Molecular Aspects of Photoaging
Background.—Extrinsic aging appears to play the major role in age- associated degeneration of the skin. Because it is mainly caused by ultraviolet (UV) irradiation, this process is known as photoaging. Photoaging is distinct from intrinsic aging of the skin from a clinical, biological, and molecular standpoint. Histologic, immunohistochemical, and ultrastructural studies identify dermal accumulation of elastotic material as a key feature of photoaging. One early event in the photoaging process is activation of elastin gene expression, with enhanced transcriptional activity of other extracellular matrix genes. A new animal model for studying the molecular basis of photoaging was described.
Animal Model.—A line of transgenic mice was developed that expresses the human elastin promoter in a tissue-specific and developmentally regulated manner. The human gene was linked to a chloramphenicol acetyl- transferase reporter gene. Elastin promoter activation was enhanced when the animals were exposed to UV radiation, especially UVB radiation. Cultured skin fibroblasts from the transgenic mice showed a significant effect of UVB but not UVA radiation. When the cultures were exposed to UVA plus 8-methoxypsoralen, there was a marked enhancement of activity.
Conclusions.—The findings in this animal model, coupled with previ-ously reported findings, show that UVB plays a key role in the development of solar elastosis. In vivo, UVA is an important contributing factor. The transgenic mouse model described will be useful for studying the mechanisms of cutaneous photoaging, as well as testing possible compounds to protect against photodamage.
This article presents an excellent review of the molecular basis of photoaging (extrinsic aging), which is clearly different from innate or chronological aging (intrinsic aging). The authors describe a new transgenic mouse model that can aid in the study of mechanisms underlying cutaneous photodamage. Such studies have helped differentiate the effects of UVA and UVB radiation on elastin promoter activation.
Pathophysiology of Premature Skin Aging Induced by Ultraviolet Light
Objective.—Characteristics of photoaged skin include wrinkles, pig-mentary alterations, and loss of skin tone. Histologic and ultrastructural studies of photoaged skin show major changes in the collagenous extra-cellular matrix of connective tissue. The matrix-degrading metalloproteinases were investigated as possible mediators of collagen damage in photoaging.
Methods.—The study included 59 white research subjects (age range, 21-58 years). All had light-to-moderate skin pigmentation and no current or previous skin disease. Buttock skin was irradiated by fluorescent ultra¬violet lights, and specimens of irradiated and nonirradiated skin were obtained. The effects of tretinoin or vehicle—applied to the skin under occlusion for 48 hours before irradiation—were studied as well. In situ hybridization, immunohistologic, and in situ zymographic studies were performed to assess the expression of matrix metalloproteinases. Radioimmunoassay of soluble cross-linked telopeptides was done to measure irradiation-induced degradation of skin collagen, and Western blotting was used to assess the protein level of tissue inhibitor of matrix metalloproteinases type 1.
Results.—After just 1 exposure to ultraviolet radiation, there were significant increases in expression of 3 matrix metalloproteinases in the skin connective tissue and outer skin layers compared with nonirradiated skin. These were collagenase, a 92-kd gelatinase, and stromelysin. Irradiated skin showed a 58% increase in degradation of endogenous type I collagen fibrils. After 4 exposures to ultraviolet irradiation, collagenase and gelat-inase activity remained at peak levels for 7 days. Tretinoin pretreatment was associated with a 70% to 80% inhibition of induction of matrix metalloproteinase proteins and activity in both connective tissue and outer skin layers. A tissue inhibitor of matrix metalloproteinases-1, which regulates the enzyme, was also induced by ultraviolet irradiation. Tretinoin had no effect on the induction of this inhibitor.
Conclusions.—Ultraviolet irradiation causes sustained increases in matrix metalloproteinases that degrade skin collagen. These metalloprotein¬ases may play an important role in photoaging. Topical tretinoin inhibits the induction of matrix metalloproteinases in response to irradiation but does not affect their endogenous inhibitor.
The authors suggest that the reported beneficial effect of topical tretinoin on repeatedly photodamaged skin might result from its inhibition of proteolytic matrix metalloproteinases, including collagenase, 92-kd gelatinase, and stromelysin-1.
Retinoic Acid Inhibits the Regulated Expression of Vascular Cell Adhesion Molecule-1 by Cultured Dermal Microvascular Endothelial Cells
Objective.–Expression of cell adhesion molecules (CAMs) plays a key role in the localization of circulating leukocytes to the endothelium, and thus in the pathogenesis of various inflammatory disorders. Previous studies of cutaneous disease have suggested that retinoic acid and its synthetic derivatives have anti-inflammatory effects, although the mechanisms of these effects remain unknown. The retinoids’ mechanisma of action in modulating inflammatory responses were examined.
Methods and Results.--Studies were performed in cultured human dermal microvascular and endothelial cells. Cells pretreated with all-trans-retinoic acid (t-RA) showed specific inhibition of tumor necrosis factor-α (TNFα)-induced expression of vascular cell adhesion molecule- 1 (VCAM-1) expression. However, there was no effect on induction of intercellular adhesion molecule-1 (ICAM-1) or E-selectin induction. In cells treated with TNFα, t-RA also reduced VCAM-1-dependent T-cell binding.The different effects of t-RA on TNFα-induced CAM expression were observed at steady-state mRNA levels and in nuclear run-on studies.
