The in vivo fluorescence of tryptophan moieties in human skin increases with UV exposure and is a marker for epidermal proliferation.

We have investigated the in vivo fluorescence of human skin with UV excitation and the effect of UV irradiation on the UV fluorescence. A particular chromophore was found to be very sensitive to suberythemogenic UV radiation. This chromophore has the spectral characteristics of tryptophan residues in proteins and is characterized by a fluorescence excitation maximum at 295 nm. The fluorescence of this chromophore in human epidermis has an excitation maximum that is coincident with the maximum of the action spectrum of most UV-induced photobiologic responses to human skin. The fluorescence of the chromophore was found to increase with UV exposure. The action spectrum was determined by following the increase of the emission at 345 nm with excitation at 295 nm as a function of skin exposure to a number of wavelengths in the UV region of the spectrum. The results show that irradiation in the UVB (290-320 nm) is more effective in producing the change in the fluorescence of tryptophan. Irradiation in the UVA (320-380 nm) was found to be capable of producing the increase but to a smaller extent. Whereas tryptophan fluorescence is found in both the epidermis and the dermis, it is only the epidermal component that increases with UV exposure. The change in 295 nm fluorescence with UV exposure was determined to be oxygen dependent. The fluorescence of tryptophan moieties measured in situ was found to increase when epidermal proliferation increases. This was verified by inducing epidermal repair after mechanical insult (tape stripping). The results suggest two possible scenarios for the UV-induced increase of the fluorescence: a prompt photooxidation of tryptophan moieties or a fast proliferation response to the insult created by UV irradiation.

Attenuated total reflection-Fourier transform infrared spectroscopy as a possible method to investigate biophysical parameters of stratum corneum in vivo.

We investigated the use of attenuated total reflection-Fourier transform infrared spectroscopy as a method to study differences in the molecular components of human stratum corneum in vivo. These variations as a function of the anatomic site and of the depth into its layered structure are important to understand the biology and physiology of the tissue. In this preliminary study we have investigated spectroscopic changes in 18 healthy individuals. Total reflection-Fourier transform infrared spectroscopy represents a potentially powerful tool to study biophysical properties of surfaces. We observed that, in vivo, biophysical parameters of the stratum corneum (such as hydration, lipid composition, and conformation of the aliphatic chains) are indeed dependent on the anatomic site. As in total reflection-Fourier transform infrared spectroscopy experiments the penetration depth of the evanescent field into the stratum corneum is comparable with the thickness of a layer of corneocytes, this technique can be used to follow the distribution of lipids, water, and proteins as a function of depth into the tissue. We found that, in vivo, these molecular components are non-uniformly distributed, in agreement with the presence of water and lipid reservoirs as observed with ex vivo ultrastructural investigations. Composition and conformational order of lipids are also a function of depth into the stratum corneum. Finally we compared the in vivo superficial hydration measured using the infrared absorption of the OH stretch of water, with the hydration measured using the Skicon hygrometer. Our results indicate that total reflection-Fourier transform infrared spectroscopy might be useful to measure important chemical and biophysical parameters of stratum corneum in vivo.

Aging and effects of ultraviolet A exposure may be quantified by fluorescence excitation spectroscopy in vivo.

