Ultraviolet-B (UVB) (290 -320 nm) radiation-induced cyclobutane pyrimidine dimers within the DNA of epidermal cells are detrimental to human health by causing mutations and immunosuppressive effects that presumably contribute to photocarcinogenesis. Conventional photoprotection by sunscreens is exclusively prophylactic in nature and of no value once DNA damage has occurred. In this paper, we have therefore assessed whether it is possible to repair UVB radiationinduced DNA damage through topical application of the DNA-repair enzyme photolyase, derived from Anacystis nidulans, that specifically converts cyclobutane dimers into their original DNA structure after exposure to photoreactivating light. When a dose of UVB radiation sufficient to induce erythema was administered to the skin of healthy subjects, significant numbers of dimers were formed within epidermal cells. Topical application of photolyase-containing liposomes to UVB-irradiated skin and subsequent exposure to photoreactivating light decreased the number of UVB radiation-induced dimers by 40 -45%. No reduction was observed if the liposomes were not filled with photolyase or if photoreactivating exposure preceded the application of filled liposomes. The UVB dose administered resulted in suppression of intercellular adhesion molecule-1 (ICAM-1), a molecule required for immunity and inflammatory events in the epidermis. In addition, in subjects hypersensitive to nickel sulfate, elicitation of the hypersensitivity reaction in irradiated skin areas was prevented. Photolyase-induced dimer repair completely prevented these UVB radiation-induced immunosuppressive effects as well as erythema and sunburn-cell formation. These studies demonstrate that topical application of photolyase is effective in dimer reversal and thereby leads to immunoprotection.

UV irradiation interferes with the induction of T cell-mediated immune responses, in part by causing cells in the skin to produce immunoregulatory cytokines. Recent evidence implicates UV-induced DNA damage as a trigger for the cascade of events leading to systemic immune suppression in vivo. However, to date, there has been no direct evidence linking DNA damage and cytokine production in UVirradiated cells. Here we provide such evidence by showing that treatment of UV-irradiated murine keratinocytes in vitro with liposomal T4 endonuclease V, which accelerates the repair of cyclobutylpyrimidine dimers in these cells, inhibits their production of immunosuppressive cytokines, including interleukin 10. Application of these liposomes to murine skin in vivo also reduced the induction of interleukin 10 by UV irradiation, whereas liposomes containing heat-inactivated T4 endonuclease V were ineffective. These results support our hypothesis that unrepaired DNA damage in the skin activates the production of cytokines that down-regulate immune responses initiated at distant sites.Excessive exposure of the skin to UV radiation has many biological consequences, including sunburn, solar keratosis, skin cancer, and immune suppression. Suppression of the immune response contributes to the formation of UV-induced skin cancers in mice by permitting the outgrowth of highly antigenic, UV-transformed cells (1). One mechanism proposed for UV-induced immune suppression is the elaboration of immunomodulatory cytokines by UV-irradiated keratinocytes (2). Studies by Streilein and Vermeer (3) implicated tumor necrosis factor a as an important mediator of the suppression of contact hypersensitivity responses in UV-irradiated mice. Ullrich and colleagues (4) demonstrated that interleukin 10 (IL-10) plays a crucial role in suppression of delayed type hypersensitivity (DTH) responses. They showed that IL-10 is produced and secreted by keratinocytes in vitro in response to UV irradiation and suggested that in vivo this cytokine diverts the DTH response toward a suppressor pathway (4, 5).What has not yet been established is the identity of the initial photobiological reaction responsible for triggering the production of immunomodulatory cytokines. In previous studies, we provided evidence that DNA damage in the form of cyclobutyl pyrimidine dimers (CPD) is an initiating event in several models of UV-induced immune suppression (6, 7). These in vivo studies showed that reducing the number of CPD in UV-irradiated skin abrogated the systemic suppression of contact hypersensitivity and DTH responses. From these results we have inferred that unrepaired DNA damage stimulates keratinocytes to produce cytokines that modify critical steps in the immunological pathway, ultimately leading to reduced cell-mediated immune responses and suppressor cell generation. However, evidence of a direct effect of DNA damage on cytokine production has been lacking. In these studies, we address a pivotal aspect of this hypothesis by demonstrating that DNA damage ...

