Hugh H. Harris

Production of Se-methylselenocysteine in transgenic plants expressing selenocysteine methyltransferase.

BackgroundIt has become increasingly evident that dietary Se plays a significant role in reducing the incidence of lung, colorectal and prostate cancer in humans. Different forms of Se vary in their chemopreventative efficacy, with Se-methylselenocysteine being one of the most potent. Interestingly, the Se accumulating plant Astragalus bisulcatus (Two-grooved poison vetch) contains up to 0.6% of its shoot dry weight as Se-methylselenocysteine. The ability of this Se accumulator to biosynthesize Se-methylselenocysteine provides a critical metabolic shunt that prevents selenocysteine and selenomethionine from entering the protein biosynthetic machinery. Such a metabolic shunt has been proposed to be vital for Se tolerance in A. bisulcatus. Utilization of this mechanism in other plants may provide a possible avenue for the genetic engineering of Se tolerance in plants ideally suited for the phytoremediation of Se contaminated land. Here, we describe the overexpression of a selenocysteine methyltransferase from A. bisulcatus to engineer Se-methylselenocysteine metabolism in the Se non-accumulator Arabidopsis thaliana (Thale cress).ResultsBy over producing the A. bisulcatus enzyme selenocysteine methyltransferase in A. thaliana, we have introduced a novel biosynthetic ability that allows the non-accumulator to accumulate Se-methylselenocysteine and γ-glutamylmethylselenocysteine in shoots. The biosynthesis of Se-methylselenocysteine in A. thaliana also confers significantly increased selenite tolerance and foliar Se accumulation.ConclusionThese results demonstrate the feasibility of developing transgenic plant-based production of Se-methylselenocysteine, as well as bioengineering selenite resistance in plants. Selenite resistance is the first step in engineering plants that are resistant to selenate, the predominant form of Se in the environment.

Intracellular mapping of the distribution of metals derived from the antitumor metallocenes

The intracellular distribution of transition metals in V79 Chinese hamster lung cells treated with subtoxic doses of the organometallic anticancer complexes Cp2MCl2, where Cp is η5 -cyclopentadienyl and M is Mo, Nb, Ti, or V, has been studied by synchrotron-based X-ray fluorescence (XRF). While significantly higher concentrations of Mo and Nb were found in treated cells compared with control cells, distinct differences in the cellular distribution of each metal were observed. Analysis of thin sections of cells was consistent with some localization of Mo in the nucleus. Studies with a noncytotoxic thiol derivative of molybdocene dichloride showed an uneven distribution of Mo in the cells. For comparison, the low levels of Ti and V in cells treated with the more toxic titanocene and vanadocene complexes, respectively, resulted in metal concentrations at the detection limit of XRF. The results agree with independent chemical studies that have concluded that the biological chemistry of each of the metallocene dihalides is unique.

Studies of glutathione transferase P1-1 bound to a platinum(IV)-based anticancer compound reveal the molecular basis of its activation.

Platinum-based cancer drugs, such as cisplatin, are highly effective chemotherapeutic agents used extensively for the treatment of solid tumors. However, their effectiveness is limited by drug resistance, which, in some cancers, has been associated with an overexpression of pi class glutathione S-transferase (GST P1-1), an important enzyme in the mercapturic acid detoxification pathway. Ethacraplatin (EA-CPT), a trans-Pt(IV) carboxylate complex containing ethacrynate ligands, was designed as a platinum cancer metallodrug that could also target cytosolic GST enzymes. We previously reported that EA-CPT was an excellent inhibitor of GST activity in live mammalian cells compared to either cisplatin or ethacrynic acid. In order to understand the nature of the drug-protein interactions between EA-CPT and GST P1-1, and to obtain mechanistic insights at a molecular level, structural and biochemical investigations were carried out, supported by molecular modeling analysis using quantum mechanical/molecular mechanical methods. The results suggest that EA-CPT preferentially docks at the dimer interface at GST P1-1 and subsequent interaction with the enzyme resulted in docking of the ethacrynate ligands at both active sites (in the H-sites), with the Pt moiety remaining bound at the dimer interface. The activation of the inhibitor by its target enzyme and covalent binding accounts for the strong and irreversible inhibition of enzymatic activity by the platinum complex.

