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[Cancer Research 59, 4591-4602, September 1, 1999]
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[Cancer Research 59, 4591-4602, September 15, 1999]
© 1999 American Association for Cancer Research

Carcinogenesis

Time Course for Early Adaptive Responses to Ultraviolet B Light in the Epidermis of SKH-1 Mice1

Yao-Ping Lu, You-Rong Lou, Patricia Yen, David Mitchell, Mou-Tuan Huang and Allan H. Conney2

Laboratory for Cancer Research, Department of Chemical Biology, College of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854-8020 [Y-P. L., Y-R. L., P. Y., M-T. H., A. H. C.], and University of Texas M. D. Anderson Cancer Center, Smithville, Texas 78957 [D. M.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (~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 (~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.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sunlight-induced, nonmelanoma skin cancer is a major cancer in the United States and in other temperate parts of the world (1, 2, 3) . UVB light (280–320 nm) and to a much lesser extent UVA (320–400 nm) are responsible for sunlight-induced cancers (4 , 5) . Molecular studies on mutations observed in the p53 tumor suppressor gene in human cancers have also implicated UV light as a major cause of human skin cancer (6, 7, 8, 9) . UVB irradiation is believed to exert its carcinogenic and cytotoxic actions mainly through the direct formation of cyclobutane pyrimidine dimers (thymine dimers) and pyrimidine (6-4) pyrimidone photodimers (6-4 photoproducts) in DNA, but UVB exposure also results in the formation of reactive oxygen species that damage DNA and non-DNA cellular targets (10, 11, 12, 13, 14, 15) .

To maintain genetic integrity after DNA damage, several cellular responses are activated, including mechanisms for removal of DNA damage, cell cycle delay, and apoptosis. The p53 tumor suppressor gene has an important role in protecting cells from DNA-damaging agents (16, 17, 18, 19, 20, 21, 22) . DNA damage triggers a rapid increase in the level of cellular wild-type p53 protein, which shuts off cell replication and DNA synthesis, thereby allowing more time for DNA repair and/or apoptosis. This block of the cell cycle by increased levels of wild-type p53 protein prevents the replication of damaged DNA templates. The increased level of p53 protein after DNA damage is also associated with enhanced programmed cell death (apoptosis), presumably in those cells that are too damaged for adequate DNA repair (23, 24, 25, 26, 27) . Several studies have shown a transient stimulatory effect of UV light on the level of wild-type p53 in cultured cells and in mouse and human epidermis (18 , 28, 29, 30, 31, 32) . The kind of DNA damage required to enhance p53 levels was investigated in cultured cells by Nelson and Kastan (33) , who concluded that DNA strand breaks were necessary to stimulate the formation of increased p53 levels.

An important function of p53 protein is to act as a transcription factor by binding to a p53-specific DNA consensus sequence in responsive genes (26 , 34) . p21(WAF1/CIP1), gadd 45, and mdm-2 genes contain a p53 binding site, and the expression of these genes is responsive to wild-type p53 protein but not to mutant p53 protein (35, 36, 37, 38) . Accordingly, UVB-induced increases in the level of wild-type p53 protein would be expected to increase the synthesis of p21(WAF1/CIP1), GADD 45, and MDM-2 proteins. Increases in p21(WAF1/CIP1) and GADD 45 inhibit the cell cycle, whereas an increase in MDM-2 inhibits p53 function and enhances its degradation. Interestingly, recent studies showed that increased MDM-2 protein is mediated by a p53-dependent increase in p300 (39) and that ARF inhibits the action of MDM-2 (40) . Recent studies have also shown that a DNA-dependent protein kinase is activated after DNA damage, and this kinase is required for p53 sequence-specific DNA binding and expression of p21(WAF1/CIP1) (41) . In addition, phosphorylation of p53 by DNA-protein kinase that is induced by ionizing radiation prevents MDM-2 from inhibiting p53-dependent transactivation (42) . These results indicate that DNA-protein kinase both activates p53 binding to DNA and blocks p53 inactivation by MDM2. It is important to note that increased p21(WAF1/CIP1) and GADD 45 protein levels can also occur by a p53-independent pathway in p53 null cells (43, 44, 45, 46) . p27, another cell cycle inhibitory protein, is formed by a p53-independent pathway (47) . It has also been suggested that p53 can play direct and indirect roles in UVB-inducible, transcription-coupled DNA repair (48) . A scheme that describes some expected early effects of UVB is shown in Fig. 1Citation .



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  		Fig. 1. Scheme for expected early effects of UVB.

 
In the present study, we have used immunohistochemical and morphological assays with skin sections or direct assays with isolated epidermal DNA to evaluate the time course for early effects of a single exposure of SKH-1 hairless mice to UVB on: (a) formation and disappearance of thymine dimers, 6-4 photoproducts, and strand breaks in epidermal DNA; (b) expression of the mismatch repair enzyme, MSH2; (c) accumulation of wild-type p53 protein; (d) expression of p21 (WAF1/CIP1); (e) incorporation of BrdUrd3 into DNA; (f) formation of morphologically distinct apoptotic sunburn cells; (g) increases in myeloperoxidase-positive epidermal cells; (h) neutrophil infiltration into the dermis (inflammatory response); and (i) increases in the number of epidermal cell layers and epidermal thickening (hyperplasia).


