Sunless skin tanning with dihydroxyacetone delays broad-spectrum ultraviolet photocarcinogenesis in hairless mice

Mutation Research 542 (2003) 129–138

Sunless skin tanning with dihydroxyacetone delays broad-spectrum ultraviolet photocarcinogenesis in hairless mice Anita B. Petersen, Renhua Na, Hans Christian Wulf∗ Department of Dermatology D92, Bispebjerg Hospital, Bispebjerg Bakke 23, DK-2400 Copenhagen, NV, Denmark Received 15 January 2003; received in revised form 8 September 2003; accepted 12 September 2003

Abstract Sunless tanning with dihydroxyacetone (DHA) is not considered to be a sunscreen although it does absorb parts of the ultraviolet (UV) spectrum. We investigated the protection with topical application of DHA against solar UV-induced skin carcinogenesis in lightly pigmented hairless hr/hr C3H/Tif mice. Broad-spectrum UV radiation, simulating the UV part of the solar spectrum was obtained from one Philips TL12 and five Bellarium-S SA-1-12 tubes. Three groups of mice were UV-exposed four times a week to a dose-equivalent of four times the standard erythema dose (SED), without or with application of 5 or 20% DHA only twice a week. Similarly, three groups of mice were treated with DHA and irradiated with a high UV dose (8 SED), simulating a skin burn. Two groups (controls) were not irradiated, but either left untreated or treated with 20% DHA alone. The UV-induced skin pigmentation by melanogenesis could easily be distinguished from DHA-induced browning and was measured by a non-invasive, semi-quantitative method. Application of 20% DHA reduced by 63% the pigmentation produced by 4 SED, however, only by 28% the pigmentation produced by 8 SED. Furthermore, topical application of 20% DHA significantly delayed the time to appearance of the first tumor ≥1 mm (P = 0.0012) and the time to appearance of the third tumor (P = 2 × 10−6 ) in mice irradiated with 4 SED. However, 20% DHA did not delay tumor development in mice irradiated with 8 SED. Application of 5% DHA did not influence pigmentation or photocarcinogenesis. In conclusion, this is the first study to show that the superficial skin coloring generated by frequent topical application of DHA in high concentrations may delay skin cancer development in hairless mice irradiated with moderate UV doses. © 2003 Elsevier B.V. All rights reserved. Keywords: Dihydroxyacetone; Self tanning; Non-enzymatic glycosylation; Skin browning; Photocarcinogenesis; Hairless mice; Ultraviolet light

1. Introduction Sunless or self-tanning lotions contain dihydroxyacetone (DHA) that darkens the skin by a chemical reaction [1,2]. DHA is a physiological product of the ∗ Corresponding author. Tel.: +45-3531-3156; fax: +45-3531-3950. E-mail address: [email protected] (H.C. Wulf).

body formed and utilized in the Kreb’s cycle and is presumed to be neither toxic nor carcinogenic [3,4]. The site of action of DHA in the skin is the stratum corneum [5]. DHA induces a concentration-dependent formation of brown color complexes through an irreversible non-enzymatic glycosylation of amines or amino groups in skin proteins. This process is known as the Maillard reaction [6] and involves the formation of free radicals when DHA is applied to mouse skin

