Potential protection of skin by acute UVA irradiation--from cellular to animal models.

The UVA (320-380 nm) component of sunlight has oxidizing properties which may be deleterious to skin cells and tissue but can also lead to the strong up-regulation of the heme-catabolizing enzyme, heme oxygenase-1. This enzyme has well-established antioxidant actions in cells as well as anti-inflammatory properties in mammals. There is also evidence from rodent models that this enzyme is responsible for the UVA-mediated protection against UVB-induced immunosuppression that occurs in skin. The relevance of these findings to acute and chronic effects of sunlight including skin carcinogenesis is currently under investigation as are the potential implications for sunlight protection in humans.

Solar ultraviolet A radiation: an oxidizing skin carcinogen that activates heme oxygenase-1.

Ultraviolet A (UVA: 320-380 nm) radiation is an oxidizing carcinogen that has proved an ideal agent for demonstrating the oxidant inducibility of the mammalian heme oxygenase-1 (HO-1) gene. The UVA response in cultured human skin fibroblasts and other cell types is mediated by singlet oxygen and is strongly influenced by cellular reducing equivalents. Free heme, an entity that can be generated by UVA irradiation of cells, also appears to be a critical intermediate that can directly influence both the transcriptional activation and repression of the HO-1 gene. Heme release is likely to be of central importance to the inflammatory response in skin and its abrogation by HO.

Tea flavonoids and cardiovascular health.

Tea is rich in antioxidant polyphenols (catechins, flavonols, theaflavins and thearubigins). Epidemiological evidence relating regular consumption of tea or related polyphenols to CHD is equivocal. Catechins are absorbed from tea, but low plasma concentrations are attained. The bioavailability of theaflavins and thearubigins is unknown. Tea does not reduce blood pressure or plasma lipids in well-controlled human trials. Tea polyphenols inhibit LDL lipid peroxidation in vitro, but the effect ex vivo is small. The plasma antioxidant potential increases after drinking green but not black tea. Tea consumption tended to reduce the development of aortic atherosclerosis in rabbits. Tea polyphenols exert marked effects on cells, and inhibit neutrophil migration and inflammatory responses, sometimes at low concentrations. These diverging results suggest potential beneficial effects, but emphasize the need for good human trials of tea using early markers of CHD before firm conclusions can be drawn.

Epicatechin and its in vivo metabolite, 3'-O-methyl epicatechin, protect human fibroblasts from oxidative-stress-induced cell death involving caspase-3 activation.

There is considerable current interest in the cytoprotective effects of natural antioxidants against oxidative stress. In particular, epicatechin, a major member of the flavanol family of polyphenols with powerful antioxidant properties in vitro, has been investigated to determine its ability to attenuate oxidative-stress-induced cell damage and to understand the mechanism of its protective action. We have induced oxidative stress in cultured human fibroblasts using hydrogen peroxide and examined the cellular responses in the form of mitochondrial function, cell-membrane damage, annexin-V binding and caspase-3 activation. Since one of the major metabolites of epicatechin in vivo is 3'-O-methyl epicatechin, we have compared its protective effects with that of epicatechin. The results provide the first evidence that 3'-O-methyl epicatechin inhibits cell death induced by hydrogen peroxide and that the mechanism involves suppression of caspase-3 activity as a marker for apoptosis. Furthermore, the protection elicited by 3'-O-methyl epicatechin is not significantly different from that of epicatechin, suggesting that hydrogen-donating antioxidant activity is not the primary mechanism of protection.

An HPLC method to detect heme oxygenase activity.

This unit presents a method to calculate heme oxygenase enzymatic activity from the formation of bilirubin equivalents [biliverdin-Ix alpha (BV) and bilirubin-IX alpha (BR)]. The BV and BR generated in the reaction are separated by reversed-phase HPLC and detected using visible absorbance spectroscopy. Since both metabolites of heme degradation are directly quantifiable, the assay eliminates the requirement for biliverdin reductase supplementation.