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Exposure of Mice to UVB and the Preparation of Serial Sections of Skin
Female SKH-1 hairless mice (6–8 weeks of age) were fed a Purina Laboratory Chow 5001 diet and were irradiated with UV lamps that emit UVB (280–320 nm; 75–80% of total energy) and UVA (320–375 nm; 20–25% of total energy), as described in our earlier studies (49) . The UV lamps used (FS72T12-UVB-HO; National Biological Corp., Twinsburg, Ohio) emitted little or no radiation <280 nm or >375 nm. The dose of UVB was quantified with a UVB Spectra 305 dosimeter (Daevlin Co., Byran, OH). The radiation was further calibrated with a model IL-1700 research radiometer/photometer (International Light Inc., Neburgport, MA). Skin samples (20 mm long; 5 mm wide), which also contained associated dermis, were taken from the middle of the back and placed in 10% phosphate-buffered formalin at 4°C for 18–24 h. The skin samples were then dehydrated in ascending concentrations (80, 95, and 100%) of ethanol, cleared in xylene, and embedded in Paraplast (Oxford Labware, St. Louis, MO). Four-µm serial sections of skin containing epidermis and dermis were made, deparaffinized, rehydrated with water, and used for H&E staining or immunohistochemical staining. These sections were used for the measurement of morphologically distinct apoptotic sunburn cells and epidermal cells with DNA strand breaks, thymine dimers, MSH-2, p53, p21 (WAF1/CIP1), BrdUrd incorporation into DNA, and myeloperoxidase. In addition, the number of epidermal cell layers and the epidermal thickness were measured, and neutrophil infiltration into the dermis was also determined in the same H&E-stained skin sections. Separate groups of mice were used for measuring the effects of UVB on the levels of 6-4 photoproducts in isolated epidermal DNA and for measuring the levels of epidermal peroxidase enzyme activity, ascorbic acid, and glutathione in the epidermis. All histological and immunohistochemical determinations were performed with 400-fold magnification and scored blind by two investigators (Y-P. L. and Y-R. L.), who evaluated coded samples randomly. Good agreement was obtained between the two investigators, and the mean value obtained from the examination of multiple fields by each investigator was determined for each mouse and used in the calculation of mean ± SE for the mice in each group. Each microscope field was approximately equivalent to a 0.5-mm length of epidermis.

Thymine Dimer Detection in Situ
Thymine dimers in epidermal cells were detected by a horseradish peroxidase-labeled monoclonal anti-thymine dimer antibody (Kamiya Biomedical Co., Seattle, WA) and visualized using streptavidin-peroxidase and 3,3'-diaminobenzidine, which stains thymine dimer-containing nuclei a dark brown (50) . We used a horseradish peroxidase-labeled monoclonal anti-thymine dimer antibody that eliminates the need for a second antibody and permits the expression of strong positive staining with very low or no background. Endogenous peroxidase was blocked by incubating the sections in 3% hydrogen peroxide in methanol for 10 min at room temperature. The slides were then incubated in a moist chamber with 0.125% trypsin for 10 min at 37°C. After rinsing in distilled water and incubation at room temperature for 30 min with 1 N HCl, tissue sections were incubated with goat serum for 10 min at room temperature and covered with mouse monoclonal anti-thymine dimer antibody at room temperature for 90 min. Sections were rinsed with PBS, and color development was achieved by incubation for 5 min at room temperature with a substrate solution containing 0.02% 3,3'-diaminobenzidine tetrahydrochloride and 0.02% hydrogen peroxide. The slides were counterstained in Mayer’s hematoxylin (Sigma Chemical Co., St. Louis, MO) for 2 min, cleared with xylene, mounted with a coverslip, and scored under a light microscope. The percentage of thymine dimer-positive cells in the epidermis for each mouse was calculated from the number of stained thymine dimer-positive cells per 100 cells counted in 5–10 representative fields (400-fold magnification; 100–150 epidermal cells/field) within each skin section.

Measurement of 6-4 Photoproducts in DNA
Purification of Epidermal DNA.
The mice were killed by cervical dislocation at various times after exposure to UVB, and skins were removed. To remove the epidermis from dermis, the skins were plunged into a 56–58°C water bath for 30 s, and then the skins were quickly submerged in an ice-water bath. The epidermis was then removed from the dermis by gentle scraping with a spatula and placed in a PBS solution. Purification of epidermal DNA was done according to the procedure described in the Easy-DNA kit from Invitrogen, Inc. (San Diego, CA). About 100–200 µg of epidermal DNA in PBS were mixed with solution A and incubated at 65°C for 10 min. We added 150 µl of solution B and vortexed vigorously until the sample was uniformly viscous. The sample was extracted with chloroform and centrifuged to separate the aqueous and organic phases. The upper layer was transferred into a microcentrifuge tube, and DNA was precipitated with 100% ethanol and placed in an ice bath for 20 min. The precipitated DNA was washed with 80% ethanol three to five times and air dried. The dry DNA samples were dissolved in Tris-EDTA buffer and incubated with RNase (40 µg/ml) at 37°C for 30 min. DNA was precipitated with 100% ethanol, washed with 80% ethanol, and air dried. A portion of each DNA sample was dissolved in 0.01 M Tris-HCl/0.001 M EDTA (pH 7.3), and the 260/230 nm and 260/280 nm absorbance ratios of the purified DNA solutions were always >2.4 and >1.8, respectively. The isolated DNA samples were used for the assay of 6-4 photoproducts.

RIA for 6-4 Photoproducts in DNA.
Details of the RIA for 6-4 photoproducts in DNA and the specificity of the method are described elsewhere (51) . Antiserum was raised against DNA that was irradiated with 10,000 mJ/cm2 UVC (254 nm) light. For the RIA, 2–5 µg of heat-denatured sample DNA were incubated with 5–10 pg of poly(dA):poly(dT) (labeled to >5 x 108 cpm/µg by nick translation with [32P]dTTP) together with 10 mM Tris (pH 7.8), 150 mM NaCl, 1 mM EDTA, and 0.15% gelatin (Sigma Chemical Co.) in a final volume of 1 ml. Antiserum was added at a dilution that yielded 30–60% binding to labeled ligand, and after incubation overnight at 4°C, the immune complex was precipitated with goat anti-rabbit immunoglobulin (Calbiochem, San Diego, CA) and carrier serum from nonimmunized rabbits. After centrifugation, the pellet was dissolved in tissue solubilizer (Amersham Pharmacia Biotech Inc., Piscataway, NJ) and mixed with ScintiSafe (Fisher Scientific, Morris Plains, NJ) containing 0.1% glacial acetic acid; the 32P was quantified by liquid scintillation spectrometry. Under these conditions, antibody binding to an unlabeled competitor inhibited antibody binding to the radiolabeled ligand, and sample inhibition was extrapolated through a standard (dose-response) curve to determine the number of photoproducts in 106 bases. For standard, we used double-stranded salmon testes DNA (Sigma) irradiated with increasing doses of UVC (254 nm) light, heat denatured, aliquoted, and kept frozen at -20°C. Rates of photoproduct induction were quantified previously using nonimmunological enzymatic and biochemical techniques and determined to be 1.56 6-4 photoproducts/megabases/J/m2.