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[7]. The depth of color correlates with the thickness and compactness of stratum corneum and is thus dependent on the concentration of keratin. Microscopic studies of stripped stratum corneum and hair demonstrate DHA-pigment masses distributed irregularly in the keratin layer [5,8]. The produced brown color complexes are water and soap resistant and pigment loss occurs only through sloughing of the stratum corneum [9]. Producers of sunless tanning lotions claim that DHA does not function as a sunscreen protection factor [5,10]. However, the DHA-induced colored complexes absorb light in the UVA and visible light region [11]. Furthermore, using depth profilometry it was recently reported that DHA itself shows absorption band shapes around 270 nm, with a clear increase in absorption over the entire spectrum (200–500 nm) after topical application [12]. A progressive absorption increase in the 270 nm region is due to DHA penetration inside the different layers of the skin, whereas the increase in absorption over the 350–500 nm range corresponds to the self-tanning action of DHA inside the skin. The light absorption in the visible spectrum is responsible for skin coloration and for its appearance as tanned or pale. The strongest increase in absorption occurs over the stratum corneum, whereas a small but nonnegligible increase is observed in the deeper layer [12]. The photoprotection of DHA browning has been evaluated in an animal model with photosensitized rats [13,14]. It was shown that skin treatment with DHA protected against PUVA-induced erythema. Furthermore, DHA is being clinically useful in protection of the skin in light sensitive persons [14–16] and cosmetically useful in the visible improvement of vitiligo [3,5,17]. On the other hand, DHA has been shown to be slightly mutagenic in bacteria [18,19]. In response to the demand for a safer or more convenient way to tan, cosmetics companies have produced more self-tanning lotions with improved color acceptance. However, despite the availability of self-tanning lotions since the 1960s, and despite the knowledge of the physiochemical properties of DHA browning, there has to date not been performed a scientific investigation of whether DHA browning may provide some protection against ultraviolet (UV)-induced skin carcinogenesis. To address this issue, we investigated the broad-spectrum ultraviolet photocarcinogenesis in hairless mice after topical application of DHA.

2. Materials and methods 2.1. Animals Lightly pigmented, hairless, female hr/hr C3H/Tif mice (N = 143) were used. The animals were 27–30 weeks old at the start of the experiment. The mice were divided into eight groups (Table 1). Consecutive numbers were tattooed on the abdomen of the mice. Each group was housed in a separate box. The animals had free access to water and standard laboratory food and were kept on a 12 h light/12 h dark cycle at a temperature of 23–24 ◦ C. 2.2. Radiation source and procedure Broad-spectrum UV radiation, simulating the UV part of the solar spectrum (solar UV) was obtained from one TL12 UVB tube (Philips, Eindhoven, The Netherlands) and five Bellarium-S SA-1-12 UVA tubes (Wolff System, Atlanta, Georgia, USA). The emission spectrum of the Solar-UV source has been published before [20]. The irradiance of the radiation source was measured monthly at 8 evenly distributed points in the irradiation boxes with a Solartell research radiometer (4D Controls Ltd., Cornwall, UK). Dosimetry was expressed in terms of standard erythema dose (SED) (1 SED = 100 J/m2 at 298 nm, CIE action spectrum) [21,22]. 2.3. Experimental design The treatment schedule is shown in Table 1. Fifty microliters (25 mg) of Locobase lotion (Yamanouchi Europe, Leiderdorp, Holland) containing 5 or 20% DHA (University Pharmacy, Copenhagen, Denmark) Table 1 Treatment schedule Group no.

No. of mice

[DHA] (%)

UV dose

1 2 3 4 5 6 7 8

17 18 18 18 18 18 18 18

0 0 0 20 5 20 5 20

0 4 8 0 4 4 8 8

SED SED SED SED SED SED SED SED

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was applied twice weekly on the dorsum (2 mg/cm2 ) of the mice. Application of the lotion was randomized among groups and the mice were irradiated the same day, at least half an hour after application. The mice were also irradiated on the day following application. The lotion was kept refrigerated in air-tight containers at 4 ◦ C to avoid decomposition; new containers were opened each month. The animals were irradiated from above, with a vertical distance of 22.5 cm from the UV source to the level of exposure. Groups 2, 5 and 6 were irradiated with a moderate UV dose of 4 SED per day (6 min 10 s per day), 4 days a week. Groups 3, 7 and 8 were irradiated with an erythemogenic dose of 8 SED per day (12 min 20 s per day), 4 days a week. The mice receiving 8 SED were irradiated with gradually increasing UV doses (4, 6, 8 SED) during the first 2 weeks of the experiment. However, irradiation with 8 SED resulted in a persistent erythema, sore skin, skin thinning and small wounds for 6 weeks of the experiment as a consequence of sunburning. Therefore, the UV irradiation was interrupted for 1 week to allow healing, and the affected skin was treated with chlorohexidine to prevent infection. UV irradiation was continued thereafter with only a slight erythema in mice irradiated with 8 SED. 2.4. Registration and statistics 2.4.1. Skin tumors Since UV radiation is carcinogenic in mice [20], the different delays in tumor appearance were analyzed. The mice were examined once a week for tumor development. The criteria for identifying tumors were palpability and diameter ≥1 mm. The end-points were: the time of the appearance of the first visible growing tumor (≥1 mm) and the appearance of a third growing tumor (≥1 mm). The probability of survival without a tumor was calculated using the Kaplan–Meier method and plotted as a function of the duration of the experiment in weeks. The differences in time from the start of the experiment to the appearance of the tumors were analyzed using the log-rank test. 2.4.2. UV-induced skin pigmentation by melanogenesis UV radiation induces melanin production in the epidermis, which is seen as a pigmentation of the skin.

