Kinetics of UV light-induced cyclobutane pyrimidine dimers in human skin in vivo: an immunohistochemical analysis of both epidermis and dermis

It is well known that UV exposure of human skin induces DNA damage, and the cumulative effect of such repeated damage is an important contributor to the development of skin cancer. Here, we demonstrate UV dose- and time-dependent induction of DNA damage in the form of cyclobutane pyrimidine dimers (CPD) in skin cells following a single exposure of human skin to UV radiation. CPD+ cells were identified by an immunohistochemical technique using monoclonal antibodies to thymine dimers. The percentage of CPD+ cells was UV dose-dependent, even a suberythemal (0.5 minimal erythemal dose [MED]) dose resulted in detectable level of cells that contained pyrimidine dimers. Forty-eight hours after irradiation the percent of total epidermal cells positive for CPD ranged from 19 +/- 8, 36 +/- 10, 57 +/- 12 and 80 +/- 10, and total percent dermal cells positive for CPD ranged from 1 +/- 1, 7 +/- 3, 16 +/- 3 and 20 +/- 5, respectively, following 0.5, 1.0, 2.0 and 4.0 MED. CPD were also observed in deeper reticular dermis, which suggest the penetrating ability of UV radiation into the skin. The change in CPD+ cells from 0.5 to 240 h post-UV exposure in both epidermal and dermal compartments of the skin was also quantitated. CPD+ cells were observed in skin biopsies at early time points after UV exposure which remained elevated for 48 h, then declined significantly by 3 days post-UV. A close examination of the skin at and after 3 days following UV exposure indicates the significant removal of DNA damaged cells from the epidermis. Ten days after UV exposure the levels of CPD+ cells in both epidermis and dermis were not significantly different from that in unirradiated skin.     

MOLECULAR MECHANISMS OF ULTRAVIOLET RADIATION CARCINOGENESIS

UV radiation is a potent DNA damaging agent and a known inducer of skin cancer in experimental animals. There is excellent scientific evidence to indicate that most non-melanoma human skin cancers are induced by repeated exposure to sunlight. UV radiation is unique in that it induces DNA damage that differs from the lesions induced by any other carcinogen. The prevalence of skin cancer on sun-exposed body sites in individuals with the inherited disorder XP suggests that defective repair of UV-induced DNA damage can lead to cancer induction. Carcinogenesis in the skin, as elsewhere, is a multistep process in which a series of genetic and epigenetic events leads to the emergence of a clone of cells that have escaped normal growth control mechanisms. The principal candidates that are involved in these events are oncogenes and tumor suppressor genes. Oncogenes display a positive effect on transformation, whereas tumor suppressor genes have an essentially negative effect, blocking transformation. Activated ras oncogenes have been identified in human skin cancers. In most cases, the mutations in the ras oncogenes have been localized to pyrimidine-rich sequences, which indicates that these sites are probably the targets for UV-induced DNA damage and subsequent mutation and transformation. The finding that activation of ras oncogenes in benign and self-regressing keratoacanthomas in both humans and in animals indicates that they play a role in the early stages of carcinogenesis (Corominas et al., 1989; Kumar et al., 1990). Since cancers do not arise immediately after exposure to physical or chemical carcinogens, ras oncogenes must remain latent for long periods of time. Tumor growth and progression into the more malignant stages may require additional events involving activation of other oncogenes or deletion of growth suppressor genes. In addition, amplification of proto-oncogenes or other genes may also be involved in tumor induction or progression. In contrast to the few studies that implicate the involvement of oncogenes in UV carcinogenesis, the role of tumor suppressor genes in UV carcinogenesis is unknown. Since cancer-prone individuals, particularly XP patients, lack one or more repair pathways, one can speculate that DNA repair enzymes would confer susceptibility to both spontaneous and environmentally induced cancers. Another potential candidate that can function as a tumor suppressor gene is the normal c-Ha-ras gene. Spandidos and Wilkie (1988) have shown that the normal c-Ha-ras gene can suppress transformation induced by the mutated ras gene.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultraviolet (280-400 nm)-induced DNA damage in the eggs and larvae of Calanus finmarchicus G. (Copepoda) and Atlantic cod (Gadus morhua)