Heme oxygenase activity causes transient hypersensitivity to oxidative ultraviolet A radiation that depends on release of iron from heme.

Heme oxygenase (HO) breaks down heme to iron, biliverdin, and carbon monoxide, and activity of this enzyme increases in many tissues and cell types after exposure to oxidative stress. There is evidence that increased HO activity is involved in long-term protective mechanisms against oxidative stress. We studied the effect of artificially overexpressed HO activity on the cytotoxicity of oxidative ultraviolet A (UVA) radiation after loading human cells with the HO substrate ferric heme (hemin). In contrast to the reported long-term protection attributed to HO activity, cells overexpressing HO activity were hypersensitive to UVA radiation shortly after heme treatment when compared with control cells. Cells overexpressing HO activity showed an increased rate of heme consumption and a higher level of accumulated free chelatable iron when compared with control cells. The hypersensitivity of cells overexpressing HO to UVA radiation after heme treatment was apparently caused by the increased accumulation of chelatable iron, because the iron chelator desferrioxamine strongly reduced the hypersensitivity. One day after the heme treatment, cells overexpressing HO activity were no longer hypersensitive to UVA radiation. We conclude that increased HO activity can temporarily increase the sensitivity of cells to oxidative stress by releasing iron from heme.

Modulation of c-jun and c-fos transcription by UVB and UVA radiations in human dermal fibroblasts and KB cells.

We have previously demonstrated that the oxidizing component of ultraviolet-A (UVA) plays a central role in the activation of the nuclear oncogene and transcription factor, c-fos, in cultured human skin fibroblasts. We have now shown that expression of both c-jun and c-fos (AP-1) family of transcription factors is modulated by short and long wavelength solar ultraviolet (UV) radiation in human fibroblasts and human KB cells. UVA radiation activated c-jun and c-fos in both fibroblasts and KB cells, whereas ultraviolet-B (UVB) radiation activates such oncogenes only in KB cells. Moreover, decreasing the intracellular levels of reducing equivalents in human fibroblasts by glutathione (GSH) depletion lowered the UVA dose threshold for c-jun and c-fos activation several-fold and greatly amplified the UVA-mediated activation of such genes. A more modest effect was observed in GSH-depleted KB cells. In both GSH-depleted fibroblasts and KB cells, UVB radiation failed to amplify c-jun and c-fos activation indicating that the oxidative component of UVB plays a minor role in the modulation of such oncogene expression. These findings clearly indicate that both c-jun and c-fos are activated by the oxidizing component of UVA radiation in human fibroblasts and KB cells, while UVB-mediated modulation seems to be restricted to human epithelial cells and does not involve oxidizing intermediates.

The heme synthesis and degradation pathways: role in oxidant sensitivity. Heme oxygenase has both pro- and antioxidant properties.

The heme biosynthetic and catabolic pathways generate pro- and antioxidant compounds, and consequently, influence cellular sensitivity to oxidants. Heme precursors (delta-aminolevulinic acid, porphyrins) generate reactive oxygen species (ROS), from autoxidation and photochemical reactions, respectively. Heme, an essential iron chelate, serves in respiration, oxygen transport, detoxification, and signal transduction processes. The potential toxicity of heme and hemoproteins points to a critical role for heme degradation in cellular metabolism. The heme oxygenases (HOs) provide this function and participate in cellular defense. This hypothesis emerges from the observation that the activation of HO-1 is an ubiquitous cellular response to oxidative stress. The reaction products of HO activity, biliverdin, and its subsequent metabolite bilirubin, have antioxidant properties. Furthermore, iron released from HO activity stimulates ferritin synthesis, which ultimately provides an iron detoxification mechanism that may account for long-term cytoprotection observed after HO induction. However, such models have overlooked potential pro-oxidant consequences of HO activity. The HO reaction releases iron, which could be involved in deleterious reactions that compete with iron reutilization and sequestration pathways. Indeed, the induction of HO activity may have both pro- and antioxidant sequelae depending on cellular redox potential, and the metabolic fate of the heme iron.