Multiple protective activities of neuroglobin in cultured neuronal cells exposed to hypoxia re‐oxygenation injury

Oxidative stress is associated with the pathology of acute and chronic neurodegenerative disease. We have cloned a human neuroglobin (Nb) construct and over‐expressed this protein in cultured human neuronal cells to assess whether Nb ameliorates the cellular response to experimental hypoxia‐reoxygenation (H/R) injury. Parental cells transfected with a blank (pDEST40) vector responded to H/R injury with a significant decrease in cellular ATP at 5 and 24 h after insult. This was coupled with increases in the cytosolic Ca2+, and the transition metals iron (Fe), copper (Cu), and zinc (Zn) within the cell body, as monitored simultaneously using X‐ray fluorescence microprobe imaging. Parental cell viability decreased over the same time period with a ∼4 to 5‐fold increase in cell death (maximum ∼25%) matched by an increase in caspase 3/7 activation (peaking at a 15‐fold increase after 24 h) and condensation of β‐actin along axonal processes. Over‐expression of Nb inhibited ATP loss and except for significant decreases in the sulfur (S), chlorine (Cl), potassium (K) and Ca2+ contents, maintained cellular ion homeostasis after H/R insult. This resulted in increased cell viability, significantly diminished caspase activation and maintenance of the β‐actin cytoskeletal structure and receptor‐mediated endocytosis. These data indicate that bolstering the cellular content of Nb inhibits neuronal cell dysfunction promoted by H/R insult through multiple protective actions including: (i) maintenance of cellular bioenergetics; (ii) inhibition of Ca2+ influx; (iii) a reduction in cellular uptake of Fe, Cu and Zn at the expense of S, Cl and K; and (iv) an enhancement of cell viability through inhibiting necrosis and apoptosis.

Copper speciation and isotopic fractionation in plants: uptake and translocation mechanisms

The fractionation of stable copper (Cu) isotopes during uptake into plant roots and translocation to shoots can provide information on Cu acquisition mechanisms. Isotope fractionation ((65) Cu/(63) Cu) and intact tissue speciation techniques (X-ray absorption spectroscopy, XAS) were used to examine the uptake, translocation and speciation of Cu in strategy I (tomato-Solanum lycopersicum) and strategy II (oat-Avena sativa) plant species. Plants were grown in controlled solution cultures, under varied iron (Fe) conditions, to test whether the stimulation of Fe-acquiring mechanisms can affect Cu uptake in plants. Isotopically light Cu was preferentially incorporated into tomatoes (Δ(65) Cu(whole plant-solution ) = c. -1‰), whereas oats showed minimal isotopic fractionation, with no effect of Fe supply in either species. The heavier isotope was preferentially translocated to shoots in tomato, whereas oat plants showed no significant fractionation during translocation. The majority of Cu in the roots and leaves of both species existed as sulfur-coordinated Cu(I) species resembling glutathione/cysteine-rich proteins. The presence of isotopically light Cu in tomatoes is attributed to a reductive uptake mechanism, and the isotopic shifts within various tissues are attributed to redox cycling during translocation. The lack of isotopic discrimination in oat plants suggests that Cu uptake and translocation are not redox selective.

