetic drugs, and the former two are likely to represent their typical decomposition products in gastrointestinal media. XANES spectral speciation was performed by principal component analysis and multiple linear regression techniques, and the distribution of V between RBC and plasma fractions was measured by electrothermal atomic absorption spectroscopy. Reactions of A, C, or D with whole blood (1.0 mM V, 1-6 h at 310 K) led to accumulation of ∼50% of total V in the RBC fraction (∼10% in the case of B), which indicated that RBC act as V carriers to peripheral organs. The spectra of V products in RBC were independent of the initial V complex, and were best fitted by a combination of V(IV)-carbohydrate (2-hydroxyacid moieties) and/or citrate (65-85%) and V(V)-protein (15-35%) models. The presence of RBC created a more reducing environment in the plasma fraction of whole blood compared with those in isolated plasma or serum, as shown by the differences in distribution of V(IV) and V(V) species in the reaction products of A-D in these media. At physiologically relevant V concentrations (<50 µM), this role of RBC may promote the formation of V(III)-transferrin as a major V carrier in the blood plasma. The results reported herein have broad implications for the roles of RBC in the transport and speciation of metal pro-drugs that have broad applications across medicine.

Vanadium(V) and -(IV) complexes of anionic polysaccharides: Controlled release pharmaceutical formulations and models of vanadium biotransformation products.

Uncontrolled reactions in biological media are a main obstacle for clinical translation of V-based anti-diabetic or anti-cancer pro-drugs. We investigated the use of controlled-release pharmaceutical formulations to ameliorate this issue with a series of V(V) and (IV) complexes of anionic polysaccharides. Carboxymethyl cellulose, xanthan gum, or alginic acid formulations were prepared by the reactions of [VO4](3-) with one or two molar equivalents of biological reductants, L-ascorbic acid (AA) or L-cysteine (Cys), in the presence of excess polysaccharide at pH~7 or pH~4. XANES studies with the use of a previously developed library of model V(V), V(IV) and V(III) complexes showed that reactions in the presence of AA led mostly to the mixtures of five- and six-coordinate V(IV) species, while the reactions in the presence of Cys led predominantly to the mixtures of five- and six-coordinate V(V) species. The XANES spectra of some of these samples closely matched those reported previously for [VO4](3-) biotransformation products in isolated blood plasma, red blood cells, or cultured adipocytes, which supports the hypothesis that modified polysaccharides are major binders of V(V) and V(IV) in biological systems. Studies by EPR spectroscopy suggested predominant V(IV)-carboxylato binding in complexes with polysaccharides. One of the isolated products (a V(IV)-alginato complex) showed selective release of low-molecular-mass V species at pH~8, but not at pH~2, which makes it a promising lead for the development of V-containing formulations for oral administration that are stable in the stomach, but release the active ingredient in the intestines.

Biotransformations of Antidiabetic Vanadium Prodrugs in Mammalian Cells and Cell Culture Media: A XANES Spectroscopic Study.

The antidiabetic activities of vanadium(V) and -(IV) prodrugs are determined by their ability to release active species upon interactions with components of biological media. The first X-ray absorption spectroscopic study of the reactivity of typical vanadium (V) antidiabetics, vanadate ([V(V)O4](3-), A) and a vanadium(IV) bis(maltolato) complex (B), with mammalian cell cultures has been performed using HepG2 (human hepatoma), A549 (human lung carcinoma), and 3T3-L1 (mouse adipocytes and preadipocytes) cell lines, as well as the corresponding cell culture media. X-ray absorption near-edge structure data were analyzed using empirical correlations with a library of model vanadium(V), -(IV), and -(III) complexes. Both A and B ([V] = 1.0 mM) gradually converged into similar mixtures of predominantly five- and six-coordinate V(V) species (∼75% total V) in a cell culture medium within 24 h at 310 K. Speciation of V in intact HepG2 cells also changed with the incubation time (from ∼20% to ∼70% V(IV) of total V), but it was largely independent of the prodrug used (A or B) or of the predominant V oxidation state in the medium. Subcellular fractionation of A549 cells suggested that V(V) reduction to V(IV) occurred predominantly in the cytoplasm, while accumulation of V(V) in the nucleus was likely to have been facilitated by noncovalent bonding to histone proteins. The nuclear V(V) is likely to modulate the transcription process and to be ultimately related to cell death at high concentrations of V, which may be important in anticancer activities. Mature 3T3-L1 adipocytes (unlike for preadipocytes) showed a higher propensity to form V(IV) species, despite the prevalence of V(V) in the medium. The distinct V biochemistry in these cells is consistent with their crucial role in insulin-dependent glucose and fat metabolism and may also point to an endogenous role of V in adipocytes.