Transcriptional activation studies showed that t-RA treatment inhibited activation of the human VCAM-1 promoter by TNFα. However, the lack of effect on ICAM-1 promoter activation suggests that selective inhibition of CAM expression occurs partly at the level of gene transcription. The findings indicate that the inhibitory effect of t-RA on gene transcription is mediated by its effects on activation of nuclear factor-ĸB (NF-ĸB- dependent complex formation. On protein-DNA binding assays, specific NF-ĸB-dependent binding to the tandem NF-ĸB sites of the VCAM-1 promoter was significantly inhibited. However, binding to the functional NF-ĸB motif of the ICAM-1 promoter was unaffected.
Conclusions.—-Therefore t-RA has varying effects on regulation of CAM expression by cultured dermal microvascular endothelial cells. This retinoid preferentially inhibits TNFα-induced VCAM-1 regulation, partially through transcriptional control mechanisms involving the activation of NF-ĸB-dependent complex formation. Selective suppression of VCAM-1 expression and subsequent activity in leukocyte-endothelial cell recognition and localization may explain the mechanism by which retinoids exert their anti-inflammatory effect.
The observation that t-RA can inhibit cytokine-mediated VCAM expression is most certainly only one of a number of ways by which retinoids exert their biological effects, in vivo. In this study, pretreatment with t-RA prevented TNFα-mediated VCAM-1 but not ICAM-1 or E-selectin induction. This differential susceptibility to t-RA appears to be regulated, at least in part, at the level of gene transcription.
1,25-Dihydroxy vitamin D3 Regulates Estrogen Metabolism in Cultured Keratinocytes
Background.—Local estrogen metabolism may be important in modulating cell development in peripheral tissues, such as breast, adipose, and bone tissue. The enzyme aromatase converts C19 androgens into C18 estrogens, and the overexpression of this enzyme has been associated with breast cancer. Various isoforms of the enzyme 17β-hydroxysteroid dehy-drogenase (17βHSD) control interconversion of active estradiol (E2) to inactive estrone. The expression of these 2 enzymes in human keratinocytes was studied.
Methods and Findings.—Keratinocytes cultured in serum-free medium demonstrated aromatase activity of 2.5 fmol/hr-mg cell protein. None of the culture treatments affected this. A much greater level of 17βHSD activity was seen in the keratinocytes, primarily E2 conversion to estrone. This inactivation of E2 increased in a dose-dependent manner after cell treatment with antiproliferative doses of l,25-(OH)2D3. Simultaneous dex- amethasone treatment enhanced the effect of 1,25-(OH)2D3 on 17βHSD activity and increased the antiproliferative action of l,25-(OH)2D3. Keratinocytes expressed messenger RNA for 3 17βHSD isoenzymes in reverse transcription-polymerase chain reaction and Northern analysis. Treatment with l,25-(OH)2D3 was associated with the upregulation of messenger RNA levels for type 2 17βHSD. Estrogen receptors were found in the cultured keratinocytes on further RNA analysis and E2 binding experiments.
Conclusions.—Keratinocytes may be targets for systemically or locally produced estrogens. These may, in turn, play an important role in the development of normal skin. The 17βHSD isoenzymes are believed to be key target genes for l,25-(OH)2D3 in keratinocytes. They may also be an important feature of the antipsoriatic effects of vitamin D and its analogues.
The data presented here demonstrate considerable potential for local estrogen metabolism in the skin, and show that the expression and activity of estrogenic enzymes are regulated by 1,25-dihydroxyvitamin D3 (calcipotriene), a known antiproliferative and antipsoriatic agent. Regulation of local 17βHSD and aromatase may also be important in the homeostasis of hair follicles and sebaceous glands as well as in the pathogenesis of disorders such as acne and androgenic alopecia, which appear to involve abnormal control of androgen function. Moreover, local, as well as systemic, estrogen metabolism may play a role in the luxuriant hair growth noted during pregnancy and the diffuse postpartum hair loss recognized as telogen effluvium.
Age-related Changes in Facial Skin Contours and Rheology
Introduction.—-The use of a variety of methods for evaluating the aging of facial skin has created a number of discrepancies in the literature. The effect of age on the biomechanical properties of facial skin was assessed using computerized optical profilometry in 189 healthy white women of phototype II and III.
Methods.—Age range of research subjects was 18-67 years. All research subjects had indoor occupations and denied excessive sun exposure. Women with actinic keratoses and cutaneous cancers were excluded. There were 30 research subjects in each of the 5 decades represented. Women aged 48-57 years were allocated to 1 of 2 groups of 30, depending on use or nonuse of hormone replacement therapy (HRT). Mechanical properties of the skin were measured using a small hollow-probe suction device. Optical profilometry and computerized image analysis were used to evaluate skin contours. Skin replicas were obtained from the crowsfeet wrinkle area with a Silflo resin.
Results.—Skin aging of the face is characterized by a gradual increase in extensibility related to diminished elasticity. Tonicity loss is accompanied by an ongoing deepening of facial creases. Age-related rheologic changes seem to be limited in women taking HRT, but HRT does not have a preventive effect on wrinkling of facial skin.
Conclusion.—The aging of facial skin is similar to features already described on sun-protected areas of the forearms. The overall process of aging may be slowed with HRT, but HRT administration does not limit the number or depth of wrinkles. Computerized optical profilometry is a convenient and effective method for evaluating age-related changes in facial skin contours and rheology.
The effect of HRT on biomechanical properties of facial skin confirms previous data from the relatively sun-protected area of the inner forearms. Unfortunately, the beneficial effect on mechanical properties did not prevent wrinkling. Thus, although HRT may slow both chronologic aging and photoaging, it does not Improve all the tissue components responsible for wrinkling.
