Comparable photoreactivity of hair melanosomes, eu- and pheomelanins at low concentrations: low melanin a risk factor for UVA damage and melanoma?

Melanin is known to be photoreactive and photoprotective, but its function in skin in vivo is still debated. Data is lacking of the effects of UVA irradiation on human skin melanosomes of different pigmentation, which have not been extensively degraded by isolation procedures. We have shown previously that melanosomes isolated from human oriental and black cat hair, and synthetic eumelanins, are photoreactive producing superoxide at low concentrations when exposed to UVA irradiation comparable to UK levels of sunlight. Here we investigated the UVA-irradiation of melanosomes, isolated from different colored human hair samples, using electron spin resonance spectroscopy and spin trapping. Comparable irradiation of synthetic pheomelanins synthesized from L-dopa and L-cysteine was also studied. An alkali method (5 min NaOH at 90 degrees C) could be used to isolate oriental hair melanosomes but was not suitable for auburn and blonde hair. Dithiothreitol and proteinase K resulted in melanin release from possible over-digestion of melanosomes; however, dithiothreitol and papain resulted in no melanin release and good melanosome yields with separation from residual keratin for brown, auburn and blonde hair. Melanosomes isolated by the latter method and synthetic pheomelanins were similar in UVA-photoreactivity at low concentrations, independent of hair color, and broadly comparable to synthetic melanins. Melanosome concentration at constant fluence may be more significant with respect to photodamage and UVA photocarcinogenesis (melanoma) via superoxide radical production than pigment type.

Measuring sunscreen protection against solar-simulated radiation-induced structural radical damage to skin using ESR/spin trapping: development of an ex vivo test method.

The in vitro star system used for sunscreen UVA-testing is not an absolute measure of skin protection being a ratio of the total integrated UVA/UVB absorption. The in vivo persistent-pigment-darkening method requires human volunteers. We investigated the use of the ESR-detectable DMPO protein radical-adduct in solar-simulator-irradiated skin substitutes for sunscreen testing. Sunscreens SPF rated 20+ with UVA protection, reduced this adduct by 40-65% when applied at 2 mg/cm(2). SPF 15 Organic UVA-UVB (BMDBM-OMC) and TiO(2)-UVB filters and a novel UVA-TiO(2) filter reduced it by 21, 31 and 70% respectively. Conventional broad-spectrum sunscreens do not fully protect against protein radical-damage in skin due to possible visible-light contributions to damage or UVA-filter degradation. Anisotropic spectra of DMPO-trapped oxygen-centred radicals, proposed intermediates of lipid-oxidation, were detected in irradiated sunscreen and DMPO. Sunscreen protection might be improved by the consideration of visible-light protection and the design of filters to minimise radical leakage and lipid-oxidation.

Antibody-enzyme fusion proteins for cancer therapy.

Advances in biomolecular technology have allowed the development of genetically fused antibody-enzymes. Antibody-enzyme fusion proteins have been used to target tumors for cancer therapy in two ways. In one system, an antibody-enzyme is pretargeted to the tumor followed by administration of an inactive prodrug that is converted to its active form by the pretargeted enzyme. This system has been described as antibody-directed enzyme prodrug therapy. The other system uses antibody-enzyme fusion proteins as direct therapeutics, where the enzyme is toxic in its own right. The key feature in this approach is that the antibody is used to internalize the toxic enzyme into the tumor cell, which activates cell-death processes. This antibody-enzyme system has been largely applied to deliver ribonucleases. This article addresses these two antibody-enzyme targeting strategies for cancer therapy from concept to (pre)clinical trials.

Intensity-dependent direct solar radiation- and UVA-induced radical damage to human skin and DNA, lipids and proteins.

Skin can be exposed to high-intensity UV-radiation in hot countries and during sunbed use; however, the free-radical damage at these intensities is unknown. We used electron spin resonance spectroscopy to measure free-radical generation in ex vivo human skin/substitutes +/- the spin-trap 5,5 dimethyl-1-pyrroline N-oxide (DMPO) exposed to solar-irradiation equivalent to Mediterranean sunlight. Skin-substitutes, model DNA-photosensitizer systems, lipids and proteins were also irradiated with low-intensity UVA/visible light. Without DMPO a broad singlet was detected (using both irradiations) in skin/substitutes, nail-keratin, tendon-collagen, phospholipid and DNA+melanin or riboflavin. In addition to lipid-derived (tentatively tert-alkoxyl/acyl-) and protein radicals detected with DMPO at lower intensities, isotropic carbon-, additional oxygen- and hydrogen-adducts were detected in solar-irradiated skin/substitutes at higher intensities. Carbon-adducts were detected in UVA-irradiated human skin cells, DNA+melanin or riboflavin and soybean-phospholipid. Anisotropic protein-adducts, comparable to adducts in solar-irradiated tendon-collagen, were absent in UVA-irradiated skin fibroblasts suggesting the trapping of extracellular collagen radicals. Absence of hydrogen-adducts in fibroblasts implies formation in the extracellular compartment. We conclude damage at high intensities is part cellular (carbon- and oxygen-radicals) and part extracellular (protein- and hydrogen/H(+)+e(-) ), and skin substitutes are suitable for sunscreen testing. While UVA absorption and lipid-oxidation is direct, DNA and protein-oxidation require photosensitisation.