Measurement of DNA Strand Breaks by the TUNEL Assay
We determined the percentage of cells with DNA strand breaks in the epidermis by using the TUNEL method, which detects digoxigenin-labeled 3'-OH ends of genomic DNA (52 , 53) . Briefly, cells with DNA strand breaks were detected in situ using an immunoperoxidase ApopTag kit (Oncor, Gaithersburg, MD). Endogenous peroxidase was blocked by incubating the sections in 3% hydrogen peroxide in PBS for 5 min at room temperature, and the specimens were incubated with 20 µg/ml proteinase K (Sigma) for 15 min at room temperature. After proteinase K treatment, tissue sections were rinsed in PBS (pH 7.2) and incubated for 5 min with equilibrium buffer (Oncor). After equilibration, the sections were incubated in a humidified chamber with TdT enzyme for 1 h at 37°C (for the negative control, water was used instead of TdT). Sections were soaked in stop-wash buffer (Oncor) for 30 min and then rinsed in three changes of PBS. After rinsing, sections were covered with anti-digoxigenin-peroxidase (Oncor) and incubated at room temperature in a humidified chamber for 30 min. The brown color development was achieved by incubation for 6 min at room temperature with a substrate solution containing 0.008% 3,3'-diaminobenzidine tetrahydrochloride and 0.02% hydrogen peroxide. The slides were counterstained in a methyl green solution for 10 min and visualized and scored under a light microscope. The percentage of cells with DNA strand breaks in the epidermis (combined basal and suprabasal layer) was calculated from the number of TUNEL-positive epidermal cells in 5–10 representative fields (100–150 epidermal cells/field) in each skin section. In addition to measuring TUNEL-positive cells in the total epidermis, we also determined the percentage of TUNEL-positive cells specifically in the basal layer of the epidermis. In this study, all TUNEL-positive cells together with all TUNEL-negative cells in the basal layer of the entire skin section (20-mm length) were determined.

Measurement of Apoptotic Sunburn Cells
Identification of apoptotic sunburn cells was based morphologically on cell shrinkage and nuclear condensation due to fragmentation of the cells (6 , 54) . Earlier studies demonstrated that sunburn cells are indeed apoptotic cells (8) . Apoptotic sunburn cells were identified in the epidermis by their intensely eosinophilic cytoplasm and small, dense nuclei, which were observed in H&E-stained histological sections of the skin using light microscopy. The percentage of apoptotic sunburn cells in the epidermis (basal plus suprabasal layers) was calculated from the number of these cells per 100 cells counted from the entire 20-mm length of epidermis for each skin section.

p53, p21(WAF1/CIP1), and MSH2 Immunostaining
Polyclonal rabbit NCL-p53-CM5p antibody purchased from Novocastra Laboratories Ltd. (Newcastle upon Tyne, United Kingdom) reacts with mouse wild-type or mutated p53 proteins (32 , 55 , 56) . Polyclonal rabbit anti-p21(WAF1/CIP1) antibody was purchased from Oncogene Research Products (Cambridge, MA). Polyclonal rabbit antibody that reacts with full-length human or mouse MSH2 was purchased from Oncogene Research Products (Cambridge, MA). Skin sections were stained by the Biotin-Streptavidin Amplified System (alkaline phosphatase-conjugated streptavidin) using StrAviGen Super Sensitive Universal Immunostaining kit purchased from Biogenex (San Ramon, CA) with some modifications. Paraffin sections were first treated with 0.01 M sodium citrate buffer (pH 6.0) in a microwave oven at high temperature for 10 min for p53 or p21 staining or in a 10-pound pressure cooker for 10 min for MSH2 staining (57) . The sections were then incubated with a protein block (normal goat serum) for 10 min at room temperature (this procedure was carried out for p21 staining but omitted for p53 and MSH2 staining). The sections were incubated with p53 antibody (1:500 dilution), p21 antibody (1:100 dilution), or MSH2 antibody (1:20 dilution) for 1 h at room temperature. The samples were then incubated with a biotinylated anti-rabbit secondary antibody for 5 min at 37°C, followed by incubation with conjugated streptavidin solution for 5 min at 37°C. Color development was achieved by incubation with New Fuchsin Substrate Pack (containing 0.6 mg/ml levamisole solution) for 20 min at room temperature, except that levamisole solution was not used for studies with MSH2. The slides were then counterstained with hematoxylin and dehydrated, and coverslips were added for permanent mounting.

A positive reaction was shown as a pink to red precipitate in the nuclei of the cells. The percentage of p53-, p21-, or MSH2-positive cells in the epidermis (combined basal and suprabasal layers) was calculated from the number of stained p53-, p21-, or MSH2-positive cells per 100 cells counted from the entire 20-mm length of epidermis for each skin section. The UVB-induced transient increase in p53-positive cells was shown earlier to be caused by an increased level of wild-type p53 (32) .

BrdUrd Incorporation into DNA
BrdUrd, a thymidine analogue that is incorporated into proliferating cells during the S-phase, is detected by a biotinylated monoclonal anti-BrdUrd antibody and visualized using streptavidin-peroxidase and 3,3'-diaminobenzidine, which stains BrdUrd-containing nuclei a dark brown (staining kit from Oncogene Research Products, Cambridge, MA; Refs. 52 and 55 ). Briefly, all animals were injected with BrdUrd (50 mg/kg) i.p. and killed 1 h later. Endogenous peroxidase was blocked by incubating the tissue sections in 3% hydrogen peroxide in methanol for 10 min at room temperature. The tissue sections were then incubated in a moist chamber with 0.125% trypsin for 10 min at 37°C, rinsed in distilled water, and incubated at room temperature for 30 min with denaturing solution (Oncogene Research Products). The sections were incubated with blocking solution for 10 min at room temperature and covered with biotinylated mouse monoclonal anti-BrdUrd antibody (Oncogene Research Products) at room temperature for 90 min. Sections were rinsed with PBS and incubated with streptavidin-peroxidase for 10 min. Color development was achieved by incubation for 5 min at room temperature with a substrate solution containing 0.02% 3,3'-diaminobenzidine tetrahydrochloride and 0.02% hydrogen peroxide. The slides were weakly counterstained in Mayer’s hematoxylin (Sigma) for 2 min, cleared with xylene, mounted with a coverslip, and scored under a light microscope. The percentage of BrdUrd-labeled cells in the basal layer of the epidermis was calculated from the number of stained BrdUrd-positive cells per 100 basal cells counted from 5 to 10 representative fields (60–80 basal cells/field) for high counts (i.e., 48–72 h after UVB) or BrdUrd-positive basal layer cells from the entire 20-mm length of epidermis for low counts at 0–36 h or 96–240 h after UVB for each section (52 , 55 , 58) .

