Protection of UV-induced Suppression of Skin Contact Hypersensitivity: A Common Feature of Flavonoids after Oral Administration?

In this study we investigated the effect of the dietary ingredients fruit and vegetable, green tea phenol extract (GTP) and the specific flavonoid components quercetin and chrysin on the UV-induced suppression of the con-tact hypersensitivity (CHS) response to picryl chloride (PCl). The SKH-1 mice were fed with test diet from 2 or 4 weeks before and during the UV irradiation (daily, 95 mJ/cm²) and tested for the CHS ear-swelling response 10 weeks after the onset of the irradiation. For the CHS, mice were immunized with PCl by epicutaneous application on nonirradiated sites. Four days after sensitization all mice were challenged on both sides of each ear by topical application of one drop PCl. In addition, from mice fed with the fruit and vegetable mixture the number of Langerhans cells (LC) were scored in the skin and from mice fed with quercetin, quercetin levels in plasma were measured at week 11 after the start of UV irradiation. It was found that fruit and vegetable (19% in the diet), GTP (0.1% and 0.01% in the drinking water), quercetin (1% in the diet) and chrysin (1% and 0.1% in the diet), prevented statistically significantly the UV-induced suppression of CHS to PCl. In the skin of mice fed with fruit and vegetables combined with UV irradiation the number of LC were comparable to the control mice, whereas the number of LC were significantly diminished in mice treated with UV only. This protective effect on the presence of LC in the epidermis after UV irradiation, which was also observed in a previous study with quercetin, may play a role in the prevention of UV-induced immunosuppression by the flavonoids tested. In conclusion, we found protection of flavonoids against UV-induced effects on CHS, which may be a common feature of most flavonoids.     

Quercetin Prevents UV-lnduced Local Immunosuppression, but Does Not Affect UV-lnduced Tumor Growth in SKH-1 Hairless Mice

Abstract— Ultraviolet is thought to induce skin tumors by its dual activity as a mutagenic agent and a suppressor of cell-mediated immunity. In the present study the effects of quercetin, a flavonoid-containing compound, on carcinogenesis and immunosuppression were studied in SKH hairless mice exposed to suberythemal doses of UV for up to 17 weeks. It was found that quercetin did not affect the onset or growth of non-melanoma skin tumors resulting from UV exposure. In contrast, it prevented the suppression in contact hypersensitivity (CHS) to picryl chloride induced by UV. The mechanism of this prevention might be explained by the observation that the decreased number of epidermal Langerhans'cells is partly prevented by the quercetin. Quercetin did not alter the effects of UV in increasing numbers of spleen and lymph node cells, only partly in decreasing the CD8-positive cells in spleen cell populations and decreasing the lym-phoproliferative response of spleen cells to the mitogens concanavalin A and phytohemagglutinin. Thus oral quercetin did not prevent UV-induced carcinogenesis although it restored the skin-associated CHS response probably by protecting the antigen-presenting cells in the skin.     

Decrease in langerhans cells and increase in lymph node dendritic cells following chronic exposure of mice to suberythemal doses of solar simulated radiation

Exposure of certain strains of mice to ultraviolet radiation (UVR) causes suppression of some innate and adaptive immune responses. One such consequence of acute UVB exposure is a reduction in the number of Langerhans cells (LC) in the epidermis and an increase in dendritic cells (DC) in lymph nodes draining the irradiated skin sites. Exposure to chronic UVB irradiation also has effects on the immune system, but it is unknown what effects are caused by repeated doses of solar simulated radiation (SSR). Consequently, the main aims of the present study were to determine whether repeated exposure to low doses of SSR would lead to similar changes in these cell populations and whether chronic doses of SSR activate a protective photoadaptation mechanism. Groups of C3H/HeN mice were irradiated daily with 3.7 J/cm(2) SSR from Cleo Natural lamps for 2, 10, 20, 30 or 60 days. Further groups of mice received an additional dose of 7.4 J/cm(2) SSR on days 2, 10, 30 or 60 to test for photoadaptation. The numbers of LC in the epidermis and DC in the lymph nodes draining irradiated skin sites were counted 24 h after the final irradiation. With the exception of mice irradiated for only 2 days, LC were significantly reduced throughout the chronic irradiation protocol, and no recovery occurred. DC numbers were significantly increased in the draining lymph nodes of mice irradiated for 20 days and 60 days.     