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Dietary omega-3 polyunsaturated fatty acids (omega-3 PUFAs) protect against photocarcinogenesis in animals, but prospective human studies are scarce. The mechanism(s) underlying the photoprotection are uncertain, although omega-3 PUFAs may influence oxidative stress. We examined the effect of supplementation on a range of indicators of ultraviolet radiation (UVR)-induced DNA damage in humans, and assessed effect on basal and post-UVR oxidative status. In a double-blind randomized study, 42 healthy subjects took 4 g daily of purified omega-3 PUFA, eicosapentaenoic acid (EPA), or monounsaturated, oleic acid (OA), for 3 months. EPA was bioavailable; the skin content at 3 months showing an 8-fold rise from baseline, P < 0.01. No consistent pattern of alteration in basal and UVR-exposed skin content of the antioxidants glutathione, vitamins E and C or lipid peroxidation, was seen on supplementation. Sunburn sensitivity was reduced on EPA, the UVR-induced erythemal threshold rising from a mean of 36 (SD 10) mJ/cm(2) at baseline to 49 (16) mJ/cm(2) after supplementation, P < 0.01. Moreover, UVR-induced skin p53 expression, assessed immunohistochemically at 24 h post-UVR exposure, fell from a mean of 16 (SD 5) positive cells/100 epidermal cells at baseline to 8 (4) after EPA supplementation, P < 0.01. Peripheral blood lymphocytes (PBL) sampled on 3 successive days both pre- and post-supplementation, showed no change with respect to basal DNA single-strand breaks or oxidative base modification (8-oxo-dG). However, when susceptibility of PBL to ex vivo UVR was examined using the comet assay, this revealed a reduction in tail moment from 84.4 (SD 3.4) at baseline to 69.4 (3.1) after EPA, P = 0.03. No significant changes were seen in any of the above parameters following OA supplementation. Reduction in this range of early markers, i.e. sunburn, UVR-induced p53 in skin and strand breaks in PBL, indicate protection by dietary EPA against acute UVR-induced genotoxicity; longer-term supplementation might reduce skin cancer in humans.

Exposing skin to UVB (280-320 nm) radiation suppresses contact hypersensitivity by a mechanism that involves an alteration in the activity of cutaneous antigenpresenting cells (APC). UV-induced DNA damage appears to be an important molecular trigger for this effect. The specific target cells in the skin that sustain DNA damage relevant to the immunosuppressive effect have yet to be identified. We tested the hypothesis that UV-induced DNA damage in the cutaneous APC was responsible for their impaired ability to present antigen after in vivo UV irradiation. Cutaneous APC were collected from the draining lymph nodes of UVBirradiated, hapten-sensitized mice and incubated in vitro with liposomes containing a photolyase (Photosomes; Applied Genetics, Freeport, NY), which, upon absorption of photoreactivating light, splits UV-induced cyclobutane pyrimidine dimers. Photosome treatment followed by photoreactivating light reduced the number of dimer-containing APC, restored the in vivo antigen-presenting activity of the draining lymph node cells, and blocked the induction of suppressor T cells. Neither Photosomes nor photoreactivating light alone, nor photoreactivating light given before Photosomes, restored APC activity, and Photosome treatment did not reverse the impairment of APC function when isopsoralen plus UVA (320-400 nm) radiation was used instead of UVB. These controls indicate that the restoration of APC function matched the requirements of Photosome-mediated DNA repair for dimers and post-treatment photoreactivating light. These results provide compelling evidence that it is UVinduced DNA damage in cutaneous APC that leads to reduced immune function.Exposing skin to UV radiation induces lesions in cellular DNA. Among those lesions, cyclobutane pyrimidine dimers (subsequently referred to as ''dimers'') and photoproducts predominate and are believed to play an important role in initiating skin cancer (1). UV irradiation of murine skin also reduces certain immune responses, and this suppressed immunity plays a significant role in UV carcinogenesis by allowing highly antigenic, UV-induced skin cancers to grow unimpeded (2, 3). The mechanisms of UVB (280-320 nm)-induced immune suppression are not completely understood; evidence is accumulating, however, that the induction of dimers by UVB represents a key step in the initiation of immune suppression (4-6), although other mechanisms such as the photoisomerization of urocanic acid (7), free-radical formation (8), and signal transduction-mediated activation of transcription factors (9, 10) may play a role as well.Immunological changes induced by UVB can be manifested locally within the UV-irradiated skin or systemically at sites distant from the UV-irradiated skin, depending on the experimental conditions (11). A well studied, local immunological effect of UV irradiation is inhibition of the contact hypersensitivity (CHS) response, which occurs after a contact sensitizer is applied to UV-irradiated skin of certain strains of mice (reviewed in ref. 12). The CH...