T S Rafferty

The University of Edinburgh, Edinburgh, SCT, United Kingdom

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Publications (10)36.15 Total impact

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    ABSTRACT: Background Ultraviolet radiation (UVR), a ubiquitous environmental genotoxin for the skin, produces DNA damage. The trace element selenium induces synthesis of the glutathione peroxidase and thioredoxin reductase enzyme families. These selenoenzymes detoxify a range of toxic compounds generated by free radicals. Objectives To assess the effects of pretreatment of primary human keratinocytes with selenium on UVR-induced DNA damage. Methods Cells were irradiated with UVR from FS-20 lamps and were subjected to comet assay. Results Comet tail length due to UVR-induced T4 endonuclease V-sensitive sites (caused by cyclopyrimidine dimers, CPDs) increased to 35 +/- 4.5 microm (mean +/- SD) immediately after irradiation (time 0 h, 100%). After 4 h, 68% of the damage remained and after 24 h, 23% of the damage was still present. Treatment with up to 200 nmol L-1 selenomethionine or 50 nmol L-1 sodium selenite had no effect on CPD formation or rates of repair, or on the number of excision repair sites as measured by cytosine arabino furanoside and hydroxyurea treatment. However, selenite and selenomethionine protected against oxidative damage to DNA as measured by formation of formamidopyrimidine (FaPy) glycosylase-sensitive sites, which are indicative of 8-hydroxy-2-deoxyguanosine photoproduct formation. In this assay, irradiation of keratinocytes increased mean +/- SD glycosylase-specific comet tail length from 5 +/- 1.5 microm to 19 +/- 3.3 microm. Preincubation for 18 h with 50 nmol L-1 selenite abolished the UVR-induced increase in comet length. Preincubation with 200 nmol L-1 selenomethionine was similarly protective. Conclusions Selenite and selenomethionine protect keratinocytes from UVR-induced oxidative damage, but not from formation of UVR-induced excision repair sites.
    British Journal of Dermatology 06/2003; 148(5):1001-9. · 3.76 Impact Factor
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    ABSTRACT: Selenium (Se) is a dietary trace element that is essential for effective immunity and protection from oxidative damage induced by ultraviolet radiation (UVR). Langerhans cells (LC) represent the major antigen-presenting cells resident in the epidermis; a proportion migrate from the skin to the draining lymph nodes in response to UVR. Because it is known that Se deficiency impairs immune function, we determined what effect this has on LC numbers. CH3/HeN mice were weaned at 3 wk and placed on diets containing <0.005 ppm of Se (Se deficient) or 0.1 ppm of Se (Se adequate, control mice). After 5 wk on the diet, the epidermal LC numbers in the Se-adequate group were 966 +/- 51 cells/mm2 and LC counts in the epidermis of the Se-deficient mice were 49% lower (p<0.05). Glutathione peroxidase- I (GPx) activity was measured in the epidermis, lymph nodes, and liver. In the epidermis, the activity of GPx in the Se-deficient mice was only 39% (p<0.01) of that seen in epidermis from Se-adequate mice (1.732 U/mg protein). The mice were then irradiated with one dose of 1440 J/m2 of broadband UVB or mock irradiated. After 24 h, the decrease in LC number after UVB was greater in the Se-adequate mice, (40% decrease) compared to the Se-deficient group (10%). Thus, Se deficiency reduces epidermal LC numbers, an effect that might compromise cutaneous immunity.
    Biological Trace Element Research 05/2003; 92(2):161-72. · 1.31 Impact Factor
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    ABSTRACT: The generation of reactive oxygen species has been implicated in ultraviolet radiation (UVR)-induced skin damage. In mice, increasing dietary selenium intake protects skin from UVR-induced DNA damage and photocarcinogenesis. We sought to determine whether selenium supplementation could protect keratinocytes from apoptosis resulting from exposure to broadband (TL20W/12) UVR. Unirradiated cultures contained 6.5 +/- 1% apoptotic cells; the maximum percentage of apoptotic cells (34 +/- 5%) was seen 16 h after UVR of 600 J/m(2). Under these conditions cell death from necrosis was 15 +/- 2.5% of the total cells. A 24-h preincubation with sodium selenite (10 nm(-1) microm) or selenomethionine (50 nm(-1) microm) protected cultured human keratinocytes from UVR-induced apoptosis. In primary keratinocytes the greatest reduction in apoptosis was found with 100 nm of either selenium compound (71% reduction in the numbers of total apoptotic cells; P < 0.01). Supplementation with 100-200 nm selenite or selenomethionine prevented UVR-induced apoptosis, but did not decrease the levels of UVR-induced p53, as measured by Western blotting. Collectively, this data suggests that selenium prevents UVR-induced cell death by inhibiting p53-independent cell death pathways.