Localization of the Trace Elements Iron, Zinc and Selenium in Relation to Anatomical Structures in Bovine Ovaries by X-Ray Fluorescence Imaging.

X-ray fluorescence (XRF) was used to image 40 histological cross-sections of bovine ovaries (n=19), focusing on structures including: antral follicles at different stages of growth or atresia, corpora lutea at three stages of development (II-IV), and capillaries, arterioles, and other blood vessels. This method identified three key trace elements [iron (Fe), zinc (Zn), and selenium (Se)] within the ovarian tissue which appeared to be localized to specific structures. Owing to minimal preprocessing of the ovaries, important high-resolution information regarding the spatial distribution of these elements was obtained with elemental trends and colocalizations of Fe and Zn apparent, as well as the infrequent appearance of Se surrounding the antrum of large follicles, as previously reported. The ability to use synchrotron radiation to measure trace element distributions in bovine ovaries at such high resolution and over such large areas could have a significant impact on understanding the mechanisms of ovarian development. This research is intended to form a baseline study of healthy ovaries which can later be extended to disease states, thereby improving our current understanding of infertility and endocrine diseases involving the ovary.

Mechanisms of murine cerebral malaria: Multimodal imaging of altered cerebral metabolism and protein oxidation at hemorrhage sites.

Using a multimodal biospectroscopic approach, we settle several long-standing controversies over the molecular mechanisms that lead to brain damage in cerebral malaria, which is a major health concern in developing countries because of high levels of mortality and permanent brain damage. Our results provide the first conclusive evidence that important components of the pathology of cerebral malaria include peroxidative stress and protein oxidation within cerebellar gray matter, which are colocalized with elevated nonheme iron at the site of microhemorrhage. Such information could not be obtained previously from routine imaging methods, such as electron microscopy, fluorescence, and optical microscopy in combination with immunocytochemistry, or from bulk assays, where the level of spatial information is restricted to the minimum size of tissue that can be dissected. We describe the novel combination of chemical probe-free, multimodal imaging to quantify molecular markers of disturbed energy metabolism and peroxidative stress, which were used to provide new insights into understanding the pathogenesis of cerebral malaria. In addition to these mechanistic insights, the approach described acts as a template for the future use of multimodal biospectroscopy for understanding the molecular processes involved in a range of clinically important acute and chronic (neurodegenerative) brain diseases to improve treatment strategies.

XAS studies of Se speciation in selenite-fed rats.

The biological activity of selenium is dependent on its chemical form. Therefore, knowledge of Se chemistry in vivo is required for efficacious use of selenium compounds in disease prevention and treatment. Using X-ray absorption spectroscopy, Se speciation in the kidney, liver, heart, spleen, testis and red blood cells of rats fed control (∼0.3 ppm Se) or selenite-supplemented (1 ppm or 5 ppm Se) diets for 3 or 6 weeks, was investigated. X-ray absorption spectroscopy revealed the presence of Se-Se and Se-C species in the kidney and liver, and Se-S species in the kidney, but not the liver. X-ray absorption near edge structure (XANES) spectra showed that there was variation in speciation in the liver and kidneys, but Se speciation was much more uniform in the remaining organs. Using principal component analysis (PCA) to interpret the Se K-edge X-ray absorption spectra, we were able to directly compare the speciation of Se in two different models of selenite metabolism--human lung cancer cells and rat tissues. The effects of Se dose, tissue type and duration of diet on selenium speciation in rat tissues were investigated, and a relationship between the duration of the diet (3 weeks versus 6 weeks) and selenium speciation was observed.

Synthesis and biological evaluation of a class of mitochondrially-targeted gadolinium(III) agents.

A structure-activity relationship study of a library of novel bifunctional Gd(III) complexes covalently linked to arylphosphonium cations is reported. Such complexes have been designed for potential application in binary cancer therapies such as neutron capture therapy and photon activation therapy. A positive correlation was found between lipophilicity and cytotoxicity of the complexes. Mitochondria uptake was determined by means of inductively coupled plasma mass spectrometry (ICP-MS), and Gd uptake was determined by means of quantification using synchrotron X-ray fluorescence (XRF) imaging. A negative correlation between lipophilicity and tumour selectivity of the Gd(III) complexes was demonstrated. This study highlights the delicate balance required to minimise in vitro cytotoxicity and optimise in vitro tumour selectivity and mitochondrial localisation for this new class of mitochondrially-targeted binary therapy agents. We also report the highest in vitro tumour selectivity for any Gd agent reported to date, with a T/N (tumour/normal cell) ratio of up to 23.5±6.6.

