Green tea polyphenol treatment to human skin prevents formation of ultraviolet light B-induced pyrimidine dimers in DNA.

Cancer chemopreventive effects of polyphenols from green tea (GTP) in mouse models of photocarcinogenesis are established. The present study is extended from mouse model to human system in vivo to determine the effect of topical application of GTP to human individuals against UV light-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) in the skin. UVB-induced CPDs were detected by immunohistochemical technique using monoclonal antibodies to thymine dimers. With the gradual increase in UVB dose, both erythema response and CPD formation in the skin was increased. GTP treatment inhibited both UVB-induced erythema response as well as CPD formation. Topical treatment with GTP (approximately 1 mg/cm2 of skin area) 20 min before human buttock skin (sun-protected site) exposure to UVB inhibited CPD formation in epidermis by 81, 70, 60, and 60% at 0.5, 1.0, 2.0, and 4.0 minimal erythema dose of UV exposure, respectively. Treatment of human skin with varying doses of GTP (1-4 mg/2.5 cm2 of skin area) before a single dose of UVB exposure (4.0 minimal erythema dose) decreased dose dependently the formation of UVB-induced CPDs in both epidermis and dermis. The inhibition of UVB-induced CPDs by GTP treatment may be, at least in part, responsible for the inhibition of photocarcinogenesis. Our data suggest that GTP may be used as a novel chemopreventive candidate and possible strategy to reduce UV-induced skin cancer risk in the human population.     

Induction of oxidative DNA base damage in human skin cells by UV and near visible radiation

The premutagenic oxidative DNA base damage, 7,8-dihydro-8-oxoguanine, is induced in human skin fibroblasts by monochromatic radiation ranging from a UVB wavelength (312 nm) up to wavelengths in the near visible (434 nm). The oxidative damage is not generated by absorption of radiation in DNA but rather by activation of photosensitizers generating genotoxic singlet oxygen species. The spectrum for the yield of the oxidative damage in confluent, non-growing, primary skin fibroblasts shows that it is UVA (above 334 nm) and near visible radiations which cause almost all of this guanine oxidation by natural sunlight in the fibroblast model. We estimate that the total amount of oxidation of guanine induced by sunlight in fibroblasts in the epidermis of the skin equals or exceeds the amount of the major type of direct DNA damage, cyclobutane pyrimidine dimers. In rapidly dividing lymphoblastoid cells, no oxidative guanine damage was induced. However, in melanoma cells almost as much damage as in non-growing fibroblasts (1.1 per 10(4) guanine bases after 1200 kJ/m2 UVA) was found. We conclude that oxidative DNA base damage can probably contribute to the induction of both non-melanoma and melanoma skin cancer by sunlight.      

Sunlight and carcinogenesis: Expression of p53 and pyrimidine dimers in human skin following UVA I,UVA I + II and solar simulating radiations

DNA damage by UV radiation plays an essential role in skin cancer induction. We report that even sub-erythemal doses of solar simulating radiation, are capable of inducing substantial nuclear damage, namely pyrimidine dimers and p53 induction in human skin in situ<0R>. The quantity and distribution of p53 induced in human skin by UV radiation depended highly on the waveband and dose of UV used. Solar simulating radiation induced very high levels of p53 throughout all layers in epidermal keratinocytes 24 hr following an erythemal dose (230 ± 15.9/1000 cells), and the induction followed a dose response. Following UVA I + II and UVA I radiations, p53 expression was approximately half of that seen with equivalent biological doses of solar simulating radiation (63.5 ± 28.5 and 103 ± 15.9, respectively). Expression of p53 was seen in basal cell keratinocytes at lower doses of UVA, but all layers of the epidermis were affected at higher doses. Pyrimidine dimer induction, however, was seen to be the same for equivalent biological doses of UVA I, UVA I + II and solar simulating radiations, which coincides with previous findings that pyrimidine dimers initiate the erythemal response and are implicated in skin carcinogenesis. When equivalent biological doses of pure UVA are used with no UVB contamination, significant nuclear alterations occur in human skin in situ,<0R> which can approach those seen with UVB radiation. Our results suggest that DNA damage assessed in vivo<0R> by immunohistochemistry could provide a very sensitive endpoint for determining the efficacy of protective measures, such as sunscreens or protective clothing, against both UVB- and UVA-induced damage in human skin. Int. J. Cancer 76:201–206, 1998.© 1998 Wiley-Liss, Inc.     