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The UV-induced skin pigmentation was measured by a non-invasive, semi-quantitative method [23]. In week 25 of the experiment, the pigmentation of all the mice was scored once. Pigmentation was scored by placing each mouse under a bank of six Philips TL08 fluorescent tubes in a dark room. A Kodak gray scale (Eastman Kodak Company, 1977, publication no. Q-14) with 20 different shades from white to black was held to the back of the mice. The Philips TL08 tubes emit long-wave UVA and the radiation changed the color of the grey scale and the pigmentation of the mice into purple hues. The purple color of the back of each mouse could then be identified as identical to one of the numbered shades on the grey scale. The UV-induced tanning could be easily distinguished from the browning of the stratum corneum by DHA, which appeared orange. Therefore, the DHA browning did not influence the determination of the pigmentation using this method. The statistical analysis of the skin pigmentation was performed using the Mann–Whitney U-test. The differences between groups were considered significant when P < 0.05. 2.5. Spectrophotometric measurements To measure the absorbance of DHA in vitro, UV and visible absorbance spectra of DHA (CAS no. 62147-49-3, Sigma–Aldrich, Steinheim, Germany) and DHA browning on bovine serum albumin (BSA) (Sigma, St. Louis, MO) were obtained using an UltrospecIII® spectrophotometer (Pharmacia, Allerød, Denmark). DHA was incubated separately or mixed with BSA in distilled water at 37 ◦ C overnight and up to three following days in the dark. The pH value of DHA in solution was 5.5 and the pH value of BSA in solution with or without DHA was 7.2 measured with pH strips. Absorbance spectra were recorded in the range of 200–500 nm. To measure the absorbance of DHA browning in mice in vivo, the reflectance of DHA-treated mouse skin was measured with a transputer-integrated diode array spectrometer (TIDAS, J&M Analytische Mess-und Regeltechnik GMBH, Aalen, Germany). The light source used was a high-pressure xenon lamp (75 W). An optical Y-fiber was used to deliver the light to the skin and to collect the reflected light. The fiber probe was placed gently and perpendicularly on the skin during the measurements. The detector output

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was displayed and stored in a computer. The mouse skin was treated with topical sunless tanning lotion daily for 3 days, and the measurements were conducted during 24 h after each treatment. All measurements were performed at room temperature in the dark. 3. Results 3.1. Absorbance of DHA and sunless tanning Substances that absorb radiation in the UV region of the light spectrum may be potential sunscreens. The absorbance wavelengths of DHA and DHA-induced browning was measured in order to determine the sunscreen potential of DHA and DHA-induced browning in vitro and in vivo. In vitro non-enzymatic glycosylation reactions were performed between DHA and BSA, and spectral properties of the resulting brown color complexes were measured. A wavelength scanning revealed an absorption peak at 270 nm for DHA and an absorption peak at 280 nm for the protein (Fig. 1a, panel A). The absorption peak of DHA broadens into the UVB range with increasing concentration of DHA (Fig. 1a, panels B and C). When DHA was incubated with protein an increasing absorption in the UVA and visible range appeared in a dose- and time-dependent manner (Fig. 1a, panels B and C). In vivo measurements of skin reflection also revealed an increasing absorption of radiation in the UVA and visible range of the spectrum with consecutive applications of sunless tanning lotion (Fig. 1b). 3.2. Acute skin effects after UV irradiation and DHA application Before and after treatment, the mice in all groups were examined for any acute effects caused by either DHA application or UV irradiation. We noticed that the groups of mice irradiated with 4 SED developed a transient erythema in the beginning of the study. Furthermore, a persistent erythema was still observed after 6 weeks in most of the mice in the groups that were irradiated with 8 SED but stabilized thereafter. The DHA-induced skin color developed faster and persisted longer in the mice treated with 20% DHA lotion than in the mice treated with 5% DHA lotion.

