DNA-protection by sunscreens: p53-immunostaining

ARTICLE

Skin cancer has become a serious health problem for Caucasians all over the world. The most common forms of non-melanoma skin cancer (NMSC), i.e. basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), have an incidence almost as frequent as all other cancers combined [1]. In the development of NMSC, UV light is a well-documented and accepted factor [2]. Therefore, to help bring the increasing numbers under control, prevention of extensive sun exposure has become an important item.

Ultraviolet B (UVB, 280-315 nm), causes not only a suntan but also sunburn, photoageing and skin cancer induction. Ultraviolet A (UVA, 315-400 nm), is associated with the immediate tanning reaction, photoageing, and tumour promotion. The molecular mechanisms of these UV-induced effects are not completely understood. However, there is evidence that DNA-damage is involved in many biological effects of UV radiation.

It is known that UV radiation induces structural changes in cellular DNA, leading to an altered expression of oncogenes and tumour suppressor genes, such as p53 [3, 4]. NMSC shows a high incidence of mutations in this gene [2]. Wild-type p53 is known to block the cell-cycle, in order to allow DNA repair before its duplication. Therefore this protein is also called the "guardian of the genome" [5]. The induction of detectable levels of wild-type p53 in human epidermis after UV exposure is likely to be relevant in the prevention of skin carcinogenesis [6].

Wild-type p53 becomes immunohistochemically detectable in the epidermis and in superficial dermal fibroblasts of normal human skin after exposure to doses of UV radiation that induce mild sunburn [7]. Solar-simulated light, composed of UVA and UVB, induces a rapid upregulation of p53 expression, starting within 4 h of mild UV exposure, with a peak at 24-48 h and a fall to undetectable levels by 360 h. This mechanism of wild-type p53 induction by UV light probably relates to post-translational stabilization [8].

The pattern of the p53 staining reaction in human skin was shown to be wavelength dependent [9]: UVA gave predominantly staining in the basal cell layer, while UVB resulted in staining throughout the epidermis. Furthermore, it was found that the erythemal response and p53 protein expression were not occurring concomitantly: UVB doses that did not cause erythema, resulted in a significant increase of p53 protein levels in human epidermal cells [10].

The use of high sun protection factor sunscreens, assessed by their ability to inhibit UV-induced erythema, is advocated to reduce the skin cancer risk [11]. Many studies of sunscreens, however, report that sunscreens that completely prevented erythema were either ineffective or only partially effective in preventing UV-induced immunosuppression both in humans and in mouse models [12]. Other studies suggest that complete immunoprotection will be achieved only by using a higher sun protection factor than is necessary for the prevention of erythema [13].

So far, only the protective effect for erythema has been semi-quantitatively indicated for the different sunscreens, by the introduction of a sun protection factor (SPF), while a measure for their potential to prevent skin cancer, i.e. epidermal DNA protection or immunosuppression, has not yet been established. The SPF is defined as the UV energy needed to produce minimal erythema (the minimal erythemal dose; MED) in sunscreen protected skin, divided by the MED of unprotected skin.

The aim of this study was to investigate to what extent commercially available sunscreens provide DNA-protection, as measured by means of p53 expression.

Materials and methods

Twenty-five healthy volunteers entered this study, which was approved by the local Ethics Committee. Informed written consent was obtained from all participants. All volunteers (ages 21 to 35; 15 female, 10 male) were Caucasian; 9 with skin type 1; 9 with skin type 2; and 7 with skin type 3.

Investigations were carried out on the buttocks or lower back. For at least two weeks prior to the experiment, these areas were not exposed to UV light (artificial or natural). Three-millimetre punch biopsy specimens were taken under local anaesthesia from sunscreen protected and unprotected skin, before (controls) and 24 h after sun exposure, and were immediately snap-frozen in liquid nitrogen and stored at ­ 70° C.

Immediately before UV exposure, 2 skin areas were covered with a sunscreen. The two sunscreens (Zwitsal SPF 10, containing octyl methoxycinnemate and Zwitsal SPF 20, containing titanium dioxide) provided by Kortman-Intradal, Den Haag, The Netherlands, were applied at a dose rate of 2 mg/cm², each to one of these two skin areas. Another skin area was exposed to sunshine, without sunscreen protection.

The sunshine exposure time was 1.5 h for twenty-one volunteers. Four volunteers with skin type 1 were exposed for only 1 h. Twenty-four hours after UV exposure, punch biopsies were taken from the sunscreen-protected areas, as well as from the unprotected skin.

