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Thiocyanate potentiates antimicrobial photodynamic therapy: in situ generation of the sulfur trioxide radical anion by singlet oxygen.

ABSTRACT: Antimicrobial photodynamic therapy (PDT) is used for the eradication of pathogenic microbial cells and involves the light excitation of dyes in the presence of O2, yielding reactive oxygen species including the hydroxyl radical (OH) and singlet oxygen ((1)O2). In order to chemically enhance PDT by the formation of longer-lived radical species, we asked whether thiocyanate (SCN(-)) could potentiate the methylene blue (MB) and light-mediated killing of the gram-positive Staphylococcus aureus and the gram-negative Escherichia coli. SCN(-) enhanced PDT (10 µM MB, 5 J/cm(2) 660 nm hv) killing in a concentration-dependent manner of S. aureus by 2.5 log10 to a maximum of 4.2 log10 at 10mM (P<0.001) and increased killing of E. coli by 3.6 log10 to a maximum of 5.0 log10 at 10mM (P<0.01). We determined that SCN(-) rapidly depleted O2 from an irradiated MB system, reacting exclusively with (1)O2, without quenching the MB excited triplet state. SCN(-) reacted with (1)O2, producing a sulfur trioxide radical anion (a sulfur-centered radical demonstrated by EPR spin trapping). We found that MB-PDT of SCN(-) in solution produced both sulfite and cyanide anions, and that addition of each of these salts separately enhanced MB-PDT killing of bacteria. We were unable to detect EPR signals of OH, which, together with kinetic data, strongly suggests that MB, known to produce OH and (1)O2, may, under the conditions used, preferentially form (1)O2.

SUBMITTER: St Denis TG

PROVIDER: S-EPMC3889203 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Thiocyanate potentiates antimicrobial photodynamic therapy: in situ generation of the sulfur trioxide radical anion by singlet oxygen.

St Denis Tyler G TG Vecchio Daniela D Zadlo Andrzej A Rineh Ardeshir A Sadasivam Magesh M Avci Pinar P Huang Liyi L Kozinska Anna A Chandran Rakkiyappan R Sarna Tadeusz T Hamblin Michael R MR

Free radical biology & medicine 20130819

Antimicrobial photodynamic therapy (PDT) is used for the eradication of pathogenic microbial cells and involves the light excitation of dyes in the presence of O2, yielding reactive oxygen species including the hydroxyl radical (OH) and singlet oxygen ((1)O2). In order to chemically enhance PDT by the formation of longer-lived radical species, we asked whether thiocyanate (SCN(-)) could potentiate the methylene blue (MB) and light-mediated killing of the gram-positive Staphylococcus aureus and t ...[more]

PMID: 23969112

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Project description:Singlet oxygen ((1)O2), which is generated through metabolic reactions and oxidizes numerous biological molecules, has been a useful tool in basic research and clinical practice. However, its role as a signaling factor, as well as a mechanistic understanding of the oxidation process, remains poorly understood. Here, we show that hyperpolarization-activated, cAMP-gated (HCN) channels--which conduct the hyperpolarization-activated current (Ih) and the voltage-insensitive instantaneous current (Iinst), and contribute to diverse physiological functions including learning and memory, cardiac pacemaking, and the sensation of pain--are subject to modification by (1)O2. To increase the site specificity of (1)O2 generation, we used fluorescein-conjugated cAMP, which specifically binds to HCN channels, or a chimeric channel in which an in-frame (1)O2 generator (SOG) protein was fused to the HCN C terminus. Millisecond laser pulses reduced Ih current amplitude, slowed channel deactivation, and enhanced Iinst current. The modification of HCN channel function is a photodynamic process that involves (1)O2, as supported by the dependence on dissolved oxygen in solutions, the inhibitory effect by a (1)O2 scavenger, and the results with the HCN2-SOG fusion protein. Intriguingly, (1)O2 modification of the HCN2 channel is state dependent: laser pulses applied to open channels mainly slow down deactivation and increase Iinst, whereas for the closed channels, (1)O2 modification mainly reduced Ih amplitude. We identified a histidine residue (H434 in S6) near the activation gate in the pore critical for (1)O2 modulation of HCN function. Alanine replacement of H434 abolished the delay in channel deactivation and the generation of Iinst induced by photodynamic modification. Our study provides new insights into the instantaneous current conducted by HCN channels, showing that modifications to the region close to the intracellular gate underlie the expression of Iinst, and establishes a well-defined model for studying (1)O2 modifications at the molecular level.

Project description:The skin is the largest organ in the body and is consistently exposed to aggressive environmental attacks (biological/physical/chemical, etc.). Reactive oxygen species (ROS) are formed during the normal oxidative metabolism which enhances to a lethal level under stress conditions referred to as oxidative stress. While, under normal conditions, cells are capable of dealing with ROS using non-enzymatic and enzymatic defense system, it can lead to a critical damage to cell system via the oxidation of cellular components under stress condition. Lipid peroxidation is a well-established mechanism of cellular injury in all kinds of organisms and it is often used as an indicator of oxidative stress in cells and tissues. In the presence of metal ions, ROS such as hydrogen peroxide (H2O2) produces highly reactive hydroxyl radical (HO•) via Fenton reaction. In the current study, we have used the porcine skin (intact pig ear/skin biopsies) as an ex vivo/in vitro model system to represent human skin. Experimental results have been presented on the participation of HO• in the initiation of lipid peroxidation and thereby leading to the formation of reactive intermediates and the formation of electronically excited species eventually leading to ultra-weak photon emission (UPE). To understand the participation of different electronically excited species in the overall UPE, the effect of a scavenger of singlet oxygen (1O2) on photon emission in the visible and near-infrared region of the spectrum was measured which showed its contribution. In addition, measurement with interference filter with a transmission in the range of 340-540 nm reflected a substantial contribution of triplet carbonyls (3L=O?) in the photon emission. Thus, it is concluded that during the oxidative radical reactions, the UPE is contributed by the formation of both 3L=O? and 1O2. The method used in the current study is claimed to be a potential tool for non-invasive determination of the physiological and pathological state of human skin in dermatological research.

Dataset Information

Thiocyanate potentiates antimicrobial photodynamic therapy: in situ generation of the sulfur trioxide radical anion by singlet oxygen.

Publications

Thiocyanate potentiates antimicrobial photodynamic therapy: in situ generation of the sulfur trioxide radical anion by singlet oxygen.

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