Sunscreens by definition are topical preparations that reduce penetration of damaging solar ultraviolet (UV) wavelengths. Ideally, sunscreens should prevent acute and chronic actinic damage of sun exposure by delaying photoaging and preventing carcinomas and even melanoma induction.
Sunscreen preparations are generally assessed for their ability to protect against erythema formation. Their sun protection factor (SPF) indicates the number of minimal erythema doses a given individual can tolerate before an erythema occurs on the protected site. As the relationship of erythema to skin cancer induction is not obvious, it was recently suggested that other earlier end points are perhaps more accurate predictors of sunscreen effectiveness from cancer (Ananthaswamy et al, 1999). In this respect, mutation of the p53 tumor suppressor gene was proposed as a short-term marker for skin cancer development as it takes 1 mo to occur. It was also stated by the same authors that the inhibition of p53 mutation by sunscreens is most probably due to their protection against UV-induced DNA damage. This indeed seems logical as p53 mutations occur most probably at pyrimidine dimer sites. We have previously shown, when using a highly sensitive immunohistochemical technique, that both pyrimidine dimers and p53 are induced in human skin following UVB and UVA radiations (Applegate et al, 1997) even following suberythemal doses (Hall et al, 1993;Barnadas et al, 1996). Pyrimidine dimers alter the biologic functions of DNA and they have been shown to be a major cause of lethal and mutagenic (Harm, 1976), tumorigenic (Hart et al, 1977), and transformational (Sutherland et al, 1985) events. More specifically in human and other mammalian skin, pyrimidine dimers have been shown to be involved in many, thought to be detrimental, pathways including erythema (van Weelden, 1984;Ley, 1985), edema, keratinocyte damage (sunburn cell formation) (Ley and Applegate, 1985;1987), suppressed immunity (Applegate et al, 1989), and tumor formation (Ley et al, 1991;VandeBerg et al, 1994). These lesions have readily been shown to be present using immunohistochemistry techniques with peak protein expression at 18–24 h. Interestingly, many of the lesions remain at 48 h and even up to several weeks post-UV (Young et al, 1996;Applegate et al, 1997;Ahmed et al, 1999). Therefore, it is well established that UV radiation induces DNA damage, which has been shown to be responsible for a variety of negative effects in human skin.
Another lesion that can be induced by the generation of reactive oxygen species from ultraviolet radiation is the mutation-prone DNA base-modified product 8-hydroxy-2'-deoxyguanosine (8-OHdG). These lesions can be readily induced in human skin following UVB radiation and are still present in part 24 h postirradiation (Ahmed et al, 1999). We have also seen that high levels of 8-OHdG can be induced by UVA I and UVA I + II irradiation of human skin (unpublished results). Transversions of GC to TA have been demonstrated in the p53 and ras genes in UVB-induced skin tumors in human nonmelanoma skin cancer (Van der Schroeff et al, 1990). A high percentage of these GC to TA transversions in human skin tumors have been associated with the formation of 8-OHdG by UVA-generated reactive oxygen species (Daya-Grosjean et al, 1995).
By assessing the initial damage of DNA such as pyrimidine dimer formation (CC
TT) along with the expression of p53 protein and 8-OHdG following UV irradiation in sunscreen-protected human skin, it would be possible to provide sensitive biologic endpoints that could be used to evaluate sunscreen formulations (efficiency or possible photosensitization) targeted for UVB alone or for total block protection of UV in human skin.
Materials and methods
Volunteers
A total of 28 healthy volunteers between the ages of 20 and 45 y with skin types II-III were used in these studies. Delimited areas of the buttocks were subjected to 10 increasing doses of UV radiation and the erythema and melanin reactions were measured at 24 h post-UV on each site along with two control unirradiated sites (Dermaspectrometer). In eight volunteers where sunscreen preparations were used, both UVB and UVB + UVA sunscreens were applied to delimited sites (2 mg per cm2) at least 15 min prior to UV exposure. Biopsies of 4 mm were taken under local anesthesia (1% lidocaine) 24 h after the UV exposure to ½ and 1 minimal erythema dose (MED) and from an adjacent nonirradiated control site. A total of six biopsies were taken from each of the 28 volunteers as approved by the Hospital Ethics Committee.
For frozen sections, one half of each biopsy was immediately placed in a beaker with isopentane-2-methyl-butane chilled in liquid nitrogen for 2 min. Thereafter, the tissue was transferred to liquid nitrogen for storage until serial frozen sections of 5 
m could be prepared. The other half of each biopsy was placed in formalin for fixation and paraffin embedded for subsequent 5 m serial sections.
