Carolyn T. Dillon

Copper complexes of non-steroidal anti-inflammatory drugs: an opportunity yet to be realized

The proposed curative properties of Cu-based non-steroidal anti-inflammatory drugs (NSAIDs) have led to the development of numerous Cu(II) complexes of NSAIDs with enhanced anti-inflammatory activity and reduced gastrointestinal (GI) toxicity compared with their uncomplexed parent drug. These low toxicity Cu drugs have yet to reach an extended human market, but are of enormous interest, because many of todays anti-inflammatory drug therapies, including those based on the NSAIDs, remain either largely inadequate and/or are associated with problematic renal, GI and cardiovascular side effects. The origins of the anti-inflammatory and gastric-sparing actions of Cu-NSAIDs, however, remain uncertain. Their ability to influence copper metabolism has been a matter of debate and, apart from their frequently reported superoxide dismutase (SOD)-like activity in vitro, relatively little is known about how they ultimately regulate the inflammatory process and/or immune system. Furthermore, little is known of their pharmacokinetic and biodistribution profile in both humans and animals, stability in biological media and pharmaceutical formulations, or the relative potency/efficacy of the Cu(II) monomeric versus Cu(II) dimeric complexes. The following review will not only discuss the etiology of inflammation, factors influencing the metabolism of copper and historical overview of the development of the Cu-NSAIDs, but also outline the structural characteristics, medicinal and veterinary properties, and proposed modes of action of the Cu-NSAIDs. It will also compare the SOD, anti-inflammatory and ulcerogenic effects of various Cu-NSAIDs. If the potential opportunities of the Cu-NSAIDs are to be completely realized, a mechanistic understanding and delineation of their in vivo and in vitro pharmacological activity is fundamental, along with further characterization of their pharmacokinetic/pharmacodynamic disposition.

Studies on the genotoxicity of chromium: from the test tube to the cell

Abstract A critical review of the relevance of in vitro chemical studies to Cr-induced cancers is given. In particular, the nature of the most likely reductants, the species that stabilise intermediates and the mechanisms of DNA damage and damage to transcription proteins are discussed. It is concluded that the major damaging species responsible for the genotoxicity include Cr(V), Cr(IV) and/or reactive organic intermediates. These studies on the chemistry are also related to spectroscopic and other studies on mammalian cells.

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.

Microprobe XRF mapping and XAS investigations of the intracellular metabolism of arsenic for understanding arsenic-induced toxicity

Arsenic (As) is responsible for mass-poisonings worldwide following the ingestion of As-contaminated drinking water. Importantly, however, As toxicity is exploited in the antileukemia drug, Trisenox (As2O3), which successfully cures 65-80% of patients suffering relapsed acute promyelocytic leukemia. In an effort to determine the intracellular organelle and biomolecular targets of As, microprobe X-ray fluorescence (XRF) and X-ray absorption spectroscopy (XAS) analyses were performed on HepG2 cells following their exposure to high doses of arsenite (1 mM) or arsenate (20 mM). Microprobe XRF elemental mapping of thin-sectioned cells showed As accumulation in the euchromatin region of the cell nucleus (following arsenite exposure) synonymous with As targeting of DNA or proteins involved in DNA transcription. X-ray absorption near edge spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS) analysis of arsenite-treated cells, however, showed the predominance of an As tris-sulfur species, providing increased credence to As interactions with nuclear proteins as a key factor in As-induced toxicity.

DNA Interactions and Bacterial Mutagenicity of Some Chromium(III) Imine Complexes and their Chromium(V) Analogues. Evidence for Chromium(V) Intermediates in the Genotoxicity of Chromium(III)