Excision of pyrimidine dimers from epidermal DNA and nonsemiconservative epidermal DNA synthesis following ultraviolet irradiation of mouse skin.

Pyrimidine dimer production and excision in epidermal DNA were studied at five different dose levels of ultraviolet light in the skin of intact mice. Dimer production increased with dose up to 50,400 ergs/sq mm. Approximately 30% of the thymine-containing dimers were excised by 24 hr after irradiation at three lower dose levels of ultraviolet light. Nonsemiconservative DNA replication in ultraviolet-irradiated mouse skin was shown to continue for at least 18 hr. The rate of nonsemiconservative replication decreased with time, but did so slowly. The initial rates of nonsemiconservative replication increased uith ultraviolet light dose level up to about 4,200 ergs/sq mm, after which the initial rates were decreased. Semiconservative epidermal DNA synthesis was shown to be inhibited by hydroxyurea, but hydroxyurea had no effect on ultraviolet light-induced nonsemiconservative DNA replication. The observed pyrimidine dimer excision and nonsemiconservative DNA replication suggest that in the intact mouse the cells of the epidermis are capable of DNA excision repair after ultraviolet irradiation of mouse skin.     

The etiology of sunlight-induced melanoma in Xiphophorus hybrid fish

In contrast to sunlight-induced squamous cell carcinoma the etiology of cutaneous malignant melanoma (CMM) is not well understood. In particular, the role that sunlight exposure and DNA damage play in the initiation of this deadly form of cancer is an open question. Early UV carcinogenesis studies in the Xiphophorus backcross hybrid fish model by Richard Setlow indicated that direct DNA damage caused by exposure to the UVB component of sunlight is necessary and sufficient for melanoma formation. Subsequent studies by Setlow suggested that monochromatic UVA radiation that is not directly absorbed by DNA was also sufficient for melanoma induction in Xiphophorus and was, indeed, primarily responsible for initiating human melanoma. These results had significant public health consequences, suggesting that although sunscreens may inhibit UVB-induced erythema they may actually increase exposure to the UVA wavelengths that cause cancer. An intensive worldwide public debate on sunscreen use and "abuse" ensued. Our data do not support a major role of free radical chemistry in melanoma induction. We find evidence that the direct damage caused by the absorption of UVB wavelengths by DNA (e.g., the cyclobutane pyrimidine dimer or CPD) is required for CMM formation and that the ability to repair these lesions plays a significant role in tumor susceptibility. Using the Xiphophorus backcross hybrid fish we are currently in the process of re-evaluating the wavelength- and DNA damage-dependence of UV-induced melanoma and the role nucleotide excision repair and the genes controlling DNA repair and the UV response play in melanoma resistance. From these studies we hope to define the effective solar wavelength boundaries of melanoma, identify the class of critical DNA damage and elucidate the role of DNA repair in tumor suppression.     

Inhibition of UVB induced DNA photodamage in mouse epidermis by topically applied alpha-tocopherol