Solar-UV radiation-induced pigmentation 3 weeks into the experiment. Data on acute effects are not shown. 3.3. Skin pigmentation UV radiation induces melanin production in the epidermis, which is seen as a pigmentation of the skin. Ordinary sunscreen lotions absorb some of the UV radiation and therefore reduce the induction of melanogenesis, which is seen as a slower pigmentation compared to skin irradiated without sunscreen. In order to determine whether DHA might function as a sunscreen, the pigmentation of the irradiated mice was determined. The mean pigmentation of the irradiated groups determined with the grey scale is shown in Fig. 2. There was a clear distinction in UV-induced pigmentation between the groups that received 4 and 8 SED, the latter inducing pigmentation twice as dark as the pigmentation induced by 4 SED. The animals in group 1 (0% DHA) and group 4 (20% DHA) did not develop a pigmentation that could be determined with the grey scale. The results of the Mann–Whitney tests showed that topical application of 20% DHA significantly decreased the UV-induced pigmentation. The pigmentation was reduced by 63% in mice irradiated with 4 SED and by 28% in mice irradiated with 8 SED. However, topical application of 5% DHA did not modify the UV-induced pigmentation. 3.4. Carcinogenicity and survival of the mice Skin tumor development and survival of the mice were used as end points to evaluate the protection with topical DHA application. Out of 108 irradiated animals (Table 1), 87 mice developed at least one visible skin tumor, and 82 developed 3 or more visible tumors. None of the mice in the two unirradiated control groups (groups 1 and 4) developed any skin tumors. The probability of survival of the mice without a tumor ≥1 mm and without three tumors is shown in Figs. 3 and 4, respectively. The log-rank test showed that topical application of 20% DHA significantly delayed the time of appearance of the first tumor (P = 0.0012) (Fig. 3a) and the third tumor (P = 0.000002) (Fig. 4a) in mice irradiated with 4 SED (group 6), compared to mice irradiated with 4 SED alone (group 2).

Absorbance units / relative intensity

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3

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2 mg/ml BSA 5 % DHA

2,5

5 % DHA + 2 mg/ml BSA

2 1,5 1 0,5 0 200

250

(A)

300

350

400

450

500

Wavelength nm

3

10 mg/ml BSA 5% DHA 5% DHA + BSA (day 1)

Absorbance units

2,5

5% DHA + BSA (day 2) 5% DHA + BSA (day 3)

2 1,5 1 0,5 0 200

250

(B)

300

350

400

450

500

Wavelength nm

3

10 mg/ml BSA 20% DHA

Absorbance units

2,5

20% DHA + BSA (1 day) 20% DHA + BSA (2 days)

2

20% DHA + BSA (3 days)

1,5 1 0,5 0 200

(a) (C)

250

300

350

400

450

500

Wavelength nm

Fig. 1. Spectral properties of DHA and sunless browning. (a) Spectrophotometric measurements of DHA solutions at different concentrations after incubation with or without BSA at 37 ◦ C. Panel A shows absorption peaks for 5% DHA incubated 1 day with or without 2 mg/ml BSA. Panel B shows absorption spectra for 5% DHA incubated with or without 10 mg/ml BSA for 1–3 days. Panel C shows absorption spectra for 20% DHA incubated with or without 10 mg/ml BSA for 1–3 days. (b) Reflectance measurements of mouse skin after applications of sunless tanning lotion for three consecutive days.