Immunohistochemical Detection of Myeloperoxidase-positive Epidermal Cells
Polyclonal rabbit anti-myeloperoxidase antibody made against human myeloperoxidase but also reacting with mouse myeloperoxidase was purchased from Biodesign International (Kennebunk, ME). All samples were stained by the Biotin-Streptavidin Amplified system (alkaline phosphatase-conjugated streptavidin) using StrAviGen Super Sensitive Universal Immunostaining kit purchased from Biogenex Laboratory, Inc. (San Ramon, CA) with some modifications. Sections were incubated with anti-myeloperoxidase antibody (1:20 dilution) for 1 h at room temperature, and the sections were then incubated with a secondary antibody for 5 min at 37°C, followed by incubation with conjugated streptavidin solution for 5 min at 37°C. Color formation was obtained by placing the slides in New Fuchsin Substrate Pack (containing 0.6 mg/ml levamisole solution) for 25 min at room temperature. After that, the slides were counterstained with hematoxylin, and a coverslip was added for permanent mounting. A positive reaction was shown as a pink to red precipitate in the nuclei or cytoplasm of the cells. The percentage of myeloperoxidase-positive cells in the epidermis (combined basal and suprabasal layers) was calculated from the number of nuclear stained cells per 100 cells counted using the entire length of epidermis (20 mm) for each skin section.

Epidermal Peroxidase Enzyme Activity Assay
Epidermal peroxidase activity was measured as described elsewhere (59) . Dorsal skin samples were removed and immediately placed in buffer solution (0.5% hexadecyltrimethyl ammonium bromide in 50 mM potassium phosphate, pH 6.0) at 56–58°C for 20 s and submerged in an ice bath containing the same buffer. The epidermis was scraped off and placed in 1 ml of the same buffer. The epidermis was homogenized with a Polytron homogenizer (Brinkmann Instruments Inc., Westbury, NY) three times at 4°C (10 s/homogenization with a 10-s interval between homogenizations). The homogenates were centrifuged at 18,000 x g for 20 min at 4°C. To each polystyrene cuvette, 1.3 ml of 25 mM 4-aminoantipyrine-2% phenol solution and 1.5 ml of 1.7 mM hydrogen peroxide were added and equilibrated for 3–4 min. After establishing the basal rate of increase in absorbance at 510 nm, a 0.2-ml epidermal suspension or a known amount of human myeloperoxidase (Sigma Chemical Co.) as standard was added to cuvettes in duplicate and quickly mixed. Increases in absorbance at 510 nm for 4 min at 0.1-min intervals were recorded. The protein concentration was assayed with Coomassie Brilliant Blue G-250 dye (purchased from the Bio-Rad Laboratories, Hercules, CA) as the protein assay reagent (60) . Peroxidase activity was calculated from the linear portion of the curve and expressed as units/mg epidermal protein. One unit of peroxidase activity is defined as that which degrades 1 µmol of hydrogen peroxide/min at 25°C.

Neutrophil Infiltration into the Dermis
UVB-induced diffuse infiltration of neutrophil inflammatory cells into the dermis (comparison with control skin sections) was measured by evaluating the amount of infiltration of morphologically distinct neutrophils in the dermis. The amount of infiltration was graded as 0 (no infiltration), 1 (slight), 2 (moderate), 3 (severe), or 4 (very severe) as described earlier (61) . For this evaluation, 30–40 fields of the dermis (400-fold magnification) were examined for each section.

Epidermal Thickness and Number of Epidermal Cell Layers
Morphometric analysis was performed with a light microscope using 400-fold magnification and an ocular micrometer as described previously (61) . The number of nucleated cell layers was counted at 10 randomly selected locations per slide and averaged. The thickness of the noncornified cell layer of the epidermis was also measured in a similar manner, and the means ± SE were calculated.

Ascorbic Acid Assay
The concentration of ascorbic acid in the epidermis was measured as described previously (62 , 63) . Dorsal skin was removed and placed in 5% TCA at 56–58°C for 20 s. It was then immediately submerged in an ice bath containing 5% TCA. The epidermis was blotted dry, scraped away from the dermis, weighed, homogenized in cold 5% TCA (4°C) with a Polytron homogenizer, and centrifuged at 18,000 x g for 15 min at 4°C. The acidified supernatant fraction was removed from the denatured protein precipitate for analysis.

Five % TCA (0.3 ml) was added to cuvettes containing 0.3 ml of acidified supernatant to give a volume of 0.6 ml. Reagents were added to cuvettes in the following order: 0.04 ml of orthophosphoric acid (85%); 0.32 ml of aqueous {alpha},{alpha}'-dipyridyl (1%); and 0.04 ml of aqueous ferric chloride (3%). The contents of the cuvettes (final volume, 1.0 ml) were mixed thoroughly after each addition, and color was allowed to develop for 35 min at room temperature. Absorbance at 525 nm was measured in a spectrophotometer. The mean ± SE for each group of four mice was calculated.

Glutathione Assay
Dorsal skin was placed in 5% metaphosphoric acid at 56–58°C for 20 s. The skin sample was then immediately submerged in an ice-water bath containing 5% metaphosphoric acid. The epidermis was blotted dry, scraped away from the dermis, weighed, and homogenized in cold 5% metaphosphoric acid (4°C) with a Polytron homogenizer and centrifuged at 18,000 x g for 15 min at 4°C. The acidified supernatant fraction was removed from the denatured protein and analyzed using a commercial kit purchased from Oxis International, Inc. (Portland, OR). For each measurement, we took 20 µl of sample and brought it to a final volume of 900 µl with 5% metaphosphoric acid. Fifty µl of 0.012 M 4-chloro-1-methyl-7-trifluromethyl-quinolinium methylsulfate were added, and the sample was thoroughly mixed. Fifty µl of 30% NaOH solution were added, and the sample was thoroughly mixed. The sample was then incubated at 25°C for 10 min in the dark, and the absorbance was measured at 400 nm. The mean ± SE for each group of four mice was calculated.