Ultraviolet B (UVB, 290-320 nm) exposure results in a variety of cellular insults including induction of cyclobutane pyrimidine dimers in DNA. Accumulation of these lesions can lead to mutations in critical genes and contribute to the development of nonmelanoma skin cancer. Topically applied alpha-tocopherol (vitamin E) has previously been shown to prevent the induction of skin tumors in UVB irradiated female C3H/HeNTac mice. We hypothesized that alpha-tocopherol, which absorbs strongly in the UVB, may act as a sunscreen to prevent photodamage. To explore possible mechanisms of photoprotection, we topically applied alpha-tocopherol dispersed in a neutral cream vehicle to the dorsal epidermis of female C3H/HeNTac mice and exposed them to 2.5 J/m2/s of UVB for 60 min. Immediately after exposure, we analyzed thymine dimer levels in DNA by capillary gas chromatography with electron capture detection. Epidermal DNA from mice receiving this UVB dose contained 247 +/- 42 pmol thymine dimers/micromol thymine. Topical application of alpha-tocopherol inhibited dimer formation in a dose-dependent manner. A 1% alpha-tocopherol dispersion inhibited the formation of thymine dimers to 43% of levels in vehicle controls. Several vitamin E compounds, including alpha-tocopherol acetate, alpha-tocopherol methyl ether, gamma-tocopherol, and delta-tocopherol also inhibited thymine dimer formation, but were five- to ten-fold less potent than alpha- tocopherol. A variety of commercially available sunscreens were also less potent than alpha-tocopherol in their ability to reduce dimer formation. These results suggest that DNA photoprotection is an important mechanism by which topically applied alpha-tocopherol can inhibit UVB induced skin cancer. Alpha-Tocopherol acetate, the most common form of vitamin E in commercial skin care products, conferred less protection, perhaps due to its lower absorptivity in the UVB. Our results further underscore the importance of determining which forms of vitamin E can inhibit specific lesions involved in photocarcinogenesis.      

Protective role of histone deacetylase 4 from ultraviolet radiation-induced DNA lesions

Ultraviolet B (UVB) exposure is a core factor that leads to skin disease or carcinogenesis through the insufficient repair of DNA lesions. UVB-induced DNA lesions are mainly removed by the nucleotide excision repair (NER) mechanism. The expression of histone deacetylase 4 (HDAC4) is altered in the skin upon UVB exposure, indicating its possible implication in UVB-induced DNA lesions repair. Here, we investigated the role of HDAC4 in the NER removal of the main classes of UVB-induced DNA lesions consisting of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). We found that UVB irradiation increased HDAC4 expression at both the mRNA and protein levels. HDAC4 interacted with NER factor XPC, which played an important role in effectively removing the UVB-induced DNA lesions. This study provides an understanding of the HDAC4 function in DNA repair, which will allow the development of efficient strategies to protect the skin from UVR-induced diseases.     

Silibinin prevents ultraviolet B radiation-induced epidermal damages in JB6 cells and mouse skin in a p53-GADD45α-dependent manner

Better preventive strategies are required to reduce ultraviolet (UV)-caused photodamage, the primary etiological factor for non-melanoma skin cancer (NMSC). Accordingly, here we examined the preventive efficacy of silibinin against UVB-induced photodamage using mouse epidermal JB6 cells and SKH1 hairless mouse epidermis. In JB6 cells, silibinin pretreatment protected against apoptosis and accelerated the repair of cyclobutane pyrimidine dimers (CPD) induced by moderate dose of UVB (50 mJ/cm(2)), which we are at risk of daily exposure. Silibinin also reversed UVB-induced S phase arrest, reducing both active DNA synthesizing and inactive S phase populations. In mechanistic studies, UVB-irradiated cells showed a transient upregulation of both phosphorylated (Ser-15 and Ser-392) and total p53, whereas silibinin pretreatment led to a more sustained upregulation and stronger nuclear localization of p53. Silibinin also caused a marked upregulation of GADD45α, a downstream target of p53, implicated in DNA repair and cell cycle regulation. Importantly, under p53 and GADD45α knockdown conditions, cells were more susceptible to UVB-induced apoptosis without any significant S phase arrest, and protective effects of silibinin were compromised. Similar to the in vitro results, topical application of silibinin prior to or immediately after UVB irradiation resulted in sustained increase in p53 and GADD45α levels and accelerated CPD removal in the epidermis of SKH1 hairless mice. Together, our results show for the first time that p53-mediated GADD45α upregulation is the key mechanism by which silibinin protects against UVB-induced photodamage and provides a strong rationale to investigate silibinin in reducing the risk and/or preventing early onset of NMSC.     

Pyrimidine dimer removal enhanced by DNA repair liposomes reduces the incidence of UV skin cancer in mice.

