Julian M. Menter

PHOTOCHEMISTRY OF TYPE I ACID-SOLUBLE CALF SKIN COLLAGEN : DEPENDENCE ON EXCITATION WAVELENGTH

Although previous studies have demonstrated that the predominant photochemistry of type I collagen under 254 nm irradiation may be attributed either to direct absorption by tyrosine/phenylalanine or to peptide bonds, direct collagen photochemistry via solar UV wavelengths is much more likely to involve several age‐ and tissue‐related photolabile collagen fluorophores that absorb in the latter region. In this study, we compare and contrast results obtained from irradiation of a commercial preparation of acid‐soluble calf skin type I collagen in solution with UVC (primarily 254 nm), UVA (335–400nm) and broad‐band solar‐simulating radiation (SSR; 290^1–00nm). Excitation spectroscopy and analysis of photochemically induced disappearance of fluorescence (fluorescence fading) indicates that this preparation has at least four photolabile fluorescent chromophores. In addition to tyrosine and L‐3,4‐dihydroxyphenylalanine, our sample contains two other fluorophores. Chromophore I, with emission maximum at 360 nm, appears to be derived from interacting aromatic moieties in close mutual proximity. Chromophore II, with broad emission at430–435 nm, may be composed of one or more age‐related molecules. Collagen fluorescence fading kinetics are sensitive to excitation wavelength and to conformation. Under UVC, chromophore I fluorescence disappears with second‐order kinetics, indicating a reaction between two proximal like molecules. Adherence to second‐order kinetics is abrogated by prior denaturation of the collagen sample. A new broad, weak fluorescence band at400–420 nm, attributable to dityrosine, forms under UVC, but not under solar radiation. This band is photolabile to UVA and UVB wavelengths. Amino acid analysis indicates significant destruction of aromatic amino acids under UVC, but not under UVA or SSR. When properly understood, collagen fluorescence fading phenomena may act as a sensitive molecular probe of structure, conformation and reactivity.

High performance liquid chromatographic assay for the quantitation of total glutathione in plasma

A simple and widely used homocysteine HPLC procedure was applied for the HPLC identification and quantitation of glutathione in plasma. The method, which utilizes SBDF as a derivatizing agent utilizes only 50 microl of sample volume. Linear quantitative response curve was generated for glutathione over a concentration range of 0.3125-62.50 micromol/l. Linear regression analysis of the standard curve exhibited correlation coefficient of 0.999. Limit of detection (LOD) and limit of quantitation (LOQ) values were 5.0 and 15 pmol, respectively. Glutathione recovery using this method was nearly complete (above 96%). Intra-assay and inter-assay precision studies reflected a high level of reliability and reproducibility of the method. The applicability of the method for the quantitation of glutathione was demonstrated successfully using human and rat plasma samples.

Effect of UV irradiation on type I collagen fibril formation in neutral collagen solutions

Background: Collagens have the well‐known ability to spontaneously self‐associate to form fibrils at physiological temperature and neutral pH in vitro and in vivo. Because solar UV may photochemically alter collagen, the kinetics of fibril formation may be modified. Thus, we have begun a systematic study of the effect of various UV wavebands on fibril formation.

Effect of UV on the susceptibility of acid-soluble Skh-1 hairless mouse collagen to collagenase

Background/Purpose: Photoaging of the skin is a result of chronic exposure to environmental ultraviolet radiation (UV). The milieu provided by the extracellular matrix, which significantly influences the behaviour of resident fibroblasts, depends critically on the supermolecular collagen structure. We ask whether direct photochemical treatment of type I collagen with solar wavelengths capable of reaching the dermis can modify the substrates susceptibility to collagenase in a model in vitro system.

Protection against photodynamic therapy (PDT)‐induced photosensitivity by fabric materials

“Special” highly protective fabrics are now available that offer broad‐spectrum protection in preventing sunburn, and possibly other types of photodamage. It is important to know to what extent these fabrics are capable of protecting the wearer against skin cancer, photosensitivity disorders, and inadvertent phototoxic reactions from photodynamic therapy (PDT). We assess the ability of one such special (Solumbra) fabric and one “typical” summer fabric to provide protection against PDT phototoxicity produced in tape‐stripped Sk‐1 hairless mice by topical 5‐aminolevulinic acid (ALA) and (primarily) visible light (360–800 nm). Since ALA‐derived photosensitizers absorb most of the visible spectrum, results from these studies give a good indication of the photoprotective capability of these fabrics throughout this region. Mice were irradiated dorsally with a Kodak slide projector equipped with a 300 W tungsten‐halogen lamp (I0=48.3 mW/cm2). After determining the minimal phototoxic dose (MPD) to be 1.40±0.4 J/cm2, we irradiated the tape‐stripped ALA‐sensitized mice through the stretched test fabrics with appropriate multiples of the MPD. The special fabric provided protection against 25–30 MPD visible light between 360–800 nm in 14/14 mice. The typical fabric failed to provide protection against 2.5 MPD of the same spectrum. No phototoxic or other adverse responses were seen with either the ALA or light control. In conclusion, the Solumbra fabric is much more protective against ALA photosensitization than the typical fabric. Both appear better at blocking UV than visible light.

Photoprotection of mammalian acid-soluble collagen by cuttlefish sepia melanin in vitro.