Characterization of photosensitivity in the Smith-Lemli-Opitz syndrome: a new congenital photosensitivity syndrome.

Photosensitivity has recently been reported as a feature of the Smith-Lemli-Opitz syndrome (SLO). The aim of this study was to establish the photobiological features of this disorder and to examine the hypothesis that the photosensitivity is caused by the high levels of 7-dehydrocholesterol found in SLO. All known cases of SLO in the U.K. were reviewed and clinical details of photosensitivity were recorded in detail. The action spectrum of the photosensitive eruption was defined by monochromator light testing. Thirteen of the 23 subjects (57%) had severe photosensitivity, and in 10 there was no photosensitivity. No correlation was identified between levels of 7-dehydrocholesterol and severity of photosensitivity, suggesting that the photosensitivity in SLO is not caused by a direct phototoxic effect mediated by 7-dehydrocholesterol. A novel pattern of photosensitivity was observed, with onset of a sunburn-like erythema on sun-exposed skin within minutes of sun exposure, which persisted in most cases for up to 24-48 h before fading. Monochromator light testing in three subjects showed an ultraviolet (UV) A-mediated photosensitivity eruption with greatest photosensitivity at 350 nm. Photosensitivity is a common and prominent feature of SLO and appears to be UVA-mediated. Elucidation of its biochemical basis may provide insight into normal cutaneous protective mechanisms against UVA-induced photodamage, and also sun sensitivity in general.

The role of melanin in the induction of oxidative DNA base damage by ultraviolet A irradiation of DNA or melanoma cells.

Highly pigmented, dark skin is more resistant to the harmful effects of solar ultraviolet radiation than light-colored human skin. The extent to which tanning protects skin from harmful effects including induction of skin cancer is not known, however. We have investigated whether the skin pigment, melanin, sensitizes or protects isolated DNA or nuclear DNA in melanoma cells from the induction of the premutagenic oxidative DNA base damage, 8-hydroxy-deoxyguanosine, by ultraviolet A irradiation. Synthetic eumelanin sensitized isolated DNA to induction of the oxidative DNA base damage by ultraviolet A, but it also induced the oxidative DNA base damage in the dark. To study the role of natural melanin in mammalian melanoma cells in the induction of oxidative DNA base damage, melanin synthesis was modulated 5-7-fold in the human melanoma cells GLL19 and IGR1 (which contain both pheomelanin and eumelanin) as well as in the mouse melanoma cells B16 (which contain mainly eumelanin). Increased melanin synthesis clearly did not protect against ultraviolet A-induced oxidative DNA base damage in cells. On the contrary, the human melanoma cells with high melanin content accumulated two times more 8-hydroxy-deoxyguanosine after ultraviolet A irradiation than cells with low melanin content. Furthermore, preirradiation of the human melanoma cells, IGR1, with ultraviolet A 4 h before a second ultraviolet A exposure produced an altered amount of induced 8-hydroxy-deoxyguanosine dependent on the melanin content of the cells. We conclude that stimulation of melanin synthesis, but probably not melanin itself, increases the susceptibility of human melanoma cells to induction of premutagenic oxidative DNA base damage by ultraviolet A irradiation.

Heme oxygenase induction mediates the photoimmunoprotective activity of UVA radiation in the mouse.