Sunscreens Protect from UV-Promoted Squamous Cell Carcinoma in Mice Chronically Irradiated with Doses of UV Radiation Insufficient to Cause Edema

Previously we reported that the broad-spectrum sunscreen microfine titanium dioxide (MTD) could completely protect C3H/HeJ mice from UV radiation-induced immunosuppression to a contact sensitizer. In contrast, 2-ethylhexyl p-methoxycinnamate (2-EHMC), a UVB-absorbing sunscreen, only partially protected the skin immune system. In this study we investigated further this differential protection of the skin immune system by comparing the ability of 2-EHMC and MTD to protect these mice from the promotion phase of tumorigenesis. The mice were initiated using a single subcarcinogenic dose of 7,12-dimethylbenz(a)anthracene (DMBA) followed by promotion with chronic low-dose solar-simulated UV radiation for 32 weeks. We used doses of UV insufficient to cause edema in order to simulate daily human exposure to solar UV radiation. Mice were observed for the appearance of squamous cell carcinomas for 48 weeks. The DMBA-initiation alone and DMBA-initiated, sunscreen-treated groups did not develop tumors. Ultraviolet alone induced the appearance of tumors in 46% of mice at week 48 and therefore some tumors were initiated by UV. Initiation with DMBA prior to UV irradiation enhanced tumorigenesis such that 87% of mice at week 48 had tumors. Both 2-EHMC and MTD completely protected these mice from UV-induced promotion as well as from complete carcinogenesis despite the different UV-absorption spectra of the sunscreens and their differential abilities to protect from UV-induced immunosuppression. Furthermore, we have shown that, if UV exposure is not increased to compensate for tolerance to edema, protection from tumorigenesis is afforded by sunscreens.     

UV protective effects of DNA repair enzymes and RNA lotion

Solar UV radiation is known to cause immune suppression, believed to be a critical factor in cutaneous carcinogenesis. Although the mechanism is not entirely understood, DNA damage is clearly involved. Sunscreens function by attenuating the UV radiation that reaches the epidermis. However, once DNA damage ensues, repair mechanisms become essential for prevention of malignant transformation. DNA repair enzymes have shown efficacy in reducing cutaneous neoplasms among xeroderma pigmentosum patients. In vitro studies suggest that RNA fragments increase the resistance of human keratinocytes to UVB damage and enhance DNA repair but in vivo data are lacking. This study aimed to determine the effect of topical formulations containing either DNA repair enzymes ( Micrococcus luteus ) or RNA fragments (UVC-irradiated rabbit globin mRNA) on UV-induced local contact hypersensitivity (CHS) suppression in humans as measured in vivo using the contact allergen dinitrochlorobenzene. Immunohistochemistry was also employed in skin biopsies to evaluate the level of thymine dimers after UV. Eighty volunteers completed the CHS portion. A single 0.75 minimum erythema dose (MED) simulated solar radiation exposure resulted in 64% CHS suppression in unprotected subjects compared with unirradiated sensitized controls. In contrast, UV-induced CHS suppression was reduced to 19% with DNA repair enzymes, and 7% with RNA fragments. Sun protection factor (SPF) testing revealed an SPF of 1 for both formulations, indicating that the observed immune protection cannot be attributed to sunscreen effects. Biopsies from an additional nine volunteers showed an 18% decrease in thymine dimers by both DNA repair enzymes and RNA fragments, relative to unprotected UV-irradiated skin. These results suggest that RNA fragments may be useful as a photoprotective agent with in vivo effects comparable to DNA repair enzymes.     

Isoflavonoid compounds from red clover (Trifolium pratense) protect from inflammation and immune suppression induced by UV radiation

