David H. Petering

The role of redox-active metals in the mechanism of action of bleomycin

Belomycin is a glycopeptide antibiotic routinely used to treat human cancer. It is commonly thought to exert its biological effects as a metallodrug, which oxidatively damages DNA. This review systematically examines the properties of bleomycin which contribute to its reaction with DNA in vitro and may be important in the breakage of DNA in cells. Because strand cleavage results from the reductive activation of dioxygen by metallobleomycins, the mechanism of this process is given primary attention. Current understanding of the structures of the coordination sites of various metallobleomycins, their thermodynamic stabilities, their propensity to form adduct species, and their properties in ligand substitution reactions provide a foundation for consideration of the chemistry of dioxygen activation as well as a basis for thinking about the metal-speciation of bleomycin in biological systems. Oxidation-reduction pathways of iron-bleomycin, copper-bleomycin, and other metal-bleomycin species with O2 are then examined, including information on photochemical activation. With this background, structural and thermodynamic features of the binding interactions of DNA with bleomycin, its metal complexes, and adducts of metallobleomycins are reviewed. Then, the DNA cleavage reaction involving iron-bleomycin is scrutinized on the basis of the preceding discussion. Particular emphasis is placed on the constraints which the presence of DNA places on the mechanism of dioxygen activation. Similarly, the reactions of other metalloforms of bleomycin with DNA are reviewed. The last topic is an analysis of current understanding of the relationship of bleomycin-induced cellular DNA damage to the model developed above, which has evolved on the basis of chemical experimentation. Consideration is given to the question of the importance of DNA strand breakage caused by bleomycin for the mechanism of cytotoxic activity of the drug.

Interaction of 2-formylpyridine thiosemicarbazonato copper (II) with ehrlich ascites tumor cells

Abstract The reaction of 2-formylpyridine thiosemicarbazonato copper (II) with Ehrlich cells was studied. The complex was readily taken up and bound by cells. Little efflux of copper was observed. Electron paramagnetic resonance studies were consistent with the complex having bound to thiol groups furnished by glutathione. The chelate was quite stable in cells, having a first-order rate constant for reaction of 4.5 × 10−5 sec−1. However, this was apparently a reflection of a steady-state redox process in which thiols were being oxidized and oxygen reduced. A model reaction between the complex and reduced glutathione showed that the complex reached a steady state as oxygen was consumed in the process. Cellular DNA synthesis was inhibited at low concentration by this copper chelate, whereas RNA synthesis was much less sensitive. Although isolated mitochondria were inhibited by the complex, any cellular reaction was obscured by the rapid oxygen reduction that occurred in the thiol oxidation process.

Inhibition of oxidative phosphorylation in tumor cells and mitochondria by daunomycin and adriamycin.

Abstract The binding of copper to daunomycin has been investigated. It is concluded that the strenght of binding is not large enough for the 1:2 copper-daunomycin complex to exist in vivo . Daunomycin and adriamycin inhibit glutamate- and pyruvate-malate-dependent oxidative phosphorylation in bovine heart mitochondria and adriamycin uncouples this process as well. No inhibition of Ehrlich ascites tumor cell or mitochondrial respiration by daunomycin is observed at concentrations much larger than those used for heart. In conjunction with the work of others, these results suggest a role for the inhibition of oxidative phosphorylation in the cardiac toxicity of these anthracycline drugs.

Basal metallothionein in tumors: Widespread presence of apoprotein

A survey has been conducted of solid and ascites tumors from mice and solid tumors in rats for the presence of metallothionein or metallothionein-like protein. In most tumors, a positive identification was made on the basis of Sephadex G-75 and HPLC-DEAE chromatography followed by competitive radioimmunoassay for metallothionein. Apometallothionein was revealed in a number of tumors for the first time by comparing the Sephadex G-75 chromatographic profiles of Zn in native cytosol and Cd in cytosol incubated briefly with CdCl2 to saturate free binding sites on the protein before Sephadex G-75 chromatography. In two cases unsaturation of metallothionein was correlated with a lack of zinc in the ascites fluid which supplies the tumor with zinc.

Structure-reactivity relationships of metallothionein, a unique metal-binding protein

Abstract Metallothionein is an intensively studied protein, which binds a variety of essential, toxic, and pharmacologically active metals, including Zn, Cd, Au, and Pt. Until recently, attention focused primarily on its biological properties and static features of its chemistry. It is now apparent that metallothionein is a remarkably reactive protein in metal exchange and ligand substitution reactions and in its interactions with a number of electrophilic compounds, which are both metallic and organic in nature. This unique behavior finds its basis in the dynamic character of its metal—ligand structure, which is sensitively probed by 113Cd NMR techniques. In an effort to relate the chemistry of metallothionein to its cellular activities, it is shown that the kinetic reactivity of the metal binding sites of metallothionein distinguishes it from other typical metalloproteins involved in enzyme catalysis. The rich inorganic chemistry of this structure is clearly important for some of its known functions and...