Systemic Therapy With Estrogen or Estrogen With Progestin Has No Effect on Skin Collagen in Postmenopausal Women
Background.—Skin collagen content is reduced after menopause. Ac-cording to previous studies, estrogen can reverse this decline. Collagen synthesis rate and skin collagen content were compared in postmenopausal women who received estrogen alone, estrogen plus progestin, or no hormone replacement therapy.
Methods.—The study included 43 postmenopausal women, 29 of whom desired treatment for climacteric symptoms. Fifteen women with an intact uterus received continuous oral estrogen plus progestin therapy for 1 year; dosages were 2 mg/day of 17-β-estradiol and 1 mg/day of norethisterone acetate. Fourteen women who had been hysterectomized—most because of bleeding problems or pain related to myoma—were treated with estradiol valerate, 2 mg/day. Another 15 women who had menopausal symptoms but did not desire treatment were studied as controls. The 3 groups were compared for histologic skin findings; types I and III procollagen skin immunohistochemistry findings; skin thickness; skin total collagen, as determined by colorimetry; and synthesis of type I and III collagen, deter-mined by measuring procollagen peptides in suction blister fluids. All measurements were made at baseline and after 6 and 12 months of treatment. Computerized image analysis to assess the proportional area of elastic fibers and epidermal thickness was performed before and after 12 months of treatment.
Results.—There were no significant differences in skin thickness, capillary blood flow, or capacitance measurements. Neither were there any differences in the amount or rate of collagen synthesis. None of the 3 groups had any changes in skin histologic findings during the 1-year study, and the findings were no different in the hormone-treated group than in the control group. On computerized digital image analysis, hormone treat¬ment was shown to have no effect on the proportional area of elastic fibers in the papillary or reticular dermis or on epidermal thickness.
Conclusion.—One year of hormone replacement therapy—whether with estrogen alone or estrogen plus progestin—has no effect on collagen synthesis or on total skin collagen content. The findings conflict with previous reports that hormone replacement therapy increases skin thick¬ness and collagen content after menopause. More research is needed to determine how hormone replacement therapy affects skin collagen turn¬over rate and degradation.
The authors report that 1 year of treatment with systemic estrogen, alone or combined with progestin, does not change the amount of skin collagen or the rate of collagen synthesis in recently menopausal Women. It is possible that earlier initiation of treatment or more prolonged treatment might have yielded more promising results; conversely, because there are no marked changes in skin collagen synthesis up to age 60-70 years, it is also possible that the amount of skin collagen and collagen synthesis were still at an optimum level, and hormone replacement therapy could have increased them no more.
Yet another possibility is that hormone replacement therapy induces qual-itative, rather than quantitative, changes in skin collagen; however, none were dectected using the methodologies of these researchers. Based on these studies and the lack of convincing experimental data, there is little evidence to support the contention that systemic hormone therapy or topical treatment with hormone-containing creams improves symptoms of skin aging.
Evidence for UV-associated Activation of Telomerase in Human Skin
Introduction.—The finding of telomerase activity in most benign (60%), premalignant (89%), and malignant (91%) skin tumors suggests that telomerase activation is involved in the process of human skin carcinogenesis. A study was conducted to examine a possible correlation between telomerase activity, ultraviolet (UV) exposure (the major etiologic factor in human skin cancer), and p53 mutations (the most frequent genetic alter-ations found in human skin tumors thus far).
Methods.—Skin biopsy specimens examined were from normal skin, normal skin distant from premalignant tumors or skin cancers, normal skin adjacent to benign skin tumors, and normal skin adjacent to premalignant tumors and skin cancers. Also examined were a variety of benign, premalignant, and malignant skin tumors. Data for chronically sun-ex-posed body sites were compared with those for covered sites.
Results.—Telomerase activity, present in 39% of all normal skin samples, was unrelated to neighboring lesions but strongly associated with the level of sun exposure. Normal skin samples from chronically sun- exposed sites were more likely to be telomerase-positive (54%) than samples from covered sites (12%); the difference in incidence was statis¬tically significant. Australian patients with skin cancer have a substantial number of cells with UV-specific, CC to TT p53 gene mutations in mor¬phologically normal skin. Analysis of the frequency of UV-specific CC to TT p53 mutations at codon 247/8 suggested that telomerase activation may precede mutation of the p53 gene in human skin carcinogenesis. Only 7 of 16 telomerase-positive normal skin samples at sun-exposed sites contained this mutation, whereas 7 of 7 samples with UV-specific p53 mutations showed telomerase activity.
Conclusion.—Findings suggest that either activation of telomerase or clonal expansion of telomerase-positive cells by chronic sun exposure is an early feature of skin carcinogenesis, and this activity may precede the acquisition of UV-specific p53 mutations. A proposed model of UV-associated human skin carcinogenesis begins with the proliferation of telomerase-positive cells, followed by the acquisition of UV-specific p53 mutations, clonal expansion, and development of the precancerous lesion.
The molecular events of cutaneous carcinogenesis are gradually being elucidated. The telomerase enzyme appears to play a critical role in the pathogenesis of malignancy in general, and its cloning last year may have been a landmark event that will christen a new generation of antineoplastic agents.
