We have shown that UVA (320-380 nm) radiation, hydrogen peroxide, and sodium arsenite induce a stress protein of %32 kDa in human skin fibroblasts. The synthesis and cloning of cDNA from arsenite-induced mRNA populations have now allowed us to unequivocally identify the 32-kDa protein as heme oxygenase. By mRNA analysis we have shown that the heme oxygenase gene is also induced in cultured human skin fibroblasts by UVA radiation, hydrogen peroxide, cadmium chloride, iodoacetamide, and menadione. The known antioxidant properties of heme catabolites taken together with the observation ofa high level ofinduction ofthe enzyme in cells from an organ not involved in hemoglobin breakdown strongly supports the proposal that the induction of heme oxygenase may be a general response to oxidant stress and constitutes an important cellular defense mechanism against oxidative damage.Mammalian cells respond to a wide variety of adverse conditions, both chemical and physical, by inducing the synthesis of a number of stress proteins. The most widely studied of these responses is that induced by heat shock (for review see refs. 1 and 2). Although certain insults other than heat shock itself induce heat shock proteins-for example, treatment of cells with amino acid analogues (3)-others induce stress proteins unrelated to those inducible by heat shock. Both glucose deprivation and anoxic stress are good examples of the latter (4, 5). We have reported (6) the induction of a 32-kDa stress protein in human skin fibroblasts by both near UV radiation (UVA) and the oxidizing agent hydrogen peroxide. Neither of these treatments induced the major heat shock proteins. In addition, we were unable to induce the 32-kDa protein by heat shock in these cells, indicating that the mechanism of induction of this protein is different from that involved in heat shock. Interestingly, a protein of30-35 kDa had been reported (7-10) as inducible by certain chemical treatments, including heavy metal salts and the sulfhydryl reagent sodium arsenite in rodent, avian, and human cells. In all of these cases the heat shock proteins were also induced. A comparison of the 32-kDa proteins induced by UVA, hydrogen peroxide, and sodium arsenite in human skin fibroblasts by partial peptide mapping indicated that they were identical (6).We now describe the isolation ofcDNA clones corresponding to the mRNA species encoding this stress protein and identify the inducible gene as coding for heme oxygenase. We speculate that this enzyme participates in an inducible protective mechanism against oxidative stress induced by UVA and hydrogen peroxide in human skin cells. MATERIALS AND METHODSCell Culture and Treatment with UVA and Chemicals. The normal human skin fibroblast cell line EK4 was derived from a foreskin explant, in this laboratory, and cultured routinely as described (11). Cells were seeded into plastic culture dishes (30-50% confluence) 2-4 days prior to either chemical treatment or UVA irradiation. To treat cells with chemicals, the growth medium was rem...