Isoflavones derived from many edible plants have been reported to possess significant antioxidant, estrogenic and tyrosine kinase inhibitory activity. Genistein has been found previously to provide protection from oxidative damage induced by UV radiation both in vitro and following dietary administration. We have therefore examined the potential of a number of isoflavones from red clover (Trifolium pratense) and some metabolically related compounds to offer protection from UV irradiation in hairless mice by topical application after UV exposure. We show that whereas the primary isoflavones, daidzein, biochanin A and formononetin, were inactive, 20 microM lotions of genistein and the metabolites equol, isoequol and the related derivative dehydroequol had powerful potential to reduce the inflammatory edema reaction and the suppression of contact hypersensitivity induced by moderate doses of solar-simulated UV radiation. For equol the protection was concentration dependent and 5 microM equol markedly reduced the UV-induced inflammation but abrogated the UV-induced immunosuppression. Equol protected similarly from immunosuppression induced by the putative epidermal mediator, cis-urocanic acid (UCA), indicating a potential mechanism of action involving inactivation of this UV-photoproduct. Since immunosuppression induced by both UV radiation and by cis-UCA appears to be an oxidant-dependent response our observations support the actions of these topically applied isoflavones and their metabolites as antioxidants. They also indicate that lotions containing equol, unlike topical UV sunscreens, more readily protect the immune system from photosuppression than from the inflammation of the sunburn reaction, even when applied after exposure, and thus such compounds may have a future role as sun-protective cosmetic ingredients.     

Chronic Low-Dose UVA Irradiation Induces Local Suppression of Contact Hypersensitivity, Langerhans Cell Depletion and Suppressor Cell Activation in C3H/HeJ Mice

Abstract— It has previously been demonstrated that chronic low-dose solar-simulated UV radiation could induce both local and systemic immunosuppression as well as tolerance to a topically applied hapten. In this study, we have used a chronic low-dose UV-irradiation protocol to investigate the effects of UVA on the skin immune system of C3H/HeJ mice. Irradiation with UVA+B significantly suppressed the local and systemic primary contact hypersensitivity (CHS) response to the hapten 2,4,6-trinitrochlo-robenzene. Furthermore UVA+B reduced Langerhans cell (LC) and dendritic epidermal T cell (DETC) densities in chronically UV-irradiated mice. Ultraviolet A irradiation induced local, but not systemic, immunosuppression and reduced LC (32%) but not DETC from the epidermis compared to the shaved control animals. Treatment of mice with both UVA+B and UVA radiation also induced an impaired secondary CHS response, and this tolerance was transferable with spleen cells. These results suggest that depletion of LC, but not DETC, may be involved in UVA-induced local immunosuppression in our model, and that tolerance was induced in the presence of normal numbers of DETC. Hence exposure of C3H/HeJ mice 5 days per week for 4 weeks with UVA can induce local immunosuppression and tolerance.     

Suppression of Delayed and Contact Hypersensitivity Responses in Mice Have Different UV Dose Responses

Although acute exposure to UV radiation suppresses the induction of delayed-type (DTH) and contact (CHS) hypersensitivity in mice, it is not clear whether the photo-biological mechanism(s) involved in suppressing these closely related immune reactions is the same. A careful examination of the UV dose responses and wavelength dependencies involved in suppressing CHS and DTH may provide important insights into the mechanisms involved. We compared the UV dose-response curves for suppressing four closely related immune reactions, local and systemic suppression of CHS to dinitrofluorobenzene, systemic suppression of DTH to Candida albicans and systemic suppression of DTH to alloantigen using three different UV spectra (FS40 sunlamps, Kodacel-filtered FS40 sunlamps and solar-simulated light). For each immune response studied, the amount of UVB radiation required to induce 50% immune suppression was lowest when FS40 sunlamps were used, highest with solar-simulated light and intermediate when Kodacel-filtered FS40 sunlamps were used, but the differences observed were not statistically significant. The UV dose-response curves for immune suppression differed significantly depending on the assay used, the site of antigenic sensitization and the antigen used. These findings suggest that the mechanisms by which UV radiation induces immune suppression differ for the four immunological reactions studied.     

Viability of the antigen determines whether DNA or urocanic acid act as initiator molecules for UV-induced suppression of delayed-type hypersensitivity