Induction of Mutagenic DNA Damage in Human Fibroblasts After Exposure to Artificial Tanning Lamps
Purpose.—Artificial ultraviolet (UV) tanning beds are increasingly popular, raising concern about their ability to induce skin cancer, as well as other adverse health effects. For skin tumors to develop, mutagenic DNA damage must occur. Cyclobutane pyrimidine dimers (CPDs) and other type of direct damage induced by UV light are known to lead to mutations in tumor suppressor genes and oncogenes. Exposure to UV radiation may also cause indirect but still mutagenic DNA damage. The types of mutagenic DNA damage induced by commercial tanning beds were examined using assays for DNA repair enzymes T4 endonuclease V and endonuclease III, and the comet assay.
Findings.—Experiments were performed with primary human fibroblast cell cultures exposed to commercial tanning bed lamps for periods of 15 seconds to 20 minutes. The assays confirmed the presence of CPDs, oxidized or hydrated pyrimidine bases, and single-strand breaks in the cultured cells. The 2 different lamps studied showed considerable variation in the ratio of endonuclease III to T4 endonuclease V sensitive sites. The more powerful of the 2 lamps induced CPDs to the same extent as natural sunlight.
Conclusions.—Commercial UV tanning beds induce the same type of DNA damage responsible for induction of skin cancer in human cells. Because tanning lamps cause the same type of DNA damage as sunlight, a known carcinogen, it is highly probable that tanning lamps are carcinogenic as well. The findings support epidemiologic studies suggesting an increased skin cancer risk associated with tanning bed use.
This article provides excellent objective data to substantiate the deleterious effects of commercial tanning lamps. Surprisingly, this study appears to have received very little attention in the United States.
Epidermal Proliferation But Not the Quantity of DNA Photodamage Is Correlated With UV-induced Mouse Skin Carcinogenesis
Background.—The main cause of skin cancer in humans is exposure to ultraviolet (UV) radiation, and the major lesion caused by UV exposure is the cyclobutane pyrimidine dimer (CPD). Experiments have shown that, after exposure to UV radiation, hairless SKH-1 mice have a higher incidence of skin tumors, a shorter latency period to tumor development, and a higher tumor yield than SENCAR mice. These 2 strains were compared for susceptibility to UV damage by measurement of DNA photodamage level and epidermal proliferation.
Methods.—Female SENCAR and hairless SKH-1 mice were exposed to chronic UV irradiation for 9 weeks. The total cumulative UV dose was 6.975 J/cm2 for the SENCAR strain and 5.52 J/cm2 for the SKH-1 strain. At intervals from 1 to 9 weeks, DNA photodamage and repair were measured. Parameters associated with proliferation, including epidermal hyperplasia, DNA synthesis, and p53 expression, were assessed as well.
Results.—The SKH-1 mice had significantly greater induction rates for CPDs and pyrimidine (6-4) pyrimidone photoproducts ([6-4] PDs) than the SENCAR mice. However, there were no differences in repair kinetics for CPDs or (6-4) PDs. In response to chronic UV exposure, the 2 strains showed equal amounts of DNA photodamage. By 6 weeks, there were significantly more (6-4) PDs than CPDs. The SKH-1 mice had a 1.5-fold increase in the number of epidermal cells. They also showed a greater number of basally and suprabasally located cycling cells, as measured by 5-bromo-2′-deoxyuridine (BrdU). There was no difference between strains in the number of cells with immunoreactivity to p53. However, at the end of 9 weeks of UV exposure, immunoreactive cells were located suprabasally in the SKH-1 mice.
Conclusions.—These mouse studies suggest that UV-induced tumorigenesis may not be entirely dependent on accumulation of CPDs and PDs, and thus on the amount of DNA photodamage in the epidermis. The degree of epidermal hyperplasia associated with DNA synthesis appears to play a significant role. Some of the differences observed in the SKH-1 strain, which has enhanced susceptibility to UV-induced skin cancer, may reflect an enhanced susceptibility to mutation.
These observations suggest that, at least in the strains of mice tested here, the induction of UV tumorigenesis is not entirely dependent on DNA photodamage but also is dependent on the degree of epidermal hyperplasia associated with DNA synthesis.
The Inhibition of Antigen-presenting Activity of Dendritic Cells Resulting From UV Irradiation of Murine Skin Is Restored by in vitro Photo¬repair of Cyciobutane Pyrimidine Dimers
Introduction.—Exposing skin to UV radiation causes lesions in cellular DNA. When skin is exposed to UVB (280-320 nm), it suppresses contact hypersensitivity by a mechanism involving an alteration in the activity of cutaneous antigen-presenting cells (APCs). The UV-induced DNA damage seems to be a crucial molecular trigger for this effect. The specific target cells in the skin that undergo DNA damage relevant to the immunosup-pressive effect are not yet identified. Experiments were conducted to determine whether UV-induced DNA damage in the cutaneous APCs was responsible for their impaired ability to present antigen after in vivo UV irradiation.
Findings.—Cutaneous APCs were collected from the draining lymph nodes of UVB-irradiated, hapten-sensitized mice. They were incubated in vitro with liposomes containing a photolyase that divides UV-induced cyclobutane pyrimidine dimers upon absorption of photoreactivating light. Photosome treatment followed by photoreactivating light decreased the number of dimer-containing APCs, restored the in vivo antigen-presenting activity of the draining lymph node cells, and obstructed the induction of suppressor T cells. Antigen-presenting cells were not restored with photosomes alone, photoreactivating light alone, or photoreactivating light given before photosomes. Photosome treatment did not reverse impairment of APC function when isopsoralen plus UVA (320-400 nm) radiation was used rather than UVB. The restoration of APC function matched the requirements of photosome-mediated DNA repair for dimers and posttreatment photoreactivating light.