Radiation sources and exposure conditions
A 15S single port Solar UV Simulator filtered with Schott WG320/1 mm and UG 11/1 mm filters (290–400 nm) emitting energy similar to that of the overhead sun up to 400 nm was used at a dose rate of approximately 108 MED per h, extrapolated by the manufacturer to be 13–16 W per m2 (Solar Light, Philadelphia, PA) as indicated by the manufacturer. Radiation exposure (intensity and dose) was continuously controlled by a Dose Control System (Solar Light) directly attached to the 15S Solar Simulator, which automatically operates a shutter system that closes for a preset dose. Irradiation periods were of approximately 30–60 s duration (without sunscreen preparations) to obtain an MED on type II-III skin.
Sunscreen preparations
Sunscreens with SPF 15 for UVB only or UVB + UVA absorbers were provided by Givaudan (Geneva) and are similar to many formulations that are available on the market today. Oil in water emulsions containing Parsol MCX (8%) and Eusolex (2%) were used for the UVB only preparation and Uvinul (9%), Eusolex (0.3%), Mexoryl (0.7%) and Parsol 1789 (3%) for the UVB + UVA sunscreen preparation.
Immunohistochemistry
Frozen sections (pyrimidine dimers) and fixed tissue sections (p53 and 8-OHdG) of 5 m thickness were used for the immunohistochemistry. All incubations were done in a humidified chamber in the dark unless otherwise specified.
For detection of pyrimidine dimers, frozen sections were subjected to a mild alkaline hydrolysis (0.7 N NaOH for 4 min) followed by digestion with proteinase K (1 g per ml for 10 min).
For detection of 8-OHdG, tissue sections were treated with a Target Retrieval Solution (Dako, Zug, Switzerland) in a 95°C water bath followed by a 20 min cooling period and all washings were with Tris-buffered saline with Tween (TBST) at 95°C.
For both frozen and fixed tissue, the sections were fixed with 4% paraformaldehyde for 30 min at 25°C and washed with phosphate-buffered saline (PBS)/TBST three times for 10 min each. Tissue sections were incubated with 0.1% phenylhydrazine in PBS for 60 min at 37°C to block endogenous peroxidases and washed twice for 5 min each. This blocking step was accomplished with the Dako blocking system for 8-OHdG and was followed by an amplification with a Dako catalyzed signal amplification system as suggested by the supplier. The antibody was from the Japan Institute for the Control of Aging and was diluted at 1:20,0000 with Dako antibody diluent with background-reducing components incorporated.
For p53 and dimers, nonspecific binding was blocked by an incubation for 2 h at 25°C with a solution of PBS containing 5% fetal bovine serum (FBS), 7% normal goat serum (NGS), and 0.1% Triton X 100. Tissue sections were then incubated overnight at 4°C with H3 monoclonal specific antibodies against thymine dimers (provided by Dr. Len Roza, TNO, The Netherlands) at a 1:50 dilution in PBS containing 5% FBS, 5% NGS, and 0.1% Triton X 100, or for 30 min with DO7 antibodies against wildtype p53 protein (Dako). For control samples, the primary antibodies were replaced with a nonspecific IgG antibody. Immediately following this incubation (p53) or the following morning (pyrimidine dimers), tissue sections were washed three times for 10 min each in PBS and treated with biotinylated goat antirabbit antibody at 1:200 in a solution of PBS with 5% FBS, 1% NGS, and 0.1% Triton X 100 for 3 h at 25°C. Tissue sections were washed four times for 5 min each in PBS and then treated with Vectastain ABC (Vector, Burlingame, CA) as indicated by the company for 3 h at 25°C. After this incubation, tissue sections were washed three times for 10 min each in PBS and treated with 0.5 mg per ml 3,3'-diaminobenzidine with 0.32 l of 30% H2O2 added just before an incubation of 1–2 min. All samples were treated at the same time. Control samples were included with each experiment, using negative control reagents that were routinely applied to histologic sections of each control and test specimen to avoid nonspecific interpretation. The primary antibody was replaced with an isotype IgG. The antibody staining for thymine dimers, p53, or 8-OHdG is represented by the brown coloration. The samples were washed for 5 min under running water. They were counterstained with Papanicolaou (Harris's Haematoxylin solution), dehydrated, and mounted with Merckoglas (Merck, Switzerland).
