- Top of page
- Literature Cited
- Supporting Information
Ascorbic acid (AA) and copper have been increasingly employed in flow cytometry (FCM) and high content analysis (HCA) since the introduction of “click chemistry” as a non-destructive alternative to classical 5-bromo-2′-deoxyuridine (BrdU) immunodetection for DNA synthesis and proliferation assays. Mixtures of ascorbate and catalytic copper, under certain experimental conditions, act as oxidizing agent, catalyzing the formation of reactive hydroxyl radicals through hydrogen peroxides decomposition via Fenton reaction. We developed a procedure for BrdU incorporation detection based on the use of AA and cupric ions as DNA damaging agents. Optimal DNA damaging conditions were identified and found to provide results comparable with “click” 5-ethynyl-deoxyuridine (EdU) cycloaddition approach and classical BrdU immunodetection. Scavenger agents were found to prevent hydroxyl-induced DNA damages, providing the proof-of-concept for the use of this procedure for DNA denaturation prior to BrdU detection. We demonstrated hydroxyl radicals' reaction to be readily applicable to HCA and FCM assays, for both classical BrdU immunostaining and EdU cycloaddition procedure. This technique was successfully employed for BrdU pulse-chase experiments and in multiparametric immunofluorescence assays for the simultaneous detection of labile phosphoproteins in intact cells. The use of AA/Cu prior to immunodetection for BrdU incorporation assays is a viable alternative to chemical/physical DNA denaturing agents (acids or heat), since it allows preservation of labile epitopes such as phosphoproteins, and over enzymatic agents (digestion with DNases) for its lower cost. © 2013 International Society for Advancement of Cytometry
l-Ascorbic acid (Vitamin C, AA) is a water-soluble antioxidant, which efficiently scavenges free radicals and other radical oxygen species (ROS) produced by cell metabolism. ROS generation is associated with several forms of tissue damage and plays a role in the development of a multitude of chronic diseases [1-3]. AA acts as donor/acceptor in redox reactions, providing a protective activity against free radicals. A mechanism able to control ROS generated during aerobic metabolism is essential for cell viability and AA and glutathione work together to prevent accumulation of oxidative damages that can lead to cell death .
Despite its role as radical scavenger, in the presence of free metal ions AA acts as a pro-oxidant and can damage biological molecules, such as nucleic acids. AA reduces cupric (Cu++) to cuprous (Cu+) ion, which is capable of catalyzing the formation of reactive hydroxyl radicals through hydrogen peroxides decomposition via the so-called Fenton's reaction [5-7]. The hydroxyl radicals efficiently react with deoxyribose backbone of DNA : hydroxyl radicals abstract an hydrogen atom from deoxyribose, leading to the formation of a free radical intermediate, and eventually triggering extensive damages such as backbone cleavages, abasic sites, and nucleotide aldehydes [9, 10].
In the last years, AA and cupric ions were employed together in cell-based assays as catalytic agents for “click chemistry” reactions to detect protein, RNA, and DNA synthesis. A common application of click chemistry in cell biology is the quantification of DNA synthesis in cells, which incorporate 5-ethynyl-deoxyuridine (EdU) during S phase: a copper-catalyzed azide-alkyne cycloaddition (CuAAC) has been exploited to covalently bind azide-conjugated fluorescent probes to EdU, without the need of prior DNA denaturation . In previous works, we used 5-bromo-2′-deoxyuridine (BrdU)-azide derivatives followed by anti-BrdU immunodetection and fluorescence amplification by secondary antibody to analyze cells in S phase [12, 13].
Here we show that, at concentrations similar to those employed to catalyze click chemistry reactions in intact cells, AA/copper mixtures also act as DNA damaging agents and introduce double-strand breaks, which render incorporated halogen deoxyuridine derivatives accessible for immunodetection. We exploited this unexpected reactivity to develop a novel procedure for BrdU/EdU detection in multiplexed flow cytometry (FCM) and high content assays.
- Top of page
- Literature Cited
- Supporting Information
The use of cuprous ions and AA has been described in FCM and HCA for the detection of cells that incorporated EdU or BrdU azides through a click chemistry approach [12, 13]. Nevertheless, the use of EdU has been so far limited to few applications in cellular assays due to some drawbacks: (i) EdU inhibits cell proliferation at lower doses than BrdU [13, 16, 34]; (ii) the CV of DNA profiles using Cu/AA catalysis is higher than those obtained with BrdU ; (iii) presence of some aspecific staining using alkyne labeling and CuAAC . Cupric ions (Cu++) in the presence of AA or other reducing agents was demonstrated to cause DNA strand scission following site-specific formation of hydroxyl radicals (Fenton reaction), with production of cuprous ions (Cu+) and oxidized AA , as shown in Figure 1. The hydroxyl radicals may react by hydrogen abstraction, electron transfer, and addition reactions.