Conclusion.—These findings indicate that UV-induced DNA damage in cutaneous APCs leads to diminished immune function.
The authors establish a causal relationship between the presence of persistent dimers in cutaneous antigen-presenting cells and their altered immune function. Photorepair of these dimers led to restoration of antigen- presenting function. Could this same approach be used to repair DNA damage in other cell systems?
Immunosuppression Induced by Acute Solar-simulated Ultraviolet Exposure in Humans: Prevention by a Sunscreen With a Sun Protection Factor of 15 and High UVA Protection
Introduction.—Sunscreens are effective in preventing sunburn, erythema, and solar keratoses, but results vary regarding their effectiveness in preventing UVB induced immunosuppression in animal experiments. It has recently been demonstrated that high sun protection factor (SPF) sunscreens prevent localized UVB-induced suppression of contact hyper-sensitivity (CHS) in humans. Irritant and CHS responses to dinitrochlo- robenzene (DNCB) were analyzed to determine whether solar-simulated exposure of human skin is responsible for impairment of CHS responses and whether the preapplication of an SPF-15 sunscreen with high UVA protection could prevent the locally UV-induced immunosuppression.
Methods.—A total of 160 volunteers, were randomly assigned to 1 of 8 groups to be evaluated for irritant and CHS responses to DNCB. These groups included volunteers with or without UV exposure and with or without an SPF 15 sunscreen with high UVA protection. The DNCB sensitization was done 3 days after acute UV irradiation corresponding to 3 minimal erythema doses.
Results.—The rate of positive responses to DNCB sensitization fell from 95% to 50% after solar-simulated UV exposure; however, such a decrease did not occur with pre-application of the sunscreen formulation.
Conclusion.—The sunscreen tested was able to prevent the reduction of CHS responses after acute solar-simulated UV exposure. These findings add further evidence that a localized sunburn can impair the afferent arm of the CHS reaction and alter T-cell responses at distant, unirradiated cutaneous sites. It is not known if this locally induced UV suppression causes increased susceptibility to skin cancers, cutaneous infections, and vaccination failure.
It has long been known that the immune system plays an important role in UV carcinogenesis by contributing to host resistance against tumor growth. Conversely, UVB immunosuppression increases the risk of skin cancer. Although sunscreens can decrease UV-induced erythema, their protective effect on the immune response is not well quantified. This study demonstrates that the use of a sunscreen with an SPF of 15 or more, along with high protection in the UVA spectrum, seems to protect against the suppression of cutaneous hypersensitivity that occurs after acute solar- simulated UV exposure. Because it has not yet been determined whether locally induced UV suppression increases the susceptibility to skin cancer, one cannot conclude from these data that the immunoprotective effect of sunscreens will contribute to a decreased incidence of skin cancer.
Sunscreen Lotions Prevent Ultraviolet Radiation-induced Suppression of Antitumor Immune Responses
Objective.—Ultraviolet (UV) radiation both induces skin cancers and promotes their emergence and growth. Previous experiments have suggested that tumor immunity in mice is suppressed by exposure to subcar-cinogenic UV doses, leading to the emergence and growth of highly immunogenic skin cancers. Though sunscreens are known to prevent induction of tumors by UV, it is unknown whether they can block UV-induced tumor immune suppression. Various sunscreens were compared for their tumor immune protective capacity in terms of UV spectrum and dose.
Methods.—The study compared 4 commercially available sunscreens with sun protection factors (SPF) ranging from 8 to 45. The sunscreens were compared for their effective tumor immune suppression dose (TISD), defined as the lowest dose to induce outgrowth of transplanted nonmelanoma skin tumors in 100% of UV-exposed C3H mice. For each sunscreen, the TISD was determined for each of 3 UV sources: unfiltered and Ko-dacel-filtered UVB-type FS20 sunlamps vs. a filtered xenon arc lamp solar stimulator.
Results.—A significantly higher dose of UV radiation was needed to induce tumor immune suppression with the solar simulator vs. the filtered and unfiltered sunlamps. In response to filtered sunlamp light, tumor incidence was significantly reduced to 54% by the SPF 15-sunscreen, 46% by the SPF 30 sunscreen, and 0% by the SPF 45 sunscreen; no significant reduction was achieved with the SPF 8 sunscreen. With solar simulator radiation, tumor outgrowth was reduced to 40% with the SPF 8 and 15 sunscreens and 20% with the SPF 30 and 45 sunscreens. When the effects of unfiltered UV radiation were tested, none of the sunscreens provided significant tumor immune protection. In tests using the solar simulator, measurements of the sunscreens’ tumor immune protective levels were consistent with the levels predicted by the labeled SPF. In addition, the SPF 30 and 45 sunscreens blocked activation of tumor antigen-specific sup-pressor T lymphocytes in mice exposed to solar simulator radiation.
Conclusions.—Under appropriate testing conditions, the tumor immune protection conferred by sunscreens is directly correlated with their labeled SPF. Significant protective effects are noted in tests with filtered sunlamp and solar simulator UV radiation, though not with unfiltered UV radiation. The findings are consistent with evidence that sunscreens effectively block photocarcinogenesis in human skin.
