Induction and repair of UVB-induced cyclobutane pyrimidine dimers and (6-4) photoproducts in organ-cultured normal human skin

Summary To examine the induction and repair of UV-induced DNA damage, indirect immunofluorescence was performed on UVB-irradiated organ-cultured normal human skin using monoclonal antibodies specific for either cyclobutane pyrimidine dimers or (6-4) photoproducts. Nuclear immunofluorescence of cyclobutane pyrimidine dimers and (6-4) photoproducts were observed in a dosedependent manner after UVB irradiation. The intensity of nuclear immunofluorescence of the upper epidermal layers was stronger and clearer than that of the lower epidermal layers. DNA repair time-course studies showed that both types of DNA damage could be repaired within 24 h after UVB irradiation.     

In situ repair of cyclobutane pyrimidine dimers and 6-4 photoproducts in human skin exposed to solar simulating radiation

DNA repair is crucial to the integrity of the human genome. The ultraviolet radiation portion of solar radiation is responsible for the rising incidence of skin cancer, one of the most common types of cancer in humans. We applied a recently developed 32P-postlabeling technique to measure the in situ DNA repair efficiency of solar-simulated radiation induced cyclobutane pyrimidine dimers and 6-4 photoproducts in the skin of nine healthy volunteers with skin type II. Our results show about 6-fold interindividual variations in the level of DNA damage after exposure to an equal biologic dose - 2 minimal erythema doses. The kinetics of DNA repair indicated a base sequence dependence of the repair process. The DNA repair efficiency showed a 20-fold difference in volunteers. An age-related decrease of DNA repair capacity was observed; however, the data are limited due to a small number of subjects and a narrow age range. The variable response in DNA damage levels and individual differences in DNA repair efficiency suggest a susceptible subgroup of people probably with a higher skin cancer risk.     

Cyclobutane pyrimidine dimer formation and p53 production in human skin after repeated UV irradiation

Substantial differences in DNA damage caused by a single UV irradiation were found in our previous study on skin with different levels of constitutive pigmentation. In this study, we assessed whether facultative pigmentation induced by repeated UV irradiation is photoprotective. Three sites on the backs of 21 healthy subjects with type II-III skin were irradiated at 100-600 J/m(2) every 2-7 days over a 4- to 5-week period. The three sites received different cumulative doses of UV (1900, 2900 or 4200 J/m(2)) and were biopsied 1 day after the last irradiation. Biomarkers examined included pigment content assessed by Fontana-Masson staining, melanocyte function by expression of melanocyte-specific markers, DNA damage as cyclobutane pyrimidine dimers (CPD), nuclear accumulation of p53, apoptosis determined by TUNEL assay, and levels of p21 and Ser46-phosphorylated p53. Increases in melanocyte function and density, and in levels of apoptosis were similar among the 3 study sites irradiated with different cumulative UV doses. Levels of CPD decreased while the number of p53-positive cells increased as the cumulative dose of UV increased. These results suggest that pigmentation induced in skin by repeated UV irradiation protects against subsequent UV-induced DNA damage but not as effectively as constitutive pigmentation.     

Variations in excision repair of UVB-induced pyrimidine dimers in DNA of human skin in situ

The excision repair kinetics of UVB (280-320 nm)*-induced pyrimidine dimers in DNA of human skin in situ was determined for seventeen volunteers using a dimer-specific endonuclease from Micrococcus luteus in conjunction with agarose gel electrophoresis. Removal of pyrimidine dimers from human skin could be detected within 6 h after irradiation and the average half-life for removal of pyrimidine dimers was 11.0 h (+/- 4.3 h). However, there was significant inter-individual variability of repair as indicated by a half-life coefficient of variation of 38%.     

Production of pyrimidine dimers in DNA of human skin exposed in situ to UVA radiation

Cyclobutyl pyrimidine dimers, measured as sites recognized by the dimer-specific ultraviolet (UV) endonuclease from Micrococcus luteus, were produced in DNA of human skin exposed in situ to UVA (320-400 nm) radiation. The dimer yields produced by a broadband UVA source, by broadband UVA filtered to remove all light of wavelength less than 340 nm, and by narrow band radiation centered at 365 nm were similar, indicating that UVA radiation, and not stray shorter wavelength radiation, was responsible for dimer production. The identity of the UVA-induced DNA lesions was confirmed as pyrimidine dimers by photoreactivation of approximately 100% of the endonuclease-sensitive sites in vitro with the 40,000 dalton Escherichia coli photoreactivating enzyme.     