Video image analysis of immunohistochemical treated skin
Sections stained for each antibody were analyzed with a Leica DC100 video imaging system (offered by SPIRIG, Switzerland). For each experiment, four histologic sections were stained by each antibody and used only on analysis and sections without counterstaining. Six regions from each section were selected and the intensity of staining for a total area was measured and quantified by the analysis program designed for these studies. The frank quantification of dimers and 8-OHdG was not determined because of the treatments necessary to permeabilize cells for staining. The antigen retrieval can damage the normal histology of the section and more frequent holes are seen in the tissue sections than in paraffin-embedded samples. Therefore we semiquantified these results from visual assessment on a scale of – (no to very little staining) to ++++ for increasing staining intensity with the aid of video image analysis. We could successfully quantify p53 from paraffin-embedded tissues and stained nuclei were counted for a total of 1000 cells. Results are reported as the number of p53-positive nuclei for 1000 cells.
Results
Erythema induction and protection by sunscreens
In all 28 individuals, there was a dose-dependent increase in erythema and the tested MED varied from 20 to 35 mJ per cm2 Figure 1. Both UVB and UVB + UVA sunscreen preparations (SPF 15) were able to protect against erythema induction in all individuals irradiated with 7.5 MED and in five of the eight individuals following 15 MED. Three individuals had a slight, uneven erythema in the 15 MED irradiated sites (one in the UVB alone group and two in the UVB + UVA sunscreen group).
Figure 1.
Induction of erythema in UV-irradiated human skin. Erythema was measured by a dermaspectrometer on irradiated buttock sites of 28 individuals. The erythema was reported in arbitrary units once the background coloration was subtracted for each individual. Points are an average of 10 irradiation sites on each of 28 individuals with the associated standard deviation of the mean.
Full figure and legend (61K)Pyrimidine dimer, p53, and 8-OHdG expression in human skin following UV irradiation
Eight individuals were irradiated with the Solar Light Simulator at doses of 0, ½, and 1 MED (four for pyrimidine dimers and four for p53 and 8-OHdG). In all individuals, there was no expression of pyrimidine dimers in nonirradiated skin. Following increasing doses of UV radiation there was a concomitant augmentation of pyrimidine dimer induction Figure 2. Even following suberythemal UV doses, approximately 25%-45% of the epidermal cells showed staining for pyrimidine dimers in any given individual tested.
Figure 2.
Induction of pyrimidine dimers, p53, and 8-OHdG in UV-irradiated human skin in situ. Pyrimidine dimers, p53, and 8-OHdG expression were assessed by enhanced avidin-biotin immunohistochemical methods in skin irradiated with 0, ½, and 1 MED of solar simulator radiation (Solar Light Simulator) and in skin treated with UVB or UVB + UVA sunscreen preparations and exposed to 7.5 and 15 times the MED of each individual. Scale bar: 40 
m.
For p53, all unirradiated biopsies from individuals showed an occasional one to four stained nuclei for 1000 cells and likewise, as with pyrimidine dimer induction, p53 stained epidermal cells increased concomitantly with higher doses of UV radiation Figure 2. Suberythemal doses of UV also showed already significant increases in p53 epidermal staining Figure 2. All individuals showed some staining for 8-OHdG in normal skin and staining of epidermal cells increased in a dose-dependent manner following UV radiation. Maximal levels of 8-OHdG were seen at ½ MED doses whereas there was a continued increase in nuclear staining for both p53 and dimers at 1 MED.
Differential protection of UVB and UVB + UVA sunscreen preparations against pyrimidine dimers, p53, and 8-OHdG expression in UV- irradiated human skin
Both sunscreen preparations showed good protection against erythema even after the 15 MED exposure and there was measurable damage in all protected skin for pyrimidine dimers, p53, and 8-OHdG.
The preparations were effective in reducing the number of cells stained for pyrimidine dimers and 8-OHdG to a level of damage similar to that induced by ½ to 1 MED with no protection (Table I, Figure 3). The quantification of dimers and of 8-OHdG is rendered difficult because of the treatment of the tissue to have access to the antibody sites. The UVB and UVB + UVA sunscreen protected skin revealed damage comparable to that observed in nonprotected skin irradiated with ½ to 1 MED for 7.5 and 15 MED, respectively Figure 3.
Figure 3.
Quantity of p53 protein induction in UV-irradiated human skin and the degree of protection provided by UVB and UVB + UVA sunscreens. p53 expression was assessed by enhanced avidin-biotin immunohistochemical methods in biopsies at 24 h in skin exposed to 0, ½, and 1 MED and in sunscreen-protected skin (UVB alone and UVB + UVA cream preparations) following 7.5 and 15 MED from the Solar Light Simulator (dose ½ and 1, respectively). Expression of p53 in human skin assessed by the number of p53-positive cells for 1000 cells counted as a function of dose of UV radiation from the Solar Light and Oriel Solar Simulators. Data points are an average of eight individuals for no protection (
) and for the sunscreen preparations UVB alone sunscreen (
) and UVB + UVA sunscreen (
) with associated standard deviations of the mean.
