Effect of Sunscreen Application on UV-Induced Thymine Dimers

DURING THE past 2 decades, there has been a substantial increase in the incidence of skin cancer. The annual incidence of malignant melanoma per 100 000 individuals almost tripled among American men, from 6.7 in 1973 to 19.3 in 1997, and more than doubled among American women, from 5.9 to 13.8 for the same period.¹ Skin cancer incidence is undoubtedly linked to UV irradiation,² but it is also linked to aging³ and increases exponentially independent of insolation.⁴ Nonmelanoma skin cancer accounts for 40% of all cancers diagnosed in the United States, more than 1 million cases annually.^{5 - 6} The link between UV exposure and nonmelanoma skin cancer is widely recognized, and UV irradiation appears to be the principal environmental cause of at least 65% of melanomas worldwide in fair-skinned individuals.⁷

An increase in outdoor leisure activities has led to an increase in skin exposure to UV radiation. As a result, fair-skinned individuals encounter photodamage and ultimately develop skin cancer. Several authorities have run educational campaigns that encourage the general population to limit their solar UV exposure. An important part of these campaigns has been the adoption of measures to limit sun exposure, such as sunscreen applications and protective clothing. Some studies indicate that regular use of sunscreens can prevent long-term carcinogenic effects of solar exposure such as squamous cell carcinoma and its precursor, actinic keratosis.^{8 - 10} However, other studies have challenged the concept that sunscreen use provides adequate protection against the formation of skin cancer, in particular melanoma.^{11 - 13}

Ultraviolet radiation, primarily the UV-B range (280-320 nm), is directly absorbed by DNA and leads to the formation of pyrimidine dimers, of which more than 75% are thymine dimers¹⁴ (the remainder being pyrimidine [6-4] pyrimidone photoproducts [(6-4) PPs]).^{15 - 17} Because thymine dimers are more abundant than (6-4) PPs, and their removal rate is considerably slower,^{18 - 19} thymine dimers have been postulated by some authorities to be more mutagenic.²⁰ Indeed, increased levels of thymine dimers and/or a decreased rate of removal are statistically linked to increased mutation rates and development of skin cancer.^{21 - 23} The importance of adequate DNA repair is demonstrated in patients with xeroderma pigmentosum, a family of hereditary diseases characterized by defective repair of DNA photoproducts and a more than 1000-fold increased risk of developing skin cancer.²⁴ In this context, decreased ability to repair DNA damage is associated with aging. Through an indirect method to introduce damaged reporter plasmid into cells derived from donors of different ages, an age-associated decrease in plasmid repair was observed.^{21 ,25 - 26} Also, the rate of removal of (6-4) PPs is slower in fibroblasts from older adult donors than in those from young adult donors, in part because of decreased levels of proteins that participate in DNA damage repair.¹⁹ Hence, aging alone is a risk factor for developing skin cancer.

Ultraviolet A radiation (320-400 nm) is far more prevalent than UV-B in terrestrial sunlight, but is less erythemogenic and is thought to contribute proportionately less to the development of skin cancer.^{27 - 28} Nevertheless, UV-A irradiation leads to the formation of oxygen free radicals that generate modified DNA bases, in particular 8-oxo-7,8 dihydroguanine, which has been implicated in cancer development.²⁹

Besides its damaging effect, UV irradiation causes immunosuppression (reviewed in Kripke³⁰ ), at least in part through DNA damage that weakens the cell-mediated cutaneous cancer surveillance systems and allows the development of tumors. With regard to immunosuppression, UV-B is known to contribute to weakened immune response,³⁰ but recent studies suggest that UV-A may also be a major player in this process. Ultraviolet A1 irradiation (340-400 nm) leads to decreased numbers of epidermal Langerhans cells and reduced antigen-presenting activity,^{31 - 32} inducing local immunosuppression that may contribute to the development of skin cancer.

In addition to the above, UV irradiation has been implicated in photoaging (reviewed in Gilchrest and Bohr³³ ). It induces cumulative DNA damage through random errors during DNA replication and through oxidative damage, affecting epidermal components as well as structural dermal macromolecules (reviewed in Ma et al³⁴ and Kang et al³⁵ ). Cumulative photodamage causes fine and coarse wrinkles, mottled pigmentation, sallowness, and roughness, all characteristic of chronically sun-exposed skin.