Repair of cyclobutyl pyrimidine dimers in human skin: variability among normal humans in nucleotide excision and in photorepair

BACKGROUND/AIMS: Photoreactivation (PR) of cyclobutyl pyrimidine dimers (CPD) in human skin remains controversial. Recently Whitmore et al. (1) reported negative results of experiments using two photorepair light (PRL) sources on UV-irradiated skin of volunteers. However, their PRL sources induced substantial levels of dimers in skin, suggesting that the additional dimers formed could have obscured PR. We met a similar problem of dimer induction by a PRL source. We designed and validated a PRL source of sufficient intensity to catalyse PR, but that did not induce CPD, and used it to measure photorepair in human skin. METHODS AND RESULTS: Using a solar simulator filtered with three types of UV-filters, we found significant dimer formation in skin, quantified by number average length analysis using electrophoretic gels of isolated skin DNA. To prevent scattered UV from reaching the skin, we interposed shields between the filters and skin, and showed that the UV-filtered/shielded solar simulator system did not induce damage in isolated DNA or in human skin. We exposed skin of seven healthy human volunteers to 302 nm radiation, then to the improved PRL source (control skin areas were kept in the dark for measurement of excision repair). CONCLUSIONS: Using a high intensity PRL source that did not induce dimers in skin, we found that three of seven subjects carried out rapid photorepair of dimers; two carried out moderate or slow dimer photorepair, and three did not show detectable photorepair. Excision repair was similarly variable in these volunteers. Subjects with slower excision repair showed rapid photorepair, whereas those with rapid excision generally showed little or no photoreactivation.     

Effect of photoreactivating light on UV radiation-induced alterations in human skin

BACKGROUND/AIMS: Photoreactivating light (PRL) after ultraviolet radiation (UVR) exposure causes photoreversal of cyclobutane pyrimidine dimers through the activation of photolyase. Although photoreversal has been demonstrated in the "three kingdoms of life," its existence in man remains controversial. We sought evidence for photoreversal in man. METHODS AND RESULTS: Seven subjects were spot-irradiated at two sites with 4 minimal erythema doses (MED) of solar-simulating UVR. Of the two sites, one was then immediately exposed to a PRL source. Epidermal biopsies were taken immediately after exposure. No significant difference in the quantity of pyrimidine dimers was detected comparing the "UVR only" site to the "UVR, PRL-exposed" site. Biopsies were repeated 24 h later and no significant difference in p53 protein expression or dendritic cell number was detected. However, the "UVR, PRL-exposed" site showed a greater reduction in pyrimidine dimer quantity. CONCLUSIONS: We found no evidence for a direct effect of PRL causing photoreversal of UVR-induced pyrimidine dimers in man. Our results do, however, suggest that some indirect effect of PRL may enhance pyrimidine dimer repair in the 24-h period following UVR exposure.     

Effect of ultraviolet adaptation on the ultraviolet absorption spectra of human skin in vivo

Background: The absorption spectrum of human skin provides a basis for the estimation of the possible photobiological impact of ultraviolet (UV) radiation. The optical properties of human skin in the UV spectral range have so far mainly been measured ex vivo due to a lack of an appropriate in vivo technique and the change of optical properties during the course of adaptation to higher UV doses has hardly been addressed. Methods: We have determined the absorption spectra of human skin in vivo in the wavelength range from 290 to 341 nm in 3 nm steps using laser optoacoustics. In this technique, optical properties are derived from the pressure profile generated by absorbed light energy in the sample. Spectra from the volar and dorsal aspects of the forearm of 20 subjects were compared, i.e. sites with native and various facultative pigmentation. Results: UV adaptation shows as an increase in absorption coefficients over the entire measured UV range and especially in short‐range UVB. Subject groups with high vs. low UV exposure can be discriminated by analyzing the difference absorption spectra between dorsal and volar aspects of the forearm. No dependence on the subject's phototype was seen in the degree of adaptation. Conclusion: The difference between native and facultative pigmentation may be explained by the absorption properties of the two prime chromophores responsible for adaptation to higher UV exposure: melanin and keratin. Stronger pigmentation, i.e. a higher melanin concentration, is found as an increase of absorption coefficients over the entire UVA‐II/UVB range. The thickening of the horny layer and accordingly, a higher influence of keratin on the absorption spectra is prominent especially in the UVB region.     