Table I - Induction of pyrimidine dimers, p53, and 8-OHdG in human skin by UV radiation and the protection provided by topical sunscreens (UVB only versus UVB + UVA formulations).
Full table Skin samples stained for p53 could be quantified using the video imaging analysis microscope. A total of 1000 cells were counted for each biopsy and the number of p53-positive nuclei was assessed. There was a dose-dependent increase in p53 staining. The UVB sunscreen preparation showed protection of damage that would normally be induced by 1 MED without protection Figure 3. The UVB + UVA preparation showed protection against p53 induction following 7.5 and 15 MED doses of UV where damage was similar to nonprotected sites at 1 MED.
Discussion
Skin carcinogenesis depends strongly on DNA repair systems available to excise DNA lesions. Excision repair can remove the pyrimidine dimer and some adjacent nucleotides, and the integrity of the DNA molecule is restored through repair synthesis. Excision repair has been reported to occur in the epidermis of marsupials and humans whereas mouse and rat cells have no or markedly lower rates of excision repair (Ley et al, 1977;Vijg et al, 1984;Applegate and Ley, 1987). This perhaps is an important aspect when using the mouse model for carcinogenesis (Ananthaswamy et al, 1997) or assessing chronically accumulated damage in rodent tissue.
In general, it is agreed that the initial step in UV carcinogenesis is damage to the DNA molecule and this can be assessed to a high degree of sensitivity in human skin in vivo. Pyrimidine dimers, p53, and 8-OHdG have been shown to be induced by UVA I, UVA I + II, and solar simulating radiation and therefore are interesting markers for assessing protective measures in human skin.
Protection against acute sunburn is well substantiated by extensive human experience. The development of other short-term markers for SPF testing are those shown to be involved in carcinogenesis (as this endpoint would be impossible to test in humans). Several studies in human skin show that sunscreens protect against DNA damage in UV-exposed human skin by reducing pyrimidine dimers and 6–4 photoproducts and also the induction of p53 (Freeman et al, 1988;Van Praag et al, 1993;Ponten et al, 1995;Ananthaswamy et al, 1997;Bykov et al, 1998). In addition, markers for photo-immunosuppression and photoaging have also been tested with various protocols and with various degrees of success. Unfortunately, many of the testing procedures did not test an SPF 15 cream with 15 times the dose of UV to induce an erythema and therefore the full possibilities of protection were not truly exploited (Beasley et al, 1998;Berne et al, 1998).
We have chosen to assess several lesions detectable in DNA following oxidative stress that have been induced following both UVB and UVA radiation in human skin. The observed inferior protection in human skin obtained with the UVB + UVA sunscreen compared to that with the UVB sunscreen is interesting. Whether this is due to some chemical interaction causing a sensitivity increase or the possibility that UVA radiation enhances the protection capacity of human skin is not known. Photosensitivity of many sunscreen components is well known (Kaidbey and Kligman, 1978;Sutherland and Griffen, 1984;Thune, 1984) and this important possibility cannot be eliminated at present. It has also been shown, however, that UVA radiation and/or visible light can attenuate certain damage induced by UVB in human skin. It could therefore be possible that, because these wavelengths are prevented with sunscreen protection, more damage is permitted in human skin. This possibility is supported by several studies where UVB-induced erythema in human skin was decreased by a combination of UVA radiation (van Weelden et al, 1984); moreover, UVB-induced thymine dimers were decreased when human skin was exposed to UVA and/or visible light (Sutherland et al, 1980;D'Ambrosio et al, 1981;Eggset et al, 1983;Roza et al, 1991).
In our study, we have seen that high levels of DNA damage can be induced even without the first warning of an erythema present. Does this mean that sunscreens offer false safety? They do prevent actinic erythema but they do not seem to prevent all effects of UV. DNA lesions can be induced at very high rates even in protected skin and these lesions represent an initial indicator for carcinogenic pathways. New generations of sunscreens are available (i.e., Tinosorb M, methylene-bis-benzotriazolyl-tetramethyl-butylphenol; and Tinosorb S, bis-thylhexyloxyphenol-methoxyphenyl-triazine) that cover both the UVB and UVA spectrum with just one active ingredient. New technologies for both chemical and physical protection should aim to eliminate direct and oxidative DNA damage.
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Acknowledgments
The authors wish to thank Franca Labidi and Simone Pradervand for help in tissue sectioning. These studies were funded by grants from the Swiss National Fund (FNS 31.49120.96) and the Swiss League Against Cancer (KFS-695–7-1998). We also wish to thank SP1R1G for the generous gift of the video analyzer.
