Triplet Excited Carbonyls and Singlet Oxygen Formation During Oxidative Radical Reaction in Skin

Abstract

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.

Keywords: singlet oxygen; skin; triplet excited carbonyl; two-dimensional photon imaging; ultra-weak photon emission.

FIGURE 1

Mechanism of the formation of electronically excited species by oxidative metabolic processes via oxidation of polyunsaturated fatty acid initiated by Fenton’s reagent. The Fenton’s reagent generates hydroxyl radical (HO^•) at different locations within the vicinity of phospholipid bilayer.

FIGURE 2

(A) Electron paramagnetic resonance (EPR) spectra of POBN-OH adduct was detected in solutions containing 100 μM FeSO₄ in the absence (a) and presence of 20 μM (b) of H₂O₂. Bar represent 5000 relative units. (B) The data are presented as the mean and standard deviation of three measurements.

FIGURE 3

Schematic illustration of the experimental setup for detection of kinetics of ultra-weak photon emission (UPE) using PMT (A) and two-dimensional imaging of UPE using CCD camera (B).

FIGURE 4

Fenton’s reagent-induced UPE measured using visible PMT from the porcine skin sample. (A) Kinetics of UPE was measured after the topical application of Fenton’s reagent (100 μM H₂O₂ containing 500 μM FeSO₄). (B) Kinetics of UPE was measured after the topical application of Fenton’s reagent (1 mM H₂O₂ containing 500 μM FeSO₄). (C) Kinetics of UPE was measured in the presence of sodium ascorbate (5 mM) applied to the skin prior to topical application of Fenton’s reagent (1 mM H₂O₂ containing 500 μM FeSO₄). The decay curve was measured for a duration of 30 min. The arrow indicates the application of chemicals.

FIGURE 5

Transmission spectrum of interference filter type 644 (Schott & Gen., Jena, Germany) (A) and kinetics of UPE measured after the topical application of Fenton’s reagent (containing 1 mM H₂O₂ and 500 μM FeSO₄) in the presence of interference filter type 644 (340–540 nm) (B). Other experimental conditions as described in Figure 4.

FIGURE 6

Two-dimensional Fenton’s reagent-induced UPE measured using CCD camera from the porcine ear/skin biopsies. (A) Photograph of pig ear (circle represents the area of the porcine ear where Fenton’s reagent was topically applied) and two-dimensional UPE imaging measured after the topical application of Fenton’s reagent (1 mM H₂O₂ containing 500 μM FeSO₄). (B) Photographs (a, d, and g) and corresponding images of UPE of spontaneous (b), induced with Fenton’s reagent (1 mM H₂O₂ containing 500 μM FeSO₄) (e) and induced with Fenton’s reagent (1 mM H₂O₂ containing 500 μM FeSO₄) in the presence of sodium ascorbate (5 mM) (h). Figure (B) (c, f, and i) shows the spatial profile of the photon emission in a single strip of the image (dashed line) in spontaneous (c), Fenton’s reagent-induced (f) and Fenton’s reagent-induced in the presence of sodium ascorbate (i). Y-axis reflects the number of photon counts accumulated after 30 min, whereas the X-axis denotes the pixel of the image.

FIGURE 7

Fenton’s reagent-induced UPE measured using near-infrared PMT from the porcine skin sample. (A) Kinetics of UPE was measured after the topical application of Fenton’s reagent (100 μM containing 500 μM FeSO₄). (B,C) Kinetics of UPE was measured after the topical application of Fenton’s reagent (1 mM H₂O₂ containing 500 μM FeSO₄) added between 30 s and 1 min of the start of measurement (indicated by arrow) in the absence (B) and presence (C) of sodium ascorbate (5 mM), respectively added prior to treatment with Fenton’s reagent. The decay curve was measured for a duration of 10 min.