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4. Discussion

Fig. 1. (Continued ).

The log-rank test showed that the time to appearance of the first tumor in the mice irradiated with 4 SED (group 2) and 8 SED (group 3) was identical (P = 0.125). The time to appearance of the third tumor was shorter in the mice irradiated with 4 SED (group 2) than in those irradiated with 8 SED (group 3) (P = 0.001) (Fig. 4). Furthermore, in mice treated with 5% DHA, the time of appearance of the third tumor was also shorter after irradiation with 4 SED (group 5) than with 8 SED (group 7) (P = 0.01) (Fig. 4).

Sunless tanning with dihydroxyacetone has been used since the 1960s. Skin treatment with DHA has been shown to be non-toxic and non-carcinogenic [4]. This is the first study conducted concerning the potential protective effect of topical DHA application against UV-induced skin carcinogenesis. The brown-colored complex produced by the non-enzymatic glycosylation of epidermal proteins with DHA is known to absorb light in the UVA and visible spectrum [11]. This we have confirmed using reflectance measurements of DHA-treated skin. Furthermore, DHA itself was recently shown to absorb light in the UVB region [12]. This absorption was confirmed in this study. The reduction in UV-induced pigmentation observed in mice treated with 20% DHA might thus result from less penetration of UV light into the epidermis, due to absorption in the UVB range by DHA itself and in the UVA range by DHA-generated superficial coloring in the skin. Therefore, DHA may be considered to be a sunscreen substance. The time of appearance of the first tumor was independent of the UV dose, however, the mice irradiated with 4 SED developed skin tumors faster than mice irradiated with 8 SED. This is unexpected, since in general a higher UV dose is more carcinogenic [20]. The high dose of solar UV was chosen to simulate sun-

Fig. 2. Mean skin pigmentation index ± S.D. of the mice exposed to solar UV determined using a Kodak gray scale. Pigmentation was scored once in the 25th week of the experiment. P-values were determined by the Mann–Whitney U-test. The difference between groups was considered significant when P < 0.05.

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Probability of survival without a tumor 1 mm

1

135

Gr.2 (4SED) Gr.5 (5% DHA + 4SED) Gr.6 (20% DHA + 4SED)

0,8

0,6

0,4

0,2

0 15

20

(a)

25

30

40

Time, wk

1

Probability of survival without a tumor 1 mm

35

Gr.3 (8SED) Gr.7 (5% DHA + 8SED) Gr.8 (20% DHA + 8SED)

0,8

0,6

0,4

0,2

0 15

(b)

20

25

30

35

40

Time, wk

Fig. 3. Kaplan–Meier plot showing time to first tumor with a diameter ≥1 mm for groups irradiated with (a) 4 SED and (b) 8 SED.

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Gr.2 (4SED)

1

Probability of y survival without 3 tumors

Gr.5 (5% DHA + 4SED) Gr.6 (20% DHA + 4SED) 0,8

0,6

0,4

0,2

0 15

20

(a)

25

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40

Time, wk

1

Gr.3 (8SED)

Probability of survival without 3 tumors

Gr.7 (5% DHA + 8SED) Gr.8 (20% DHA + 8SED)

0,8

0,6

0,4

0,2

0 15 (b)

20

25

30

35

40

Time, wk

Fig. 4. Kaplan–Meier plot showing time to the appearance of the third tumor with a diameter ≥1 mm for groups irradiated with (a) 4 SED and (b) 8 SED.

