James G. Muller

Formation of DNA adducts using nickel(II) complexes of redox-active ligands: a comparison of salen and peptide complexes

Abstract A series of water-soluble Ni(salen) complexes have been synthesized and studied under oxidative conditions with DNA. Experiments involving gel electrophoretic analysis, substituent effect studies, and mass spectral characterization point to the formation of covalent adducts of the salen ligand with guanine nucleobases. The mechanism of this novel reactivity of transition metal complexes with nucleic acids is discussed in comparison with nickel(II) complexes of peptides and other ligands.

Structural context effects in the oxidation of 8-Oxo-7,8-dihydro-2′- deoxyguanosine to hydantoin products: Electrostatics, base stacking, and base pairing

8-Oxo-7,8-dihydroguanine (OG) is the most common base damage found in cells, where it resides in many structural contexts, including the nucleotide pool, single-stranded DNA at transcription forks and replication bubbles, and duplex DNA base-paired with either adenine (A) or cytosine (C). OG is prone to further oxidation to the highly mutagenic hydantoin products spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) in a sharply pH-dependent fashion within nucleosides. In the present work, studies were conducted to determine how the structural context affects OG oxidation to the hydantoins. These studies revealed a trend in which the Sp yield was greatest in unencumbered contexts, such as nucleosides, while the Gh yield increased in oligodeoxynucleotide (ODN) contexts or at reduced pH. Oxidation of oligomers containing hydrogen-bond modulators (2,6-diaminopurine, N(4)-ethylcytidine) or alteration of the reaction conditions (pH, temperature, and salt) identify base stacking, electrostatics, and base pairing as the drivers of the key intermediate 5-hydroxy-8-oxo-7,8-dihydroguanine (5-HO-OG) partitioning along the two hydantoin pathways, allowing us to propose a mechanism for the observed base-pairing effects. Moreover, these structural effects cause an increase in the effective pK(a) of 5-HO-OG, following an increasing trend from 5.7 in nucleosides to 7.7 in a duplex bearing an OG·C base pair, which supports the context-dependent product yields. The high yield of Gh in ODNs underscores the importance of further study on this lesion. The structural context of OG also determined its relative reactivity toward oxidation, for which the OG·A base pair is ~2.5-fold more reactive than an OG·C base pair, and with the weak one-electron oxidant ferricyanide, the OG nucleoside reactivity is >6000-fold greater than that of OG·C in a duplex, leading to the conclusion that OG in the nucleoside pool should act as a protective agent for OG in the genome.

METAL-MEDIATED OXIDATION OF GUANINES IN DNA AND RNA : A COMPARISON OF COBALT(II), NICKEL(II) AND COPPER(II) COMPLEXES

Nickel(II) complexes of certain strong field tetradentate ligands have been found to mediate guanine-specific modification of DNA and RNA in the presence of the strong oxidant KHSO5. Reaction occurs specifically at solvent exposed guanines, i.e. those that are not part of a Watson-Crick B helix. Whereas the ion [Ni(H2O)6]2+ is completely inactive, the simple aqueous cation [Co(H2O)6]2+ can mediate oxidative DNA modification, also with guanine specificity. However, the [Co(H2O)6]2+-mediated DNA cleavage occurs at all guanine residues, with some preference for those in single-stranded regions. In both the nickel and cobalt reaction systems strand cleavage is only observed after piperidine treatment, suggestive of base rather than sugar damage. Mechanistic studies, including radical quenching data, imply that different active species are formed by the two systems. The nickel-mediated cleavage possibly occurs via a caged radical species, while the cobalt-mediated cleavage appears to occur through the formation of freely diffusible sulfate radicals. Copper(II) complexes can also mediate DNA damage in the presence of monoperoxysulfate, however the utility of this system is hampered by the higher concentrations of complex and KHSO5 required.

Metallodrug complexes that mediate DNA and lipid damage via sulfite autoxidation: copper(II) famotidine and iron(III) bis(salicyglycine)

Random DNA damage via the autoxidation of sulfite is observed to be mediated by micromolar concentrations of copper(II) and iron(III) metallodrug complexes. Importantly, these results are in contrast to observations made for nickel(II) complexes, where guanine-specific modification of DNA was observed. While the exact mechanism is not known, alcohol-quenching studies suggest the possible formation of sulfuroxyl radicals (SO3−, SO5−). Furthermore, these complexes are also observed to be effective catalysts for lipid oxidation. This study represents the first observation of DNA and lipid damage by sulfite and copper(II) and iron(III) coordination compounds and may lend insight to the biological toxicity of these transition metals.