UV exposure has been linked to skin cancer in humans by epidemiology and the rare genetic disease xeroderma pigmentosum. However, UV produces multiple photoproducts in DNA, and their relative contribution is uncertain. An enzyme which specifically repairs cyclobutane pyrimidine dimers in DNA, T4 endonuclease V, was encapsulated in liposomes for topical delivery into mouse and human skin. In both species, liposomes applied after UV exposure localized in the epidermis and stimulated the removal of cyclobutane pyrimidine dimers. UV-irradiated mice treated with these liposomes had a dose-dependent decrease in the incidence of squamous cell carcinoma compared to controls. The results demonstrate that unrepaired cyclobutane pyrimidine dimers in DNA are a direct cause of cancer in mammalian skin.     

Cyclobutane pyrimidine dimers form preferentially at the major p53 mutational hotspot in UVB-induced mouse skin tumors

The most prevalent DNA lesion induced by UV irradiation is the cyclobutane pyrimidine dimer (CPD) which forms at positions of neighboring pyrimidines. In mouse skin tumors induced by irradiation with UVB (280-320 nm) lamps or solar UV simulators, a major mutational hotspot occurs at codon 270 (Arg-->Cys) involving a sequence change from 5'-TCGT to 5'-TTGT. We have shown previously that CPD formation by UVB or sunlight is enhanced up to 10-fold at 5'-CCG and 5'-TCG sequences due to the presence of 5-methylcytosine bases. Sequence analysis showed that the CpG at codon 270 is methylated in mouse epidermis at a level of approximately 85%. Irradiation of mouse skin or mouse cells in culture produced the strongest CPD signal within exon 8 at the 5'-TCG sequence which is part of codon 270. Time course experiments showed that CPDs at this particular sequence persist longer than at several neighboring positions. The data suggest that formation of CPDs is responsible for induction of the major p53 mutational hotspot in UV-induced mouse skin tumors.     

DNA mismatch repair proteins promote apoptosis and suppress tumorigenesis in response to UVB irradiation: an in vivo study

DNA mismatch repair (MMR) proteins are integral to the maintenance of genomic stability and suppression of tumorigenesis due to their role in repair of post-replicative DNA errors. Recent data also support a role for MMR proteins in cellular responses to exogenous DNA damage that does not involve removal of DNA adducts. We have demonstrated previously that both Msh2- and Msh6-null primary mouse embryonic fibroblasts are significantly less sensitive to UVB (ultraviolet B)-induced cytotoxicity and apoptosis than wild-type control cells. In order to ascertain the physiological relevance of the data we have exposed MMR-deficient mice to acute and chronic UVB radiation. We found that MMR-deficiency was associated with reduced levels of apoptosis and increased residual UVB-induced DNA adducts in the epidermis 24-h following acute UVB exposure. Moreover, Msh2-null mice developed UVB-induced skin tumors at a lower level of cumulative UVB exposure and with a greater severity of onset than wild-type mice. The Msh2-null skin tumors did not display microsatellite instability, suggesting that these tumors develop via a different tumorigenic pathway than tumors that develop spontaneously. Therefore, we propose that dysfunctional MMR promotes UVB-induced tumorigenesis through reduced apoptotic elimination of damaged epidermal cells.     

Pyrimidine dimer formation and repair in human skin

Cyclobutyl pyrimidine dimers have been detected in the DNA of human skin following in vivo irradiation with suberythemal doses of ultraviolet (UV) radiation from FS-20 sun lamp fluorescent tubes. Dimers were assayed by treatment of extracted DNA with Micrococcus luteus UV-specific endonuclease, alkaline agarose electrophoresis, and ethidium bromide staining. This technique, in contrast to conventional dimer assays, can be used with nonradioactive DNA and is optimal at low UV light doses. M. luteus endonuclease-sensitive sites were determined after exposure of untanned skin in two volunteers to UV light (0.97, 1.94, or 3.88 X 10(3) J/sq m; lambda, 290 to 360 nm). At 20 min postirradiation (dose, 1.94 X 10(3) J/sq m), fewer M. luteus endonuclease-sensitive sites were found in the DNA than immediately after the irradiation. Even fewer endonuclease-sensitive sites were found at 20 min when the UV-irradiated skin was subsequently irradiated with visible light than when the area was kept in the dark. These data suggest that some dimer disappearance by excision repair occurs within 20 min of UV irradiation and that photoreactivation of dimers can make a contribution to the total repair process.     

