Thomas Herrling

UV-induced free radicals in the skin detected by ESR spectroscopy and imaging using nitroxides

Reactive free radicals and reactive oxygen species (ROS) induced by ultraviolet irradiation in human skin are strongly involved in the occurrence of skin damages like aging and cancer. In the present work an ex vivo method for the detection of free radicals/ROS in human skin biopsies during UV irradiation is presented. This method is based on the Electron Spin Resonance (ESR) spectroscopy and imaging and uses the radical trapping properties of nitroxides. The nitroxides 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO), 3-Carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCM), and 3-Carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCA), were investigated for their applicability of trapping reactive free radicals and reactive oxygen species in skin during UV irradiation. As a result of the trapping process the nitroxides were reduced to the EPR silent hydroxylamins. The reduction rate of TEMPO was due to both the UV radiation and the enzymatic activity of the skin. The nitroxides PCM and PCA are sufficiently stable in the skin and are solely reduced by UV-generated free radicals/ROS. The nitroxide PCA was used for imaging the spatial distribution of UV-generated free radicals/ROS. As a result of the homogeneous distribution of PCA in the skin, it was possible to estimate the penetration of UVA and UVB irradiation: The UV irradiation decreased the PCA intensity corresponding to its irradiance and penetration into the skin. This reduction was shown to be caused mainly by UVA radiation (320-400 nm).

ELECTRON PARAMAGNETIC RESONANCE STUDIES ON NITROXIDE RADICAL 2,2,5,5-TETRAMETHYL-4-PIPERIDIN-1-OXYL (TEMPO) REDOX REACTIONS IN HUMAN SKIN

Electron paramagnetic resonance (EPR) is currently being explored for the study of living biological systems. Among biophysical and biochemical applications, the study of nitroxide radical interactions with tissue antioxidants and oxidants is of growing interest. Skin is a target organ of the EPR methodology and is frequently exposed to oxidative stress. We investigated the piperidine-type nitroxide 2,2,5,5-tetramethyl-4-piperidin-1-oxyl (TEMPO) because it is skin permeable and readily accepts electrons in biological systems. TEMPO is readily scavenged on the surface of cultured human skin. Pretreatment of skin cultures with butylhydroperoxide, which decreases intracellular ascorbate and glutathione, causes inhibition of nitroxide scavenging. Exposure of skin cultures to dehydroascorbate, which is internalized and converted to ascorbate, leads to stimulation of nitroxide scavenging. In human keratinocytes and fibroblasts, the TEMPO radical is reversibly reduced to the hydroxylamine depending on the oxygen concentration and the availability of intracellular glutathione and ascorbate. Cell exposure to the glutathione synthetase inhibitor buthionine-sulfoximine depleted intracellular glutathione and inhibited nitroxide reduction; exposure to dehydroascorbate or glutathione-monoethylester increased intracellular ascorbate or glutathione concentration and stimulated nitroxide reduction. Quantitative considerations indicate that the major reduction site of TEMPO in skin and skin cells is the cytosol ascorbate/glutathione redox cycle. We suggest that analysis of TEMPO radical scavenging by the EPR technique is a convenient method for measuring skin ascorbate and thiol-dependent antioxidant activity in vitro and in vivo.

Integrated Sun Protection Factor: A New Sun Protection Factor Based on Free Radicals Generated by UV Irradiation

The present work uses the initial step of the whole cascade of biological effects in the skin, the creation of free radicals by means of UVA/UVB radiation, to develop a total sun protection factor. Until now, existing in vivo indices have not been fully satisfying: SPF only reflects protection from UVB light, and persistent pigment darkening is restricted to the UVA part of the sun spectrum. The quantitative measurement of free radicals generated in human skin biopsies by means of electron spin resonance X-band spectroscopy allows to determine a new total SPF. This new sun protection index covers all UVA/UVB wavelengths taking into account their effects in the epidermis as well as the dermis. Use of skin biopsies avoids exposure of human volunteers to potentially harmful radiations. The new index is always practically equal or lower than the in vivo SPF depending on the level of a product’s UVA/UVB photoprotection balance. With this, we propose to name this new protection index ‘integrated sun protection factor’.