UV radiation suppresses the immune response, and UV-induced immune suppression contributes to UV-induced photocarcinogenesis. For UV-induced immune suppression to occur, electromagnetic energy (i.e. UV radiation) must be converted to a biological signal. Two photoreceptors have been identified in the skin that serves this purpose, epidermal DNA and trans-urocanic acid (UCA). Although compelling evidence exists to support a role for each pathway (UV-induced DNA damage or photoisomerization of UCA) in UV-induced immune suppression, it is not clear what determines which photoreceptor pathway is activated. To address this question, we injected UV-irradiated mice with a monoclonal antibody with specificity for cis-UCA or applied liposomes containing DNA repair enzymes to the skin of UV-irradiated mice. The effect that each had on UV-induced suppression of delayed-type hypersensitivity was measured. We asked whether the light source used (FS-40 sunlamps vs solar-simulated UV radiation) altered whichever pathway of immune suppression was activated. Different doses of UV radiation and the viability of the antigen were also considered. Neither the dose of UV nor the light source had any influence on determining which pathway was activated. Rather, we found that the viability of the antigen was the critical determinant. When live antigens were used, UV-induced immune suppression was blocked with monoclonal anti-cis-UCA but not with T4 endonuclease V-containing liposomes. The reverse was observed when formalin-fixed or killed antigens were used. Our findings indicate that antigen viability dictates which photoreceptor pathway predominates after UV exposure.     

The topical isoflavonoid NV-07alpha reduces solar-simulated UV-induced suppression of Mantoux reactions in humans

UV radiation suppresses delayed-type hypersensitivity responses to intradermally injected tuberculin purified protein derivative in Mantoux-positive individuals. The effect of the topically administered isoflavonoid NV-07alpha, a synthetic derivative of the isoflavonoid equol, on UV-induced suppression of Mantoux reactions was assessed in 18 healthy Mantoux-positive volunteers. A single, fixed dose of solar-simulated UV radiation was delivered to the volunteers' lower backs. Different concentrations of NV-07alpha or its vehicle were applied to different sites within the irradiated field immediately after UV exposure and again 24 h later. Forty-eight hours after irradiation, Mantoux testing was performed at both the irradiated sites and adjacent, unirradiated sites. The intensity of Mantoux reactions was measured 72 h later with a reflectance erythema meter and by measuring the diameter of each reaction. Although lower concentrations of NV-07alpha (0.5 and 2 mM) did not prevent UV immunosuppression, 4 mM NV-07alpha partially but significantly attenuated UV-induced suppression of Mantoux-induced erythema. Minimal erythema doses were also determined for sites treated with NV-07alpha or its vehicle immediately after UV exposure. NV-07alpha had no significant effects on UV erythema. We conclude that 4 mM NV-07alpha prevented the suppressive effects of UV radiation on Mantoux responses in humans but did not affect UV-induced erythema at the concentrations used.     

Ultraviolet Spectral Energy Differences Affect the Ability of Sunscreen Lotions to Prevent Ultraviolet-Radiation-Induced Immunosuppression*

Acute exposure to UV radiation causes immunosuppression of contact hypersensitivity (CH) responses. Past studies conducted with unfiltered sunlamps emitting nonsolar spectrum UV power (wavelengths below 295 nm) or using excessive UV doses have suggested sunscreens may not prevent UV-induced immunosuppression in mice. This study was thus designed to evaluate critically the effects of different UV energy spectra on the immune protection capacity of sunscreen lotions. Minimum immune suppression doses (MISD), i.e. the lowest UV dose to cause~50% suppression of the CH response to dinitrofluorobenzene in C3H mice, were established for three artificial UV sources. The MISD for each UV source was 0.25 kJ/m² for unfiltered FS20 sunlamps (FS), 0.90 kJ/m² for Kodacel-filtered FS20 sunlamps (KFS), which do not emit UV power at wavelengths <290 nm, and 1.35 kJ/m² for a 1000 W filtered xenon arc lamp solar simulator. Using MISD as baseline, sunscreens with labeled sun protection factors (SPF) of 4, 8, 15 and 30 were tested with each UV source to establish their relative immune protection factors. The immune protection factor of each sunscreen exceeded its labeled SPF in tests conducted with the solar simulator, which has a UV power spectrum (295–400 nm) similar to that of sunlight. Conversely, sunscreen immune protection factors were significantly less than the labeled SPF in tests conducted with FS and KFS. Comparison of the immunosuppression effectiveness spectra showed that relatively small amounts of nonsolar spectrum UV energy, i.e. UVC (200–290 nm) and/or shorter wavelength UVB (between 290 and 295 nm), produced by FS and KFS contributes significantly to the induction of immunosuppression. For example, 36.3% and 3.5% of the total immunosuppressive UV energy from FS and KFS, respectively, lies below 295 nm. Sunscreen absorption spectra showed that transmission of immunosuppressive UV energy below 295 nm for FS was at least eight-fold higher than that for KFS. Compared to the solar simulator UV spectrum the transmission of nonsolar immunosuppressive UV energy through sunscreens was >15-fold higher for FS and ≥1.5-fold higher for KFS. These data demonstrate that relevant evaluations of sunscreen immune protection can only be obtained when tests are conducted with UV sources that produce UV power spectra similar to that of sunlight and UV doses are employed that are based on established MISD.     