Inhibition of ultraviolet light-induced oxidative events in the skin and internal organs of hairless mice by isoflavone genistein

We have previously demonstrated that soybean isoflavone genistein inhibits ultraviolet-B (UVB)-induced skin tumorigenesis in hairless mice. In the present study, we further investigated the possible mechanism(s) of action whereby genistein inhibits photocarcinogenesis with focuses on UVB-induced oxidative events, including hydrogen peroxide (H(2)O(2)) production, lipid peroxidation (as represented by malondialdehyde, MDA), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation in vivo. We demonstrated that subacute exposure to UVB substantially increased the level of H(2)O(2), lipid peroxides, and 8-OHdG in skin of hairless mice. In addition, chronic exposure to low-dose UVB (0.9-1.2 kJ/m(2) for 20 weeks) substantially increased the levels of 8-OHdG not only in the epidermis, but also in the internal organs such as liver, brain, and spleen of mice with exception of kidney. However, genistein did not affect the level of UVB-induced pyrimidine dimmers in the same UVB exposed mouse skin, indicating selective inhibition of oxidative DNA damage by genistein. Induction of H(2)O(2) was independent of UVB fluences whereas the levels of MDA and 8-OHdG were induced in an UVB fluence-dependent manner. The results suggest that H(2)O(2) be generated as an acute cutaneous response to UVB irradiation, while MDA and 8-OHdG are accumulated with increasing UVB exposure and more closely related to chronic effects of UVB radiation. Pre-treatment of animals with 10 micromol of genistein 1 h prior to UVB exposure significantly inhibited UVB-induced H(2)O(2) and MDA in skin and 8-OHdG in epidermis as well as internal organs. Suppression of 8-OHdG formation by genistein has been corroborated in purified DNA irradiated with UVA and B. In summary, our results suggest that UVB irradiation elicit a series of oxidative events, which can be substantially inhibited by isoflavonoid genistein through either direct quenching of reactive oxygen species or indirect antiinflammatory effects. Thus, the antioxidative properties of genistein may explain for the mechanisms of anti-photocarcinogenic action of genistein.     

Detection of Ultraviolet Photoproducts in Mouse Skin Exposed to Natural Sunlight

In the present study, we for the first time investigated the formation of ultraviolet (UV) photoproducts, cyclobutane pyrimidine dimers (CPDs), pyrimidine-pyrimidone (6–4) photoproducts (64PPs) and Dewar isomers, in vivo in shaved and depilated C3H/HeN mouse skin exposed to natural sunlight (NSL) at noon for 5 min to 1 h in mid-summer, using a highly sensitive immunohistochemical method. This method permits the quantitative analysis of UV-photoproducts in formalin-fixed, paraffin-embedded sections with specific antibodies against CPDs, 64PPs and Dewar isomers. We demonstrated that the induction of CPDs in vivo in mouse skin by NSL was exposure time-dependent, but the accumulation of 64PPs or Dewar isomers was comparatively low in the skin sections from mice exposed to NSL in vivo. The results indicate that CPDs are the main photoproducts in vivo induced by sunlight and that their formation and repair may be important in connection with carcinogenesis in sun-exposed areas of human skin.     

Epidermal stem and progenitor cells in murine epidermis accumulate UV damage despite NER proficiency

Ultraviolet (UV) radiation induces cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs) in DNA, which through gene mutations (e.g. in P53) may lead to skin carcinogenesis. Upon chronic low-level UV exposure, certain basal cells in mouse epidermis were reported to accumulate CPDs. These observations raised questions on whether these cells were fully DNA-repair deficient, and whether they were stem or progenitor cells, as suggested by their long residence time. We found that CPD-retaining basal cells (CRBCs) in SKH-1 hairless mice were repair proficient as accumulation of (6-4)PP, which is a hallmark for complete nucleotide excision repair-deficiency in rodents, was not observed. Accumulation of 6-4PP as well as CPD did, however, occur in basal cells in the epidermis of DNA repair-deficient Xpc-/- mice. Chronic UV exposure of DDB2 transgenic mice and DDB2 knockout mice revealed that the occurrence of CRBCs was inversely correlated with DDB2-expression, indicating that a boost in DNA repair lowered CPD accumulation. Stem cells are quiescent cells and can be identified as 5-bromo-2'-deoxyuridine-label retaining cells (BrdU-LRCs). Induction of BrdU-LRCs followed by chronic UV irradiation showed that all BrdU-label retaining stem cells were also CPD-retaining cells. As most CRBCs were not BrdU-labeled we surmized that these cells must include BrdU-negative stem cells and early progenitor cells. In confirmation of the latter, we found that CRBCs occurred among MTS24+ hair follicle progenitor cells. These findings provide the first evidence that epidermal stem and progenitor cells are prone to the accumulation of UV-induced DNA-damage and can be a prominent target in skin carcinogenesis.     