The in vitro DNA interactions and bacterial mutagenicities of cis-[CrIII(phen)2(OH2)2]3+ and trans-[CrIII(salen)(OH2)2]+ and their CrV analogues are reported. At pH 3.3, cis-[Cr(phen)2(OH2)2]3+ (0.02–2.0 mM) causes negatively supercoiled pUC9 DNA to smear on agarose gels, with substantial precipitation in the well at ≥1.0 mM. Much weaker interactions between CrIII and DNA were apparent at pH 7.4. The interactions between DNA and CrV phen complexes (0.5 mM total Cr, pH 3.3) generated by oxidation of cis-[Cr(phen)2(OH2)2]3+ (for 10–30 min) resulted in almost complete nicking of form I DNA to forms II and III DNA. Nicking of form I DNA (≥80%) was also apparent at pH 7.4 following reaction of DNA with PbO2-oxidized [Cr(phen)2(OH2)2]3+ (2 mM Cr). Interactions between trans-[CrIII(salen)(OH2)2]+ and DNA were weaker than those of the CrIII phen complex at both pH 3.3 and 7.4. The CrV salen derivative (0.5 mM total Cr) caused the disappearance of form I DNA at oxidation times of ≥10 min and at pH 3.3 with substantial cleavage. While oxidation of [Cr(salen)(OH2)2]+ by PbO2 was not observed at pH 7.4, the complex was oxidized by iodosobenzene to produce short-lived [CrO(salen)]+ that caused DNA smearing on the agarose gel. In bacterial mutagenicity assays, the CrIII imine complexes and their CrV analogues produced similar mutagenic responses, which were believed to be due to the instabilities of the CrV species in the bacterial growth medium. While the spectrum of the mutagenic activities differed between the chromium phen and salen complexes, both exhibited greatest mutagenicity in Salmonella typhimurium TA102. These data suggest that CrV species, generated in vivo by cellular oxidative enzymes, may be responsible for CrIII-induced mutagenesis.

Synchrotron Radiation Spectroscopic Techniques as Tools for the Medicinal Chemist: Microprobe X-Ray Fluorescence Imaging, X-Ray Absorption Spectroscopy, and Infrared Microspectroscopy

This review updates the recent advances and applications of three prominent synchrotron radiation techniques, microprobe X-ray fluorescence spectroscopy/imaging, X-ray absorption spectroscopy, and infrared microspectroscopy, and highlights how these tools are useful to the medicinal chemist. A brief description of the principles of the techniques is given with emphasis on the advantages of using synchrotron radiation-based instrumentation rather than instruments using typical laboratory radiation sources. This review focuses on several recent applications of these techniques to solve inorganic medicinal chemistry problems, focusing on studies of cellular uptake, distribution, and biotransformation of established and potential therapeutic agents. The importance of using these synchrotron-based techniques to assist the development of, or validate the chemistry behind, drug design is discussed.

High resolution nuclear and X-ray microprobes and their applications in single cell analysis

Abstract Investigations of uptake of different chemical compounds by single cells have become a very popular topic and have been performed with different microanalytical techniques using electron, laser, ion and X-ray beams. With the advent of nuclear microprobes with submicron resolution it has been possible to investigate distribution of different elements inside individual cell with sensitivity of parts-per-million (ppm). Recently, with X-ray beams focused to 150 nm and high intensity obtained from third generation synchrotron facilities it became possible to perform analysis on a single cell with sensitivity of parts-per-billion (ppb). Synchrotron radiation offers also a unique possibility to investigate an oxidation state for different elements. In this paper, authors will present and discuss data from experiments on single cells performed with high resolution nuclear and X-ray microprobes.

Copper uptake, intracellular localization, and speciation in marine microalgae measured by synchrotron radiation X-ray fluorescence and absorption microspectroscopy

Metal toxicity to aquatic organisms depends on the speciation of the metal and its binding to the critical receptor site(s) (biotic ligand) of the organism. The intracellular nature of the biotic ligand for Cu in microalgal cells was investigated using the high elemental sensitivity of microprobe synchrotron radiation X-ray fluorescence (SR-XRF) and X-ray absorption near-edge spectroscopy (XANES). The marine microalgae, Ceratoneis closterium, Phaeodactylum tricornutum, and Tetraselmis sp. were selected based on their varying sensitivities to Cu (72-h 50% population growth inhibitions of 8-47 μg Cu/L). Intracellular Cu in control cells was similar for all three species (2.5-3.2 × 10(-15) g Cu/cell) and increased 4-fold in C. closterium and Tetraselmis sp. when exposed to copper, but was unchanged in P. tricornutum (72-h exposure to 19, 40, and 40 μg Cu/L, respectively). Whole cell microprobe SR-XRF identified endogenous Cu in the central compartment (cytoplasm) of control (unexposed) cells. After Cu exposure, Cu was colocated with organelles/granules dense in P, S, Ca, and Si and this was clearly evident in thin sections of Tetraselmis sp. XANES indicated coexistence of Cu(I) and Cu(II) in control and Cu-exposed cells, with the Cu ligand (e.g., phytochelatin) in P. tricornutum different from that in C. closterium and Tetraselmis sp. This study supports the hypothesis that Cu(II) is reduced to Cu(I) and that polyphosphate bodies and phytochelatins play a significant role in the internalization and detoxification of Cu in marine microalgae.