Solid-state structural studies of chromium(III) nicotinato nutritional supplements.

While Cr(III) dietary supplements are widely consumed, some commercial supplements have yet to be structurally characterized. X-ray absorption spectroscopy and other spectroscopic methods were used to characterize Cr(III) nicotinato nutritional supplements that have long been used in complementary medicine. Different ratios of nicotinic acid and CrCl3·6H2O (trans-[CrCl2(OH2)4]Cl·2H2O) at different pH values gave a range of products. The local structures of Cr(III) nicotinato complexes obtained at pH 7 and of the patented complex were characterized by performing multiple-scattering analysis of their EXAFS spectra as well as EPR, UV-vis, and IR spectroscopies. For the first time, these complexes have been definitively characterized as nicotinato-bridged polymers of dihydroxido-bridged dinuclear Cr(III) cores. In the patented complex used in commercial preparations, each Cr is octahedral with an additional terminal O-bound nicotinato ligand, two bridging nicotinato (one O and one N bound), and an aqua ligand. The other species also have two or three bridging nicotinato ligands and an aqua and, in some cases, a terminal hydroxido ligand, which is dependent upon the stoichiometry of the reactants and the pH value of the solution in which they are prepared.

Reactivity-activity relationships of oral anti-diabetic vanadium complexes in gastrointestinal media: an X-ray absorption spectroscopic study.

The reactions of oral V(V/IV) anti-diabetic drugs within the gastrointestinal environment (particularly in the presence of food) are a crucial factor that affects their biological activities, but to date these have been poorly understood. In order to build up reactivity-activity relationships, the first detailed study of the reactivities of typical V-based anti-diabetics, Na3V(V)O4 (A), [V(IV)O(OH2)5](SO4) (B), [V(IV)O(ma)2] (C, ma = maltolato(-)) and (NH4)[V(V)(O)2(dipic)] (D, dipic = pyridine-2,5-dicarboxylato(2-)) with simulated gastrointestinal (GI) media in the presence or absence of food components has been performed by the use of XANES (X-ray absorption near edge structure) spectroscopy. Changes in speciation under conditions that simulate interactions in the GI tract have been discerned using correlations of XANES parameters that were based on a library of model V(V), V(IV), and V(III) complexes for preliminary assessment of the oxidation states and coordination numbers. More detailed speciation analyses were performed using multiple linear regression fits of XANES from the model complexes to XANES obtained from the reaction products from interactions with the GI media. Compounds B and D were relatively stable in the gastric environment (pH ∼ 2) in the absence of food, while C was mostly dissociated, and A was converted to [V10O28](6-). Sequential gastric and intestinal digestion in the absence of food converted A, B and D to poorly absorbed tetrahedral vanadates, while C formed five- or six-coordinate V(V) species where the maltolato ligands were likely to be partially retained. XANES obtained from gastric digestion of A-D in the presence of typical food components converged to that of a mixture of V(IV)-aqua, V(IV)-amino acid and V(III)-aqua complexes. Subsequent intestinal digestion led predominantly to V(IV) complexes that were assigned as citrato or complexes with 2-hydroxyacidato donor groups from other organic compounds, including certain carbohydrates. The absence of strong reductants (such as ascorbate) in the food increased the V(V) component in gastrointestinal digestion products. These results can be used to predict the oral bioavailability of various types of V(V/IV) anti-diabetics, and the effects of taking such drugs with food.

Speciation of copper in a range of food types by X-ray absorption spectroscopy.

Copper (Cu) is an essential element and the effects of diets deficient in it are well established. However, the effects of long-term high copper intake are less clear. The chemical form of copper from food sources and its resultant bioavailability is a potentially important factor in its biological activity. X-ray Absorption Near-Edge Structure (XANES) was used to determine the chemical forms of Cu in a range of foods that would make significant contributions to total copper absorption in a standard diet, as well as a chlorinated tap water sample. Analysis of the Cu K-edge XANES spectra suggested that Cu existed in both Cu(I) and Cu(II) forms, with the following five model compounds: Cu(I) acetate; Cu(II) acetate; Cu(I)-glutathione; Cu(I)-cysteine; and, Cu(II)-histidine being fitted to the sample spectra. This research suggested that the absorption of dietary copper could vary markedly dependent on the types of food consumed and the different bioavailability of the Cu species they contain.