Metabolism of selenite in human lung cancer cells: X-ray absorption and fluorescence studies

Selenite is an inorganic form of selenium that has a cytotoxic effect against several human cancer cell lines: one or more selenite metabolites are considered to be responsible for its toxicity. X-ray absorption spectroscopy was used to monitor Se speciation in A549 human lung cancer cells incubated with selenite over 72 h. As anticipated, selenodiglutathione and elemental Se both comprised a large proportion of Se in the cells between 4 and 72 h after treatment, which is in accordance with the reductive metabolism of selenite in the presence of glutathione and glutathione reductase/NADPH system. Selenocystine was also present in the cells but was only detected as a significant component between 24 and 48 h concomitant with a decrease in the proportion of selenocysteine and the viability of the cells. The change in speciation from the selenol, selenocysteine, to the diselenide, selenocystine, is indicative of a change in the redox status of the cells to a more oxidizing environment, likely brought about by metabolites of selenite. X-ray fluorescence microscopy of single cells treated with selenite for 24 h revealed a punctate distribution of Se in the cytoplasm. The accumulation of Se was associated with a greater than 2-fold increase in Cu, which was colocalized with Se. Selenium K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy revealed Se-Se and Se-S bonding, but not Se-Cu bonding, despite the spatial association of Se and Cu. Microprobe X-ray absorption near-edge structure spectroscopy (μ-XANES) showed that the highly localized Se species was mostly elemental Se.

Uptake, Distribution, and Speciation of Selenoamino Acids by Human Cancer Cells: X-ray Absorption and Fluorescence Methods

Selenium compounds exhibit chemopreventative properties at supranutritional doses, but the efficacy of selenium supplementation in cancer prevention is dependent on the chemical speciation of the selenium supplement and its metabolites. The uptake, speciation, and distribution of the common selenoamino acid supplements, selenomethionine (SeMet) and Se-methylselenocysteine (MeSeCys), in A549 human lung cancer cells were investigated using X-ray absorption and fluorescence spectroscopies. X-ray absorption spectroscopy of bulk cell pellets treated with the selenoamino acids for 24 h showed that while selenium was found exclusively in carbon-bound forms in SeMet-treated cells, a diselenide component was identified in MeSeCys-treated cells in addition to the carbon-bound selenium species. X-ray fluorescence microscopy of single cells showed that selenium accumulated with sulfur in the perinuclear region of SeMet-treated cells after 24 h, but microprobe selenium X-ray absorption near-edge spectroscopy in this region indicated that selenium was carbon-bound rather than sulfur-bound. X-ray absorption and X-ray fluorescence studies both showed that the selenium content of MeSeCys-treated cells was much lower than that of SeMet-treated cells. Selenium was distributed homogeneously throughout the MeSeCys-treated cells.

Selenium metabolism in cancer cells: The combined application of XAS and XFM techniques to the problem of selenium speciation in biological systems

Determining the speciation of selenium in vivo is crucial to understanding the biological activity of this essential element, which is a popular dietary supplement due to its anti-cancer properties. Hyphenated techniques that combine separation and detection methods are traditionally and effectively used in selenium speciation analysis, but require extensive sample preparation that may affect speciation. Synchrotron-based X-ray absorption and fluorescence techniques offer an alternative approach to selenium speciation analysis that requires minimal sample preparation. We present a brief summary of some key HPLC-ICP-MS and ESI-MS/MS studies of the speciation of selenium in cells and rat tissues. We review the results of a top-down approach to selenium speciation in human lung cancer cells that aims to link the speciation and distribution of selenium to its biological activity using a combination of X-ray absorption spectroscopy (XAS) and X-ray fluorescence microscopy (XFM). The results of this approach highlight the distinct fates of selenomethionine, methylselenocysteine and selenite in terms of their speciation and distribution within cells: organic selenium metabolites were widely distributed throughout the cells, whereas inorganic selenium metabolites were compartmentalized and associated with copper. New data from the XFM mapping of electrophoretically-separated cell lysates show the distribution of selenium in the proteins of selenomethionine-treated cells. Future applications of this top-down approach are discussed.