Statistical Analysis
Statistical analysis of all data were done by Student’s t test. Four or five mice were used for each data point (n = 4 or 5), as indicated in the tables and figures. For all histological measurements, multiple fields from each skin section were examined to obtain a mean value for each mouse prior to determining the mean ± SE for the different mice for each data point.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Irradiation of SKH-1 mice with UVB (180 mJ/cm2) resulted in erythema at 3–24 h. The time course for mechanistically important early effects of UVB was evaluated predominantly by the use of immunohistochemical and morphological methods (Tables 1Citation and 2Citation ; Figs. 2Citation and 3Citation ). The use of these methods allowed multiple measurements on serial sections from the same skin samples that were embedded in paraffin blocks. Some typical examples of the use of morphology and immunohistochemical methods are shown in Fig. 4Citation .


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  		Table 1 Time course for epidermal DNA damage and repair after exposure of SKH-1 mice to UVB

 

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  		Table 2 Time course for adaptive responses in the skin after exposure of SKH-1 mice to UVB

 


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  		Fig. 2. Time course for UVB-induced DNA damage and repair in the epidermis of SKH-1 mice. Female SKH-1 mice (7–8 weeks of age) were exposed to UVB (180 mJ/cm2), and the mice were sacrificed before and at various times after the treatment. The data for TUNEL-positive cells, thymine dimer-positive cells, and MSH2-positive cells were obtained from a single experiment where each data point was from four mice. The data for 6-4 photoproducts in isolated DNA were obtained from a different experiment where each value was from five mice. Measurements were made as described in "Materials and Methods." Each value represents the mean from four or five mice; bars, SE.

 


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  		Fig. 3. Time course for UVB-induced adaptive responses in the skin of SKH-1 mice. Female SKH-1 mice (7–8 weeks of age) were exposed to UVB (180 mJ/cm2), and the mice were sacrificed before and at various times after the treatment. All measurements were made in the epidermis except for infiltration of neutrophils into the dermis. Measurements were made as described in "Materials and Methods." Each value represents the mean from four mice; bars, SE.

 


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  		Fig. 4. Immunohistochemical and morphological assays for thymine dimer-positive cells (A and B), apoptotic sunburn cells (C and D), p53-positive cells (E and F), and BrdUrd incorporation into the DNA of epidermal cells (G and H). Female SKH-1 mice (7–8 weeks of age) were exposed to UVB (180 mJ/cm2) and were sacrificed 10 h later (B, D, and F). Other mice were sacrificed 48 h after 180 mJ/cm2 of UVB (H). A, C, E, and G, untreated mice. All panels represent a 400-fold magnification of the epidermis and associated dermis. A description of apoptotic sunburn cells and the immunohistochemical measurement of thymine dimer-positive cells, p53-positive cells, and BrdUrd incorporation into DNA in the epidermis is described in "Materials and Methods" and in other parts of the text. D, arrows, representative sunburn cells.

 
UVB-induced DNA Damage and Repair.
Exposure of SKH-1 mice to UVB (180 mJ/cm2) resulted in easily measurable increases in strand breaks (TUNEL assay) and thymine dimers in the DNA of epidermal cells (total epidermis) at 0.05–0.5 h. At 1 h after UVB exposure, 85% of the epidermal cells were thymine dimer positive, and 76% of the epidermal cells had DNA strand breaks and were TUNEL positive (Table 1Citation ; Fig. 2Citation ). Although the percentage of thymine dimer-positive cells was similar in the basal and suprabasal layers, more intense staining for thymine dimers was observed in the suprabasal layer than in the basal layer. Fewer stained cells and less intense staining for thymine dimers were observed in the dermis than in the epidermis (Fig. 4)Citation . The number of epidermal cells with DNA strand breaks and the number of thymine dimer-positive cells fell markedly between 12 and 36 h (Table 1Citation ; Fig. 2Citation ). The level of 6-4 photoproducts in isolated epidermal DNA was high immediately after exposure of the mice to UVB, and the levels of 6-4 photoproducts fell rapidly between 3 min and 3 h, and the levels of 6-4 photoproducts fell further during the subsequent 3-h interval.

A separate examination of TUNEL-positive cells only in the basal layer of the epidermis revealed that UVB irradiation caused a 5–6-fold increase in the number of cells with DNA strand breaks at 3 min, a 7-fold increase at 30 min, and a maximum 12-fold increase in cells with DNA strand breaks at 6 h. DNA strand breaks in the basal layer of the epidermis decreased gradually between 6 and 24 h and then decreased more markedly between 24 and 36 h. (Table 1Citation ; Fig. 2Citation ). The mean number of TUNEL-positive cells in the total epidermis or in the basal layer of the epidermis was decreased below the control values at 96–240 h after UVB, but these decreases were not statistically significant.

Exposure of SKH-1 mice to UVB caused a rapid, severalfold increase in the number of epidermal cells with the mismatch repair protein, MSH2 (within 30–60 min), and the number of MSH2-positive cells remained elevated for at least 48 h, followed by a decrease toward control values at 72–96 h (Table 1Citation ; Fig. 2Citation ). MSH-2-positive cells were observed mostly in the basal layer, but some were also observed in the suprabasal layer. Most of the increased MSH-2-positive cells at early times after exposure to UVB exhibited only weak nuclear staining, and the proportion of cells with intense MSH-2 staining increased markedly at later times. The percentage of epidermal cells that were strongly positive for MSH-2 was ~1% in control epidermis and at 3 h after exposure to UVB. The percentage of strongly positive epidermal cells, however, was increased by 13- and 20- fold, respectively, at 12 and 24 h after UVB exposure.

UVB-induced Increase in Wild-Type p53 Protein.
Irradiation of SKH-1 mice with UVB (180 mJ/cm2) resulted in a rapid increase in the number of epidermal cells, with wild-type p53 protein starting at 1–2 h after UVB treatment (Table 2Citation ; Fig. 3Citation ). Peak increases in the number of p53-positive epidermal cells (>200-fold higher than for control nonirradiated mice) occurred at 8–12 h after exposure to UVB (Table 2Citation ; Fig. 3Citation ). p53-positive cells were seen primarily in the basal layer, but some were also observed in the suprabasal layer of the epidermis near the basal layer (Fig. 4)Citation . The number of p53-positive cells fell rapidly between 24 and 48 h after UVB irradiation, and p53-positive cells were not observed after 48 h (Table 2Citation ; Fig. 3Citation ). Lower doses of UVB also increased the number of p53-positive cells in the epidermis. A dose-response relationship for the effect of exposure of mice to 30 or 60 mJ/cm2 of UVB on the number of p53-positive epidermal cells was observed at 10 h after treatment with UVB (Fig. 5)Citation .