SUPPRESSION OF CONTACT HYPERSENSITIVITY BY UV RADIATION AND ITS RELATIONSHIP TO UV-INDUCED SUPPRESSION OF TUMOR IMMUNITY

Abstract— In this study, we examine some of the photobiologic and immunologic characteristics of the suppression of contact hypersensitivity (CHS) by UV radiation. BALB/c mice were irradiated on the shaved dorsal skin with FS40 sunlamps and sensitized 5 days later by applying a contact sensitizer lo the shaved abdomen. The suppression of CHS resulting from exposure to a given total dose of UV radiation was unaffected by changes in dose fractionation over a 5-day period and by changes in dose-rate over a 10-fold range. Elimination of wavelengths below 315 nm with a mylar filter abrogated the suppressive effect of the sunlamps, even when the same total energy was administered. Irradiation of unshaved mice required 14 times more energy to produce 50% suppression than was required for shaved mice, suggesting that the exposed skin is the primary target of this effect. Contact sensitization of UV-irradiated, but not unirradiated, mice induced the appearance of antigen-specific suppressor T lymphocytes in their spleen. The photobiologic and immunologic similarities between the suppression of CHS by UV radiation and the UV-mediated suppression of tumor rejection that we described previously suggest that these two immunosuppressive effects of UV exposure share certain steps in their pathways.     

25 years of UV-induced immunosuppression mediated by T cells-from disregarded T suppressor cells to highly respected regulatory T cells

For more than 25 years it is known that UV radiation, in particular the UVB range suppresses the immune system. In contrast to conventional immunosuppression by immunosuppressive drugs, UV radiation does not compromise the immune system in a general but rather in an antigen-specific fashion via induction of immunotolerance. This effect is mostly mediated via regulatory T cells (Treg) induced by UV. Several subtypes of UV-induced Treg may exist, the best characterized are those which inhibit contact hypersensitivity. Induction of these Tregs by UV radiation is an active process which requires antigen presentation by UV-damaged but still alive Langerhans cells (LC) in the lymph nodes. UV-induced Treg have recently been characterized as expressing CD4 and CD25 and as releasing upon activation the immunosuppressive cytokine interleukin (IL)-10. Once activated in an antigen-specific manner, they suppress immune responses in a general fashion via the release of IL-10, a phenomenon called bystander suppression. The further phenotypic and functional characterization of these cells will not only contribute to a better understanding of the impact of UV radiation on the immune system but will also determine whether they can be applied in the future therapeutically with the final aim of achieving specific immunosuppression.     

PROTECTION AGAINST ULTRAVIOLET-B RADIATION-INDUCED LOCAL and SYSTEMIC SUPPRESSION OF CONTACT HYPERSENSITIVITY and EDEMA RESPONSES IN C3H/HeN MICE BY GREEN TEA POLYPHENOLS

Abstract— Exposure of skin to UV radiation can cause diverse biological effects, including induction of inflammation, alteration in cutaneous immune cells and impairment of contact hypersensitivity (CHS) responses. Our laboratory has demonstrated that oral feeding as well as topical application of a poly-phenolic fraction isolated from green tea (GTP) affords protection against the carcinogenic effects of UVB (280–320 nm) radiation. In this study, we investigated whether GTP could protect against UVB-induced immunosuppression and cutaneous inflammatory responses in C3H mice. Immunosuppression was assessed by contact sensitization with 2,4-dinitrofluorobenzene applied to UVB-irradiated skin (local suppression) or to a distant site (systemic suppression), while double skin-fold swelling was used as the measure of UVB-induced inflammation. Topical application of GTP (1–6 mg/animal), 30 min prior to or 30 min after exposure to a single dose of UVB (2 kj/m²) resulted in significant protection against local (25–90%) and systemic suppression (23–95%) of CHS and inflammation in mouse dorsal skin (70–80%). These protective effects were dependent on the dose of GTP employed; increasing the dose (1–6 mg/animal) resulted in an increased protective effect (25–93%). The protective effects were also dependent on the dose of UVB (2–32 kJ/m²). Among the four major epicatechin derivatives present in GTP, (‐)-epigallocatechin-3-gallate, the major constituent in GTP, was found to be the most effective in affording protection against UVB-caused CHS and inflammatory responses. Our study suggests that green tea, specifically polyphenols present therein, may be useful against inflammatory dermatoses and immunosuppression caused by solar radiation.     