In previous work, we evaluated the effects of ultraviolet (UV = 280-400 nm) radiation on the early life stages of a planktonic Calanoid copepod (Calanus finmarchicus Gunnerus) and of Atlantic cod (Gadus morhua). Both are key species in North Atlantic food webs. To further describe the potential impacts of UV exposure on the early life stages of these two species, we measured the wavelength-specific DNA damage (cyclobutane pyrimidine dimer [CPD] formation per megabase of DNA) induced under controlled experimental exposure to UV radiation. UV-induced DNA damage in C. finmarchicus and cod eggs was highest in the UV-B exposure treatments. Under the same spectral exposures, CPD loads in C. finmarchicus eggs were higher than those in cod eggs, and for both C. finmarchicus and cod embryos, CPD loads were generally lower in eggs than in larvae. Biological weighting functions (BWF) and exposure response curves that explain most of the variability in CPD production were derived from these data. Comparison of the BWF revealed significant differences in sensitivity to UV-B: C. finmarchicus is more sensitive than cod, and larvae are more sensitive than eggs. This is consistent with the raw CPD values. Shapes of the BWF were similar to each other and to a quantitative action spectrum for damage to T7 bacteriophage DNA that is unshielded by cellular material. The strong similarities in the shapes of the weighting functions are not consistent with photoprotection by UV-absorbing compounds, which would generate features in BWF corresponding to absorption bands. The BWF reported in this study were applied to assess the mortality that would result from accumulation of a given CPD load: for both C. finmarchicus and cod eggs, an increased load of 10 CPD Mb(-1) of DNA due to UV exposure would result in approximately 10% mortality.     

FORMATION OF THYMINE DIMERS IN MAMMALIAN SKIN BY ULTRAVIOLET RADIATION IN VIVO

Abstract— Ultraviolet radiation of 220–300 nm is known to produce cyclobutyl pyrimidine dimers in extracellular DNA, in bacteria, and in mammalian cells in culture. The formation in vivo of such dimers in mammalian skin has remained inferential. We report that one of the important and recognizable biologic events that occurs in mammalian skin during irradiation is the formation of thymine dimers. [³H]-labelled thymidine was applied to the epilated skin of guinea pigs to label their DNA. Animals were irradiated individually, using wavelengths of either 254, 285–350, or 320–400 nm. Immediately after irradiation, epidermis was separated from the rest of the skin and homogenized; DNA and RNA were isolated. Irradiation with wavelengths of 285–350 nm, which included the sunburn-producing spectrum (i.e., 290–320 nm), produced thymine dimers (1·7–2·6 per cent of the total [³H]-thymine incorporated into DNA). Irradiation with 254nm also produced fewer dimers (0·46–1·2 percent); and 320–400 nm produced none. The dimer could be cleaved by 250 nm radiation to form thymine. The epidermal cell damage by ultraviolet radiation, particularly by the sunburn-producing spectrum (290–320 nm), may be related to the formation of such dimers.     

Structure and dynamics of poly(T) single-strand DNA: implications toward CPD formation

The formation of cyclobutane pyrimidine dimers between adjacent thymines by UV radiation is thought to be the first event in a cascade leading to skin cancer. Recent studies showed that thymine dimers are fully formed within 1 ps of UV irradiation, suggesting that the conformation at the moment of excitation is the determining factor in whether a given base pair dimerizes. MD simulations on the 50 ns time scale are used to study the populations of reactive conformers that exist at any given time in T18 single-strand DNA. Trajectory analysis shows that only a small percentage of the conformations fulfill distance and dihedral requirements for thymine dimerization, in line with the experimentally observed quantum yield of 3%. Plots of the pairwise interactions in the structures predict hot spots of DNA damage where dimerization in the ssT18 is predicted to be most favored. The importance of hairpin formation by intra-strand base pairing for distinguishing reactive and unreactive base pairs is discussed in detail. The data presented thus explain the structural origin of the results from the ultrafast studies of thymine dimer formation.     

Impact of the Circadian Clock on UV‐Induced DNA Damage Response and Photocarcinogenesis

The skin is in constant exposure to various external environmental stressors, including solar ultraviolet (UV) radiation. Various wavelengths of UV light are absorbed by the DNA and other molecules in the skin to cause DNA damage and induce oxidative stress. The exposure to excessive ultraviolet (UV) radiation and/or accumulation of damage over time can lead to photocarcinogenesis and photoaging. The nucleotide excision repair (NER) system is the sole mechanism for removing UV photoproduct damage from DNA, and genetic disruption of this repair pathway leads to the photosensitive disorder xeroderma pigmentosum (XP). Interestingly, recent work has shown that NER is controlled by the circadian clock, the body's natural time‐keeping mechanism, through regulation of the rate‐limiting repair factor xeroderma pigmentosum group A (XPA). Studies have shown reduced UV‐induced skin cancer after UV exposure in the evening compared to the morning, which corresponds with times of high and low repair capacities, respectively. However, most studies of the circadian clock–NER connection have utilized murine models, and it is therefore important to translate these findings to humans to improve skin cancer prevention and chronotherapy.     