Here, we show that AA at high concentrations in the presence of copper salts is able to induce damage of both plasmidic DNA [7, 33] or genomic DNA extracted from cell lysates, generating small DNA fragments in both cases. Generation of hydroxyl radicals in proximity to DNA causes base damages and DNA strand breaks; this effect is proportional to the accessible surface areas of the hydrogen atoms of the DNA backbone . To note, alternative bioconjugation methods can be employed to avoid undesirable effects on DNA damage, such as difluorocyclooctyne coupling using strain-promoted alkyne-azide cycloaddition chemistry  or redox-protected reactions [38, 39].
We verified the appearance of DNA base modification after exposure to AA/Cu mixtures of live cells, by means of 8-hydroxy-guanosine and ARP assays . Experiments with pre-stained DNA using dyes including PI, Hoechst 33342, and 7AAD showed fluorescent bleaching using dyes with affinity for T and G sequences [40-42]. In order to explain the effect of Cu/AA treatment on DNA stainability (Fig. 3), we explored different experimental conditions for CuAAC reaction.
Several forms of DNA damage, including strand scission, base oxidation, and base liberation, can contribute to the loss of fluorescence. DNA damage after exposure to ROS generated by AA/Cu mixture resulted in decreased mean fluorescence because of physical dissociation of the dye from its binding site on DNA and fluorescence bleaching is an indicator of DNA damage . The extent of DNA damage determined through FCM analysis of PI staining corroborated DNA gel electrophoresis data, which showed massive DNA fragmentation . No base specificity for DNA cleavage was found, since DNA-binding fluorochromes, Hoechst 33342 (which preferentially binds A-T sequences), and PI (a DNA intercalating agent pairs without base specificity) were equally affected by the fluorescence decrease . A similar effect on fluorescence bleaching was observed for TO-PRO-3 and DRAQ5, and to a lesser extent for 7-AAD and Vybrant Dye Cycle Green. Since anthraquinone and diazo dyes can be easily oxidized by hydroxyl radicals, it can be postulated that the observed fluorescence quenching represents the result of DNA damage (loss of stainability) and dye alteration (fluorescence bleaching) .
Most Fenton reactions generate non specific DNA cleavage; however, there is evidence that cuts preferentially occur at thymidine residues .
Cleavage effects by copper chemistry, however, are well known: copper complexes, such as bis-(1,10-phenanthroline)copper(I) complex, are responsible for direct DNA strand scission [45, 46] through hydrogen atom H-1′ abstraction from the deoxyribose moiety. Moreover, hydroxyl radicals are employed for RNA and DNA footprinting assays [47-49], where the [Fe(EDTA)]2− complex is oxidized by hydrogen peroxide in the presence of AA, to abstract hydrogen from each deoxyribose carbon of B-form DNA. Hydroxyl radicals are highly reactive, with a half-life in aqueous solution of less than 1 ns: when produced in vivo, they react very close to the site of formation such ; cupric ions were found to interact with phosphate groups of DNA backbone , and specific binding at N-7 guanine bases over adenine bases was demonstrated in double-stranded DNA . Traces of transitions metals such as copper and iron are found in buffer solutions , cell culture media, syringes and laboratory tools, making possible that Fenton reactions might occur in intact cells in the presence of AA. In addition to copper and iron, the list of metals potentially involved in Fenton reactions include chromium, titanium, cobaltum, rhodio, nickel, manganese, and palladium . Rare earth from the lanthanide group, such as cerium and terbium, possess similar redox proprieties and have already been described to cause DNA cleavage in live cells [54, 55].
Additional methods to introduce DNA strand breaks for BrdU incorporation detection include ultraviolet light [56, 57] (DNA photolysis). Two disadvantages of DNA photolysis are the need of transilluminators or sensitizing agents such as Hoechst 33342 and the poor reliability of UV lamps, as compared to approaches like Fenton or click chemistry. On the other hand, Fenton reaction has the drawback of causing massive DNA degradation: this implies that experimental conditions should carefully set to allow simultaneous DNA content analysis using fluorescent dyes.
Our results are in agreement with a those obtained by Koberna and coworkers , who also demonstrated oxidative attack at the deoxyribose moiety by copper(I) in the presence of oxygen and AA and exploited Fenton reactions to track BrdU incorporation. In this article, we examine in depth the molecular mechanism of Fenton reactions in intact cells and show that they can occur as side reaction of click CuAAC cycloaddictions. Moreover, we demonstrate that hydroxyl radicals' reaction can be combined with click chemistry for BrdU/EdU pulse-and-chase experiments and can be employed in multiplexed immunofluorescence assays both for HCA and for FCM.
It is known that transition metals play a biological role in ROS signal transduction , but there is increasing evidence that they might represent a valuable tool in bioinorganic research and many bioanalytical applications . Additional analytical approaches involving the use of transition metals to generate hydroxyl radicals include nucleic acid-protein interaction studies  and pure proteomics . Here, we demonstrate that Fenton reactions occur under certain circumstances alongside click chemistry-based BrdU analysis, providing a novel tool for BrdU/EdU pulse-and-chase experiments and for multiplexed analysis of DNA replication and immunofluorescence of labile epitopes.