    Clinical and Experimental Dermatology 05/2003; 28(3):294-300. · 1.33 Impact Factor
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    ABSTRACT: Background Ultraviolet radiation (UVR), a ubiquitous environmental genotoxin for the skin, produces DNA damage. The trace element selenium induces synthesis of the glutathione peroxidase and thioredoxin reductase enzyme families. These selenoenzymes detoxify a range of toxic compounds generated by free radicals.Objectives To assess the effects of pretreatment of primary human keratinocytes with selenium on UVR-induced DNA damage.Methods Cells were irradiated with UVR from FS-20 lamps and were subjected to comet assay.Results Comet tail length due to UVR-induced T4 endonuclease V-sensitive sites (caused by cyclopyrimidine dimers, CPDs) increased to 35 ± 4·5 μm (mean ± SD) immediately after irradiation (time 0 h, 100%). After 4 h, 68% of the damage remained and after 24 h, 23% of the damage was still present. Treatment with up to 200 nmol L−1 selenomethionine or 50 nmol L−1 sodium selenite had no effect on CPD formation or rates of repair, or on the number of excision repair sites as measured by cytosine arabino furanoside and hydroxyurea treatment. However, selenite and selenomethionine protected against oxidative damage to DNA as measured by formation of formamidopyrimidine (FaPy) glycosylase-sensitive sites, which are indicative of 8-hydroxy-2-deoxyguanosine photoproduct formation. In this assay, irradiation of keratinocytes increased mean ± SD glycosylase-specific comet tail length from 5 ± 1·5 μm to 19 ± 3·3 μm. Preincubation for 18 h with 50 nmol L−1 selenite abolished the UVR-induced increase in comet length. Preincubation with 200 nmol L−1 selenomethionine was similarly protective.Conclusions Selenite and selenomethionine protect keratinocytes from UVR-induced oxidative damage, but not from formation of UVR-induced excision repair sites.
    British Journal of Dermatology 04/2003; 148(5):1001 - 1009. · 3.76 Impact Factor
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    ABSTRACT: Selenium is an essential trace nutrient necessary for the normal function of the immune system. Selenium compounds protect mice against ultraviolet (UV) B-induced tumours, probably by preventing oxidative damage to the host skin cells and to the host immune system. One possible mechanism of protection is that selenium can prevent oxidative stress-induced release of cytokines such as interleukin (IL)-10, which could suppress cell-mediated immunity. To determine whether selenium compounds can inhibit UVB induction of IL-10 protein in murine keratinocytes. The murine keratinocyte cell line PAM 212 was treated with or without selenomethionine (50-200 nmol L-1) or sodium selenite (1-50 nmol L(-1)) for 24 h before exposure to 200 J m(-2) UVB. The cells were stained with an antibody to IL-10, 24 h after irradiation. Preincubation with both selenium compounds inhibited UVB induction of IL-10 immunostaining, although selenomethionine was more effective. Pretreatment with 200 nmol L(-1) selenomethionine decreased IL-10 immunostaining to levels seen in the unirradiated controls. The protective effects of selenium against UVB-induced skin cancer in murine models may result, in part, from its ability to inhibit release of cytokines that are capable of suppressing cell-mediated immunity.
    British Journal of Dermatology 04/2002; 146(3):485-9. · 3.76 Impact Factor
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    ABSTRACT: trans-Urocanic acid (UCA) acts as a chromophore for UV radiation in the epidermis and isomerizes to cis-UCA which then initiates some of the changes leading to UV-induced immunosuppression. The mechanism of the immunomodulation by cis-UCA is unknown at present, but one possibility is that the interaction between cis-UCA and keratinocytes causes the release of immunosuppressive cytokines locally. To test this hypothesis, PAM-212 cells, a murine keratinocyte cell line, were incubated with 0.10-100 micrograms/mL trans- and cis-UCA for 6 or 24 h, respectively. The expression of interleukin (IL)-10, transforming growth factor (TGF)-beta and tumor necrosis factor (TNF)-alpha messenger RNA (mRNA) was then measured by reverse transcription-polymerase chain reaction in comparison with the mRNA for the house-keeping gene, beta-actin. No change or significant induction of any of the cytokine messages occurred. However, the expression of IL-10 messenger RNA (mRNA) was induced 24 h after UVB irradiation (300 J/m2) and that of TNF-alpha mRNA occurred 6 h after treatment with phorbol myristate acetate. The expression of IL-10 protein was also examined by immunostaining in both PAM-212 cells and B16-F10 murine melanoma cells between 3 and 48 h after incubation with 10 and 100 micrograms/mL cis- and trans-UCA. No alteration was seen with either isomer at either concentration. In contrast, UVB irradiation of both cell lines resulted in a marked increase in intracellular IL-10 protein at 24 and 48 h. Therefore the upregulation of the immunosuppressive cytokines, IL-10, TNF-alpha and TGF-beta, in keratinocytes is unlikely to be the mechanism by which cis-UCA induces immunosuppression in mice.