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burn and it induced a persistent erythema 6 weeks into the experiment. This over-exposure may have induced cell death and apoptosis in the epidermis. This resulted in peeling of the skin and thereby removal of potential initiated or mutant cells that otherwise would have been transformed (promoted) into tumor cells. This may have resulted in a delay of visible tumors in the groups irradiated with sunburn doses (8 SED). Sunburn doses may thus be expected to result in lack of a normal dose response of the cancer incidence because of change in acute biological consequence of radiation. This may only be valid for squamous cell carcinomas. In mice and man, development of UV-induced squamous cell carcinoma is a cumulative process. Reducing the amount of UV radiation reaching the basal layer will retard that process. The delayed appearance of tumors in mice after treatment with 20% DHA may be explained by a reduction of the UV dose reaching the basal layer of the skin due to absorption by DHA itself and DHA browning. This may have resulted in less UV-induced DNA damage of the keratinocyte stem cells and thus, decreased mutational activation of oncogenes or inactivation of tumor suppressor genes. A decreased level of DNA damage may also have caused the reduction of the UV-induced pigmentation [24,25]. The reason why protection against tumor development was not significant in the mice irradiated with a high UV dose (8 SED), may be due to ‘over’-exposure and subsequent very high cell turn over, as this dose was chosen to give a high degree of erythema. Most commercial sunless tanning lotions contain up to 5% DHA [10]. In our study, 5% DHA was not sufficient to provide any protection against tumor development in mice. However, the stratum corneum is thicker in human skin (10–15 ␮m) than in murine skin (2–3 ␮m), and therefore, a darker DHA browning might be expected with 5% DHA on human skin because of the larger amount of keratin in the stratum corneum being available for non-enzymatic glycosylation. A sunless tanning lotion may eventually provide some protection in human skin that may not be seen in mice. Regardless of the mechanism responsible for the inhibitory effect of DHA browning on tumor development after Solar-UV exposure, our results point out an interesting new aspect of the use of sunless tanning lotion. However, before it is possible to devise a

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more general model concerning the protective effects of DHA browning in humans, a number of different aspects, such as dosimetry, time-dose responses and species/strain specificity between mice and man, have to be investigated. In conclusion, this study is the first to show that the skin coloring generated by frequent topical application of DHA in high concentrations may delay skin cancer development in hairless mice at moderate UV exposure. Topical application of DHA and the resulting DHA-produced skin coloring may be a beneficial skin protection against UV light.

Acknowledgements We thank Matas A/S for financial support and Trine Raun, Maria Clausen and Eva Hoffmann for animal handeling. References [1] E. Wittgenstein, H.K. Berry, Staining of skin with dihydroxyacetone, Science 132 (1960) 894–895. [2] E. Wittgenstein, H.K. Berry, Reaction of dihydroxyacetone (DHA) with human skin callus and amino compounds, J. Invest. Dermatol. 36 (1961) 283–286. [3] L. Goldman, D. Blaney, J. Goldman, Topical therapy with dihydroxyacetone, Acta Dermatol.-Venerol. 40 (1960) 500– 503. [4] F.J. Akin, E. Marlowe, Non-carcinogenicity of dihydroxyacetone by skin painting, JEPTO 5 (4–5) (1984) 349–351. [5] H.I. Maibach, A.M. Kligman, Dihydroxyacetone: a suntansimulating agent, Arch. Dermatol. 82 (1960) 73–75. [6] S. Blau, N.B. Kanof, L. Simonson, Dihydroxyacetone (DHA)—a keratin coloring agent, Arch. Dermatol. 82 (1960) 501–504. [7] R.V. Lloyd, A.J. Fong, R.M. Sayre, In vivo formation of Maillard reaction free radicals in mouse skin, J. Invest. Dermatol. 117 (2001) 740–742. [8] L. Goldman, J. Barkoff, D. Blaney, T. Nakai, R. Suskind, Investigative studies with the skin coloring agents dihydroxyacetone and glyoxal, J. Invest. Dermatol. 35 (1960) 161–164. [9] J.A. Johnson, R.M. Fusaro, Therapeutic potential of dihydroxyacetone, J. Am. Acad. Dermatol. 29 (2) (1993) 284–285. [10] S.B. Levy, Dihydroxyacetone-containing sunless or selftanning lotions, J. Am. Acad. Dermatol. 27 (1992) 989–993. [11] A. Meybeck, A spectroscopic study of the reaction products of dihydroxyacetone with amino acids, J. Soc. Cosmet. Chem. 28 (1977) 25–35. [12] G. Puccetti, R.M. Leblanc, A sunscreen-tanning compromise: 3D visualization of the actions of titanium dioxide particles

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