SUPPRESSION OF THE INDUCTION OF DELAYED-TYPE HYPERSENSITIVITY REACTIONS IN MICE BY A SINGLE EXPOSURE TO ULTRAVIOLET RADIATION

Abstract— After a single exposure of mice to UV radiation, their ability to generate a contact hypersensitivity (CHS) response to contact sensitizers applied epicutaneously to distant, unirradiated skin is severely impaired. It is not clear, however, if the classic delayed type hypersensitivity (DTH) reponse to exogenous antigens, injected into the subcutaneous (s.c.) space, can also be modulated by UV radiation. We report here that a single exposure of mice to UV radiation suppressed the induction of DTH to both erythrocyte and soluble protein antigens injected s.c., but did not suppress the elicitation of the response. The suppressive effect was abrogated by cyclophosphamide treatment. In addition, antigen-specific suppressor cells were found in the spleens of the mice with a decreased DTH response. Since the ability to mount a DTH response has been linked with the resistance to certain pathogenic microorganisms, we suggest that the suppression of DTH by UV radiation may have the potential to compromise host resistance to such infectious agents.     

INHIBITION OF THE IMMUNE RESPONSE TO ALLOANTIGEN IN THE RAT BY EXPOSURE TO ULTRAVIOLET RADIATION

Exposure of mice to ultraviolet radiation (UV) followed by alloantigen sensitization can suppress the immune response to that alloantigen. In order to assess the applicability of using UV-induced immunosuppression in organ transplantation, the effectiveness of UV in prolonging the survival of vascularized organ allografts must be determined. Because, for technical reasons, rats are better suited than mice for such experiments, we first wanted to determine whether UV suppresses the immune response of inbred rats to alloantigens. The data presented here demonstrate that exposure of rats to UV (115-129 kJ/m2) prior to alloantigenic sensitization decreases the mixed lymphocyte response to alloantigen. The depression of the proliferative response to alloantigen was selective in that spleen cells from the UV-treated rats could respond to mitogenic stimulation. In contrast to previous results with mice, suppressor cells could not be demonstrated in the spleens of the UV-treated rats. In addition, UV treatment after sensitization inhibited the response to alloantigen. These data suggest that treatment of the recipient with UV before or after alloantigenic sensitization may provide a novel method of inhibiting immune responses to allogeneic antigens.     

Inverse relationship between increased apoptosis and decreased skin cancer in UV-irradiated CD1d-/- mice

We previously demonstrated that CD1d knockout mice were resistant to ultraviolet (UV)-induced immunosuppression. Because immune suppression is a critical factor in the development of UV-induced skin cancers, we investigated the response of wild type (WT) and CD1d-/- mice to UV carcinogenesis. We found that although 100% of WT mice developed skin tumors after 45 weeks of UV irradiation, only 60% of CD1d-/- mice developed skin tumors. To investigate the mechanisms involved in the resistance of CD1d-/- mice to UV-induced carcinogenesis, we determined the time course and kinetics of keratinocyte cell death after UV irradiation. After acute UV exposure, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL)-positive keratinocytes were eliminated from the skin of WT mice by 72 h post-UV, but they still persisted until 96 h in CD1d-/- mice. The kinetics of p53 protein expression closely followed the kinetics of apoptotic cell death. Chronic UV irradiation resulted in induction of a significantly higher number of apoptotic keratinocytes in CD1d-/- than WT mice. In addition, epidermis and dermis from chronically UV-irradiated CD1d-/- mice harbored significantly fewer p53 mutations than WT mice. These results indicate that the resistance of CD1d-/- mice to UV carcinogenesis may be due to increased cell death and elimination of keratinocytes and fibroblasts containing DNA damage and p53 mutations.     