To determine the effectiveness of sun protection factor (SPF) 15 sunscreen in preventing UV-induced DNA damage, volunteers were exposed daily to 2 minimal erythema doses (MEDs) of UV radiation over 4 consecutive days. Before the irradiation, SPF 15 sunscreen was applied each time to 1 of the irradiated areas and was applied 3 of the 4 times to other areas. There was no significant difference in the level of thymine dimers between nonirradiated skin and skin consistently protected with SPF 15 sunscreen. However, omitting sunscreen application prior to even 1 of the 4 irradiations resulted in significant induction of thymine dimers. Also, more than 75% of the dimers remaining 24 hours after irradiation were removed within 48 hours after irradiation. Our study underscores the importance of using sunscreen on a daily basis and suggests that consistent use of sunscreen should markedly reduce UV-induced DNA damage that leads to photocarcinogenesis and photoaging.

A total of 24 healthy women, aged 20 to 55 years (mean age, 31 years), skin type I through IV, were enrolled after providing written informed consent. None had a history of photosensitivity or was taking any photosensitizing medication. The study was approved by the institutional review board of Boston University Medical Center.

The UV radiation source was a 150-W compact xenon arc solar simulator equipped with a 1-mm Schott WG-320 and 1-mm UG-11 filter and a dichromic mirror (Solar Light Co Inc, Philadelphia, Pa) to approximate the solar UV spectrum (290-400 nm). Spectral power distribution was measured before the study with a UV spectroradiometer (model 754; Optronics Co, Orlando, Fla). Each irradiated skin site was approximately 1 cm in diameter.

The MED for each subject was determined by exposing skin sites on the buttock area to simulated solar radiation and noting the smallest dose at which confluent pink macular erythema was observed 20 to 24 hours after irradiation. One week following the MED determination, 5 sites were randomly delineated on the opposite buttock. A commercial sunscreen preparation with SPF 15, containing the active ingredients octyl methoxycinnamate and zinc oxide, was applied homogeneously according to the manufacturer's recommendations (2 mg/cm²) to 4 of these sites. The fifth site served as the untreated control and was also not irradiated. One of the 4 irradiated sites was treated with SPF 15 daily, and to simulate intermittent product use, the remaining 3 irradiated sites were treated with sunscreen on 3 of the 4 irradiation days, skipping application on day 2, 3, or 4. Approximately 30 minutes after sunscreen application, each active site was exposed to 2 MEDs of simulated solar UV radiation. This sequence was repeated on 4 consecutive days.

Twenty-four hours after the last UV exposure, a 4-mm punch biopsy specimen was obtained from the center of each site. Specimens were fixed in 10% neutral buffered formalin for 24 hours, dehydrated, and embedded according to standard histologic protocols. Immunohistochemical studies were performed on 4-µm paraffin sections using an automated stainer and the mouse monoclonal anti–thymine dimer IgG1 antibody (Kamiya Biomedical Co, Seattle, Wash) at 1:40 dilution. The secondary antibody procedure used a prediluted biotinylated antibody followed by prediluted alkaline phosphatase–streptavidin conjugate and Fast Red stain. No counterstain was used. Because of technical problems during tissue processing, specimens from 6 subjects were removed from the study.

Stained sections were examined under ×20 magnification. The number of nuclei that displayed antibody binding was determined throughout the entire available epidermis. Epidermal area was measured by computer-image analysis as previously described,³⁶ and the number of positive nuclei per square millimeter was then calculated.

Sections from 18 volunteers were analyzed and the number of nuclei per square millimeter positive for thymine dimer immunoreactivity was recorded for the nonirradiated skin as well as for the skin treated with SPF 15 sunscreen. The statistical differences among the 5 conditions were determined (SPSS statistical package, version 10 GLS module with post hoc analysis; SPSS Inc, Chicago, Ill).

Biopsy specimens obtained from each subject 24 hours after the last irradiation were processed for thymine dimer immunoreactivity. As expected, in nonirradiated control skin, no thymine dimers were detected (Figure 1 and Figure 2). Interestingly, although most volunteers using daily SPF 15 sunscreen displayed no detectable nuclei positive for thymine dimers, 7 of the 18 volunteers showed thymine dimer formation. One of these displayed approximately 500 positive nuclei/mm², most likely due to unexpected removal or inappropriate application of the sunscreen. The mean ± SEM number of positive cells in the remaining volunteers of this group was 21 ± 7 cells/mm², and among all daily sunscreen users, 7 ± 3 cells/mm². Overall, there was no statistically significant difference in concentrations of UV-induced thymine dimers between irradiated skin samples protected by daily sunscreen application and nonirradiated controls.

Biopsy specimens from irradiated sites that were not protected by sunscreen were taken 24, 48, and 72 hours after sunscreen application was skipped (Figure 1 and Figure 2), in each case 24 hours after the fourth irradiation. The mean ± SEM number of nuclei showing positive staining for thymine dimers was highest in samples taken 24 hours after irradiation of an area not protected by sunscreen (495 ± 52 cells/mm²) (Figure 1 and Figure 2). There was a progressive decrease in the number of nuclei positive for thymine dimers within 48 hours and 72 hours after irradiation of nonprotected area (251 ± 71 and 87 ± 39 cells/mm², respectively). There was no evidence of epidermal hyperplasia in any of the sections.