An investigation into the interactions of gold nanoparticles and anti-arthritic drugs with macrophages, and their reactivity towards thioredoxin reductase

Gold(I) complexes are an important tool in the arsenal of established approaches for treating rheumatoid arthritis (RA), while some recent studies have suggested that gold nanoparticles (Au NPs) may also be therapeutically efficacious. These observations prompted the current biological studies involving gold(I) anti-RA agents and Au NPs, which are aimed towards improving our knowledge of how they work. The cytotoxicity of auranofin, aurothiomalate, aurothiosulfate and Au NPs towards RAW264.7 macrophages was evaluated using the MTT assay, with the former compound proving to be the most toxic. The extent of cellular uptake of the various gold agents was determined using graphite furnace atomic absorption spectrometry, while their distribution within macrophages was examined using microprobe synchrotron radiation X-ray fluorescence spectroscopy. The latter technique showed accumulation of gold in discrete regions of the cell, and co-localisation with sulfur in the case of cells treated with aurothiomalate or auranofin. Electrospray ionization mass spectrometry was used to characterize thioredoxin reductase (TrxR) in which the penultimate selenocysteine residue was replaced by cysteine. Mass spectra of solutions of TrxR and aurothiomalate, aurothiosulfate or auranofin showed complexes containing bare gold atoms bound to the protein, or protein adducts containing gold atoms retaining some of their initial ligands. These results support TrxR being an important target of gold(I) drugs used to treat RA, while the finding that Au NPs are incorporated into macrophages, but elicit little toxicity, indicates further exploration of their potential for treatment of RA is warranted.

Biological evaluation of bismuth non-steroidal anti-inflammatory drugs (BiNSAIDs): stability, toxicity and uptake in HCT-8 colon cancer cells.

Recent studies showed that the metal-coordinated non-steroidal anti-inflammatory drug (NSAID), copper indomethacin, reduced aberrant crypt formation in the rodent colon cancer model, while also exhibiting gastrointestinal sparing properties. In the present study, the stability and biological activity of three BiNSAIDs of the general formula [Bi(L)3]n, where L=diflunisal (difl), mefenamate (mef) or tolfenamate (tolf) were examined. NMR spectroscopy of high concentrations of BiNSAIDs (24h in cell medium, 37°C) indicated that their structural stability and interactions with cell medium components were NSAID specific. Assessment of cell viability using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium]bromide (MTT) assay showed that the toxicity ranking of the BiNSAIDs paralleled those of the respective free NSAIDs: diflH<mefH<tolfH. While the IC50 values of the BiNSAIDs (ranging between 16 and 81μM) were lower than the free NSAIDs, it was apparent that the toxicity of the BiNSAIDs was due to the molar ratio of the three NSAID molecules contained in the BiNSAIDs, with the exception of [Bi(difl)3]. The highest cellular bismuth content was observed following treatment with [Bi(tolf)3]. Since NMR studies indicated that [Bi(tolf)3] was the most stable BiNSAID and that cellular uptake of bismuth correlated with structural stability it appears that bismuth uptake is assisted by the NSAID. Microprobe SR-XRF imaging showed that the intracellular fate of bismuth was independent of the specific BiNSAID treatment whereby all BiNSAID-treated cells showed bismuth accumulation in the cytoplasm within 24-h exposure. The size and location of the hot spots (0.3-5.8μm(2)), were consistent with cellular organelles such as lysosomes.