The Fe-heme structure of met-indoleamine 2,3-dioxygenase-2 determined by X-ray absorption fine structure.

Multiple-scattering (MS) analysis of EXAFS data on met-indoleamine 2,3-dioxygenase-2 (IDO2) and analysis of XANES have provided the first direct structural information about the axial donor ligands of the iron center for this recently discovered protein. At 10K, it exists in a low-spin bis(His) form with Fe-Np(av)=1.97Å, the Fe-NIm bond lengths of 2.11Å and 2.05Å, which is in equilibrium with a high-spin form at room temperature. The bond distances in the low-spin form are consistent with other low-spin hemeproteins, as is the XANES spectrum, which is closer to that of the low-spin met-Lb than that of the high-spin met-Mb. The potential physiological role of this spin equilibrium is discussed.

XAS and XFM studies of selenium and copper speciation and distribution in the kidneys of selenite-supplemented rats.

Dietary selenium has been implicated in the prevention of cancer and other diseases, but its safety and efficacy is dependent on the supplemented form and its metabolites. In this study, X-ray absorption spectroscopy (XAS) and X-ray fluorescence microscopy (XFM) have been used to investigate the speciation and distribution of Se and Cu in vivo. In kidneys isolated from rats fed a diet containing 5 ppm Se as selenite for 3 weeks, Se levels increased 5-fold. XFM revealed a strong correlation between the distribution of Se and the distribution of Cu in the kidney, a phenomenon that has previously been observed in cell culture (Weekley et al., JBIC, J. Biol. Inorg. Chem., 2014, DOI: 10.1007/s00775-014-1113-x). However, X-ray absorption spectra suggest that most of the Se in the kidney is found as Se-Se species, rather than Cu-bound, and that most of the Cu is bound to S and N, presumably to amino acid residues in proteins. Furthermore, SOD1 expression did not change in response to the high Se diet. We cannot rule out the possibility of some Cu-Se bonding in the tissues, but our results suggest mechanisms other than the formation of Cu-Se species and SOD1 upregulation are responsible for the highly correlated distributions of Se and Cu in the kidneys of rats fed high selenite diets.

Parkinson's disease-linked human PARK9/ATP13A2 maintains zinc homeostasis and promotes α-Synuclein externalization via exosomes.

α-Synuclein plays a central causative role in Parkinson's disease (PD). Increased expression of the P-type ATPase ion pump PARK9/ATP13A2 suppresses α-Synuclein toxicity in primary neurons. Our data indicate that ATP13A2 encodes a zinc pump; neurospheres from a compound heterozygous ATP13A2(-/-) patient and ATP13A2 knockdown cells are sensitive to zinc, whereas ATP13A2 over-expression in primary neurons confers zinc resistance. Reduced ATP13A2 expression significantly decreased vesicular zinc levels, indicating ATP13A2 facilitates transport of zinc into membrane-bound compartments or vesicles. Endogenous ATP13A2 localized to multi-vesicular bodies (MVBs), a late endosomal compartment located at the convergence point of the endosomal and autophagic pathways. Dysfunction in MVBs can cause a range of detrimental effects including lysosomal dysfunction and impaired delivery of endocytosed proteins/autophagy cargo to the lysosome, both of which have been observed in cells with reduced ATP13A2 function. MVBs also serve as the source of intra-luminal nanovesicles released extracellularly as exosomes that can contain a range of cargoes including α-Synuclein. Elevated ATP13A2 expression reduced intracellular α-Synuclein levels and increased α-Synuclein externalization in exosomes >3-fold whereas ATP13A2 knockdown decreased α-Synuclein externalization. An increased export of exosome-associated α-Synuclein may explain why surviving neurons of the substantia nigra pars compacta in sporadic PD patients were observed to over-express ATP13A2. We propose ATP13A2's modulation of zinc levels in MVBs can regulate the biogenesis of exosomes capable of containing α-Synuclein. Our data indicate that ATP13A2 is the first PD-associated gene involved in exosome biogenesis and indicates a potential neuroprotective role of exosomes in PD.

High mitochondrial accumulation of new gadolinium(III) agents within tumour cells.

The first bifunctional Gd(III) complexes covalently bound to arylphosphonium cations and the first tumour-cell selective mitochondrial agents designed for potential application in binary cancer therapies are reported. The highest in vitro cellular uptake for any Gd complex reported to date is described, with levels exceeding 10(10) Gd atoms per tumour cell.

Key role of bismuth in the magnetoelastic transitions of Ba3BiIr2O9 and Ba3BiRu2O9 as revealed by chemical doping.

