Aged human skin removes UVB-induced pyrimidine dimers from the epidermis more slowly than younger adult skin in vivo
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- Yamada, M., Udono, M.U., Hori, M. et al. Arch Dermatol Res (2006) 297: 294. doi:10.1007/s00403-005-0618-0
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Although many studies have been reported on the repair of ultraviolet light (UV)-induced cyclobutane-type pyrimidine dimers (CPDs) in DNA, the effects of aging on the removal of UV-induced CPDs from the human skin epidermis in vivo remains uncertain. Therefore, we employed immunoblotting and immunohistochemical methods using monoclonal antibodies (TDM-2) to CPDs to study age-related differences in the time required for the in vivo removal of UVB-induced CPDs. The flexure surfaces of the upper arms of five young men were exposed to UVB light at a fluence of 35 and 700 mJ/cm2, and four older men were also irradiated with the same doses of UVB mentioned above. Each area of skin was biopsied before and immediately after irradiation, and at 4, 24 h, 2 and 4 days after irradiation in the younger group; and before and immediately after irradiation, and at 24 h, 4, 7, and 14 days after irradiation in the older group. A total of 108 DNA samples were taken from the epidermis of 108 biopsied specimens. These samples were immunoblotted using TDM-2 and the intensities of the immunoprecipitates were measured by photodensitometer. Our results show that the CPDs had been removed from the epidermis at 4 days after irradiation at either dose in the younger group, and between 7–14 days after irradiation in the aged group. The results of our immunohistochemical studies were consistent with those of our immunoblotting studies, and indicated that basal cells repair CPDs more quickly than prickle cells in the epidermis except the amounts at 24 h after UVB irradiation, and that the CPDs were removed by epidermal turnover after the nucleotide excision repair (NER). Our results showed age-associated decline in the NER in vivo, indicating high risk of UV-associated skin cancer.
KeywordsPyrimidine dimer TDM-2 UVB Epidermal cell Aging
It has been reported that ultraviolet (UV) light irradiation to DNA induces photoproducts in DNA such as cyclobutane-type thymine dimers, (6–4) photoproducts and other photoproducts [4, 14, 30, 39, 41, 44, 45]. The predominant UV-induced photoproducts are cyclobutane-type pyrimidine dimers (CPDs), of which thymine dimers account for 75–80% of total DNA lesions . These photoproducts have been detected by HPLC , and alkaline gel electrophoresis using dimer-specific endonuclease [15, 40], as well as by other methods [38, 46]. In addition, monoclonal antibodies to cyclobutane-type thymine dimer and (6–4) photoproducts have been developed [25, 26, 27, 35, 42]. Thymine dimers in DNA were shown electron-microscopically using monoclonal antibodies (TDM-1) to cyclobutane-type thymine dimer , and a decrement over a time course in the amount of UV-induced CPDs in monkey skin in vivo using monoclonal antibodies (TDM-2) to CPDs were shown immunohistochemically . More recently, the development of a 32P-postlabeling method to detect CPDs and (6–4) photoproducts has been reported . Thymine dimer, as well as (6–4) photoproduct, is known to be mutagenic to mammalian cells [22, 33, 47, 50]; this type of DNA damage is removed by nucleotide excision repair (NER) [37, 51]. A deficiency of this repair capacity in cases of xeroderma pigmentosum (XP) has been reported to be associated with cancer . The consequences of a defect in one of the NER proteins are apparent in three rare recessive syndromes: XP, Cockayne syndrome (CS), and the photosensitive form of the brittle hair disorder trichothiodystrophy (TTD). Sun-sensitive skin is associated with skin cancer predisposition in cases of XP, but remarkably not in cases of CS or TTD . Additionally, actinic keratosis (AK) and squamous cell carcinoma originating from AK are UV-associated skin tumors, and a relationship between errors in the repair of DNA photodamage and the development of actinic cancer has been proposed . It has been reported that a defect in the repair of UV-induced DNA damage is observed in the peripheral lymphocytes  and cultured fibroblasts  of AK patients, and the unrepaired CPDs in the DNA from AK lesions have been shown . The capacity to repair UV light-induced DNA damage in a reporter gene in the peripheral lymphocytes of 88 patients with primary basal cell carcinoma and 135 cancer-free controls were studied and it was reported that basal cell carcinoma patients with red hair and light skin (type I) had a DNA repair capacity 10–20% lower than that of control subjects . Given that AK develops on the exposed skin of the elderly, the effects of aging on DNA repair and skin carcinogenesis are very important. It was reported that