Copper(II) oxide nanoparticles (NPCuO) have many industrial applications, but are highly cytotoxic because they generate reactive oxygen species (ROS). It is unknown whether the damaging ROS are generated primarily from copper leached from the nanoparticles, or whether the nanoparticle surface plays a significant role. To address this question, we separated nanoparticles from the supernatant containing dissolved copper, and measured their ability to damage plasmid DNA with addition of hydrogen peroxide, ascorbate, or both. While DNA damage from the supernatant (measured using an electrophoresis assay) can be explained solely by dissolved copper ions, damage by the nanoparticles in the presence of ascorbate is an order of magnitude higher than can be explained by dissolved copper and must therefore depend primarily upon the nanoparticle surface. DNA damage is time-dependent, with shorter incubation times resulting in higher EC50 values. Hydroxyl radical is the main ROS generated by NPCuO/hydrogen peroxide as determined by EPR measurements; NPCuO/hydrogen peroxide/ascorbate conditions generate ascorbyl, hydroxyl, and superoxide radicals. Thus, NPCuO generate ROS through several mechanisms, likely including Fenton-like and Haber-Weiss reactions from the surface or dissolved copper ions. The same radical species were observed when NPCuO suspensions were replaced with the supernatant containing leached copper, washed NPCuO, or dissolved copper solutions. Overall, NPCuO generate significantly more ROS and DNA damage in the presence of ascorbate than can be explained simply from dissolved copper, and the NPCuO surface must play a large role.

The redox activity of metal ions can lead to the formation of highly reactive species that damage DNA, producing different oxidation products and types of damage depending upon the redox potentials of the DNA bases, formation of intermediate adducts, and identity of the reactive species. Other factors are also important in determining the degree of metal-mediated DNA damage, such as localization and redox chemistry of the metal ions or complexes and lifetimes of the reactive oxygen species generated. This review examines the types of DNA damage mediated by first-row transition metals under oxidative stress conditions, with emphasis on work published in the past ten years. Similarities and differences between DNA damage mechanisms of the first-row transition metals in vitro and in E. coli and human cells are compared and their relationship to disease development are discussed. Methods to detect this metal-mediated DNA damage, including backbone breakage, base oxidation, inter- and intra-strand crosslinking, and DNA-protein crosslinking are also briefly reviewed, as well as detection methods for reactive oxygen species generated by these metal ions. Understanding the conditions that cause metal-mediated DNA damage and metal generation of reactive oxygen species in vitro and in cells is required to develop effective drugs to prevent and treat chronic disease.

Concentrations of labile iron and copper are elevated in patients with neurological disorders, causing interest in metal-neurotransmitter interactions. Catecholamine (dopamine, epinephrine, and norepinephrine) and amino acid (glycine, glutamate, and 4-aminobutyrate) neurotransmitters are antioxidants also known to bind metal ions. To investigate the role of metal binding as an antioxidant mechanism for these neurotransmitters, L-dihydroxyphenylalanine (L-DOPA), and curcumin, their abilities to prevent iron- and copper-mediated DNA damage were quantified, cyclic voltammetry was used to determine the relationship between their redox potentials and DNA damage prevention, and UV-vis studies were conducted to determine iron and copper binding as well as iron oxidation rates. In contrast to amino acid neurotransmitters, catecholamine neurotransmitters, L-DOPA, and curcumin prevent significant iron-mediated DNA damage (IC(50) values of 3.2 to 18 μM) and are electrochemically active. However, glycine and glutamate are more effective at preventing copper-mediated DNA damage (IC(50) values of 35 and 12.9 μM, respectively) than L-DOPA, the only catecholamine to prevent this damage (IC(50) = 73 μM). This metal-mediated DNA damage prevention is directly related to the metal-binding behaviour of these compounds. When bound to iron or copper, the catecholamines, amino acids, and curcumin significantly shift iron oxidation potentials and stabilize Fe(3+) over Fe(2+) and Cu(2+) over Cu(+), a factor that may prevent metal redox cycling in vivo. These results highlight the disparate antioxidant activities of neurotransmitters, drugs, and supplements and highlight the importance of considering metal binding when identifying antioxidants to treat and prevent neurodegenerative disorders.

UV-like DNA damage is created in the dark by chemiexcitation, in which UV-activated enzymes generate reactive oxygen and nitrogen species that create a dioxetane on melanin. Thermal cleavage creates an electronically excited triplet-state carbonyl whose high energy transfers to DNA. Screening natural compounds for the ability to quench this energy identified polyenes, polyphenols, mycosporine-like amino acids, and related compounds better known as antioxidants. To eliminate false positives such as ROS and RNS scavengers, we then used the generator of triplet-state acetone, tetramethyl-1,2-dioxetane (TMD), to excite the triplet-energy reporter 9,10-dibromoanthracene-2-sulfonate (DBAS). Quenching measured as reduction in DBAS luminescence revealed three clusters of 50% inhibitory concentration, ~50 μM, 200–500 μM, and >600 μM, with the former including sorbate, ferulic acid, and resveratrol. Representative triplet-state quenchers prevented chemiexcitation-induced “dark” cyclobutane pyrimidine dimers (dCPD) in DNA and in UVA-irradiated melanocytes. We conclude that (i) the delocalized pi electron cloud that stabilizes the electron-donating activity of many common antioxidants allows the same molecule to prevent an electronically excited species from transferring its triplet-state energy to targets such as DNA and (ii) the most effective class of triplet-state quenchers appear to operate by energy diversion instead of electron donation and dissipate that energy by isomerization.

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In DNA, electron excitation allows adjacent pyrimidine bases to dimerize by [2 + 2] cycloaddition, creating chemically stable but lethal and mutagenic cyclobutane pyrimidine dimers (CPDs). The usual cause is ultraviolet radiation. Alternatively, CPDs can be made in the dark (dCPDs) via chemically mediated electron excitation of the skin pigment melanin, after it is oxidized by peroxynitrite formed from the stressinduced radicals superoxide and nitric oxide. We now show that the dark process is not limited to the unusual structural molecule melanin: signaling biomolecules such as indolamine and catecholamine neurotransmitters and hormones can also be chemiexcited to energy levels high enough to form dCPDs. Oxidation of serotonin, dopamine, melatonin, and related biogenic amines by peroxynitrite created triplet-excited species, evidenced by chemiluminescence, energy transfer to a triplet-state reporter, or transfer to O 2 resulting in singlet molecular oxygen. For a subset of these signaling molecules, triplet states created by peroxynitrite or peroxidase generated dCPDs at levels comparable to ultraviolet (UV). Neurotransmitter catabolism by monoamine oxidase also generated dCPDs. These results reveal a large class of signaling molecules as electronically excitable by biochemical reactions and thus potential players in deviant mammalian metabolism in the absence of light.

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Modified nucleotides, including phosphoramidates and mesyl nucleotides, are very effective in inactivating gene expression in bacteria. Gyr A is the target gene in several organisms, including Plasmodium falciparum. Antisense reactions with bacteria infecting citrus plants are promising but incomplete. Human tissue culture cells assayed with a different target are also susceptible to the presence of mesyl oligonucleotides.