If, as suspected, virtually all dimer formation resulted from the single unprotected sun exposure, these findings suggest that only half the damage present at 24 hours after irradiation is repaired by 48 hours, and only 83% of this 24-hour damage is repaired by 72 hours. The number of positive nuclei in the skin 72 hours after unprotected UV exposure was not statistically greater than in skin 24 hours after the fourth of 4 protected exposures using a sunscreen expected to block 14/15 (93%) of dimer-inducing UV radiation (P = .29). Pairwise comparisons showed significant differences in the number of thymine dimer–positive nuclei between nonirradiated skin and skin tested 24 and 48 hours after the last irradiation and between the latter two and skin protected daily with SPF 15 sunscreen. By 72 hours after unprotected UV irradiation, there was no significant difference in the number of thymine-dimer positive nuclei in irradiated vs nonirradiated skin or skin protected daily with SPF 15.

Thymine dimers are the major DNA photoproduct formed in human skin after UV exposure.³⁷ They are important because of their potential mutagenicity.³⁸ In the present study we provide histologic evidence of the protective effect of a broad-spectrum sunscreen against the formation of UV-induced thymine dimers when the sunscreen is used on a daily basis. Our study complements and expands previous studies showing that sunscreen application results in significant reduction of pyrimidine dimer and 6-4 photoproduct formation.^{39 - 40} Freeman et al³⁹ reported that immediately after UV irradiation, the number of epidermal pyrimidine dimers was 32 per 10⁷ bases in unprotected skin vs 0.8 per 10⁷ bases in skin protected with SPF 15 sunscreen. Young et al⁴⁰ found that the use of SPF 4 sunscreen followed by exposure to 4 MEDs of UV radiation gave rise to the same number of DNA photoproducts as no sunscreen followed by UV irradiation with 1 MED, suggesting that the DNA protective capacity of the preparation is analogous to its SPF. This is not surprising because the action spectra for erythema and thymine dimer formation are nearly identical.^{27 - 28} Daily use of SPF 15 sunscreen has been reported to reduce the formation of sunburn cells and the degree of inflammation and abrogate the UV effect on Langerhans cells.⁴¹

The present study is the first to examine the protective effect of daily SPF 15 application on thymine dimer formation after repeated daily UV irradiations. The findings indicate that daily application of SPF 15 sunscreen to human skin at the manufacturer's recommended concentration of 2 mg/cm² followed by exposure to simulated solar light results in practically complete protection against the deleterious effects of UV irradiation as measured by the formation of thymine dimers. Gratifyingly, excluding 1 subject who displayed no protection, we found that the SPF 15 sunscreen seemed to prevent all but 1.4% (7/495) of positive nuclei.

It is not clear why 7 of 18 subjects using daily SPF 15 sunscreen showed thymine dimer formation. It is possible that part of the sunscreen (or all of the sunscreen in 1 subject) was washed away from these areas during the 30 minutes before UV exposure. Nevertheless, thymine dimer formation even in these areas was low, and the average number of positive nuclei was 7/mm² for the entire group, excluding the subject who displayed no protection. Statistically, there was no difference between skin protected with daily sunscreen application and nonirradiated skin. That daily application of broad-spectrum SPF 15 sunscreen offers this degree of protection against UV-B–induced DNA damage is consistent with the known virtual identity between the action spectra for erythema and thymine dimer formation in human skin.^{27 - 28}

It has been suggested that stratum corneum hyperplasia after chronic UV irradiation may produce a shielding effect on the epidermis.^{42 - 44} In the present 5-day study, there was no detectable hyperplasia of the stratum corneum or the epidermis in any of the histologic sections; hence, this did not contribute to the observed UV protection.

The present results show that sunscreen is effective within 30 minutes of application, as determined during formal SPF treating,⁴⁵ but provides hardly any protection if applied 24 hours earlier, at least when no precautions are taken to prevent loss by washing or other means. In the past decade, controversy has arisen over the use of sunscreens, thereby challenging the concept of sunscreen use for protection against the development of skin cancer. Some epidemiologic studies relying on subject recall found an association between sunscreen use and increased risk of melanoma.^{11 - 13} However, those results have been challenged on the basis of study design. Other epidemiologic studies provide strong evidence for a critical role of sunburn in childhood in the pathogenesis of malignant melanoma.^{46 - 55} More recently, Gallagher et al⁵⁶ found that regular use of SPF 30 sunscreen in children over a 3-year period was associated with a decrease in nevus count and reduced development of new nevi. Because a large number of nevi is considered a risk factor for melanoma development,⁵⁷ the study suggests that sunscreen application is protective. A possible cause for the reported association between sunscreen use and increased risk of melanoma is longer duration of sun exposure by people who use high SPF sunscreens.⁵⁸