UV irradiation conditions

All spectral UV-measurements were performed on location at Scheveningen beach with a Brewer MKIII (double monochromator) by the Royal Dutch Meteorological Institute, between 12:15 and 15:15, local time (local solar noon: 13:40 local time). The sunlight exposure started at 13:00 and lasted until 14:30. The temperature reached a maximum of 33° C.

All measured UV-spectra (from 286.5 to 365 nm, 0.5 nm increment) were weighted with the CIE or McKinlay-Diffey action spectrum [14] and the so-called damaging UV (DUV) in mJ^{­ 2}s^{­ 1} was obtained. The amount of DUV for each spectrum is given in Table I.

Immunohistochemistry

Serial, 5-µm thick sections were cut from the frozen skin samples, using a cryostat at ­ 30° C. The frozen sections were air dried overnight and fixed by dipping in acetone at room temperature. The sections were incubated with 0.3% H₂O₂ in phosphate buffered saline (PBS), pH 7.2, for 20 min to block the endogenous peroxidase activity. The frozen sections were then incubated with a primary p53 antibody for 1 h at room temperature. As primary p53 antibodies, we used DO7, a mouse monoclonal antibody against recombinant human p53 (Novocastra Laboratories Ltd. Newcastle, UK) and the mouse monoclonal antibody Bp53 (BioGenex, San Ramon, CA), diluted according to the manufacturer's instructions. Nonspecific binding of antibodies was blocked by incubation with 5% normal goat serum. A rabbit peroxidase-conjugated, anti-mouse secondary antibody was used (DAKO A/S, Glostrup, Denmark). The peroxidase reaction was developed using 3-3'-diaminobenzidine as a substrate for 8 min. In negative controls the primary antibodies were omitted.

The evaluation of the staining patterns was as follows: the staining intensity of 200 epidermal nuclei per biopsy was assessed by two independent observers and scored as ­ (no staining); + (weak staining); ++ (moderate staining); and +++ (dark staining).

Results

Twenty-hours after sun exposure, distinct erythema was observed in the unprotected skin of 19 of the 25 volunteers (9 with skin type 1, 8 with skin type 2 and 2 with skin type 3), while the skin sites pretreated with a sunscreen showed no erythema.

P53 immunoreactivity patterns of the biopsy specimens are summarized in Table II and Figure 1. Examples of the staining reactions are depicted in Figure 2.

The non-exposed skin biopsies showed only weak p53 expression, in a few scattered cells in the basal cell compartment (Fig. 1a; Fig. 2A). On the other hand the unprotected, sun-exposed sites of all 25 volunteers, including the six cases without visible skin reddening, showed a variable p53 immunostaining in up to approximately 50% of the epidermal cells, with an average of 25% p53-positive epidermal cells. Intense staining activity was seen in the basal and suprabasal cells (Fig. 1b; Fig. 2B) and a decreasing fraction of positive cells towards the skin surface. p53 expression in the sunscreen protected areas was comparable to our observations in non-exposed skin (Fig. 1c and d, Fig. 2C).

It is evident from Table II, that the fraction of p53-positive nuclei was significantly increased in the unprotected cells after sun exposure, as compared to the non-exposed skin.

The sunscreens provided highly significant protection as concluded from the fact that p53 levels were not elevated as compared to the control.

Discussion

Sunlight is a carcinogen to which everyone is exposed. Its UV component is the major epidemiological risk factor for NMSC. Of the multiple steps in tumor progression, those that are sunlight-related, would be revealed if they contained mutations specific to UV. Involvement of UV light in mutations in the p53 tumour suppressor gene is indicated by the presence of a CC –>TT double base change, which is only known to be induced by UV. These UV-like p53 mutations have been shown in NMSC [2, 4].

Hall et al. [7] have shown earlier, an expression of wild-type p53 in the basal layer of the epidermis after exposure to doses of UV irradiation that induced mild sunburn. Thereafter Campbell et al. [9], showed that this p53 expression in the epidermis is wavelength specific. Recently, Pontén et al. [15] studied a group of 5 volunteers with skin type 2-3 under solar simulation and showed that application of a sunscreen to the epidermis largely prevented p53 expression. We have elaborated on these findings by examining the DNA protective potential of sunscreens in twenty-five volunteers with skin types 1-3 under natural sunshine conditions.