Ultraviolet light probably induces skin cancer formation through 2 separate mechanisms: a direct carcinogenic effect, and cutaneous immunosuppression. Sunscreens have been shown to prevent UV light-induced DNA damage and the expression of tumor suppressor genes. However, despite clinical observations that suggest sunscreens are effective in preventing skin cancer, it is uncertain whether sunscreens prevent cutaneous immunosuppression. These conflicting observations may result from using different sunscreens that vary in their spectrum of protection or the utilization of light sources that may not duplicate the UV rays reaching the earth’s surface. In this systematic study the authors demonstrate that, when used in conjunction with UV light sources that simulate solar radiation, broad-spectrum sunscreens are effective in preventing cutaneous immunosuppression and the generation of T-suppressor cells.
Effects of Tea, Decaffeinated Tea, and Caffeine on UVB Light-induced Complete Carcinogenesis in SKH-1 Mice: Demonstration of Caffeine as a Biologically Important Constituent of Tea
Objective.—Oral administration of a green-tea polyphenol fraction has been shown to inhibit ultraviolet-B (UVB) light-induced complete tumor-igenesis in the skin of SKH-1 mice. Decaffeinated teas are less effective than regular teas in inhibiting tumors initiated with 7,12-dimethylbenz- [a]anthracene. The effects of orally administered green tea, black tea, decaffeinated green tea, decaffeinated black tea, and caffeine on UVB- induced complete carcinogenesis in the skin of SKH-1 mice were evaluated.
Methods.—Solutions of caffeine, solutions of lyophilized tea solids— both caffeinated and decaffeinated—1.25% tea infusions, and a green-tea polyphenol fraction were prepared. Female SKH-1 female mice were given either water or tea to drink and received twice weekly UVB treatments for 35 weeks to induce tumorigenesis. Skin tumor volumes were measured, and tumors were analyzed histopathologically.
Results.—The mice had an average of 8.9 tumors, with an average tumor volume of 351 mm3. Lyophilized green tea (0.3% and 0.9%) reduced the number of tumors per mouse by 35% and 94%, respectively. The decaffeinated versions did not decrease the number of tumors but did slightly and significantly reduce tumor volume. Administration of 0.24 and 0.72 mg of caffeine/mL water reduced the number of tumors per mouse by 53% and 61%, respectively, at 35 weeks and by 30% to 31% at 39 weeks. Oral administration of 0.24 and 0.72 mg of caffeine/mL water reduced tumor volume by 55% and 82%. The decaffeinated products did not inhibit tumorigenesis or reduce the number of tumors or tumor volume. At high doses, some animals showed enhanced tumorigenic activity. Adding caffeine to these teas restored their inhibitory effects. When a green-tea polyphenol fraction was applied topically immediately after UVB treatment, tumorigenesis was inhibited. Whereas this study demonstrated an inhibitory effect of caffeine on tumorigenesis, other studies have shown a stimulatory effect of caffeine on carcinogenesis.
Conclusion.—More research is needed to identify the complex effects of green and black tea. The caffeine in green and black teas appears to be an inhibitor of UVB-induced tumorigenesis.
The authors demonstrate that oral administration of caffeine has an inhibitory effect on UVB-induced carcinogenesis. In this experimental model, caffeine appears to be an important factor in the biologic activity of green and black tea. It is unclear from this study whether caffeine alone is an effective antineoplastic agent. Perhaps caffeinated tea use should be examined in epidemiologic studies of human skin cancer.
Suppression of UV-induced Erythema by Topical Treatment With Melatonin (N-Acetyl-5-Methoxytryptamine): Influence of the Application Time Point
Purpose.—Free radicals appear to play a role in acute sunburn reactions, and previous studies have tried to suppress formation of ultraviolet (UV)-induced free radicals through the use of free-radical scavengers. Previously, the authors have found that topically applied melatonin 0.5% significantly suppresses UV-induced erythema in human skin in a dose- dependent fashion. A randomized, controlled trial was performed to assess the effect of the time at which melatonin is applied on its antierythema effect.
Methods.—Test areas on the lower back of 20 healthy volunteers were treated with melatonin, 0.6 mg/cm2, dissolved in a nanocolloid gel carrier. Melatonin treatment was applied 15 min before or 1, 30, or 240 min after the subjects received UV irradiation at twice their individual minimal erythema dose. A Multiport Solar UV Simulator was used to deliver both UVA and UVB radiation. After 24 hours, erythematous reactions were scored visually and by chromametry.
Results.—In areas pretreated with melatonin, UV-induced erythema was suppressed almost entirely. Later application did not significantly protect against erythema.
Conclusion.-—Topical melatonin pretreatment of human skin protects against the development of erythema in response to UV irradiation. As melatonin has no direct sunscreen effect, its radical scavenger mechanism is the most likely explanation for this finding. Later application does not inhibit erythema development.
This study shows that topical melatonin, 0.6 mg/cm2, before irradiation significantly inhibits the development of UV-induced erythema. A direct sunscreen effect can be excluded, as melatonin, which is not metabolized in human skin, does not absorb UVA or UVB. The authors suggest that topical application of melatonin most likely reduces the number of acutely generated hydroxyl radicals, thus reducing or inhibiting the development of inflammatory reactions and erythema. An effect on arachidonic acid metabolism is also possible.