the excision repair of pyrimidine dimers induced by simulated solar radiation to the skin of patients with basal cell carcinoma was decreased compared to that of age-matched cancer-free subjects . An age-related decline in post-UV DNA repair capacity and an age-related increase in post-UV mutability have been reported by many authors [16, 29, 49]; however, in contrast to these reports, a number of other papers report no age-associated decline in the DNA repair capacity for UV-induced damage [8, 28, 52]. Nevertheless, all of these studies were carried out using fibroblasts or lymphocytes as materials, and, as the majority of UV-induced skin tumors originate from epidermal cells, it is important to study the removal of UV-induced CPDs from the DNA of human skin epidermal cells in vivo with respect to aging. The concept of multistage carcinogenesis has been proposed in previous studies [3, 21], and we also speculate that the first step of multistage carcinogenesis is an age-related deficiency of the repair mechanisms of these photoproducts. Indeed, a decline in NER protein, XP group A protein and p53 protein, which participate in NER, is seen in UV-irradiated human fibroblasts with age .
In the present study, we employed immunohistochemical and immunoblotting techniques using monoclonal antibodies (TDM-2) to CPDs to address three questions concerning the removal of CPDs from the epidermis in vivo: (1) whether or not there is an age-related difference in the amount of time needed to remove CPDs from the epidermis; (2) whether or not there is a difference in removal time correlated with differences in the specific dose of UVB irradiation; (3) whether both basal cells and prickle cells are able to repair CPDS.
Materials and methods
Chemicals, antibodies, and membrane
Monoclonal antibodies (TDM-2) to CPDs were supplied by Mori et al. . Peroxidase-conjugated goat anti-mouse IgG was purchased from Tago Inc. (Burlingame, CA, USA). Nitrocellulose membranes were obtained from Bio-Rad Laboratories Inc. (Rahway, NJ, USA), and LSAB universal kits from Dako (Carpinteria, CA, USA).
A sun-lamp (FL-20SE, Toshiba, Tokyo Japan; wavelength: 275–410 nm, maximum 312) was used as the UVB source. Wavelengths of 280–340 nm include approximately 90% of the total energy output of the lamp. The fluence of the sun-lamp was measured at 312 nm by photodosimeter.
The subjects for the present study were selected from a pool of 20 healthy volunteers with approximately the same skin type (types III–IV), of whom ten were young men (age range, 22–26 years) and ten were old men (age range, 70–78 years). The study was approved by the Ethical Committee of Nagasaki University School of Medicine, and all volunteers provided their informed consent. We confirmed that all volunteers were very healthy and did not take photosensitive medicine or food. The minimal erythema dose (MED) of each volunteer was measured by graded UVB exposure to six sites of untanned flexure surface of the upper arm. Based on our results, six young men and six old men, whose MEDs were approximately 70 mJ/cm2, were selected to participate in this study.
Relationship between UVB dose and amount(%) of CPD formation
Each of six young men and six old men was irradiated at three different sites on the flexure surface of the upper arm with UVB fluences of 70, 350, and 700 mJ/cm2. Each area of irradiated skin, a total of six specimens, was biopsied immediately after irradiation and frozen with liquid nitrogen, and then stored at −80°C in a freezer until it was used for DNA extraction.
UVB irradiation and biopsies
In order to determine if the differences in the removal times of CPDs are dependent on the specific dose of UVB-irradiation, we used doses of 35 mJ/cm2 (1/2 MED) and 700 mJ/cm2 (10 MED). Five each of the six young men and the six old men were exposed to UVB with fluences of 35 and 700 mJ/cm2 on the flexure surface of their right and left upper arms, respectively. Based on the results of our preliminary study, we speculated that older men would need a longer amount of time to remove epidermal CPDs than younger men. Therefore, biopsies were conducted at different times for the different group. For younger subjects (younger group), each irradiated area of skin was biopsied before and immediately after irradiation, and at 4, 24 h, 2 and 4 days after irradiation; in older subjects (older group), biopsy was conducted before and immediately after irradiation, and at 24 h, 4, 7, and 14 days after irradiation. One member of the older group was eliminated from the study because of a bacterial infection of the irradiated site. Each biopsied specimen was divided into two pieces, one of which was fixed with 10% formalin for immunohistochemical study with TDM-2, and the other of which was frozen with liquid nitrogen and stored at −80°C in freezer until it was used for DNA extraction.