Several important clinical conditions can result in close association between the pigment melanin and dermal collagen. Because melanin and its precursors can be chemically reactive in ground and excited states, it is important to know whether the resulting melanin‐collagen interaction results in photoprotection or photoaggression. Acidic and neutral air‐saturated collagen suspensions (0.033%) were irradiated with0–2.6 times 104 J/m2 UVC or with0–83 times 104 J/m2 solar‐simulating UV radiation (SSR). Photochemical destruction of a photolabile collagen fluorophore (δem 360 nm) and collagen chain degradation were monitored as functions of irradiation time in the presence and absence of added (0–100μg) sepia eumelanin. Melanin retarded collagen photodamage but did not qualitatively alter the fluorescence fading kinetics. Both H202 and 02 can be produced by UV irradiation of eumelanin. Added H202 and K02 destroyed collagen fluorescence and caused 50% chain degradation at ca10–20‐fold molar excess. Previous studies have demonstrated that eumelanins efficiently scavenge 02 . We demonstrated that eumelanin also efficiently scavenges H202 as evidenced by its ability to (a) compete with scopoletin for peroxide uptake and (b) directly take up H202 through a dialysis bag. The latter observation suggests that peroxide scavenging could occur in vivo by melanin sequestered in melanophages. Thus, neither UV‐generated 02 nor H202 are likely to be present in concentrations high enough to cause measurable collagen damage. Absorption and/or scattering of excitation radiation away from the target chromophore appears to be the primary photoprotection mechanism, although scavenging of active 02 intermediates may play an important, if subtle role.

EFFECT OF CHRONIC UV EXPOSURE ON EPIDERMAL FORWARD SCATTERING-ABSORPTION INSK–1 HAIRLESS MOUSE SKIN

Abstract— Clinical and histological precancerous responses to UV irradiation are complicated dynamic functions of total dose, dose fractionation, fluence rate, and spectral distribution. This may be due, in large part, to the ability of UV to decrease epidermal‐stratum corneum transmission by stimulation of hyperplasia. This work provides quantitative measurement of dose‐ and wavelength‐dependent optical changes inSK–1 hairless mouse epidermis‐stratum corneum occurring under irradiation with “monochromatic” UV wavebands, at 280, 290, 300, 307, and 313 nm. Mice were irradiated 5 days per week with a filtered Xenon‐Hg high‐intensity grating monochromator, starting with 0.9 minimal erythemal dose (MED), followed by incremental increases in the radiation dose by 20% of the original dose every tenth irradiation day, for2–8 consecutive weeks. Subsequent irradiations (for longer experiments) were followed by 30% incremental increases after the 8th week every 10th irradiation day until cessation of radiation at the end of 14 weeks. Irradiated and control full‐thickness epidermis/ stratum corneum were examined histologically and by forward‐scattering absorption spectroscopy. Chronic irradiation of hairless mice resulted in significant hyperplasia which was optically manifested by a general increase in forward‐scattering absorbance. At moderate local doses (7.2 MED), the absorbance increase per MED was approximately the same for all excitation wavelengths, whereas at large total doses (≅ 100 MED) the optical increase per delivered MED progressively decreased in the order 313> 307> 300≅ 290> 280 nm. The increase in skin thickening, expressed as observed increase in absorption at 320 nm, correlated well with histological and clinical data. We propose that optical changes induced by UV‐induced thickening can account in large part, if not entirely, for dynamic changes in action spectra for (pre) cancerous processes under chronic irradiation conditions.

UV-induced elevation of skin glycosaminoglycan levels I: rapid measurement of skin glycosaminoglycans by titration with acridine orange.

Abstract— We have developed a simple, rapid, sensitive procedure for measuring polyanionic glycosaminoglycans (GAG) in unfixed defatted lyophilizedSK–1 hairless mouse skin by means of quantitative measurement of uptake of the fluorescent dye acridine orange (AO), which was shown to be GAG‐specific under the conditions of these experiments. This method is suitable for detecting and estimating elevations in steady skin GAG levels due to UV irradiation.

Melanin is a Double-Edged Sword

Melanin is known to photoprotect by physical absorption/scattering of UV and by electron transfer/free radical scavenging. In this work we show that melanin can also facilitate potentially harmful redox reactions in vitro. By kinetic spectrometry and 02uptake, we show that both synthetic eumelanin (Sigma) and Sepia melanin extracted from cuttlefish markedly accelerate the tyrosinase-catalyzed oxygenation of the cytotoxic phenol p-hydroxyanisole MMEH to the cytotoxic 4-methoxy-1,2-benzoquinone. These studies indicate that a substrate-melanin complex transfers electrons to tyrosinase to regenerate Cu(I) necessary for reduction of molecular 02.Prior irradiation of melanin with solar-simulating UV results in evanescent changes in the kinetics of quinone formation which are consistent with reversible photooxidation of melanin. The latter effect vis-a-vis photoprotection is ambivalent at best. Thus, melanin protects with one hand, and, perhaps unwittingly, may place surrounding cells at risk with the other.

Fluorescence of putative chromophores in Skh-1 and citrate-soluble calf skin collagens

Background/purpose: Fluorescence of Skh‐1 hairless mouse and calf skin acid‐soluble type I collagens are envelopes of several bands putatively due to tyrosine (excitation/emission peak at 275/300 nm), dihydroxyphenylalanine (dopa; 280/325 nm), tyrosine aggregate (285/360 nm), dityrosine 325/400 nm), and advanced glycation end (AGE) product (370/450 nm), respectively. As these fluorophores can be markers of pathological conditions, we wish to present further evidence for or against these assignments.