Evidence That Singlet Oxygen-induced Human T Helper Cell Apoptosis Is the Basic Mechanism of Ultraviolet-A Radiation Phototherapy
Introduction.—Ultraviolet (UV) light is of value for the treatment of atopic dermatitis. The pathogenesis of that disease involves a T cell- mediated immune response directed against inhalant allergens. Little is known about the mechanisms of action underlying the effectiveness of UV phototherapy of atopic dermatitis. Targets for UV phototherapy may be T helper cells present in skin lesions of patients with atopic dermatitis. Short wavelength ultraviolet B radiation was shown to be inferior to long wave length ultraviolet A (UVA) radiation, which penetrates the dermal layers of human skin and affects intradermal T cells. Depletion of skin-infiltrating T helper cells may be occurring with UVA phototherapy. In eczematous skin of patients with atopic dermatitis, UVA phototherapy was found to induce apoptosis in T helper cells.
Methods.—Five patients with atopic dermatitis were treated with UVA phototherapy as a monotherapy, with daily exposures for 10 consecutive days. In each patient, sequential biopsies were taken from chronic, lichenified, eczematous skin lesions before and after days 1, 2, 3, 4, and 10 of UVA radiation exposure.
Results.—There was a significant improvement in skin symptoms in all patients. Numerous CD4+ cells were present intradermally in eczematous skin before therapy, but not a significant number of apoptotic cells. After the UVA radiation therapy, CD4+ apoptotic cells were detected, and subsequent treatments doubled the number of positive cells and decreased the number of CD4+ cells. There was a significant decrease of CD4+ T cells after 10 exposures, and most remaining cells showed signs of apop¬tosis. Human T cell apoptosis was induced by UVA radiation, and was mediated through the FAS/FAS-ligand system, which was activated in irradiated T cells as a consequence of singlet oxygen generation.
Conclusion.—For induction of human T cell apoptosis, singlet oxygen is a potent trigger. In phototherapy, singlet oxygen generation is a fundamental mechanism of action.
The data suggest that generation of singlet oxygen within human skin may represent one of the mechanisms underlying the effect of phototherapy when used for inflammatory skin diseases. Singlet oxygen is an important intermediary in several biologic processes, and can be produced by a variety of biologic systems. The observation that singlet oxygen can induce human T cell apoptosis adds a previously unrecognized biologic activity to its known list of biologic effects. An extensive review of apoptosis was recently published.
An Open Clinical Trial of a New Mouth-PUVA Variant in the Treatment of Oral Lichenoid Lesions
Background.—Mouth PUVA was initially introduced in 1987 as a new treatment for oral mucosal lesions. To date, only perorally administered methoxsalen capsules have been used for mouth-PUVA photosensitization. The feasibility of topical psoralen plus ultraviolet A (PUVA) was investigated.
Methods.—Sixteen patients with oral lichenoid lesions (OLL) were given a 0.01% trioxsalen ointment and ultraviolet A (UVA) doses in a range of 0.09 to 1.8 J/cm2. Five patients were diagnosed as having oral lichen planus (OLP) and 11 as having oral lichenoid reaction (OLR). Patients were treated 2-3 times per week for 2-4 weeks. The mean number of sessions was 8.7, and the mean cumulative irradiation dose was 4.25 J/cm2.
Findings.—Nineteen percent of the patients had marked-to-complete healing immediately after treatment. Twenty-nine percent evidenced such healing after 3 months, and 38% after 14 months. The post-PUVA ame-lioration rate was 80% in patients with genuine OLP, compared with only 11% in those with OLR.
Conclusions.—Topical trioxsalen photosensitization can be used effectively in mouth-PUVA treatment. The main indication for this therapy is lichen planus.
Given the recalcitrant nature of oral lichen planus, any new treatment option would be welcome. Although I have had disappointing results with hydroxychloroquine, I have found low-dose oral retinoids to be quite helpful in managing this condition.
Evaluation of PUVA Bath Phototoxicity
Introduction.—Systemic psoralen plus ultraviolet light (PUVA) photo- chemotherapy is an effective treatment for psoriasis, but this therapeutic modality is associated with systemic side effects. A successful alternative, the administration of 8-methoxypsoralen (8-MOP) in a dilute water bath solution in combination with ultraviolet A (UVA) irradiation, avoids these side effects and yields significantly lower plasma levels of 8-MOP than systemic PUVA photochemotherapy. A study was conducted to determine the optimal time interval between 8-MOP bath and UVA irradiation, as well as and the persistence of photosensitivity in normal skin after PUVA bath treatment.
Methods.—Study participants were 11 volunteers (9 men and 2 women) ranging in age from 22 to 45 years. None had a history of skin diseases. After the right or left forearm was immersed for 20 minutes in a 5-mg/L water solution of 8-MOP, doses of 0.25, 0.5, 1, 1.25, and 1.5 J/cm2 UVA were applied on test sites immediately and at 20, 40, 60, and 120 minutes after the 8-MOP bath. Sites were evaluated 72 hours after irradiation for the Minimal Phototoxicity Dose (MPD). Using a 5-point rating scale, the degree of skin erythema was assessed 72 hours after the PUVA bath.
Results.—Skin areas irradiated immediately after PUVA bath had MPDs ranging from 0.25 to 1 J/cm2. The MPD range was 0.5 to 1.25 J/cm2 at 20 minutes and 0.5 to 1.5 J/cm2 (in 6 volunteers) at 40 minutes. Only 2 volunteers had a relevant phototoxic reaction (1.5 and 1.25 J/cm2) when irradiated 60 minutes after PUVA bath, indicating a rapid loss of 8-MOP activity. The erythema sum score showed the highest values in sites irradiated immediately after PUVA bath. No PUVA. erythema was induced when irradiation was applied 2 hours after the 8-MOP bath.