Preparation of epidermal nuclear DNA for immunoblotting
Each frozen specimen was incubated with water at 60°C for 1 min. The epidermis was separated gently from the dermis using tweezers, frozen in liquid nitrogen, and then pulverized in a mortar with a pestle. DNA was extracted following a modified version  of the technique described by Blin and Stafford . DNA was recovered from the aqueous phase by ethanol precipitation and dissolved in 50 μl of TE buffer. The optical density of each DNA sample was measured by spectrophotodensitometer (Shimadzu, Kyoto, Japan) at wavelengths of 280 and 260 nm, and the concentration of the DNA was calculated. A total of 114 DNA specimens, which is composed of six specimens to study UVB-dose dependent induction of CPDs and a total of 108 specimens to study age-related repair capacity of CPDs, were examined.
Measurement of CPD by immunoblotting method using TDM-2 
On every subject, 2 μg of each DNA sample from each of the different testing times was dotted on nitrocellulose membranes and incubated overnight with TDM-2 diluted to 1:1,000 with defatted dry milk solution (5 g defatted dry milk, 5.84 g NaCl, 20 ml of 1 M Tris–HCl, pH 8.0, 980 ml of distilled water) at room temperature. After incubation with peroxidase-conjugated anti-mouse IgG antibody, the reaction products were visualized using H2O2 and 4-chloro-1-naphthol. The intensity of each reaction product on every subject was measured by photodensitometer at 590 nm, which is the maximum wavelength at which the color of the reaction products is absorbed. To study the UVB dose-dependent induction of CPDs, each sample was also immunoblotted and measured three times.
Immunohistochemical staining using TDM-2
Biopsied specimens of UV-exposed skin were fixed in 10% formalin and embedded in paraffin blocks. Sections 2 μm in thickness were placed on glass slides coated with poly-L-lysine. After deparaffinization, they were autoclaved in 10 mM citrate buffer, pH 6.0, at 90°C for 15 min. Universal LSAB Kits, Peroxidase (Dako, CA, USA) were used for immunostaining. After air drying, the specimens were incubated in 3% H2O2 for 10 min to inactivate endogenous peroxidase and then washed with distilled water and rinsed with 50 mM Tris–HCl and 0.15 M NaCl, pH 7.6 (Tris-buffered saline, TBS). Endogenous biotin was inactivated with 0.1% avidin solution. After blocking non-specific reaction with 1% bovine serum albumin (BSA) solution, each section was treated with TDM-2 (diluted at 1:1,000 with TBS/1% BSA) at room temperature for 20 min. After washing with TBS three times for 5 min each time, the specimens were incubated with biotin-conjugated anti-mouse IgG antibody at room temperature for 10 min. They were then incubated with peroxidase-conjugated streptavidin and visualized with 0.03% H2O2 and 0.05% DAB in 50 mM Tris–HCl, pH 7.6. Counterstaining was carried out using Meyer’s hematoxylin.