UVB DNA Dosimeters Analyzed by Polymerase Chain Reactions

Abstract— Purified bacteriophage Λ DNA was dried on a UV-tran-sparent polymer film and served as a UVB dosimeter for personal and ecological applications. Bacteriophage Λ. DNA was chosen because it is commercially available and inexpensive, and its entire sequence is known. Each dosimeter contained two sets of DNA sandwiched between UV-transparent polymer films, one exposed to solar radiation (experimental) and another protected from UV radiation by black paper (control). The DNA dosimeter was then analyzed by a polymerase chain reaction (PCR) that amplifies a 500 base pair specific region of Λ DNA. Photoinduced damage in DNA blocks polymerase from synthesizing a new strand; therefore, the amount of amplified product in UV-exposed DNA was reduced from that found in control DNA. The average lesion frequency per 500 base pair per strand at 16 PCR cycles was –1.22, 1.00, 0.70 and 0.50 for 30 ng, 50 ng, 100 ng and 150 ng of dried DNA, respectively, after a total dose of 60 kj m 2 delivered with a solar UVB simulator. Although the average lesion frequency increases linearly with increasing doses for four different amounts of template DNA, the lesion frequency seems to be averaged by the amplified products from the protected Λ DNA molecules below the top few layers. The average daily dose, equivalent to the UV dose applied with the solar UVB simulator, was 10.2±0.4 kj m2 with the 50 ng containing DNA dosimeter in September 1995 in Melbourne, FL. Both 50 ng and 150 ng containing DNA dosimeters produced the same average daily dose within experimental error in January 1996, which was 5.2±0.3 kj m 2 at the same location. The dried Λ DNA dosimeter is compact, robust, safe and transportable, stable over long storage times and provides the total UVB dose integrated over the exposure time.     

Analysis of Compact Fluorescent Lights for Use by Patients with Photosensitive Conditions

Ultraviolet radiation (UVR) is hazardous to patients with photosensitive skin disorders, such as lupus erythematosus, xeroderma pigmentosum and skin cancer. As such, these patients are advised to minimize their exposure to UVR. Classically, this is accomplished through careful avoidance of sun exposure and artificial tanning booths. Indoor light bulbs, however, are generally not considered to pose significant UVR hazard. We sought to test this notion by measuring the UV emissions of 19 different compact fluorescent light bulbs. The ability to induce skin damage was assessed with the CIE erythema action spectrum, ANSI S(λ) generalized UV hazard spectrum and the CIE photocarcinogenesis action spectrum. The results indicate that there is a great deal of variation amongst different bulbs, even within the same class. Although the irradiance of any given bulb is low, the possible daily exposure time is rather lengthy. This results in potential daily UVR doses ranging from 0.1 to 625 mJ cm⁻², including a daily UVB (290–320 nm) dose of 0.01 to 15 mJ cm⁻². Because patients are exposed continually over long time frames, this could lead to significant cumulative damage. It would therefore be prudent for patients to use bulbs with the lowest UV irradiance.     

Inflammation Due to Voriconazole‐induced Photosensitivity Enhanced Skin Phototumorigenesis in Xpa‐knockout Mice

Voriconazole is an antifungal agent and used as a prophylactic measure, especially in immunocompromised patients. However, there have been several reports of its adverse reactions, namely photosensitivity with intense inflammatory rashes and subsequent skin cancer development. To assess the effects of photosensitizing drugs voriconazole and hydrochlorothiazide (HCTZ ) on the enhancement of UV ‐induced inflammatory responses and UV ‐induced tumorigenesis, we utilized Xpa ‐knockout mice, which is DNA repair‐deficient and more susceptible to UV ‐induced inflammation and tumor development than wild‐type mice. Administration of voriconazole prior to broadband UVB exposure significantly upregulated multiple inflammatory cytokines compared with the vehicle‐ or HCTZ ‐administered groups. Voriconazole administration along with chronic UVB exposure produced significantly higher number of skin tumors than HCTZ or vehicle in Xpa ‐knockout mice. Furthermore, the investigation of UVB ‐induced DNA damage using embryonic fibroblasts of Xpa ‐knockout mice revealed a significantly higher 8‐oxo‐7,8‐dihydroguanine level in cells treated with voriconazole N‐oxide, a voriconazole‐metabolite during UV exposure. The data suggest that voriconazole plus UVB ‐induced inflammatory response may be related to voriconazole‐induced skin phototumorigenesis.     