The fluorescence properties of skin chromophores such as tryptophan and collagen cross-links might be useful markers of aging and photoaging. As the fluorescence of pepsin-digestible collagen cross-links was found to increase with aging and decrease with photoaging we investigated the characteristics of this dependence. In vivo fluorescence excitation spectra (emission at 380 nm) of SKH hairless mouse model skin are characterized by two bands centered near 295 nm and 335 nm due, respectively, to epidermal tryptophan moieties and pepsin-digestible collagen cross-links. Several groups of hairless mice were followed over a period of 18 mo to document changes in skin fluorescence with aging. Other groups of animals were exposed to either broad band or narrowband ultraviolet A radiation to determine the effects of ultraviolet A exposure on the fluorescence of the dermal collagen cross-links and to determine an action spectrum for the induced changes. We also found that the intensity of pepsin-digestible collagen cross-links in vivo increases linearly with age and that the fluorescence of epidermal tryptophan decreases linearly with age. We found that the fluorescence of pepsin-digestible collagen cross-links decreases immediately following exposure to ultraviolet A whereas epidermal tryptophan fluorescence increases. Both changes were dose dependent but the increase in tryptophan fluorescence occurred exclusively in young animals (2--6 mo old). We found that the ultraviolet-induced fluorescence decrease of pepsin-digestible collagen cross-links is wavelength specific. The action spectrum for the ultraviolet A effect on the in vivo fluorescence of pepsin-digestible collagen cross-links shows a distinct maximum at 335 nm that corresponds to the maximum in the fluorescence excitation spectrum due to pepsin-digestible collagen cross-links. Our results seem to indicate that in vivo fluorescence of epidermal tryptophan moieties and collagen cross-links in the dermal matrix may serve as markers for skin aging, for photoaging, and for immediate assessment of exposure to ultraviolet A radiation.

The effect of photofrin on DNA strand breaks and base oxidation in HaCaT keratinocytes: a comet assay study.

Photodynamic therapy (PDT) kills cells via the production of singlet oxygen and other reactive oxygen species. PDT causes chromosomal damage and mutation to cultured cells. However, DNA damage does not contribute to the phototoxic effect. To study the effect of Photofrin-PDT-induced DNA damage, we used the comet assay in combination with endonuclease III and formamidopyrimidine DNA glycosylase and a human keratinocyte cell line to investigate photogenotoxicity and its prevention by tocopherol (TOC). This study shows that PDT induced DNA damage in HaCaT cells at doses allowing cells to survive 7 days after irradiation. alpha-TOC did not prevent the acute cell lysis caused by Photofrin-PDT but did prevent Photofrin-PDT-induced DNA damage. However, the concentration of TOC that conferred protection (100 microM) was higher than is detected in human serum. Base oxidation was also measured using the comet assay. Although TOC could prevent frank DNA strand breaks caused by PDT, it was unable to decrease the level of base oxidation as revealed by enzyme-sensitive sites. It is suggested that the potential genotoxic risk from laser-PDT could be low, and that topical micro-TOC at a high concentration may be useful in preventing some types of DNA damage without preventing acute photolysis after Photofrin-PDT.

In vivo fluorescence spectroscopy of nonmelanoma skin cancer.

In vivo and ex vivo tissue autofluorescence (endogenous fluorescence) have been employed to investigate the presence of markers that could be used to detect tissue abnormalities and/or malignancies. We present a study of the autofluorescence of normal skin and tumor in vivo, conducted on 18 patients diagnosed with nonmelanoma skin cancers (NMSC). We observed that both in basal cell carcinomas (BCC) and squamous cell carcinomas (SCC) the endogenous fluorescence due to tryptophan residues was more intense in tumor than in normal tissue, probably due to epidermal thickening and/or hyperproliferation. Conversely, the fluorescence intensity associated with dermal collagen crosslinks was generally lower in tumors than in the surrounding normal tissue, probably because of degradation or erosion of the connective tissue due to enzymes released by the tumor. The decrease of collagen fluorescence in the connective tissue adjacent to the tumor loci was validated by fluorescence imaging on fresh-frozen tissue sections obtained from 33 NMSC excised specimens. Our results suggest that endogenous fluorescence of NMSC, excited in the UV region of the spectrum, has characteristic features that are different from normal tissue and may be exploited for noninvasive diagnostics and for the detection of tumor margins.

Photodynamic therapy in dermatology: Dundee clinical and research experience.