黄芩甙对中波紫外线照射HaCaT细胞后光产物的影响

目的 探讨黄芩甙对UVB照射后HaCaT细胞光产物环丁烷嘧啶二聚体(CPD)的产生和清除的影响.方法 以一定剂量UVB照射HaCaT细胞,在照光后不同时间点采用免疫组织化学法检测CPD的产生和清除;以黄芩甙预先孵育HaCaT细胞,再观察黄芩甙对CPD的影响.结果 HaCaT细胞损伤程度随UVB照射剂量增大而加重.30mJ/cm²UVB照射后HaCaT光产物CPD的产生在0.5h左右达到高峰,同时细胞开始清除CPD,照射后4h内清除速率较快,至24h时基本清除CPD.黄芩甙预处理组UVB照射后细胞的CPD产生少于非加药组(U=2.324,P<0.05).结论 UVB照射对HaCaT细胞光损伤程度呈剂量递增性,HaCaT细胞对CPD的清除存在快速清除期及慢速清除期;黄芩甙可减少光产物生成.     

Effects and interactions of increased environmental temperature and UV radiation on photoageing and photocarcinogenesis of the skin

Solar radiation is one of fundamental elements sustaining and maintaining life on earth. Previous studies on health effects from the sun exposure mostly focused on ultraviolet (UV) radiation. Although exposure to the solar radiation likely occurs in an environment with elevated temperature, the effects and interactions of elevated environmental temperature with UV radiation on the skin, especially in the context of ageing and carcinogenesis, have not been carefully examined. It is known that UVA radiation results in reduced production and increased degradation of dermal collagen, contributing to photoageing of the skin. Previous studies showed conflicting results regarding the effects of increased environmental temperature on dermal collagen. Additionally, we demonstrated that solar‐simulated radiation and increased environmental temperature have similar impacts on dermal fibroblasts through activation of distinct pathways. UVB radiation is well known for its carcinogenic capacity. Previously, it was reported that exposure to heat treatment before UVB radiation reduces epidermal keratinocyte cell death. We demonstrated that exposure to elevated environmental temperature prior to UVB radiation reduces UVB‐induced skin tumor formation. We proposed that alterations in molecular dynamics and quantum mechanics were involved for the observed increased environmental temperature‐induced protective effect against UVB damage. This review emphasizes that both environmental temperature and solar radiation are important elements in nature that have significant impacts on the human health, and future studies should focus on the biological effects and interactions of environmental temperature and solar radiation since this scenario is most relevant to the real‐world setting.     

Skin cancer in organ transplant recipients: effects of immunosuppressive medications on DNA repair

UV-induced skin cancers comprise a major problem in organ transplant recipients (OTRs). Cyclosporin A, a calcineurin inhibitor, is used as a standard immunosuppressant and clearly increases the skin cancer risk. Azathioprine does not appear to result in such an increase in skin cancer risk, and mTOR inhibitors are associated with an even lesser skin cancer risk. The underlying molecular mechanisms of these clinically important differences among immunosuppressants are still unclear and may relate to other than immunological effects. Insights may be gained by the multistep skin cancer theory and xeroderma pigmentosum, where defective nucleotide excision repair (NER) results in a cellular mutator phenotype and cutaneous carcinogenesis. This viewpoint assay summarizes current knowledge about the influence of the most commonly used immunosuppressive drugs in OTRs on DNA repair. Calcineurin inhibition results in a 200-fold increased skin cancer risk compared with the normal population and inhibits NER. The skin cancer risk under azathioprine is threefold less compared with calcineurin inhibitors, which may relate to inhibition of only the last step of NER, i.e. gap filling. mTOR inhibitors do not reduce NER in the global genome and can inhibit the growth of already initiated tumors, which may account for the markedly reduced skin cancer risk compared with calcineurin inhibitors. We conclude that OTRs may benefit from treatment regimens other than calcineurin inhibitors and speculate that a targeted modulation of calcineurin-dependent signalling may prevent UV-induced tumor formation by enhancing NER not only in OTRs but also in the general population, at least in part.     