In this study, we show that a brief exposure to natural sunlight causes an increase in p53 expression in the epidermis of Caucasians. No significant differences in p53 expression could be found between the various skin types. There was no increase in p53 expression with the two tested sunscreens (Zwitsal SPF 10 and Zwitsal SPF 20), i.e. they have a protective effect with respect to UV-induced DNA damage. From the fact that in the unprotected and UV-exposed areas of six volunteers, high levels of p53 were found, without erythema, we conclude that these two phenomena are independent and that p53-expression is a more sensitive marker of UV-induced epidermal damage. In all the cases (19) where erythema was visible after unprotected UV-exposure, increased p53-expression was found as well.

There are several indications that the acute erythema response following UV irradiation is independent of the p53 response. For example, erythema involves, release of arachidonic acid from the cell membrane and its subsequent catabolism in the cyclo-oxygenase pathway [16]. Indomethacin, a potent inhibitor of cyclo-oxygenase, has been shown to cause significant suppression of acute phase erythema, but fails to alter UVB induction of p53 protein [17].

Based on the foregoing, we suggest expressing the DNA-protective properties of sunscreens as the degree to which they prevent wild-type p53 expression. So far, the studies that applied this parameter have chosen a semi-quantitative approach, but in the future, flow cytometric analysis of p53 expression in the epidermis could provide a more accurate estimate of sunscreen effectiveness.

REFERENCES

1. Miller DL, Weinstock MA. Nonmelanoma skin cancer in the United States: incidence. J Am Acad Dermatol 1994; 30: 774-8.

2. Brash DE, Rudolph JA, Simon JA, et al. A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma. Proc Natl Acad Sci USA 1991; 88: 10124-8.

3. Tornaletti S, Pfeifer GP. Slow repair of pyrimidine dimers at p53 mutation hotspots in skin cancer. Science 1994; 263: 1436-8.

4. Ziegler A, Jonason AS, Leffel DJ, et al. Sunburn and p53 in the onset of skin cancer. Nature 1994; 372: 773-6.

5. Lane DP. P53, guardian of the genome. Nature 1992; 358: 15-6.

6. Kamp A. Sun protection factor p53. Nature 1994; 372: 730-1.

7. Hall PA, McKee PH, du Menage P, et al. High levels of p53 protein in UV-irradiated normal human skin. Oncogene 1993; 8: 203-7.

8. Fritsche M, Haessler C, Brandner G. Induction of nuclear accumulation of the tumor-suppressor protein p53 by DNA-damaging agents. Oncogene 1993; 8: 307-18.

9. Campbell C, Quinn AG, Angus B, et al. Wavelength specific patterns of p53 induction in human skin following exposure to UV radiation. Cancer Res 1993; 53: 2697-9.

10. Healy E, Reynolds NJ, Smith MD, et al. Dissociation of erythema and p53 protein expression in human skin following UVB irradiation, and induction of p53 protein and mRNA following application of skin irritants. J Invest Dermatol 1994; 103: 493-9.

11. Mackie RM, Elwood JM, Hawk JLM. Links between exposure to ultraviolet radiation and skin cancer. J R Coll Physicians Lond 1987; 21: 91-6.

12. Young AR, Walker SL. Photoprotection from UVR-induced immunosuppression. In: Krutmann J, Elmets CA, eds. Photoimmunology. London: Blackwell Science Ltd, 1995: 285-97.

13. Walker SL, Young AR. Sunscreens offer the same UVB protection factors for inflammation and immunosuppression in the mouse. J Invest Dermatol 1997; 108: 133-8.

14. McKinlay AF, Diffey BL. A reference action spectrum for ultra-violet induced erythema in human skin. In: Passchier WF, Bosnajakovid BFM, eds. Human exposure to ultraviolet radiation: risks and regulations. Elsevier, 1987: 83-7.

15. Pontén F, Berne B, Ren Z-P. Ultraviolet light induces expression of p53 and p21 in human skin: effect of sunscreen and constitutive p21 expression in skin appendages. J Invest Dermatol 1995; 105: 402-6.

16. Black AK, Greaves MW, Hensby CN, Plummer NA. Increased prostaglandins E2 and F2alpha in human skin at 6 and 24 h after ultraviolet B irradiation (290-320 nm). Br J Clin Pharmacol 1978; 5: 431-6.

17. Farr PM, Diffey BL. A quantitative study of the effect topical indomethacin on cutaneous erythema induced by UVB and UVC radiation. Br J Dermatol 1986; 115: 453-66.