Cutaneous Tolerance to Nitroxide Free Radicals in Human Skin

No data are available on the irritant effect of nitroxide free radicals in human skin. Nitroxides are important biomedical skin probes used in Electron Paramagnetic Resonance spectroscopy and imaging. Our purpose was to study the skin irritation potential of different nitroxide free radical structures in skin of healthy human subjects. We investigated the following nitroxides: Tempo (2,2,6,6-tetramethyl-1-piperidinoxy), Doxo (2,2,5,5-tetramethyl-3-oxazolidinoxy), Proxo (2,2,5,5-tetramethyl- -dihydro-pyrrolinoxy), and Imidazo (2,2,3,4,5,5-hexamethyl-imidazoline-1-yloxyl). Cutaneous irritation was determined in human skin following a single application and after repetitive applications in comparison to the standardized irritant sodium lauryl sulfate (SLS). The response was evaluated clinically as well as by a bioengineering method analyzing transepidermal water loss (TEWL) and skin hydration (capacitance). The nitroxides were classified clinically from nonirritant (Imidazo, Proxo), to slightly irritant (Doxo, 100 mM), or moderately irritant (Tempo 100 mM) after a single application. The TEWL values were significantly increased by Doxo and Tempo, but capacitance values were not changed significantly. In the cumulative irritation test Tempo was scored as a slight irritant (10 mM). TOLH (2,2,6,6-tetramethyl-1-hydroxypiperidin), the hydroxylamine of Tempo, which is the major skin metabolite, did not cause skin irritation after a single or repetitive applications. This may indicate that a loss of cellular reducing equivalents may be involved in the inflammation process caused by Tempo. The order of nitroxide irritation potency (Tempo > Doxo >> Imidazo = Proxo) is inverse to the order of nitroxide biostability in human skin (Imidazo = Proxo >> Doxo > Tempo). In conclusion, nitroxide free radicals are classified as nonirritant to moderately irritant in human skin. Particularly, the pyrrolidine and imidazoline type nitroxides have a low potential to cause acute or subacute skin toxicity.

[28] In vivo measurement of oxidative stress status in human skin

Publisher Summary The chapter describes an electron paramagnetic resonance- (EPR) based assay to evaluate the oxidative stress status in human skin before and after exposure to solar-simulated light. For EPR in vivo measurements in human skin, two different experimental approaches have been used. The microwave penetration depth in the X-band region is about 0.5–1.0 mm in human skin and about 5 mm for S-band frequency. Thus, the X band is restricted to the upper layer of the skin (the human epidermis), whereas the S band is suited for deeper layers (the human dermis and subcutis). At the S band, the probe head is a 90° bent surface coil with an electronically matched system. Ultraviolet radiation is well known to cause oxidative stress that decreases the antioxidant capacity and the radical-scavenging activity of skin. Uncharged piperidine-type nitroxides are used preferentially, because of their redox properties, to measure tissue antioxidant activity. They possess the right reduction potential, polarity, and permeability through the wall of membranes and the horny layer of the skin. The chapter mentions the successful imaging of the distribution and metabolism of nitroxides in human skin in vivo . The chapter concludes by stating that this technique and application hold great promise in the study of cutaneous redox state, skin diseases, and development of topical pharmaceutical and cosmetic products.

In vivo electron paramagnetic resonance imaging of skin.

Publisher Summary Electron paramagnetic resonance imaging (EPRI) is a unique method for visualizing the spatial distribution of paramagnetic centers, for example, free radicals. The natural free radical concentration in biological systems is usually too low to give signal intensity sufficient for detection by EPRI. The paramagnetic species observed to date are nitroxide free radicals, but new labels with narrow linewidths and trapping capabilities might be used to image the generation of free radicals. One of the driving forces for the development of EPRI is the potential use of nitroxide free radicals. Nitroxides are sensitive to motion, polarity, structural order and fluidity, oxygen tension, redox processes, bioenergetic factors, and the chemical reactivity of their ultimate biophysical surroundings. In the biomedical field, interest has been directed toward in vivo measurements. Application of the EPRI technique to intact animals and humans is limited by two factors. The first is that the conventional EPR frequency of 9 GHz does not permit the use of large aqueous samples, because of the high dielectric loss. Use of low microwave frequencies (0.5–3.0 GHz) solves this problem. The second limiting factor is that stable concentrations of nitroxide radicals are difficult to achieve in tissues, because of nitroxide metabolism and diffusion of the probes. Using highly biostable nitroxides or encapsulating nitroxides in liposomes helps overcome this difficulty.

Penetration of spin-labeled retinoic acid from liposomal preparations into the skin of SKH1 hairless mice measurement by EPR tomography

Abstract 13- trans -Retinoic acid (vitamin A acid) was spin-labeled by forming its carboxylamide with 3-aminomethylproxyl. The spin-labeled derivative is slightly more hydrophobic than the original retinoic free acid (log p = 1.35− > 1.52). The substance was incorporated into liposomes prepared from various phospholipids including an archaebacterial tetraether lipid, and from phospholipid mixtures. With these liposomes pharmaceutical gels were prepared. The penetration of the spin-labeled retinoic acid from these liposomal gel preparations into the skin of hairless mice was determined by means of EPR imaging and compared to that from a non-liposomal hydrogel and from a fatty ointment. A new EPR imaging of the penetration of the spin label into the skin of hairless mice is presented. The advantage of a liposomal gel preparation for the penetration of spin-labeled vitamin A acid is demonstrated.