Topical photodynamic therapy (PDT) is increasingly accepted and used as a highly effective treatment for superficial non-melanoma skin cancer and dysplasia. We describe the developments in topical PDT for the treatment of skin diseases in our own PDT Centre in Dundee, both clinically and from a research base. Improvements in PDT could be achieved by optimisation of photosensitiser and light delivery, and these goals underpin the aims of our centre. We hope to facilitate the dissemination of use of PDT in dermatology throughout Scotland and outline some of the progress in these areas.

Porphyrin-melanin interaction: effect on fluorescence and non-radiative relaxations.

Optical techniques and pulsed-laser, time-resolved photoacoustics (PA) were employed to obtain information on the mechanism of interaction between cationic zinc tetrabenzilpyridilporphyrin (ZnTBzPyP) and synthetic L-Dopa melanins. Synthetic eumelanin and pheomelanin strongly quench the fluorescence of ZnTBzPyP, but Stern-Volmer plots suggest a mechanism of interaction quite different for the two pigments. This diversity was confirmed by PA: for eumelanin no thermal relaxation was observed other than prompt heat, whereas for the complexed form of ZnTBzPyP with pheomelanin we were able to detect a heat-emitting species with a non-radiative lifetime in the microsecond range. The involvement of oxygen in the photophysics of the complexes formed between the cationic porphyrin and the two pigments was demonstrated, but its role has yet to be described.

Porphyrin profile in four human cell lines after supplementation with 5-aminolaevulinic acid and its methyl ester.

Multiple factors can affect the synthesis of the prodrugs aminolaevulinic acid and its methyl ester to protoporphyrin. These may ultimately influence the efficacy of ALA-induced porphyrin as a photosensitiser for photodynamic therapy or fluorescence diagnosis. This study demonstrates the variation in total amount of porphyrin produced and cellular porphyrins synthesised in four different human cell lines after supplementation with these prodrugs. A non-invasive optical biopsy system was able to detect spectral changes associated with the more carboxylated porphyrins accumulating in oesophageal (OE19) and bladder (HT1197) carcinoma cells, and to a lesser extent neuroblastoma (SH-SY5Y) cells after a 24h incubation with the prodrugs. If the porphyrin profile changes during disease progression, or between normal and cancerous cells clinically, then the demonstrated non-invasive spectral analysis may be exploitable in distinguishing between normal, dysplastic and tumour tissue. Finally, the OE19 cell line was insensitive to photo-inactivation under the experimental conditions used, despite accumulating more porphyrin than the other cells lines.

Clinical and research applications of photodynamic therapy in dermatology: experience of the Scottish PDT Centre.

BACKGROUND AND OBJECTIVES: The Scottish PDT Centre has carried out 3,442 treatments on 762 patients with superficial skin lesions, especially superficial basal cell carcinoma (sBCC), Bowen's disease (BD) and actinic keratosis (AK). STUDY DESIGN MATERIALS AND METHODS: The article reviews our experience of various light sources and associated dosimetry; thereafter we discuss clinical outcome followed by some of our research studies in clinically important areas. RESULTS: We show that improved dosimetry is required to ensure an optimal light dose is delivered to the tumour. We have shown that photosensitizers and proteins interact in such a way that their photophysical and photochemical properties are modified. We have also demonstrated the presence of DNA strand breaks with two different photosensitizers, but there is no evidence that PDT is significantly mutagenic in clinical practice. CONCLUSIONS: In our experience, topical PDT is generally well tolerated and is an effective treatment of sBCC, BD, AK, field change and lesions at sites of poor healing.

Laser and non-laser light sources for photodynamic therapy.

Photodynamic therapy (PDT) is an anticancer combination therapy, which requires a photosensitiser, which tends to accumulate preferentially in the tumour, and light. Historically large, complex lasers have been used to carry out PDT treatment. Nowadays there is a wide range of coherent and non-coherent sources that can be used. This paper considers the important characteristics of light sources for PDT, including dye lasers pumped by argon or metal vapour lasers and frequency-doubled Nd:YAG lasers. Non-laser sources including tungsten filament, xenon arc, metal halide and fluorescent lamps are also discussed. New exciting developments such as LEDs and femtosecond lasers are also reviewed. The relative merits of laser and non-laser sources are critically examined.

