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Background: Although the photoprotective effects of β-carotene are thought to originate from its antioxidant properties, some studies documented pro-oxidant effects of β-carotene. Objective: Our purpose was to determine the effects of 2 different doses of dietary β-carotene on wrinkles and elasticity, procollagen gene expression and ultraviolet (UV)-induced DNA damage in human skin. Methods: Thirty healthy female subjects over the age of 50 years were randomized and received 2 different doses (30 and 90 mg/day) of β-carotene for 90 days. The baseline status was used as control. At baseline and completion of the study, facial wrinkles and elasticity were measured objectively. Buttock skin was taken to determine the type I procollagen, matrix metalloproteinase-1 and fibrillin-1 mRNA levels, and UV-induced thymine dimer and 8-hydroxy-2′-deoxyguanosine formation. Results: β-Carotene improved facial wrinkles and elasticity significantly only in the low-dose group. The minimal erythema dose decreased significantly only in the high-dose group. Type I procollagen mRNA levels were significantly increased to 4.4 ± 1.6 times the baseline level only in the low-dose group, and procollagen immunostaining increased accordingly. UV-induced thymine dimer staining was reduced in the low-dose group but tended to increase in the high-dose group. 8-hydroxy-2′-deoxyguanosine staining was significantly reduced in the low-dose group. Conclusions: 30 mg/day of β-carotene supplementation is demonstrated to prevent and repair photoaging.

Access to DNA packaged in nucleosomes is critical for gene regulation, DNA replication and DNA repair. In humans, the UV-damaged DNA-binding protein (UV-DDB) complex detects UV-light-induced pyrimidine dimers throughout the genome; however, it remains unknown how these lesions are recognized in chromatin, in which nucleosomes restrict access to DNA. Here we report cryo-electron microscopy structures of UV-DDB bound to nucleosomes bearing a 6–4 pyrimidine–pyrimidone dimer or a DNA-damage mimic in various positions. We find that UV-DDB binds UV-damaged nucleosomes at lesions located in the solvent-facing minor groove without affecting the overall nucleosome architecture. In the case of buried lesions that face the histone core, UV-DDB changes the predominant translational register of the nucleosome and selectively binds the lesion in an accessible, exposed position. Our findings explain how UV-DDB detects occluded lesions in strongly positioned nucleosomes, and identify slide-assisted site exposure as a mechanism by which high-affinity DNA-binding proteins can access otherwise occluded sites in nucleosomal DNA. Cryo-electron microscopy structures reveal that the DNA-repair factor UV-DDB exposes inaccessible nucleosome lesions for binding by inducing a translational shift in the nucleosome position.

Ultraviolet B (UVB) radiation induces mutagenic DNA photoproducts, in particular cyclobutane pyrimidine dimers (CPDs), in epidermal keratinocytes (KC). To prevent skin carcinogenesis, these DNA photoproducts must be removed by nucleotide excision repair (NER) or apoptosis. Here we report that the UVB-sensitive transcription factor aryl hydrocarbon receptor (AHR) attenuates the clearance of UVB-induced CPDs in human HaCaT KC and skin from SKH-1 hairless mice. Subsequent RNA interference and inhibitor studies in KC revealed that AHR specifically suppresses global genome but not transcription-coupled NER. In further experiments, we found that the accelerated repair of CPDs in AHR-compromised KC depended on a modulation of the p27 tumor suppressor protein. Accordingly, p27 protein levels were increased in AHR-silenced KC and skin biopsies from AHR −/− mice, and critical for the improvement of NER. Besides increasing NER activity, AHR inhibition was accompanied by an enhanced occurrence of DNA double-strand breaks triggering KC apoptosis at later time points after irradiation. The UVB-activated AHR thus acts as a negative regulator of both early defense systems against carcinogenesis, NER and apoptosis, implying that it exhibits tumorigenic functions in UVB-exposed skin. In fact, AHR −/− mice developed 50% less UVB-induced cutaneous squamous cell carcinomas in a chronic photocarcinogenesis study than their AHR +/+ littermates. Taken together, our data reveal that AHR influences DNA damage-dependent responses in UVB-irradiated KC and critically contributes to skin photocarcinogenesis in mice.