Cutaneous tolerance to nitroxide free radicals and nitrone spin traps in the guinea pig

The attempts to use nitroxide free radicals and nitrone spin traps topically in skin requires analysis of their potential cutaneous adverse effects. The objective of this study was to investigate the skin irritation and sensitizing potential of nitroxides and nitrones in the guinea pig. The following unsubstituted nitroxides were investigated: 2,2,6,6-tetramethyl-1-piperidinoxyl (Tempo), 2,2, 5,5-tetramethyl-3-oxazolidinoxyl (Doxo), 2,2,5,5-tetramethyl-1-dihydro-pyrrolinoxyl (Proxo), 2,2,3,4,5,5-hexamethyl-imidazoline-1-yloxyl (Imidazo) and the nitrones: 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and N-tert.-butyl-phenylnitrone (PBN). Cutaneous irritation was determined following the modified Draize protocol. The response was evaluated clinically as well as by a biophysical method analyzing transepidermal water loss (TEWL). The nitroxides and nitrones were classified clinically from non-irritant (Proxo, Imidazo, DMPO) to slightly irritant (Tempo, Doxo, PBN) according to the Draize protocol. In agreement with the clinical scoring, the TEWL values were significantly increased by Tempo, Doxo and PBN. TOLH, the hydroxylamine of Tempo and its major skin metabolite, did not cause skin irritation. The sensitizing effect was evaluated according to the Magnusson and Kligman test. The results showed no cutaneous hypersensitivity to all nitroxides and nitrones, indicating a weak sensitizing potential. That concludes that the nitroxides and nitrones tested in this study have a low potential of acute skin intolerance.

Effects of water-filtered infrared-A and of heat on cell death, inflammation, antioxidative potential and of free radical formation in viable skin - First results

The effects of water-filtered infrared-A (wIRA) and of convective heat on viability, inflammation, inducible free radicals and antioxidative power were investigated in natural and viable skin using the ex vivo Bovine Udder System (BUS) model. Therefore, skin samples from differently treated parts of the udder of a healthy cow were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, by prostaglandin E2 (PGE2) measurement and by electron spin resonance (ESR) spectroscopy. Neither cell viability, the inflammation status, the radical status or the antioxidative defence systems of the skin were significantly affected by wIRA applied within 30 min by using an irradiance of 1900 W m(-2) which is of relevance for clinical use, but which exceeded the maximum solar IR-A irradiance at the Earths surface more than 5 times and which resulted in a skin surface temperature of about 45 °C without cooling and of about 37 °C with convective cooling by air ventilation. No significant effects on viability and on inflammation were detected when convective heat was applied alone under equivalent conditions in terms of the resulting skin surface temperatures and exposure time. As compared with untreated skin, free radical formation was almost doubled, whereas the antioxidative power was reduced to about 50% after convective heating to about 45 °C.

Reduction in the free radical status and clinical benefit of repeated intrathecal triamcinolone acetonide application in patients with progressive multiple sclerosis.

BackgroundPrevious open trials performed repeated intrathecal application of the sustained release steroid triamcinolone acetonide every third day in patients with progressive multiple sclerosis and described enhanced walking abilities. ObjectivesThe objectives of this study were to demonstrate the efficacy of 5 triamcinolone administrations every other day and to describe their effects on the amount of inducible free radicals in cerebrospinal fluid. Subjects/MethodsClinical ratings, determinations of maximum walking distance, and execution of an instrumental peg insertion test were performed at baseline and on each day after a triamcinolone injection in 21 patients with progressive multiple sclerosis. Induction of free radicals was assessed in cerebrospinal fluid before each triamcinolone application by electron spin resonance spectroscopy. ResultsScores for multiple sclerosis improved, walking distance increased, and necessary intervals for the peg insertion procedure were shortened. The amount of inducible free radicals decreased. ConclusionsRepeat triamcinolone application improves dysfunction of upper and lower extremities even when administered 5 times only and in series every other day. The declined potential for free radical synthesis may be caused by the anti-inflammatory effect of triamcinolone. It may contribute to suppress the smoldering, chronic inflammation, particularly in spinal lesions of patients with progressive multiple sclerosis. The enhanced arm function hypothetically reflects the effect on cervical and brain lesions due to the hypobaric features of triamcinolone.