Anomalous temperature fluorescence quenching of N-Trp terminal peptides.

The photophysics of Trp-containing peptides is extremely affected by the position of the indole ring with respect to substituents. In this work an unusual temperature fluorescence quenching behavior is presented. The N-tryptophan terminal peptides (N-Trp) show an increase of the static emission intensity as rising the temperature from 10 to about 40 degrees C. the anomaly is typical of the N-Trp terminal peptides since neither tryptophan (Trp) nor glycyl-tryptophan (Gly-Trp) and alanyl-tryptophan (Ala-Trp) show the same trend; a similar behavior is not detected in the C-tryptophan terminals. The other important features are the wavelength and pH dependence of the effect. The anomaly is in fact detected only at neutral pH and for excitation wavelength near the red edge of the UVB absorption band of indole. An interpretation of the anomaly is suggested, though more sophisticated techniques are needed to better focus the problem; the model proposed involves the superimposition of a ground state effect (the temperature-induced equilibrium shift from the zwitterionic to the anionic form of the peptides) and an excited state mechanism. At present no unique interpretation can be provided about the excited state mechanism that favors the anomaly ans some suggestions are discussed.

A model for the explanation of the thermally induced increase of the overall fluorescence in tryptophan-X peptides.

In the range of temperature 10-35 degrees C, Trp-X dipeptides show an unusual increase of fluorescence intensity in solution at pH 7. This effect has been recently studied by means of steady-state fluorescence. Although a model involving the deprotonation at the ground state of the zwitterion was proposed, the activation energy for that process could not rule out the involvement of excited state. In order to understand the mechanism of the thermal-induced increase of fluorescence, we present here time-resolved fluorescence experiments on Trp-X and X-Trp dipeptides at different pH and excitation wave-length. The fluorescence lifetimes (tau i) decrease in accord to thermal quenching, with activation energies (Ei) ranging from 4.0 to 6.4 kcal/mol. Under those circumstances where the anomaly was detected the preexponential factors of the longer-lived component increased as well as their fractional fluorescence. This component can be assigned to the anion species. Because of its larger (three- to fourfold) fluorescence quantum yield, compared to that of the corresponding zwitterion, the large increase of the concentration of the anion leads to an increase of the overall emission despite the thermal quenching. Also the decay-associated spectra well account for the red shift of the emission fluorescence spectrum, which accompanies the anomaly. Our model well fits the experimental data using a simple equation which combines Van't Hoff and Arrhenius equations; it also explains the presence of the anomalous thermal quenching exclusively in Trp-X dipeptides excited above 290 nm and at pH around neutrality.

Photophysical properties of porphyrin planar aggregates in liposomes.

This paper describes studies of some photophysical properties of non-covalent planar aggregates of hematoporphyrin and protoporphyrin. This porphyrin species has been recently discovered and can be generated in lipid bilayers such as liposomes and inner mitochondrial membranes. The relative weight of this species in different media, as compared to porphyrin monomers and stacked aggregates, has been deduced by fluorescence decay studies. In contrast with what is observed for stacked aggregates, promotion of planar suprastructures can occur both in aqueous and lipid environments. The spectroscopic properties are very similar to those of the corresponding monomers, in particular as regards the shape of the absorption and emission spectra. The fluorescence decay times are generally higher than those of the monomers, and depend on the medium in which the planar aggregates are formed. The photooxidation properties of porphyrin planar aggregates, as revealed by oxygen consumption and histidine photodegradation upon irradiation at 365 nm, were compared to those of the monomers. The extent of the photooxidation process is nearly 20-30% higher in planar aggregates than in the monomers. In contrast, it is well known that cofacial aggregates are photochemically inert and only monomeric species of porphyrin are efficient photosensitizers. The biological relevance of these findings is discussed.