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  		Fig. 5. Effect of different doses of UVB on the formation of epidermal p53-positive cells, p21(WAF1/CIP1)-positive cells, and apoptotic sunburn cells in the epidermis of SKH-1 mice. Female SKH-1 mice were treated once with 0, 30, or 60 mJ/cm2 of UVB, and the animals were killed 10 h later. The percentages of p53-positive cells, p21(WAF1/CIP1)-positive cells, and apoptotic sunburn cells in the epidermis were determined as described in "Materials and Methods." Each value represents the mean from five mice; bars, SE.

 
UVB-induced Increase in p21(WAF1/CIP1) Protein.
Exposure of SKH-1 mice to UVB (180 mJ/cm2) resulted in a rapid increase in the number of epidermal cells, with p21(WAF1/CIP1) protein starting at 1–2 h after UVB (Table 2Citation ; Fig. 3Citation ). p21(WAF1/CIP1)-positive cells were observed mostly in the basal layer, but some were also observed in the suprabasal layer close to the basal layer. These results are similar to those observed with p53-positive cells. Although the number of epidermal p21(WAF1/CIP1)-positive cells was elevated >1000-fold at 8–12 h after exposure to UVB (time of peak p53 elevation), the maximum number of p21(WAF1/CIP1)-positive cells occurred at 24 h (>2500-fold increase). p21(WAF1/CIP1)-positive cells decreased to low levels between 36 and 96 h after irradiation with UVB (Table 2Citation ; Fig. 3Citation ). A dose-response relationship for the effect of 30 or 60 mJ/cm2 of UVB on the number of p21(WAF1/CIP1)-positive epidermal cells was observed at 10 h after exposure of the mice to UVB (Fig. 5)Citation .

UVB-induced Changes in DNA Synthesis.
Exposure of SKH-1 mice to UVB (180 mJ/cm2) resulted in a rapid doubling in the number of BrdUrd-positive cells in the basal layer of the epidermis (within 30–60 min), and this was followed by a >90% inhibition in BrdUrd-positive cells at 6–12 h after irradiation of the mice with UVB (Table 2Citation ; Fig. 3Citation ). The time course for the inhibitory effect of UVB irradiation on the incorporation of BrdUrd into DNA of cells in the basal layer of the epidermis (% BrdUrd-positive cells) paralleled very closely the time course for UVB-induced increases in the number of p53-positive cells (Table 2Citation ; Fig. 3Citation ). A large increase in the number of BrdUrd-positive cells (about 30-fold) occurred after the number of p53- and p21(WAF1/CIP1)-positive cells returned toward control values (at 48–72 h after UVB irradiation), which is when epidermal thickness and the number of epidermal cell layers were increased (hyperplasia; Table 2Citation ). BrdUrd-positive cells were mainly localized in the basal layer of the epidermis (Fig. 4)Citation , although there was some labeling of the hair follicles (data not shown).

UVB-induced Increase in Apoptotic Sunburn Cells.
Although irradiation of SKH-1 mice with UVB (180 mJ/cm2) resulted in a very rapid (within 3 min) increase in the number of TUNEL-positive cells with DNA strand breaks, the start of an increase in the number of morphologically distinct apoptotic sunburn cells was not observed until 4 h after UVB (Table 2Citation ; Fig. 3Citation ). Peak levels of apoptotic sunburn cells (>200-fold higher than in nonirradiated control mice) were observed at 8–12 h after UVB irradiation. These cells were seen in both the basal and suprabasal layers of the epidermis (Fig. 4)Citation . The time course for UVB induction of apoptotic sunburn cells paralleled very closely the time course for the increase in p53-positive cells (Table 2Citation ; Fig. 3Citation ). A dose-response relationship for the effect of 30 or 60 mJ/cm2 of UVB on the formation of apoptotic sunburn cells in the epidermis was observed at 10 h after irradiation with UVB (Fig. 5)Citation .

UVB-induced Increases in Epidermal Myeloperoxidase-positive Cells and Neutrophil Infiltration into the Dermis.
Exposure of SKH-1 mice to UVB (180 mJ/cm2) caused the appearance of occasional patches of myeloperoxidase-positive epidermal cells within 30 min, and these cells become increasingly more prominent at later times (Fig. 6)Citation . A maximum 18-fold increase in the number of myeloperoxidase-positive epidermal cells was observed by 2 h after UVB administration (Table 2Citation ; Figs. 3Citation and 6Citation ). Epidermal myeloperoxidase-positive cells remained elevated for at least 24 h. No UVB-induced neutrophil infiltration into the epidermis was observed (Fig. 6)Citation .



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  		Fig. 6. Immunohistochemical measurement of UVB-induced increases in myeloperoxidase-positive cells in the epidermis of SKH-1 mice. Female SKH-1 mice (7–8 weeks of age) were sacrificed before UVB (A) and at 0.5 h (B), 1 h (C), or 2 h (D) after exposure to UVB (180 mJ/cm2).

 
Additional SKH-1 mice were exposed once to 180 mJ/cm2 of UVB. Epidermal homogenates were made at 0, 0.5, 1, 2, 4, 6, 8, 10, 24, 48, and 96 h after UVB irradiation and assayed for peroxidase enzyme activity as described in "Materials and Methods." Enzyme activity (mean ± SE, four mice/group) was 0.21 ± 0.03 (before UVB exposure), 0.27 ± 0.04 (0.5 h), 0.37 ± 0.04 (1 h), 0.59 ± 0.12 (2 h), 0.78 ± 0.09 (4 h), 0.69 ± 0.04 (6 h), 0.68 ± 0.05 (8 h), 0.62 ± 0.07 (10 h), 0.49 ± 0.08 (24 h), 0.33 ± 0.05 (48 h), and 0.19 ± 0.03 (96 h) units per mg protein, respectively, at the indicated times after exposure of the mice to UVB. Epidermal peroxidase activity was significantly increased (P < 0.05) at 1, 2, 4, 6, 8, 10, 24, and 48 h after exposure of the mice to UVB. Although no neutrophil infiltration into the epidermis was observed at early time intervals after UVB exposure, neutrophil infiltration into the dermis (inflammatory response) was observed from 4 to 144 h after UVB irradiation (Table 2)Citation . We anticipated that the UVB-induced inflammatory response described above would deplete cellular antioxidants and enhance the oxidation of DNA and other cellular constituents.