Enhanced Expressions of Sodium-Glucose Cotransporters in the Kidneys of Diabetic Zucker Rats

Diabetes-mediated changes in mRNA expressions of kidney glucose transporters SGLT1 and SGLT2 were investigated in Zucker rats. SGLTs expressions in pre-diabetic obese rats were similar to leans. SGLT1 and SGLT2 levels in diabetic obese rats were 1.6 (P<0.03) and 4.8 (P<0.002) folds higher than age-matched leans, respectively.

Binding of cis-dichlorodiammine platinum(II) to metallothionein in Ehrlich cells.

The antitumor agent, cis-dichlorodiammine Pt(II), is cytotoxic to Ehrlich cells in culture. These cells contain a substantial amount of metallothionein in the absence of inducers of the protein. At concentrations of drug which cause 60% inhibition of cell proliferation, most of the platinum is found in the cytosol. Of this about 30% is bound in the metallothionein fraction. Isolated rat liver metallothionein reacts slowly with hydrolyzed cis-dichlorodiammine Pt(II). Thus, metallothionein is a major cellular site of binding of the platinum complex at concentrations which inhibit tumor growth.

Mammalian metallothionein in toxicology, cancer, and cancer chemotherapy

The present paper centers on mammalian metallothionein 1 and 2 in relationship to cell and tissue injury beginning with its reaction with Cd2+ and then considering its role in the toxicology and chemotherapy of both metals and non-metal electrophiles and oxidants. Intertwined is a consideration of MTs role in tumor cell Zn2+ metabolism. The paper updates and expands on our recent review by Petering et al. (Met Ions Life Sci 5:353–398, 2009).

A Fluorescence Method for Measurement of Glucose Transport in Kidney Cells

BACKGROUND Diabetes may alter renal glucose reabsorption by sodium (Na(+))-dependent glucose transporters (SGLTs). Radiolabeled substrates are commonly used for in vitro measurements of SGLT activity in kidney cells. We optimized a method to measure glucose uptake using a fluorescent substrate, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). METHODS Uptake buffers for 2-NBDG were the same as for (14)C-labeled α-methyl-d-glucopyranoside ([(14)C]AMG). Cell lysis buffer was optimized for fluorescence of 2-NBDG and Hoechst DNA stain. Uptake was performed on cultures of primary mouse kidney cells (PMKCs), the LLC-PK(1) proximal tubule cell line, or COS-7 cells transiently overexpressing mouse SGLT1 or SGLT2 by incubating cells at 37°C in buffer containing 50-200 μM 2-NBDG. Microscopy was performed to visualize uptake in intact cells, while a fluorescence microplate reader was used to measure intracellular concentration of 2-NBDG ([2-NBDG](i)) in cell homogenates. RESULTS Fluorescent cells were observed in cultures of PMKCs and LLC-PK(1) cells exposed to 2-NBDG in the presence or absence of Na(+). In LLC-PK(1) cells, 2-NBDG transport in the presence of Na(+) had a maximum rate of 0.05 nmol/min/μg of DNA. In these cells, Na(+)-independent uptake of 2-NBDG was blocked with the GLUT inhibitor, cytochalasin B. The Na(+)-dependent uptake of 2-NBDG decreased in response to co-exposure to the SGLT substrate, AMG, and it could be blocked with the SGLT inhibitor, phlorizin. Immunocytochemistry showed overexpression of SGLT1 and SGLT2 in COS-7 cells, in which, in the presence of Na(+), [2-NBDG](i) was fivefold higher than in controls. CONCLUSION Glucose transport in cultured kidney cells can be measured with the fluorescence method described in this study.

Apo-Metallothionein Emerging as a Major Player in the Cellular Activities of Metallothionein

Observations of apo-metallothlonein (apo-MT) have been made under a variety of physiologic circumstances, including zinc deficiency in cell culture and in rodents, cellular induction of MT by dexamethasone with concurrent Zn deficiency, a variety of tumors under normal Zn conditions, MT induction by Zn and Bi citrate, induction of hepatic MT after tumor cell Injection into nude mice, and overexpression of cardiac MT in MT transgenic mice. Experiments demonstrating both the lability of Zn and Cu bound to MT and the cellular stability of apo-MT are described to help rationalize the widespread presence of this metal-depleted species. Finally, comparative in vitro and cellular experiments examined the relative reactivity of Zn- and apo-MT with nitric oxide species, showing that apo-MT is much more reactive chemically and that in cells it may be a principal reactive species within the MT pool.