Time course for early adaptive responses to ultraviolet B light in the epidermis of SKH-1 mice.

Hairless SKH-1 mice were exposed once to UVB light (180 mJ/cm2), and mechanistically important early adaptive responses in the epidermis were evaluated by immunohistochemical and morphological methods. Interrelationships in the time course for these UVB-induced responses were examined. The number of epidermal cells with DNA strand breaks (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells) or with thymine dimers increased to maximal levels within 30 min after UVB. The number of cells with DNA strand breaks located specifically in the basal layer of the epidermis was increased substantially by 3-30 min after UVB and gradually increased further over the next 5.5 hours. DNA strand breaks specifically in the basal layer of the epidermis were increased maximally at 6 h after UVB. The number of epidermal cells with DNA strand breaks or thymine dimers decreased markedly between 12 and 36 h. Pyrimidine (6-4) pyrimidone photodimers (6-4 photoproducts) in isolated epidermal DNA were increased immediately after irradiation of the mice with UVB and decreased markedly during the next 6 h. Exposure to UVB caused a rapid 8-fold increase in the number of epidermal cells with the DNA mismatch repair protein, MSH2 (within 30-60 min), and the level of MSH2-positive cells remained elevated for at least 48 h. These observations suggest a possible role of MSH2 in the repair of UVB-induced DNA damage. The number of epidermal cells with wild-type p53 protein started to increase at 1 h after UVB exposure and reached maximal levels by 8-12 h. The number of p53-positive cells fell markedly between 24 and 48 h. The time course for UVB-induced increases in the number of p53-positive cells was paralleled very closely by the time course for UVB-induced increases in the number of cells with p21(WAF1/CIP1), increases in morphologically distinct apoptotic sunburn cells, and decreases in the number of epidermal cells with bromodeoxyuridine (BrdUrd) incorporation into DNA. Although the start of UVB-induced increases in the number of p21(WAF1/CIP1)-positive cells was similar to that for the increase in p53-positive cells and very high levels of p21(WAF1/CIP1)-positive cells were observed at 8-12 h, maximal increases in p21(WAF1/CIP1)-positive cells were not achieved until 24 h after UVB irradiation (approximately 12 h after the peak value for p53). Myeloperoxidase-positive epidermal cells started to increase by 30 min after UVB exposure, and maximal numbers of myeloperoxidase-positive epidermal cells were observed at 2 h after UVB (18-fold higher than in nonirradiated control mice). An increased level of epidermal peroxidase enzyme activity in the epidermis was also observed from 1 to 24 h after exposure of the mice to UVB. Although neutrophil infiltration into the epidermis was not seen after exposure to UVB, neutrophil infiltration into the dermis (inflammatory response) was observed from 4 to 144 h after UVB exposure. In contrast to the marked inhibitory effect of UVB on BrdUrd incorporation into the DNA of epidermal cells observed at 8-12 h after UVB irradiation (>90% inhibition), BrdUrd incorporation into the DNA of epidermal cells was markedly increased (approximately 30-fold increase in the number of BrdUrd-positive cells) at 48 h after UVB exposure, and increases in epidermal cell layers and epidermal thickness (hyperplasia) were also observed. These later effects were associated with regeneration of the damaged epidermis.     