Selective Aggregation of a Platinum-Gadolinium Complex Within a Tumor-Cell Nucleus**

Gadolinium(III) complexes are widely used in magnetic resonance imaging (MRI) as water relaxation agents to improve image contrast. Therapeutic gadolinium-containing agents are also known in which the metal complex enhances tumor response to chemotherapeutics such as cisplatin, or, more commonly, acts as a radiosensitizer in the treatment of diseases, such as cancer. Gadoliniummay also play an important role in therapeutic techniques, such as synchrotron stereotactic radiotherapy (SSR), in which the selective delivery of gadolinium to the cell nucleus would significantly enhance the efficacy of the treatment. Indeed, De Stasio and co-workers have demonstrated that motexafinGd, a gadolinium(III) complex of the pentadentate texaphyrin ligand, was accumulated by approximately 90% of glioblastoma cell nuclei in vitro, and its potential exploitation as a GdSSR agent is warranted. In recent years, gadolinium complexes have also been explored as potential agents in an experimental anti-cancer treatment known as gadolinium neutron-capture therapy (GdNCT), which is closely related to the well-established boron neutron-capture therapy (BNCT). GdNCTutilizes the non-radioactive Gd isotope (natural abundance 15.7%) in a highly effective thermal neutron-capture reaction to destroy tumor cells. Gd possesses the largest effective nuclear cross-section of all naturally-occurring elements (2.55 10 barns); this value is approximately 66 times greater than that of the B nucleus. Gd undergoes neutron capture to give the products of internal conversion, accompanying Auger and Coster–Kronig (ACK) electron emission and 7.94 MeVof energy. However, the very limited range of ACK electrons means that the gadolinium complex must be localized in close proximity to critical cellular components, such as the cell nucleus, if the neutron capture reaction is to be exploited effectively. The use of gadolinium(III) complexes as potential GdNCT delivery agents to brain tumors has been described, although the feasibility of using archetypal MRI agents such as Gd-DTPA (DTPA= diethylenetriaminepentaacetic acid) in a clinical context for GdNCT is considered unlikely owing to the limited number of tumor-cell nuclei that have been shown to incorporate gadolinium. Indeed, the number of gadolinium compounds reported to date that have a capacity to aggregate selectively in tumor-cell nuclei, for example, is very limited, and the search for new types of gadolinium(III) complexes with high nuclear affinity has recently been proposed. Herein, we present a new Pt-Gd complex that can effectively target the nuclei of tumor cells by means of a functionalized dtpa ligand linked to two {Pt(terpy)} (terpy= 2,2’:6’,2’’-terpyridine) units that have the capacity to bind DNA in an intercalative manner. Based on prior work with analogous Pt–Ln complexes (Ln=La, Nd, Eu), which were designed to act as luminescent probes for DNA recognition, we reasoned that the related Pt-Gd species 1 would have the capacity to deliver gadolinium to this important biomolecule. In this work, we report the first unequivocal example of gadolinium delivery to a tumor-cell nucleus by a platinum complex. Complex 1was prepared in good yield by a similar manner to that described for the analogous Pt-Ln species (Ln=La, Nd, Eu; Scheme 1). The convenient one-pot synthesis of 1 demonstrates the high affinity of the soft Pt and hard Gd cations for the soft and hard Lewis bases (S and N/O, respectively) that are present in the functionalized DTPA ligand. The purple Pt-Gd complex has excellent solubility and stability in aqueous solution, and no evidence was found for the loss of Pt or Gd ions from 1, even after 24 h of standing in a buffered pH 7.4 solution at room temperature. Preliminary DNA thermal denaturation (DNA melting) experiments involving calf-thymus DNAwere performed on 1 at pH 7.4 (Supporting Information, Figure S1). There exists a significant difference in the melting temperatures between the freeand drug-treated DNA samples (DTm= 4.5 0.5 8C), [*] Dr. E. L. Crossley, Dr. J. B. Aitken, Prof. L. M. Rendina School of Chemistry, The University of Sydney Sydney, NSW 2006 (Australia) Fax: (+61)2-9351-3329 E-mail: rendina@chem.usyd.edu.au

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.