As DNA damage induced by ultraviolet radiation plays an essential role in skin cancer induction, we pursued the measure of several DNA lesions induced by ultraviolet radiation in human skin for determining the efficacy of different topical photoprotectors. Non-exposed skin (buttocks from 20 individuals) was exposed to 10 doses of ultraviolet, which corresponded to three to four minimal erythema doses of solar-simulating radiation, and biopsies were taken at 24 h within the half and one minimal erythema dose sites and a nonirradiated, adjacent control area. We report that even suberythemal doses of ultraviolet radiation are capable of inducing substantial DNA damage, namely pyrimidine dimers, p53 induction, and the DNA base-modified product generated by oxidative stress, 8-hydroxy-2′-deoxyguanosine. All three lesions are induced in a dose-dependent manner. An additional eight individuals were treated with either ultraviolet B or ultraviolet B + ultraviolet A sunblock (sun protection factor 15) and exposed to 7½ and 15 times the minimal erythema dose on each individual, with biopsies taken at 24 h post-ultraviolet. Pyrimidine dimer and p53 expression were rarely seen in nonirradiated skin but occasional staining was seen in all normal skin for 8-hydroxy-2′-deoxyguanosine. Applications of sunscreens to human skin before irradiation were shown to attenuate erythema but did not completely eliminate all three types of cellular damage when tested up to their sun protection factor 15. Furthermore, ultraviolet B + ultraviolet A sunscreens were less efficient than the ultraviolet B alone formulation for protection against all three lesions. These results suggest that DNA damage assessed in vivo by immunohistochemistry provides a very sensitive endpoint for determining the efficacy or photosensitivity of possible different protective measures in human skin.

Solar UV radiation is the most important environmental factor involved in the pathogenesis of skin cancers. The well known genotoxic properties of UVB radiation (290-320 nm) mostly involve bipyrimidine DNA photoproducts. In contrast, the contribution of more-abundant UVA radiation (320-400 nm) that are not directly absorbed by DNA remains poorly understood in skin. Using a highly accurate and quantitative assay based on HPLC coupled with tandem mass spectrometry, we determined the type and the yield of formation of DNA damage in whole human skin exposed to UVB or UVA. Cyclobutane pyrimidine dimers, a typical UVBinduced DNA damage, were found to be produced in significant yield also in whole human skin exposed to UVA through a mechanism different from that triggered by UVB. Moreover, the latter class of photoproducts is produced in a larger amount than 8-oxo7,8-dihydro-2'-deoxyguanosine, the most common oxidatively generated lesion, in human skin. Strikingly, the rate of removal of UVA-generated cyclobutane pyrimidine dimers was lower than those produced by UVB irradiation of skin. Finally, we compared the formation yields of DNA damage in whole skin with those determined in primary cultures of keratinocytes isolated from the same donors. We thus showed that human skin efficiently protects against UVB-induced DNA lesions, whereas very weak protection is afforded against UVA. These observations emphasize the likely role played by the UVA-induced DNA damage in skin carcinogenesis and should have consequences for photoprotection strategies.

UVB readily induces cyclobutane pyrimidine dimers, mainly thymine dimers (TTs), and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) in DNA. These lesions result in “UVB signature mutations” found in skin cancers. We have investigated the induction of TTs and 6-4PPs in human skin in vivo by broadband UVA1, and have compared this with comparable erythemal doses of monochromatic UVB (300nm). In vitro and ex vivo studies have shown the production of TTs, without 6-4PPs, by UVA1. We show that UVA1 induces TTs, without 6-4PPs, in the epidermis of healthy volunteers in vivo, whereas UVB induced both photoproducts. UVB induced more TTs than UVA1 for the same level of erythema. The level of UVA1-induced TTs increased with epidermal depth in contrast to a decrease that was seen with UVB. UVA1- and UVB-induced TTs were repaired in epidermal cells at a similar rate. The mechanism by which UVA1 induces TTs is unknown, but a lack of intra-individual correlation between our subjects’ UVB and UVA1 minimal erythema doses implies that UVA1 and UVB erythema occur by different mechanisms. Our data suggest that UVA1 may be more carcinogenic than has previously been thought.