The ability of sunscreen to prevent UV-induced damage to the extent implied by its indicated SPF depends on its application in sufficient quantities. The international consensus and Food and Drug Administration recommendations state that the amount of sunscreen to be applied is 2 mg/cm², as in the present study. However, a Danish study found that sunbathers applied only about 25% of the amount of sunscreen needed to give the stated SPF,⁵⁹ and in a recent multicenter European study it was found that students during their summer vacation applied approximately 20% of the required quantity.⁶⁰ If half of the recommended amount of an SPF 15 sunscreen is applied, the SPF factor falls to 5.⁶¹ To achieve the advertised SPF, a typical adult should apply an ounce (30 mL) per application for whole-body coverage. With reapplication, a 4-oz bottle would not last long on a sunny day at the beach.⁶² It is thus likely that insufficient sunscreen application combined with ample sun exposure, rather than inherent limitations of sunscreens, accounts for the failure of sunscreen use to impact the skin cancer epidemic.

Ultraviolet irradiation induces DNA damage (photoproducts) that, if not appropriately repaired, may lead to mutations.³⁸ These mutated cells may progress to become cancerous. In the present study, we demonstrate that daily application of a broad-spectrum sunscreen greatly reduces photoproduct formation.

Although we have not examined the formation of the less prevalent (6-4) PPs,³⁷ thymine dimers and (6-4) PPs have similar action spectra,⁶³ and we assumed that the sunscreen also protected against formation of the latter. Nonetheless, further experiments using anti–(6-4) PPs are required to prove this point. Together, thymine dimers and (6-4) PPs constitute the overwhelming majority of UV-B–induced DNA damage.⁶³

The rate of repair of thymine dimers in human epidermis in vivo has been studied by several groups. Xu et al⁶⁴ reported substantial interdonor repair rate variation (25%-46% within the first 24 hours and 54%-80% within 48 hours after 40 mJ/cm² of simulated solar irradiation) as measured by the presence of cyclobutane pyrimidine dimers, specifically thymine dimers and thymine cytosine dimers. It should be noted that thymine dimers constitute most of the cyclobutane pyrimidine dimers⁶³ that form after UV irradiation. Furthermore, dividing the irradiated subjects into 2 age groups (<50 years vs ≥50 years), the authors reported a significant decrease in the percentage of repaired thymine dimers in the older age group 24 hours after irradiation. Bykov et al⁶⁵ showed that 50% of cyclobutane pyrimidine dimers are removed within less than 18 hours after a 2-MED dose of simulated solar irradiation. In another study using immunohistochemical analysis, Young et al⁶⁶ reported a thymine dimer half-life of 33 hours after exposure to 290 to 400 nm. However, it is important to remember that the technique of immunohistochemical staining most likely does not detect every dimer but produces positive findings only when a threshold number of dimers have accumulated in the nucleus. Hence, the actual burden of thymine dimers in the epidermis is most likely higher than that recorded by this detection method.

Because the level of thymine dimers was not tested immediately after irradiation in the present study, it is likely that it varied among the different volunteers. Although the initial level of thymine dimers would have an impact on the rate of repair, our study design allows examination of removal rate for thymine dimers beyond the first 24 hours after irradiation, a period critical for mutation because of resumed DNA replication. Several in vitro studies using different UV irradiation doses have reported cell cycle arrest for 24 to 72 hours, depending on the UV dose, cell type, and culture conditions.^{67 - 72} Irradiation of mouse epidermis (100 mJ/cm²) was associated with suppression of scheduled DNA synthesis for 24 hours.⁷³ Using Ki-67 as a measure for cell proliferation, Ueda et al⁷⁴ reported proliferation of lower epidermal cells in human skin 48 to 72 hours after irradiation, suggesting cell cycle arrest for that period. However, even if cells are arrested in vivo for as long as 72 hours after irradiation, our data suggest that there are ample remaining photoproducts that might give rise to mutations.

In summary, this is the first study to provide in vivo evidence that sufficient sunscreen application is protective against the formation of UV-B–induced DNA damage after repeated UV irradiations. Our study strongly supports the concept that regular sunscreen use reduces the risk of developing skin cancer and other manifestations of photodamage.

Accepted for publication July 16, 2002.

This work was supported by a grant from Procter & Gamble, Cincinnati, Ohio (Dr Phillips).

Corresponding author: Barbara A. Gilchrest, MD, Boston University School of Medicine, Department of Dermatology, 609 Albany St, Boston, MA 02118 (e-mail: bgilchre@bu.edu).