Noninvasive Assessment of UV-induced Skin Damage: Comparison of Optical Measurements to Histology and MMP Expression

Acute exposure to UV radiation (UVR) causes visible skin damage such as erythema and results in local and systemic immunosuppression while chronic exposure can result in photocarcinogenesis. These deleterious effects can be quantified by histology and by bioassays of key biological markers, including matrix metalloproteinases (MMPs), or tryptophan moieties. We now report our results in quantifying UV skin damage with noninvasive optical methods based on reflectance and fluorescence spectroscopy and compare these noninvasive measurements to histopathology and MMP-13 expression. A solar simulator with spectral output nearly identical to that of solar radiation was developed and used in our experiments. SKH1 hairless mice were exposed to solar-simulated UVR at a total dose of 21 MED delivered over 10 weeks. Changes in oxygenated and deoxygenated hemoglobin were measured by diffuse reflectance spectroscopy, and tryptophan changes were monitored via a fluorescence monitor. Our results show that there is an increase in erythema, skin fluorescence, sunburn cells and MMP-13 after a series of suberythemal doses of UV irradiation on a hairless mouse animal model. Increased skin fluorescence is observed with increasing UV exposure. The levels of MMP-13 increase as the cumulative UV dose increases but their increase does not correspond to noninvasively measured changes.     

Measurement of in vivo sunscreen immune protection factors in humans

This study investigates the level of protection provided by sunscreens against solar-simulated UV radiation-induced immunosuppression in humans. The in vivo immune protection factors (IPF) of two broad-spectrum sunscreens were determined by assessing their ability to prevent UV-induced suppression of nickel contact hypersensitivity (CHS) in 15 nickel-allergic volunteers. Each volunteer was irradiated on unprotected skin of the back with different doses of UV daily for 4 days. Multiples of these UV doses were concurrently delivered to sunscreen-treated sites on the contralateral back. Nickel patches were then applied to both irradiated sites and adjacent, unirradiated control sites. Nickel-induced erythema at each site was measured 72 h later with a reflectance spectrometer. Comparison of the nickel reactions of irradiated and unirradiated skin revealed linear UV dose-responses for immunosuppression in both unprotected and sunscreen-treated skin. The minimum level of immunosuppression that can be reliably detected with this method is 20%. Therefore, the UV dose that reduces mean nickel CHS by 20% is the minimal immune suppression dose (MISD). Sunscreen IPF were determined by dividing the mean MISD of sunscreen-treated skin by that of unprotected skin. The sunscreens, with sun protection factors of 9 and 24, had IPF of 6.5 and > 25, respectively.     

Risk Assessment of UVB Effects on Resistance to Infectious Diseases

The aim of this study was to develop a qunatitiative risk assessment of lowered resistance to Infections in humans due to (solar) ultraviolet B (UVB) exposure. We followed the steps for risk assessment as defined by the U. S. National Academy of Sciences: (1) hazard identification, (2) dose-response assessment, (3) exposure assessment, and (4) risk characterization. For step 1, the suppressory effects of UVB radiation on the immune system have been reviewed, suplemented with new data, and analyzed. Experiments on UV-induced immunosuppression cannot be performed with humans for ethical reasons, but herpes simplex virus infection appears to be the human paradigm. Thus, UVB radiation appears to be a potential hazard to immunologic functions, Step 2 is crucial, but dose-response relationships for infections have never been measured in humans. We used our earlier doseresponse rat data for suppression of lymphocyte stimulation and computed that the UVB dose resulting in a 50% reduction of lymphocyte stimulation by Listeria monocytogenes is 6.800 J/m². Using mixed skin lymphocyte response assays we found that humans are 3.8 times less sensitive than rats (interspeices variation (IEVI). To account for the 2.5 percentile of most susceptible individuals in a population, and additional factor (intraspecies varations (IAV) was introduced (0.5 for humans). Using these data, we computed that 13.100 J/m² of UVB radiation emitted by FS40 lamps would suppress 50% of the proliferative response of lymphocytes to L. monocytogenes in most sensitive skin type 2 humans. In step 3, we assumed the action spectrum for the responses analyzed by us as indentical to an action spectrum for suppression of contact hypersensitivity that is available in the literature. This led us to step 4, where we calculated that approximately 100 min of solar exposure at around noon in Italy or Spain would suppress the resistance to infections by L. monocytogenes in the most sensitive humans.