Fate of selected estrogens in two laboratory scale sequencing batch reactors fed with different organic carbon sources under varying solids retention times

This study compared the performances of two laboratory-scale sequencing batch reactors to remove 17β-estradiol and 17α-ethinyl estradiol. Both SBRs were operated to achieve organic carbon oxidation and nitrification. However, the overall bacterial population in both SBRs was targeted to be different by feeding the SBRs with peptone and glucose. Furthermore, the reactors were also run at different solid retention times (SRTs) to evaluate the effect of SRT on estrogen removal. The more diverse heterotrophic and ammonia oxidizing bacterial community in the peptone fed SBR1 had superior estrogen removal than the glucose fed SBR 2 which enriched less diverse community, particularly for 17α-ethinyl estradiol. Under a solids retention time (SRT) of 40days, the total 17β-estradiol mass was 30% of the amount under the SRT of 20days, and the total 17α-ethinyl estradiol mass was likewise 40% of the amount under the shorter SRT.

Synthesis and DNA binding properties of C3-, C12-, and C24-substituted amino-steroids derived from bile acids.

Seven new amino- and guanidino-substituted steroids have been synthesized from bile acid precursors, either deoxycholic acid or lithocholic acid. Their DNA binding properties have been examined using an ethidium displacement assay, through studies of hyperchromicity and thermal denaturation, and by circular dichroism. Comparison is made to simple aliphatic polyamines such as putrescine, 1,12-diaminododecane, spermidine, and spermine.

Electronic structure of DNA-unique properties of 8-oxoguanosine

8-Oxo-7,8-dihydroguanosine (8-oxoG) is among the most common forms of oxidative DNA damage found in human cells. The question of damage recognition by the repair machinery is a long standing one, and it is intriguing to suggest that the mechanism of efficiently locating damage within the entire genome might be related to modulations in the electronic properties of lesions compared to regular bases. Using laser-based methods combined with organizing various oligomers self-assembled monolayers on gold substrates, we show that indeed 8-oxoG has special electronic properties. By using oligomers containing 8-oxoG and guanine bases which were inserted in an all thymine sequences, we were able to determine the energy of the HOMO and LUMO states and the relative density of electronic states below the vacuum level. Specifically, it was found that when 8-oxoG is placed in the oligomer, the HOMO state is at higher energy than in the other oligomers studied. In contrast, the weakly mutagenic 8-oxo-7,8-dihydroadenosine (8-oxoA) has little or no effect on the electronic properties of DNA.

Rates of chemical cleavage of DNA and RNA oligomers containing guanine oxidation products.

The nucleobase guanine in DNA (dG) and RNA (rG) has the lowest standard reduction potential of the bases, rendering it a major site of oxidative damage in these polymers. Mapping the sites at which oxidation occurs in an oligomer via chemical reagents utilizes hot piperidine for cleaving oxidized DNA and aniline (pH 4.5) for cleaving oxidized RNA. In the present studies, a series of time-dependent cleavages of DNA and RNA strands containing various guanine lesions were examined to determine the strand scission rate constants. The guanine base lesions 8-oxo-7,8-dihydroguanine (OG), spiroiminodihydantoin (Sp), 5-guanidinohydantoin (Gh), 2,2,4-triamino-2H-oxazol-5-one (Z), and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) were evaluated in piperidine-treated DNA and aniline-treated RNA. These data identified wide variability in the chemical lability of the lesions studied in both DNA and RNA. Further, the rate constants for cleaving lesions in RNA were generally found to be significantly smaller than for lesions in DNA. The OG nucleotides were poorly cleaved in DNA and RNA; Sp nucleotides were slowly cleaved in DNA and did not cleave significantly in RNA; Gh and Z nucleotides cleaved in both DNA and RNA at intermediate rates; and 2Ih oligonucleotides cleaved relatively quickly in both DNA and RNA. The data are compared and contrasted with respect to future experimental design.

Oxidative DNA damage mediated by metal-peptide complexes

Peptides and proteins are able to form coordination complexes with nickel(II) and copper(II) through ligation of deprotonated amide nitrogens, the amino terminus, and the side chains of such residues as histidine and the amino terminus. These complexes are reactive with either 0, alone or 0, in the presence of other oxidants or reductants to generate reactive intermediates capable of DNA damage. Both DNA strand scission and cross-linking have been observed.

Reactivity of Bulged Bases in Duplex DNA with Redox-active Nickel and Cobalt Complexes

The square-planar, macrocyclic complex NiCR (CR=(2,12-dimethyl-3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),2,11,13,15-pentaenato)) as well as CoCl 2 have been investigated as catalysts for the site-specific oxidation of bulged T, C, and A nucleotides in duplex DNA oligomers. Previous studies of NiCR using KHSO 5 as oxidant indicated that this combination of reagents successfully probes the accessibility of N7 of guanine residues in non-canonical DNA and folded RNA structures. In the present study, the order of reactivity of bulged bases in synthetic oligodeoxynucleotides with NiCR/KHSO 5 is shown to be G>C>T∼A. Although the nickel complex generates a less-reactive oxidant than does cobalt, its selectivity for bulged bases is much greater, rendering it a useful probe of exposed Cs and Ts in addition to Gs in DNA and RNA structure.