Conclusion.—The optimal time for UVA irradiation was found to be immediately after the PUVA bath. Because a relevant phototoxic reaction occurred in only 6 of 11 volunteers irradiated 40 minutes after the PUVA bath, UVA should not be delayed more than 20 minutes. Remaining toxicity is minimal 2 hours after PUVA bath therapy.
Bath PUVA has the obvious advantage that 2 hours after therapy, the remaining phototoxicity is minimal. However, because the optimal time for PUVA irradiation is immediately after the bath, the usefulness of this treatment is probably limited to either inpatient situations or a well-equipped psoriasis outpatient treatment center.
Selectivity of Protoporphyrin IX Fluorescence for Condylomata After Topical Application of 5-Aminolaevulinic Acid: Implications for Photodynamic Treatment
Background.—Photodynamic therapy (PDT) has been used effectively for various inflammatory and neoplastic skin diseases. Dual selectivity— which is determined by preferential accumulation of the photosensitizer and by delivery of light to the target—is the key advantage of PDT over other forms of therapy. One small study has suggested that PDT with topical 5-aminolevulinic acid (ALA) is an effective treatment for condylo-mata acuminata. The distribution and kinetics of protoporphyrin IX (PpIX) formation in condylomata after topical ALA application were examined.
Methods.—Participants were 19 men with a clinical diagnosis of condyloma acuminatum. A total of 25 condylomata were studied. Each patient underwent topical application of ALA, followed by hourly measurement of PpIX fluorescence spectra. The gross fluorescence imaging studies were followed by biopsy and fluorescence microscopic studies. The study assessed the in vivo kinetics of PpIX accumulation and its localization in condylomata and adjacent normal skin.
Results.—Each of the 3 fluorescence studies suggested that PpIX selectively formed in condylomata after topical ALA application. More than two thirds of condylomata studied showed significantly increased fluorescence compared with normal skin. The ratio of lesional to normal skin fluorescence peaked 2 hours after ALA application. The mechanism of preferential lesional PpIX formation appeared to involve enhanced stratum corneum permeability.
Conclusions.—Photodynamic therapy with ALA is a promising approach to treatment for patients with multiple condylomata acuminata. The effect of PDT in condylomata, compared with normal skin, may be optimized by exploiting the selective accumulation of PpIX early after ALA application. The authors plan to confirm their kinetic finding and assess optimal power densities and fluences in further trials.
The Variable Response of Plaque Psoriasis After a Single Treatment with Topical 5-Aminolaevulinic Acid Photodynamic Therapy
Introduction.—Although psoralens/ultraviolet A (PUVA) is an effective therapy for psoriasis, long-term treatment increases the risk of squamous cell carcinoma. Previous reports have shown that the protoporphyrin IX (PpIX) precursor 5-aminolevulinic acid (5-ALA) selectively localizes to plaque psoriasis lesions, and that photodynamic therapy (PDT) with ALA is an effective treatment for psoriasis. Information on the clinically effec¬tive values of incident light dose and dose rate are needed before ALA-PDT can become a useful treatment for psoriasis. The results of ALA-PDT using a single exposure to broad-band visible radiation were reported.
Methods.—A total of 22 patients with chronic plaque psoriasis were studied.Psoriasis lesions were treated with topical ALA, followed by light doses of 2-16 J/cm2 given at dose rates of 10-40 mW/cm2 4 hours after ALA application. Before, during, and after treatment, PpIX fluorescence was measured to assess PpIX photo-oxidation and photodynamic dose.
Results.—The response of psoriasis lesions to ALA-PDT was variable, with 35% of patients having clearance at some sites. There was also variation in the intensity of fluorescence before illumination—this was so between patients and within individual patients. For up to 2 weeks after treatment, persistent PpIX fluorescence was noted—in one fourth of sites, fluorescence was greater at follow-up than before illumination. During treatment, PpIX photo-oxidation occurred at a rate proportional to the initial fluorescence intensity and the incident light dose rate. Photo-oxidation was nearly complete after 16 J/cm2. However, only patients in whom treatment produced clearance of psoriasis lesions showed a significant relationship between photodynamic dose and clinical response. The patients had discomfort ranging from stinging to burning. These reactions tended to be more severe at higher photodynamic doses, but not predictably so.
Conclusions.—Photodynamic therapy with ALA is a potentially useful treatment for plaque psoriasis, producing lesion clearance after a single treatment in at least some patients. There are significant problems with patient discomfort, and the response may be variable. Approaches to increasing the efficacy of ALA-PDT for psoriasis may include achieving consistent PpIX levels or giving multiple treatments.
The uses and limitations of PDT continue to evolve. Previous studies have centered on premalignant and malignant lesions of the skin; the 2 studies presented here (Abstracts 14-18 and 14-19) extend the possible spectrum of activity to infectious and inflammatory diseases. The study by Ross et al. demonstrates localization of PpIX in virally infected cells, suggesting that PDT might be a reasonable alternative for patients with multiple condylomata where destructive therapies would be impractical. Obviously, extensive clinical trials will be necessary to demonstrate the benefit-risk ratio of such treatment. The data reported by Collins et al. are remarkable in that certain sites cleared after a single treatment. Obviously, many details need to be worked out to achieve consistent PpIX levels and to lessen the incidence of acute side effects. Future studies will likely explore different protocols, including some using different ALA concentrations and others using multiple treatment exposures, perhaps at lower energy levels. The long-term adverse effects of PDT are unknown. Nevertheless, this is an innovative form of phototherapy that will likely be a part of our therapeutic armamentarium for a variety of skin diseases in the not too distant future.