The key role played by bismuth in an average intermediate oxidation state in the magnetoelastic spin-gap compounds Ba3BiRu2O9 and Ba3BiIr2O9 has been confirmed by systematically replacing bismuth with La(3+) and Ce(4+). Through a combination of powder diffraction (neutron and synchrotron), X-ray absorption spectroscopy, and magnetic properties measurements, we show that Ru/Ir cations in Ba3BiRu2O9 and Ba3BiIr2O9 have oxidation states between +4 and +4.5, suggesting that Bi cations exist in an unusual average oxidation state intermediate between the conventional +3 and +5 states (which is confirmed by the Bi L3-edge spectrum of Ba3BiRu2O9). Precise measurements of lattice parameters from synchrotron diffraction are consistent with the presence of intermediate oxidation state bismuth cations throughout the doping ranges. We find that relatively small amounts of doping (∼10 at%) on the bismuth site suppress and then completely eliminate the sharp structural and magnetic transitions observed in pure Ba3BiRu2O9 and Ba3BiIr2O9, strongly suggesting that the unstable electronic state of bismuth plays a critical role in the behavior of these materials.

Influence of equatorial and axial carboxylato ligands on the kinetic inertness of platinum(IV) complexes in the presence of ascorbate and cysteine and within DLD-1 cancer cells.

The rapid and premature reduction of platinum(IV) complexes in vivo is a significant impediment to these complexes being successfully employed as anticancer prodrugs. This study investigates the influence of the platinum(IV) coordination sphere on the ease of reduction of the platinum center in various biological contexts. In the presence of the biological reductants, ascorbate and cysteine, platinum(IV) complexes with dicarboxylato equatorial ligands were observed to exhibit lower reduction potentials and slower reduction rates than analogous platinum(IV) complexes with dichlorido equatorial ligands. Diaminetetracarboxylatoplatinum(IV) complexes exhibited unusually long half-lives in the presence of excess reductants; however, the complexes exhibited moderate potency in vitro, indicative of rapid reduction within the intracellular environment. By use of XANES spectroscopy, trans-[Pt(OAc)2(ox)(en)] and trans-[PtCl2(OAc)2(en)] were observed to be reduced at a similar rate within DLD-1 cancer cells. This large variability in kinetic inertness of diaminetetracarboxylatoplatinum(IV) complexes in different biological contexts has significant implications for the design of platinum(IV) prodrugs.

Chemically synthesised atomically precise gold clusters deposited and activated on titania. Part II.

Synchrotron XPS was used to investigate a series of chemically synthesised, atomically precise gold clusters Au(n)(PPh3)y (n = 8, 9 and 101, y depending on the cluster size) immobilized on anatase (titania) nanoparticles. Effects of post-deposition treatments were investigated by comparison of untreated samples with analogues that have been heat treated at 200 °C in O2, or in O2 followed by H2 atmosphere. XPS data shows that the phosphine ligands are oxidised upon heat treatment in O2. From the position of the Au 4f(7/2) peak it can be concluded that the clusters partially agglomerate immediately upon deposition. Heating in oxygen, and subsequently in hydrogen, leads to further agglomeration of the gold clusters. It is found that the pre-treatment plays a crucial role in the removal of ligands and agglomeration of the clusters.

X-ray fluorescence imaging of single human cancer cells reveals that the N-heterocyclic ligands of iodinated analogues of ruthenium anticancer drugs remain coordinated after cellular uptake.

Analogues of KP1019 containing iodinated indazole ligands were prepared to investigate the biological fate of the Ru-N-heterocycle bond in this class of anticancer agents. The new complexes, 5-iodoindazolium trans-tetrachloridobis(5-iodoindazole)ruthen(III)ate (1) and 5-iodoindazolium trans-tetrachlorido(dimethyl sulfoxide)(5-iodoindazole)ruthen(III)ate (3), were characterized by elemental analysis, mass spectrometry and UV-vis spectrophotometry. Tetramethylammonium salts of these complexes (2 and 4) were synthesized and characterized in a similar manner. Half-maximum inhibitory concentrations of 2 and 4 with regard to A549 cells at 24 h were determined on the basis of the dose-response curves derived from real-time cell adhesion impedance measurements and were shown to be in the same range as those determined for KP1019 and NAMI-A using the same method. X-ray fluorescence imaging of single cultured A549 cells treated with 2 or 4 showed that, in both cases, the distribution of ruthenium and iodine was identical, indicating that the Ru-N bonds in the anionic complexes remained intact after incubation in culture medium and subsequent cellular uptake and processing.