• Premise of the study: Although ultraviolet radiation (UV) is known to have negative effects on plant growth, there has been no direct evidence that plants growing at higher elevations are more severely affected by ultraviolet‐B (UV‐B) radiation, which is known to increase with elevation. We examined damage to DNA, a primary target of UV‐B, in the widespread species Polygonum sachalinense (Fallopia sachalinensis) and Plantago asiatica at two elevations. • Methods: We sampled leaves of both species at 300 and 1700 m above sea level every 2 h for 11 d across the growing season and determined the level of cyclobutane pyrimidine dimer (CPD), a major product of UV damage to DNA. • Key results: The CPD level was significantly influenced by the time of day, date, elevation, and their interactions in both species. The CPD level tended to be higher at noon or on sunny days. DNA damage was more severe at 1700 m than at 300 m: on average, 8.7% greater at high elevation in P. asiatica and 7.8% greater in P. sachalinense. Stepwise multiple regression analysis indicated that the CPD level was explained mainly by UV‐B and had no significant relationship with other environmental factors such as temperature and photosynthetically active radiation. • Conclusions: UV‐induced DNA damage in plants is greater at higher elevations.

The induction of apoptosis in keratinocytes by ultraviolet (UV)-irradiation is considered to be a protective function against skin cancer. UV-induced DNA damage is a crucial event in UVB- and UVC-mediated apoptosis. However, the differences between the UVB- and UVC-induced apoptotic pathways remain unclear. Here we examine the differential mechanisms by which UVB and UVC irradiations induce keratinocyte apoptosis using human keratinocyte HaCaT cells. Differences in the production of (6-4)photoproducts ((6-4)PPs) and cyclobutane pyrimidine dimers (CPDs) were measured following irradiation with UVB and UVC at doses causing the same extent of apoptotic cell death. In addition, main apoptotic features, such as caspase activation and its regulation, were compared between UVB- and UVC-induced apoptosis. Exposures of 500 J/m(2) UVB and 100 J/m(2) UVC resulted in apoptosis to almost the same extent. At these apoptotic doses, the amounts of both (6-4)PPs and CPDs were significantly larger in the case of UVC irradiation than UVB irradiation; in parallel, the release of cytochrome c and Smac/DIABLO and the activation of caspases-9 following UVC irradiation were greater than after UVB irradiation. Importantly, caspase-8 activation occurred only in UVB-irradiated cells. Furthermore, the activation of caspase-8 was not inhibited by caspases-9 and -3 specific tetrapeptide inhibitors, indicating that the caspase-8 cleavage is not due to feedback from activation of caspases-9 and -3. Thus, these results clearly suggest that the reason apoptosis is induced to the same extent by UVB irradiation as by UVC irradiation, despite the lower production of photoproducts in DNA by UVB irradiation, is attributable to the additional activation of the caspase-8 pathway. Thus, UVB irradiation induces apoptosis through both mitochondrial (intrinsic) and caspase-8 activation (extrinsic) pathways, while UVC induces apoptosis only via the intrinsic pathway.

A lesion-specific enzyme-induced DNA strand break assay was developed for an oligonucleotide chip for the determination of UVB-induced cyclobutane pyrimidine dimers (CPDs). A 20-mer of fluorophore-labeled and biotinylated oligonucleotide was immobilized on the chip. CPDs in DNA on the chip were formed by UVB irradiation (312 nm). T4 endonuclease V (T4N5) was used to excise the CPD site as T4N5 sensitively and specifically detects CPDs. The fluorophore-labeled DNA fragments were detected by a laser-induced fluorescence (LIF) detection system. The number of CPDs induced by UVB was determined based on a mathematical equation obtained from a predetermined calibration curve. The yield of UVB-induced CPDs was 1.73 CPDs per megabase per (kJ/m²). The reliability of this value was proved by its similarity to reference values obtained from gel electrophoresis. The developed assay has strong potential to quantify most kinds of UV-induced DNA lesions.