Protective effect of topically applied olive oil against photocarcinogenesis following UVB exposure of mice

Reactive oxygen species have been shown to play a role in ultraviolet light (UV)-induced skin carcinogenesis. Vitamin E and green tea polyphenols reduce experimental skin cancers in mice mainly because of their antioxidant properties. Since olive oil has also been reported to be a potent antioxidant, we examined its effect on UVB-induced skin carcinogenesis in hairless mice. Extra-virgin olive oil was applied topically before or after repeated exposure of mice to UVB. The onset of UVB-induced skin tumors was delayed in mice painted with olive oil compared with UVB control mice. However, with increasing numbers of UVB exposures, differences in the mean number of tumors between UVB control mice and mice pretreated with olive oil before UVB exposure (pre-UVB group) were lost. In contrast, mice that received olive oil after UVB exposure (post-UVB group) showed significantly lower numbers of tumors per mouse than those in the UVB control group throughout the experimental period. The mean number of tumors per mouse in the UVB control, pre-UVB and post-UVB groups was 7.33, 6.69 and 2.64, respectively, in the first experiment, and 8.53, 9.53 and 3.36 in the second experiment. Camellia oil was also applied, using the same experimental protocol, but did not have a suppressive effect. Immunohistochemical analysis of DNA damage in the form of cyclobutane pyrimidine dimers (CPD), (6-4) photoproducts and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in samples taken 30 min after a single exposure of UVB showed no significant difference between UVB-irradiated control mice and the pre-UVB group. In the post-UVB group, there were lower levels of 8-OHdG in epidermal nuclei, but the formation of CPD and (6-4) photoproducts did not differ. Exposure of olive oil to UVB before application abrogated the protective effect on 8-OHdG formation. These results indicate that olive oil topically applied after UVB exposure can effectively reduce UVB-induced murine skin tumors, possibly via its antioxidant effects in reducing DNA damage by reactive oxygen species, and that the effective component may be labile to UVB.     

Accelerated ultraviolet radiation-induced carcinogenesis in hepatocyte growth factor/scatter factor transgenic mice

The dramatic rise in incidence of malignant melanoma experienced by populations both within the United States and throughout the world over the last several decades has been attributed to enhanced exposure to the UV spectrum of sunlight radiation. This hypothesis can now be tested using genetically engineered mouse models predisposed to malignant melanoma. Here we use melanoma-prone transgenic mice inappropriately expressing hepatocyte growth factor/scatter factor (HGF/SF) in the skin as an experimental model system to ascertain the consequences of a chronic regimen of suberythemal UV radiation on melanoma genesis. HGF/SF is a multifunctional regulator capable of stimulating growth, motility, invasiveness, and morphogenetic transformation in cells, including melanocytes, expressing its receptor tyrosine kinase Met. HGF/SF transgenic mice demonstrate ectopic interfollicular localization and accumulation of melanocytes within the truncal dermis, epidermis, and junction and if untreated develop primary cutaneous melanoma with a mean onset age of approximately 21 months. Transgenic mice and their wild-type littermates subjected to UV radiation three times weekly using FS40 sunlamps (60% UVB and 40% UVA), with daily UV doses graded from 2.25 to 6.0 kJ/m2, developed skin tumors with a mean onset age of 26 and 37 weeks, respectively (P < 0.001, Kaplan-Meier log rank test). However, the repeated doses of suberythemal UV radiation used in this study failed to accelerate melanoma genesis, instead inducing the development of nonmelanoma tumors that included squamous cell carcinomas, squamous papillomas, and sarcomas. The conspicuous absence of melanocytic tumors occurred despite the immunohistochemical detection of a significant stimulation (P < 0.001) in melanocyte-specific bromodeoxyuridine incorporation in response to only 2 weeks of UV irradiation (total UV dose of 13.5 kJ/m2), resulting in 2.6- and 4.6-fold increases in the number of melanocytes in the dermis and epidermis, respectively. These data indicate that chronic suberythemal UV radiation preferentially favors the development of nonmelanocytic over melanocytic neoplasms in this transgenic animal, consistent with the pathogenesis proposed for sun exposure-associated skin cancer based on retrospective studies in the human population. Our findings suggest that the HGF/SF transgenic mouse will be useful as an experimental model for determining the consequences of exposure to various regimens of UV radiation and for elucidating the mechanisms by which such consequences are realized.