Effects and Mechanism of Nicotinamide Against UVA‐ and/or UVB‐mediated DNA Damages in Normal Melanocytes

Melanoma incidences are increasing rapidly, and ultraviolet (UV) radiation from the sun is believed to be its major contributing factor. UV exposure causes DNA damage in skin which may initiate cutaneous skin cancers including melanoma. Melanoma arises from melanocytes, the melanin‐producing skin cells, following genetic dysregulations resulting into hyperproliferative phenotype and neoplastic transformation. Both UVA and UVB exposures to the skin are believed to trigger melanocytic hyperplasia and melanomagenesis. Melanocytes by themselves are deficient in repair of oxidative DNA damage and UV‐induced photoproducts. Nicotinamide, an active form of vitamin B3 and a critical component of the human body's defense system has been shown to prevent certain cancers including nonmelanoma skin cancers. However, the mechanism of nicotinamide's protective effects is not well understood. Here, we investigated potential protective effects and mechanism of nicotinamide against UVA‐ and/or UVB‐ induced damage in normal human epidermal melanocytes. Our data demonstrated an appreciable protective effect of nicotinamide against UVA‐ and/or UVB‐ induced DNA damage in melanocytes by decreasing both cyclobutane pyrimidine dimers and 8‐hydroxy‐2′‐deoxyguanosine levels. We found that the photoprotective response of nicotinamide was associated with the activation of nucleotide excision repair genes and NRF2 signaling. Further studies are needed to validate our findings in in vivo models.     

INDUCTION OF SKIN TUMORS IN THE RAT BY SINGLE EXPOSURE TO ULTRAVIOLET RADIATION

Abstract— The dose response for tumor induction in albino rat skin by single exposures of UV radiation has been characterized. The shaved dorsal skin of 202 animals was exposed to either of two sources: one emitting a broad spectrum of wavelengths from 275 to 375 nm, and the other emitting at 254 nm. Skin tumors began to appear within 10 weeks of exposure and continued to appear for 70 weeks. The highest tumor yield was 5.5 tumors per rat and occurred when the rats were exposed to 13.0 times 10⁴ J/m² of the 275–375 nm UV. The 275–375 nm UV was about eight times as effective as the 254 nm UV for the induction of tumors throughout the exposure range from 0.8 times 10⁴ to 26.0 times 10⁴J/m². Tissue destruction and hair follicle damage was found at the highest exposure to 275–375 nm UV but at none of the exposures to 254 nm UV. Repeated weekly exposures to 275–375 nm UV proved less effective than an equivalent single exposure for inducing tumors, even though the multiple exposures caused more severe skin damage. The transmission of the UV through excised samples of rat epidermis indicated that the exposure to the basal cell layer was about 3% of the surface exposure at 254 nm and about 15% of the surface exposure between 275 and 320 nm. The dependence of tumor yield on UV exposure was linear for 254 nm UV but was more complex for the 275–375 nm UV. For the latter more tumors were produced per unit exposure at lower exposures than at higher exposures.     

UV protective effects of DNA repair enzymes and RNA lotion

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

In Situ Action Spectra Suggest that DNA Damage is Involved in Ultraviolet Radiation-induced Immunosuppression in Humans

Abstract— The mixed epidermal cell lymphocyte reaction (MECLR) is a commonly used method to study the immunomodulatory effects of UV radiation. The in vitro action spectrum for the MECLR showed that the UV-induced suppression of the MECLR responses is associated with UV-induced DNA damage. To investigate whether in vivo DNA damage also leads to the abrogation of the MECLR, in situ action spectra were made for the MECLR and the induction of thymine dimers (T<>T). Human skin, obtained from plastic surgery, was exposed to monochromatic light of 254, 297, 302 and 312 nm. After irradiation, epidermal cells were isolated and used as stimulator cells in the MECLR or processed for flow cytometric detection of T<>T. On the basis of dose-response curves for each wavelength, the action spectra for suppression of the MECLR and the induction of T<>T were calculated. These spectra showed close similarities, suggesting that, also in situ, UV-induced DNA damage is involved in the UV-induced suppression of the MECLR. Both action spectra showed a small decline from 254 nm to 302 nm, followed by a steep decline to 312 nm. These data show that, in situ, UVC can efficiently induce DNA damage and modulate cutaneous immune responses.     