UVB-induced Decreases in Epidermal Glutathione and Ascorbic Acid.
Epidermal homogenates were made at 0, 0.5, 1, 2, 4, 6, 10, and 24 h after exposure of SKH-1 mice to UVB (180 mJ/cm2), and they were analyzed for glutathione and ascorbic acid as described in "Materials and Methods." Exposure of the mice to UVB resulted in partial depletion of reduced glutathione and ascorbic acid in the epidermis. The concentration of epidermal glutathione was 0.91 ± 0.01 (before UVB) and 0.78 ± 0.02 (0.5 h), 0.78 ± 0.08 (1 h), 0.79 ± 0.04 (2 h), 0.76 ± 0.03 (4 h), 0.70 ± 0.03 (6 h), 0.55 ± 0.05 (8 h), 0.55 ± 0.03 (10 h), and 0.51 ± 0.02 (24 h) µg/mg wet weight epidermis (mean ± SE, four mice/group) at the indicated times after exposure to UVB. The concentration of epidermal ascorbic acid was 99 ± 6 (before UVB), 104 ± 8 (0.5 h), 71 ± 5 (1 h), 66 ± 4 (2 h), 70 ± 2 (4 h), 71 ± 10 (6 h), 91 ± 5 (10 h), and 101 ± 13 (24 h) µg/g wet weight epidermis (mean ± SE, four mice/group) at the indicated times after exposure to UVB. A maximum decrease in epidermal glutathione concentration of 40–44% occurred at 10–24 h after UVB exposure, and a maximum decrease in epidermal ascorbic acid concentration of 28–33% occurred at 1–6 h after exposure of the mice to UVB. UVB-induced decreases in epidermal glutathione concentration were statistically significant (P < 0.05) at 0.5, 2, 4, 6, 8, 10, and 24 h. UVB-induced decreases in epidermal ascorbic acid concentration were statistically significant (P < 0.05) at 1, 2, 4, and 6 h.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, SKH-1 mice were exposed to 180 mJ/cm2 of UVB, and epidermal DNA damage was observed 3–30 min later as measured by strand breaks (TUNEL assay) and thymine dimers in epidermal cells and by 6-4 photoproducts in isolated epidermal DNA. DNA strand breaks and thymine dimers were observed in both the suprabasal and basal layers of the epidermis (Fig. 4)Citation . It was of interest that more intense staining of cells for DNA strand breaks and thymine dimers occurred in the suprabasal layer than in the basal layer (Fig. 4)Citation . The less intense staining in basal cells may have resulted from the loss of energy from the UVB radiation as it passed through the suprabasal cell layer before reaching the basal layer. Although most of the 6-4 photoproducts were rapidly removed from DNA in the epidermis (within 6 h), the number of cells with thymine dimers and DNA strand breaks remained elevated for a more prolonged time interval and then fell markedly between 12 and 36 h after UVB irradiation. In studies with UVB-induced DNA strand breaks specifically in the basal layer of the epidermis, substantial numbers of cells with strand breaks were observed at 3–30 min after UVB exposure, and the number of basal cells with strand breaks increased further during the next few hours, which is associated with the time-dependent increase in morphologically distinct apoptotic sunburn cells that starts at 4–6 h after UVB exposure. As was observed for DNA strand breaks, apoptotic sunburn cells were observed in both the basal and suprabasal layers of the epidermis (Fig. 4)Citation . Although it has been thought for many years that sunburn cells are apoptotic cells (6 , 54 , 64) , it has been demonstrated only recently that sunburn cells are indeed apoptotic cells (8) . TUNEL-positive staining is a reflection of cells with free 3'-hydroxyl ends in DNA (DNA strand breaks or nicks), which is often associated with apoptosis. It is likely that DNA strand breaks are a precursor to the formation of morphologically distinct apoptotic sunburn cells. Our data indicate that UVB-induced DNA strand breaks precede the formation of morphologically distinct apoptotic sunburn cells by about 4 h. It appears from our data that only a small fraction of epidermal cells with DNA strand breaks immediately after exposure of mice to UVB progresses to morphologically distinct apoptotic sunburn cells.

The time course for the formation and removal of UVB-induced thymine dimers and 6-4 photoproducts in epidermal DNA is similar to that reported earlier in mouse skin (51) . Our results indicate a similarity in the time course for the formation and removal of UVB-induced thymine dimers and DNA strand breaks. To the best of our knowledge, the present study is the first to show a time course for the early formation and removal of UVB-induced DNA strand breaks in mouse epidermis. It is of considerable interest that treatment of mouse skin with UVB caused a rapid 8-fold increase in the number of epidermal cells with the mismatch repair enzyme, MSH2. The UVB-induced rapid increase in the number of epidermal cells with MSH2 has not been reported previously and suggests that MSH2 may play a role in the repair of UVB-induced DNA damage.

One of the earliest effects of UVB irradiation in our study was a rapid increase in the number of myeloperoxidase-positive epidermal cells and an increase in epidermal peroxidase enzyme activity. An increase in the number of myeloperoxidase-positive epidermal cells started within 30 min after exposure of mice to UVB and reached a maximum value by 2 h. The number of myeloperoxidase-positive epidermal cells remained elevated for at least 24 h. The reason why UVB exposure increased myeloperoxidase-positive protein in some epidermal cells but not in others (Fig. 6)Citation is not known and is an area for further research. The UVB-induced rapid increase in patches of myeloperoxidase-positive epidermal cells occurred in the absence of neutrophil infiltration into the epidermis (Fig. 6)Citation . The biological significance of the UVB-induced increase in epidermal peroxidase activity is not known. The very rapid UVB-induced increase in the number of myeloperoxidase-positive epidermal cells may have an important role in inactivating UVB-induced hydrogen peroxide or in triggering cellular responses to UVB by enhancing peroxidative reactions, which may function in cell signaling (65) . It should also be noted that an increased level of myeloperoxidase activity has been associated with increased generation of hypochlorous acid (HOCl-), a potent oxidizing agent, and increased levels of this substance would be expected to increase the oxidation of DNA and other cellular constituents. UVB-induced increases in myeloperoxidase in epidermal cells have been reported previously in mice (66) and are thought to be associated with an inflammatory response. Treatment of mouse skin with 12-O-tetradecanoylphorbol-13-acetate has been shown to increase the levels of myeloperoxidase enzyme activity in the epidermis (59) . It is not known whether UVB-induced increases in epidermal peroxidase activity is a protective response to UVB irradiation or whether it plays a role in peroxidative reactions leading to the oxidative damage of DNA and other cellular constituents.