Aged human skin removes UVB-induced pyrimidine dimers from the epidermis more slowly than younger adult skin in vivo

Although many studies have been reported on the repair of ultraviolet light (UV)-induced cyclobutane-type pyrimidine dimers (CPDs) in DNA, the effects of aging on the removal of UV-induced CPDs from the human skin epidermis in vivo remains uncertain. Therefore, we employed immunoblotting and immunohistochemical methods using monoclonal antibodies (TDM-2) to CPDs to study age-related differences in the time required for the in vivo removal of UVB-induced CPDs. The flexure surfaces of the upper arms of five young men were exposed to UVB light at a fluence of 35 and 700 mJ/cm², and four older men were also irradiated with the same doses of UVB mentioned above. Each area of skin was biopsied before and immediately after irradiation, and at 4, 24 h, 2 and 4 days after irradiation in the younger group; and before and immediately after irradiation, and at 24 h, 4, 7, and 14 days after irradiation in the older group. A total of 108 DNA samples were taken from the epidermis of 108 biopsied specimens. These samples were immunoblotted using TDM-2 and the intensities of the immunoprecipitates were measured by photodensitometer. Our results show that the CPDs had been removed from the epidermis at 4 days after irradiation at either dose in the younger group, and between 7–14 days after irradiation in the aged group. The results of our immunohistochemical studies were consistent with those of our immunoblotting studies, and indicated that basal cells repair CPDs more quickly than prickle cells in the epidermis except the amounts at 24 h after UVB irradiation, and that the CPDs were removed by epidermal turnover after the nucleotide excision repair (NER). Our results showed age-associated decline in the NER in vivo, indicating high risk of UV-associated skin cancer.     

Similar UV responses are seen in a skin organ culture as in human skin in vivo

Ultraviolet radiation (UVR) plays an important role in the development of non-melanoma skin cancer. Most tumors develop in chronically sun-exposed skin, most often in cosmetically sensitive locations, where in vivo experiments may be difficult to perform. In this study, we describe a skin organ culture model with preserved normal morphology and intact response to UVR. Skin explants from chronically sun-exposed and non-sun-exposed skin were irradiated with artificial UVA+UVB with and without topical sunscreen. UV-induced DNA damage, epidermal p53 response and repair kinetics were analyzed using immunohistochemistry. Four hours after UV-irradiation epidermal keratinocytes showed a strong immunoreactivity for thymine-dimers. Gradual repair during an incubation time resulted in few residual thymine-dimers after 48 h. Repair appeared to be more efficient in chronically sun-exposed skin compared with non-sun-exposed skin. There was also an accumulation of p53 protein in epidermal keratinocytes, peaking at 4-24 h after irradiation. Large interindividual differences with respect to formation and repair of thymine-dimers as well as induction and duration of the p53 response were observed. Skin explants treated with topical sunscreen prior to UV-irradiation showed a clear reduction of thymine-dimers and p53 expression. The epidermal UV-responses and repair kinetics in organ-cultured skin were similar to what was found in vivo. Our data suggest that organ-cultured skin provides a valuable tool for studies of UV-induced epidermal responses in chronically sun-exposed skin.     

European ancestry and polymorphisms in DNA repair genes modify the risk of melanoma: a case-control study in a high UV index region in Brazil

Background

UV radiation is the major environmental factor related to development of cutaneous melanoma. Besides sun exposure and the influence of latitude, some host characteristics such as skin phototype and hair and eye color are also risk factors for melanoma. Polymorphisms in DNA repair genes could be good candidates for susceptibility genes, mainly in geographical regions exposed to high solar radiation.

Objective

Evaluate the role of host characteristics and DNA repair polymorphism in melanoma risk in Brazil.

Methods

We carried out a hospital-based case-control study in Brazil to evaluate the contribution of host factors and polymorphisms in DNA repair to melanoma risk. A total of 412 patients (202 with melanoma and 210 controls) were analyzed regarding host characteristics for melanoma risk as well as for 11 polymorphisms in DNA repair genes.

Results

We found an association of host characteristics with melanoma development, such as eye and hair color, fair skin, history of pigmented lesions removed, sunburns in childhood and adolescence, and also European ancestry. Regarding DNA repair gene polymorphisms, we found protection for the XPG 1104 His/His genotype (OR 0.32; 95% CI 0.13-0.75), and increased risk for three polymorphisms in the XPC gene (PAT+; IV-6A and 939Gln), which represent a haplotype for XPC. Melanoma risk was higher in individuals carrying the complete XPC haplotype than each individual polymorphism (OR 3.64; 95% CI 1.77-7.48).

Conclusions

Our data indicate that the host factors European ancestry and XPC polymorphisms contributed to melanoma risk in a region exposed to high sun radiation.     