Glycosidic bond cleavage of thymidine by low-energy electrons

Thymidine was exposed to low-energy electrons (LEE) as a thin solid film under a high vacuum. Nonvolatile radiation products, remaining on the irradiated surface, were analyzed by HPLC/UV and GC/MS. Here, we show that exposure of thymidine to 3-100 eV electrons gives thymine as a major product with a yield of 3.2 x 10-2 per electron (about one-third of the total decomposition of thymidine). The formation of thymine indicates that LEE induces cleavage of the glycosidic bond separating the base and sugar moieties, suggesting a nonionizing resonant process involving dissociative attachment (<15 eV). In contrast, this reaction is not very efficient by DNA base ionization and does not occur by the reaction of solvated electrons with DNA. These studies introduce a new mechanism of DNA damage involving the interaction of LEE.     

Acute response of human skin to solar radiation: regulation and function of the p53 protein.

p53 is a tumor suppressor gene and mutation of p53 is a frequent event in skin cancer. The wild-type p53 encodes for a 53-kD phosphoprotein that plays a pivotal role in regulating cell growth and cell death. The wt-p53 gene is also called "guardian of the genome", for its role in preventing the accumulation of genetic alterations, observed in cancer cells. The wild-type p53 protein plays a central role in the response of the cell to DNA damage. UV, present in sunlight, is one of the most ubiquitously present DNA damage inducing stress conditions to which skin cells are exposed. The wt-p53 protein accumulates in human skin cells in vitro and in human skin in vivo upon UV irradiation. This upregulation mounts a protective response against permanent DNA damage through transactivation of either cell cycle arrest genes and DNA repair genes or genes that mediate the apoptotic response. The molecular events which regulate the activity of the wt-p53 protein activity are only beginning to be described.     

Effect of Immunosuppressants Tacrolimus and Mycophenolate Mofetil on the Keratinocyte UVB Response

Nonmelanoma skin cancer, derived from epidermal keratinocytes, is the most common malignancy in organ transplant recipients, causes serious morbidity and mortality, and is strongly associated with solar ultraviolet (UV) exposure. Preventing and treating skin cancer in these individuals has been extraordinarily challenging. Following organ transplantation, the immunosuppressants are used to prevent graft rejection. Until now, immunosuppression has been assumed to be the major factor leading to skin cancer in this setting. However, the mechanism of skin carcinogenesis in organ transplant recipients has not been understood to date; specifically, it remains unknown whether these cancers are immunosuppression‐dependent or ‐independent. In particular, it remains poorly understood what is the mechanistic carcinogenic action of the newer generation of immunosuppressants including tacrolimus and mycophenolate mofetil (MMF). Here, we show that tacrolimus and MMF impairs UVB‐induced DNA damage repair and apoptosis in human epidermal keratinocytes. In addition, tacrolimus inhibits UVB‐induced checkpoint signaling. However, MMF had no effect. Our findings have demonstrated that tacrolimus and MMF compromises proper UVB response in keratinocytes, suggesting an immunosuppression‐independent mechanism in the tumor‐promoting action of these immunosuppressants.     

光谱法研究三氯生与人类肿瘤相关DNA的相互作用

采用紫外光谱、荧光光谱和圆二色谱等多种光谱方法研究了三氯生与人类肿瘤相关DNA(抑癌基因p53 DNA和癌基因C-myc DNA)的相互作用,以期为从分子水平上评价三氯生与人类肿瘤发生的危害性提供科学依据。紫外减色效应、KI猝灭受到抑制及圆二色谱正负峰强度减弱等结果表明三氯生以弱嵌插模式与两种DNA发生作用,三氯生与DNA的结合位点数均小于1,以及DNA解链温度小幅升高等结果证实三氯生部分嵌插入DNA的沟区,影响了DNA的双螺旋结构。