    Photochemistry and Photobiology 04/2001; 73(3):238-44. · 2.29 Impact Factor
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    ABSTRACT: ABSTRACTtrans-Urocanic acid (UCA) acts as a chromophore for UV radiation in the epidermis and isomerizes to cis-UCA which then initiates some of the changes leading to UV-induced immunosuppression. The mechanism of the immunomodulation by cis-UCA is unknown at present, but one possibility is that the interaction between cis-UCA and keratinocytes causes the release of immunosuppressive cytokines locally. To test this hypothesis, PAM-212 cells, a murine keratinocyte cell line, were incubated with 0.10–100 μg/mL trans- and cis-UCA for 6 or 24 h, respectively. The expression of interleukin (IL)-10, transforming growth factor (TGF)-β and tumor necrosis factor (TNF)- messenger RNA (mRNA) was then measured by reverse transcription-polymerase chain reaction in comparison with the mRNA for the house-keeping gene, β-actin. No change or significant induction of any of the cytokine messages occurred. However, the expression of IL-10 messenger RNA (mRNA) was induced 24 h after UVB irradiation (300 J/m2) and that of TNF- mRNA occurred 6 h after treatment with phorbol myristate acetate. The expression of IL-10 protein was also examined by immunostaining in both PAM-212 cells and B16-F10 murine melanoma cells between 3 and 48 h after incubation with 10 and 100 μg/mL cis- and trans-UCA. No alteration was seen with either isomer at either concentration. In contrast, UVB irradiation of both cell lines resulted in a marked increase in intracellular IL-10 protein at 24 and 48 h. Therefore the upregulation of the immunosuppressive cytokines, IL-10, TNF- and TGF-β, in keratinocytes is unlikely to be the mechanism by which cis-UCA induces immunosuppression in mice.
    Photochemistry and Photobiology 02/2001; 73(3):238 - 244. · 2.29 Impact Factor
  • R C McKenzie, T S Rafferty, G J Beckett
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    ABSTRACT: The importance of selenium for optimal immune function is now apparent. Here, Roddie McKenzie and colleagues describe how selenium is involved in the function of immune cells, and the various immune deficiencies and diseases that result from inadequate dietary intake.
    Immunology Today 09/1998; 19(8):342-5. · 9.49 Impact Factor
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    ABSTRACT: The generation of reactive oxygen species has been implicated as part of the mechanism responsible for UVB-radiation-induced skin damage. In mice, evidence suggests that increased dietary selenium intake may protect skin from many of the harmful effects of UVB radiation. We sought to determine the selenoprotein profile of cultured human skin cells and whether selenium supplementation could protect keratinocytes and melanocytes from the lethal effects of UVB radiation. Labelling experiments using [75Se]selenite showed qualitative and quantitative differences in selenoprotein expression by human fibroblasts, keratinocytes and melanocytes. This was most noticeable for thioredoxin reductase (60 kDa) and phospholipid glutathione peroxidase (21 kDa); these proteins were identified by Western blotting. Despite these differences, we found that a 24 h preincubation with sodium selenite or selenomethionine protected both cultured human keratinocytes and melanocytes from UVB-induced cell death. With primary keratinocytes, the greatest reduction in cell death was found with 10 nM sodium selenite (79% cell death reduced to 21.7%; P<0.01) and with 50 nM selenomethionine (79% cell death reduced to 13.2%; P<0.01). Protection could be obtained with concentrations as low as 1 nM with sodium selenite and 10 nM with selenomethionine. When selenium was added after UVB radiation, little protection could be achieved, with cell death only being reduced from 88.5% to about 50% with both compounds. In all of the experiments sodium selenite was more potent than selenomethionine at providing protection from UVB radiation.
    Biochemical Journal 05/1998; 332 ( Pt 1):231-6. · 4.65 Impact Factor
  • TS Rafferty, GJ Beckett, JA Hunter, RC M
    Journal of Dermatological Science 03/1998; 16. · 3.52 Impact Factor