Cyclosporin A inhibits nucleotide excision repair via downregulation of the xeroderma pigmentosum group A and G proteins, which is mediated by calcineurin inhibition

Cyclosporin A (CsA) inhibits nucleotide excision repair (NER) in human cells, a process that contributes to the skin cancer proneness in organ transplant patients. We investigated the mechanisms of CsA-induced NER reduction by assessing all xeroderma pigmentosum (XP) genes (XPA-XPG). Western blot analyses revealed that XPA and XPG protein expression was reduced in normal human GM00637 fibroblasts exposed to 0.1 and 0.5 μm CsA. Interestingly, the CsA treatment reduced XPG, but not XPA, mRNA expression. Calcineurin knockdown in GM00637 fibroblasts using RNAi led to similar results suggesting that calcineurin-dependent signalling is involved in XPA and XPG protein regulation. CsA-induced reduction in NER could be complemented by the overexpression of either XPA or XPG protein. Likewise, XPA-deficient fibroblasts with stable overexpression of XPA (XP2OS-pCAH19WS) did not show the inhibitory effect of CsA on NER. In contrast, XPC-deficient fibroblasts overexpressing XPC showed CsA-reduced NER. Our data indicate that the CsA-induced inhibition of NER is a result of downregulation of XPA and XPG protein in a calcineurin-dependent manner.     

The 0.8% ultraviolet B content of an ultraviolet A sunlamp induces 75% of cyclobutane pyrimidine dimers in human keratinocytes in vitro

Tanning lamps, emitting predominantly ultraviolet (UV) A, are used widely throughout the U.K. and other countries, but little is known about the long-term risks associated with their use, especially with respect to skin cancer. We have exposed normal human epidermal keratinocytes to a commercial tanning lamp and used the comet assay in association with DNA repair enzymes T4 endonuclease V and endonuclease III to investigate the relative yields of directly formed cyclobutane pyrimidine dimers (CPDs) and indirectly formed types of oxidative DNA damage. To put the risk of using tanning lamps into perspective, the sunbed used in this study (five Philips Performance 80W-R UVA tubes at a distance of 35 cm) was found to be approximately 0.7 times as potent at inducing CPDs as U.K. natural sunlight around noon on a fine summer day. This compares with a relative risk for CPD induction and erythema of 0.8 and 0.7 times, respectively, calculated from the relevant action spectra of tanning lamps and British noontime sunlight. To determine the relative contribution of UVB and UVA to the induction of CPDs and oxidative DNA damage, we modified the spectral output of the tanning lamps with a series of Schott WG UVB filters. The induction of CPDs was more dependent on the UVB component of the sunbed than oxidative types of damage. Schott WG UVB filters with 50% transmission at 305 nm reduced the yield of T4 endonuclease V sites by 42% while there was only a 17% decrease in the yield of endonuclease III sites. CPD induction was not completely abolished after irradiation through WG335 and WG345 nm filters despite there being no detectable UVB. From these data, it was estimated that, although the tanning lamps emitted only 0.8% of their total output in the UVB range, these wavelengths were responsible for the induction of over 75% of CPDs and 50% of the oxidative damage to DNA.     

Production and clearance of cyclobutane dipyrimidine dimers in UV-irradiated skin pretreated with 1% pimecrolimus or 0.1% triamcinolone acetonide creams in normal and atopic patients

BACKGROUND: Ultraviolet (UV)-induced pyrimidine dimers are an early step in skin carcinogenesis, which is accelerated in the setting of long-term immunosuppression with systemic calcineurin inhibitors. It is not known whether topical application of calcineurin inhibitors exposes to a similar risk. OBJECTIVE: To assess the formation and clearance of UV-induced dipyrimidine dimers in human epidermis treated with topical pimecrolimus as compared to topical steroid, vehicle and untreated control. METHODS: Pretreated buttock skin of 20 human volunteers with (10) or without (10) atopic dermatitis was exposed to two minimal erythema doses (MED) of simulated solar radiation. DNA was extracted from epidermis 1 and 24 h postirradiation. Pyrimidine dimers were visualized by immuno slot blots and quantified by chemoluminescence image analysis. RESULTS: One-hour postirradiation, pimecrolimus-treated epidermis contains less DNA damage as compared to untreated control, but there were no statistically significant differences between pimecrolimus, triamcinolone acetonide and vehicle. Dimer levels at 24 h postirradiation showed no significant differences between different treatments. CONCLUSION: Treatment with pimecrolimus cream, triamcinolone acetonide cream and vehicle is not associated with increased epidermal DNA damage at 1 and 24 h post-UV exposure.