In contrast to the immunosuppressive potential of UVB (280-320 nm) radiation in experimental animals and humans, UVA (320-400 nm) radiation at environmentally relevant doses appears to be immunologically inert. However, such exposure to UVA radiation has been observed unexpectedly to induce resistance to UVB-induced immunosuppression in mice, by a mechanism resulting in the inactivation of cis-urocanic acid (UCA), an epidermal immunosuppressive UV photoproduct. In this study in mice, we show that the immunoprotective activity of UVA radiation, against the effects of both UVB radiation and cis-UCA, can be attributed to the induction of cutaneous heme oxygenase (HO; EC 1.14.99.3). Cell-mediated immune function was assessed in vivo by the contact hypersensitivity response induced to oxazolone at an unirradiated skin site, and HO enzyme activity was measured in cutaneous microsomal preparations from treated mice. There was a progressive increase in HO enzyme activity for at least 3 days after UVA irradiation. However HO activity, both constitutive and UVA radiation-induced, was sensitive to the effects of injecting mice with the specific HO inhibitor, tin protoporphyrin (Sn [IV] protoporphyrin IX; SnPP). We observed, in addition, that in SnPP-injected mice, the immunoprotective effect of UVA radiation against either UVB radiation or cis-UCA was abrogated. Because SnPP injection did not affect normal contact hypersensitivity responsiveness but did inhibit the constitutive HO enzyme activity, it appeared that only the inducible HO was active in modulating immune function. This finding indicates that UVA-induced HO activity is a major player in the skin defenses against UVB immunosuppression.

Ultraviolet A radiation induces immediate release of iron in human primary skin fibroblasts: the role of ferritin.

In mammalian cells, the level of the iron-storage 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.

Direct and indirect modulation of ornithine decarboxylase and cyclooxygenase by UVB radiation in human skin cells.

Exposure to solar ultraviolet (UV) B radiation is responsible for skin inflammation and tumour progression. Cyclooxygenase and ornithine decarboxylase are believed to be involved in such processes since they participate in the synthesis of mediators of inflammation and cell differentiation, respectively. We have investigated the in vitro modulation of expression of such genes by UVB radiation in different skin cell lines. We have observed that accumulation of ornithine decarboxylase mRNA is unaffected by even high UVB doses in both human epidermal keratinocytes and dermal fibroblasts, whereas cyclooxygenase-2 levels were significantly up-regulated by low UVB doses in KB human epidermoid keratinocytes. Depletion of total intracellular glutathione levels in KB cells amplified the activation, revealing a role for an oxidative component of UVB in modulating cyclooxygenase gene expression. Transfer of medium from UVB irradiated keratinocytes to fibroblasts resulted in a significant activation of cyclooxygenase expression and activity, while ornithine decarboxylase levels were unaffected. We conclude that UVB radiation can activate cyclooxygenase gene expression in human skin cells both by direct activation pathways or indirectly by inducing a paracrine mechanism.

Activation of expression of the c-fos oncogene by UVA irradiation in cultured human skin fibroblasts.

Both broad-spectrum and near-monochromatic (334 nm, 365 nm and 405 nm) UVA (320-380 nm) and near-visible radiations strongly activate accumulation of mRNA corresponding to the nuclear oncogene and transcription factor, c-fos, in cultured human skin fibroblasts within a dose-range encountered in the environment. The oxidizing component of UVA is clearly of central importance to the activation observed because the absence of reduced glutathione strongly enhances the response. In contrast to observations in rodent cells, we observe negligible activation of the gene in human cells after UVB (290-320 nm) radiation. The results of this study provide evidence that UVA radiation strongly activates c-fos gene expression in human dermal fibroblasts, a phenomena that is likely to be reflected in UVA-mediated modulation of genes containing active AP-1-based enhancer elements in the promoter region.

Cyclooxygenase dependent release of heme from microsomal hemeproteins correlates with induction of heme oxygenase 1 transcription in human fibroblasts.

Induction of heme oxygenase 1 transcription and enzymatic activity is a common response after exposure of cells to various forms of oxidative stress including ultraviolet A radiation (UVA) and hydrogen peroxide. We now show that UVA irradiation or hydrogen peroxide treatment of human skin fibroblasts leads to an immediate release of the heme oxygenase substrate, heme, from microsomal hemeproteins. The release of heme by UVA apparently involves cyclooxygenase activity because it is inhibited by the cyclooxygenase inhibitor indomethacin. We also demonstrate a high degree of correlation between the amount of heme released and the degree of subsequent induction of heme oxygenase 1 transcription following UVA and hydrogen peroxide treatment. We propose that release of heme from microsomal hemeproteins determines the degree of induction of heme oxygenase 1 transcription in human fibroblasts after oxidative stress.