UVB dose-dependent induction of CPDs
Correlations between the induction of CPDsa and UVB doses
Removal of CPDs
Amounta(%) of CPDs
Mean ± SD
Mean ± SD
Immediately after irradiation
4 h later
24 h later
Each amount(%) of CPDs cited in Table 2 was corrected by calculating the amount of the CPDs before irradiation as zero and amount of immediately after irradiation as 100
Mean ± SD
Mean ± SD
Immediately after irradiation
4 h later
24 h later
Results of immunohistochemical staining of CPD using TDM-2
In the present study, we found that 4 days were required for the younger group and 7–14 days for the older group to completely remove CPDs from the entire epidermis; this was found to be true regardless of which irradiation dose (700 or 35 mJ/cm2) was administered. Statistically significant differences were found between the younger group and the older group (p<0.01 at 700 mJ/cm2, p<0.02 at 35 mJ/cm2) in the amounts of the remaining CPDs at 4 days after the irradiation. There have been many studies on the amount of time required to remove UV-induced dimers. Sutherland et al.  report that approximately 40% of UVB-induced dimers were removed after 20 min in human skin in vivo using an alkaline agarose gel method . D’Ambrosio et al. , in a study using the same methods, described above, state that 50% of pyrimidine dimers in human skin in vivo were removed in approximately 58 min and that less than 10% remained at 24 h after 200–405 nm UV irradiation. Eggset et al.  also shows that by immunohistochemical methods using polyclonal antibodies against UV-irradiated DNA, the excision repair of pyrimidine dimers in human skin in vivo was completed by 24 h after 254 nm UV irradiation. On the other, it has been reported that the photoproducts of cultured cells have a tendency to be repaired more rapidly . Freeman  reports the variation in the repair of CPDs of in the human skin epidermis in vivo. Our results also showed stronger variation in the repair (Table 2). These differences in the removal time of CPDs reported by different researchers may be caused by differences in the experimental materials and in the methods of detection.
Immunohistochemical methods are very useful in the microscopic visualization of the repair of CPDs in cells. Figures 1, 2, 3, and 4 show that the intensities of staining of epidermal positive cells decreased gradually over time in the present study; that is, all epidermal cells (from basal cells to granular cells) are able to repair CPDs. The same findings are also seen in the pictures of changes in CPDs over time present by Qin et al. . CPDs tend to disappear more quickly in the basal cell layer than in the spinous cell layer . In the present study, CPDs disappeared more quickly in the basal cell layer than in the spinous cell layer. CPDs were scarcely seen in basal cells at 24 h after irradiation in the younger group and at 4 days after irradiation in the older group, and acanthosis was also seen in the same specimens (Figs. 1d, 3d, and 4d) except 2d. These findings suggest that epidermal turnover begins after NER of CPDs in basal cells, but not in spinous cells. As the result, remnant CPDs of prickle cells are removed completely to the stratum corneum from the prickle cells by the turnover. The present immunohistochemical studies also show that the removal of CPDs from the epidermis was completed at 4 days in the younger group and at 14 days in the older group after irradiation with doses of either 35 or 700 mJ/cm2; these results were confirmed by our immunoblotting studies. Hence, it appears that aging is a more important factor than irradiation dose in the removal of CPDs from the epidermis. It is well known that amounts of p53 and p21 protein increase in the nucleus after UVB irradiation and they work to arrest the cells at G1 stage in cell cycle [17, 20]. Basal cells arrest at G1 stage until the NER of CPDs are completed. As cell replication generally begins after NER of photoproducts, epidermal turnover begin after the repair of photoproducts in basal cells. Therefore, we suggest that the turnover of the UVB-irradiated epidermis ceases for about 24 h in younger group and for 4 days in older group after irradiation. DNA synthesis in the basal cells of mouse and human skin is suppressed until 12–201 h after UV irradiation [2, 12]. The present data are nearly consistent with these findings.
To summarize, the first 24 h in younger subjects and 4 days in older subjects after irradiation is a stage of NER of CPDs in basal cells; CPDs in prickle cells are also repaired slowly compared with basal cells at this stage. After this repair, replication of the basal cells begins and epidermal turnover starts. The CPDs in prickle cells are removed by both their own NER mechanism and the turnover of the epidermis. The time needed to repair CPDs in basal cells is longer in older subjects than in younger subjects, and the incidence of skin cancer is clearly linked to UV irradiation and increases exponentially with age. The rate of removal of thymine dimers and (6–4) photoproducts in UV-irradiated human fibroblasts derived from donors of different ages has been found to decrease significantly with age . It was reported that when CPDs are left unrepaired, they interfere with proper DNA transcription and replication and induce the release of immunosuppressive cytokines, such as interleukin-10, and over time, combination of DNA damage and immunosuppression leaves the body particularly vulnerable to cutaneous malignancies. Quick repair is important . Indeed, AK lesions become more prevalent in immunosuppressed patients , and AKs in organ-transplant recipient occur significantly earlier than in non-transplant controls (54.8 vs. 70.0 years) . AK may develop on the exposed skin of older people because of a deficiency in the repair mechanism of CPDs and because of immunosuppression in aged skin.