Epidermal wild-type p53 positive cells started to increase at 1–2 h after UVB irradiation, and peak levels were reached at 8–12 h. Most of the p53 positive cells were associated with nuclear staining of cells in the basal layer of the epidermis or in the suprabasal layer near the basal layer (Fig. 4)Citation . Western blot analysis also indicated a UVB-induced increase in the level of epidermal p53 protein (data not shown). This UVB-induced increase in p53 positive cells was paralleled very closely by a rapid increase in p21(WAF1/CIP1)-positive cells (>1000-fold increase at 10 h and a maximum increase at 24 h), an increase in apoptotic sunburn cells (peak at 8–10 h), and a decrease in BrdUrd-positive cells (maximum decrease at 6–12 h). Increased levels of p21(WAF1/CIP1) have been associated with inhibition of cyclin/CDK activity and inhibition of the cell cycle (67 , 68) . These early adaptive responses to UVB protect the organism from the effects of gene damage by blocking the cell cycle, which allows more time for the repair of DNA damage before cell division and by inducing apoptosis in those cells that are too damaged to be adequately repaired. The studies reported here are the first to show a strong association of UVB-induced increases in p53-positive cells with increases in p21(WAF1/CIP1)-positive cells and a marked decrease in BrdUrd-positive cells in mouse epidermis (Fig. 3)Citation . Our studies also show a strong association between UVB-induced increases in p53-positive cells and the formation of apoptotic sunburn cells in the epidermis (Fig. 3)Citation . Studies by Ziegler and his colleagues with p53-/- knockout mice previously indicated the importance of p53 for UVB-induced increases in apoptotic sunburn cells (6) . The relationship between UVB-induced DNA damage, increases in p53-positive cells, increases in p21(WAF1/CIP1)-positive cells, decreases in DNA synthesis, and increases in apoptosis are described here and summarized in Fig. 1Citation .

In studies by other investigators, exposure of SKH-1 mice to UVB was reported to cause a transient increase in p53 levels at 12–24 h (30 , 32) , and exposure of human skin to simulated solar UV light caused a transient increase in the expression of p53, starting at about 4 h, and peak levels were observed at 48 h (28) . In other studies, exposure of albino-haired mice to UVB (80 mJ/cm2) increased the number of morphologically distinct sunburn cells, and maximum levels were observed at 24 h (64) . A search for safe agents that enhance the levels of p53 is a worthwhile but underexplored approach to cancer chemoprevention, and this concept is also discussed elsewhere (21) . Recent studies have shown that the cancer chemopreventive agents phenethyl isothiocyanate and N-acetylcysteine stimulate p53-dependent apoptosis in cultured cells (69 , 70) . In addition, treatment of mice with green tea enhances UVB-induced increases in the number of epidermal cells with elevated wild-type p53 and the formation of apoptotic sunburn cells (71) .

The 180-mJ/cm2 dose of UVB used in the present study caused erythema at 3–24 h and may be compared with an erythemic sunburn dose of UVB in humans that is approximately 40 mJ/cm2 (72) . Outdoor occupational exposure of humans to UVB or a sunbathing exposure in the summer was reported to range from 50–100 mJ/cm2 per day (73 , 74) . Our results indicate an easily measurable dose-dependent increase in apoptotic sunburn cells and an increase in the number of p53- and p21(WAF1/CIP1)-positive cells at 10 h after exposure of mice to 30 or 60 mJ/cm2 of UVB (Fig. 5)Citation . These levels of UVB exposure are within the normal range of human exposure.

In conclusion, we have examined the time course for UVB-induced DNA damage and repair and early adaptive responses in the epidermis that occur after irradiation of SKH-1 mouse skin with UVB. In these studies, we observed: (a) DNA damage (thymine dimers, 6-4 photoproducts and strand breaks) at 3–30 min; (b) an increased level of MSH2-positive cells at 30–60 min; (c) the start of an increase in epidermal myeloperoxidase-positive cells at 3–30 min (maximum increase at 2 h); (d) the start of an increase in epidermal p53-positive cells at 1–2 h (maximum increase at ~10 h); (e) the start of an increase in epidermal p21(WAF1/CIP1)-positive cells at 1–2 h (maximum increases at 6–8 h and at 24 h); (f) the start of a decrease in BrdUrd incorporation into DNA in epidermal cells at 2–4 h (maximum inhibition at 6–12 h); (g) the start of an increase in morphologically distinct apoptotic sunburn cells at 4 h (maximum increase at 8–10 h); (h) a gradual increase in the infiltration of neutrophils into the dermis (4–48 h; inflammatory response); and (i) marked increase in BrdUrd incorporation into epidermal DNA and an increased number of epidermal cell layers and epidermal thickening (regenerative hyperplasia) at 48 h. The detailed characterization of early biomarkers for UVB-induced effects in the epidermis of mice described in the present report provides basic information on the interrelationships between mechanistically important early effects of UVB and also provides a short-term model for evaluating the effects of potential modulators of UVB-induced carcinogenesis.


   ACKNOWLEDGMENTS
 
We thank Florence Florek, Winniefred Mighty, Deborah Bachorik, and Keith Williams for help in the preparation of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by NIH Grant CA49756. A. H. C. is the William M. and Myrtle W. Garbe Professor of Cancer and Leukemia Research. Back

2 To whom requests for reprints should be addressed, at Laboratory for Cancer Research, Department of Chemical Biology, College of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Road, Piscataway, NJ 08854-8020. Phone: (732) 445-4940; Fax: (732) 445-0687; E-mail: aconney{at}rci.rutgers.edu Back

3 The abbreviations used are: BrdUrd, bromodeoxyuridine; TUNEL, terminal dideoxynucleotidyl transferase-mediated dUTP nick end labeling; TCA, trichloroacetic acid. Back

Received 11/ 3/98. Accepted 7/21/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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