No abstract

Oxidative stress of human skin fibroblasts by treatment with ultraviolet A (UVA) radiation has been shown to lead to an increase in levels of the heme catabolizing enzyme heme oxygenase 1 [heme, hydrogen-donor:oxygen oxidoreductase (a-methene-oxidizing, hydroxylating), EC 1.14.99.3] and the iron storage protein ferritin. Here we show that human skin fibroblasts, preirradiated with UVA, sustain less membrane damage during a subsequent exposure to UVA radiation than cells that had not been preirradiated. Pretreating cells with heme oxygenase 1 antsense oligonucleotide inhibited the irradiationdependent induction of both the heme oxygenase 1 enzyme and feritin and abolished the protective effect of preirradiation. Inhibition of the UVA preirradiation-dependent increase in femtin, but not heme oxygenase, with desferrioxamine also abolished the protection. This identifies heme oxygenase 1 as a crucial enzymatic intermediate in an oxidant stress-inducible antioxidant defense mechanis, involving ferritin, in human skin fibroblasts.Expression of the heme oxygenase 1 gene is enhanced by oxidative stress (1-6) including UVA radiation (320-380 nm) (7-9), a major component of sunlight. We have recently demonstrated that induction of the heme oxygenase enzyme [heme, hydrogen-donor:oxygen oxidoreductase (a-metheneoxidizing, hydroxylating), EC 1.14.99.3] by UVA irradiation of cultured human skin fibroblasts leads to an increase in ferritin (10). Ferritin constitutes the major storage site for nonmetabolized intracellular iron and therefore plays a critical role in regulating the availability of iron to catalyze such harmful reactions as the peroxidation oflipids and the Fenton reaction producing hydroxyl radicals. Recent studies have implicated intracellular ferritin in the protection of rat kidney (11) and cultured aortic endothelial cells (12) from oxidantinduced damage. This study was undertaken to determine whether a heme oxygenase-dependent increase in ferritin levels leads to an adaptive response in human skin fibroblasts. MATERIALS AND METHODS 2607The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Currently, there is no available needle-free approach for diabetics to monitor glucose levels in the interstitial fluid. Here, we report a path-selective, non-invasive, transdermal glucose monitoring system based on a miniaturized pixel array platform (realized either by graphene-based thin-film technology, or screen-printing). The system samples glucose from the interstitial fluid via electroosmotic extraction through individual, privileged, follicular pathways in the skin, accessible via the pixels of the array. A proof of principle using mammalian skin ex vivo is demonstrated for specific and 'quantized' glucose extraction/detection via follicular pathways, and across the hypo- to hyper-glycaemic range in humans. Furthermore, the quantification of follicular and non-follicular glucose extraction fluxes is clearly shown. In vivo continuous monitoring of interstitial fluid-borne glucose with the pixel array was able to track blood sugar in healthy human subjects. This approach paves the way to clinically relevant glucose detection in diabetics without the need for invasive, finger-stick blood sampling.

Ultraviolet radiation activates the expression of a wide variety of genes, by pathways which differ between the short non-solar ultraviolet C (UVC) wavelengths, which are strongly absorbed by nucleic acids, and the long solar ultraviolet A (UVA, 320-380 nm) wavelengths, which generate active oxygen intermediates. Intermediate solar ultraviolet (UV) wavelengths in the UVB (290-320 nm) range also contain an oxidative component, but more closely resemble UVC in their gene activating properties. Short wavelength UV, in common with other extracellular stimuli including growth factors, activates signal transduction events that involve both stress- and mitogen-activated protein kinase cascades. The extrapolation of the complex modulation of gene expression that ensues to the consequences of natural UV exposure requires careful attention to the details of doses and wavelength employed in the model experiments. Nevertheless, there is evidence that UVB irradiation of skin can activate the expression of proteins including immunomodulating cytokines, ornithine decarboxylase and, to a limited extent, nuclear oncogene products, as well as lead to stabilisation of p53. Non-cytotoxic doses of UVA radiation also lead to the strong activation of several genes which would be expected to have functional relevance in vivo.

In mammalian cells, the level of the ironstorage protein ferritin (Ft) is tightly controlled by the iron-regulatory protein-1 (IRP-1) at the posttranscriptional level. This regulation prevents iron acting as a catalyst in reactions between reactive oxygen species and biomolecules. The ultraviolet A (UVA) radiation component of sunlight (320-400 nm) has been shown to be a source of oxidative stress to skin via generation of reactive oxygen species. We report here that the exposure of human primary skin fibroblasts, FEK4, to UVA radiation causes an immediate release of ''free'' iron in the cells via proteolysis of Ft. Within minutes of exposure to a range of doses of UVA at natural exposure levels, the binding activity of IRP-1, as well as Ft levels, decreases in a dose-dependent manner. This decrease coincides with a significant leakage of the lysosomal components into the cytosol. Stabilization of Ft molecules occurs only when cells are pretreated with lysosomal protease inhibitors after UVA treatment. We propose that the oxidative damage to lysosomes that leads to Ft degradation and the consequent rapid release of potentially harmful ''free'' iron to the cytosol might be a major factor in UVA-induced damage to the skin.

No abstract

No abstract