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Delayed induction of pigmented spots on UVB-irradiated hairless mice
Journal of Dermatological Science 25 (2001) 29 – 35 www.elsevier.com/locate/jdermsci
Delayed induction of pigmented spots on UVB-irradiated hairless mice Masako Naganumaa a,*, Eiichiro Yagi b, Minoru Fukuda b a
Scientific Research Di6ision, Research and De6elopment Headquaters, Shiseido Co. Ltd., 3 -9 -1 Nishigotannda Shinagawa-ku, Tokyo, 141 -0031 Japan b Shiseido Research Center, Yokohama, Japan Received 5 January 2000; received in revised form 3 April 2000; accepted 5 April 2000
Abstract Human skin exposed to solar radiation for a long time subsequently develops pigmented spots, which are named solar lentigines. Since no animal model of this process is currently available, we attempted to induce similar spots in pigmented hairless mice. The mice were irradiated at 38 or 94 mJ/cm2 three times/week for various periods of time (1–8 weeks) under an ultraviolet light source (Toshiba FL-SE; UVB). Skin pigmentation of irradiated mice was visually observed and skin color was determined with a colorimeter for 78 weeks. Uniform pigmentation was induced, but persisted only during exposure, disappearing completely within 2 weeks after cessation of exposure. At about 28 weeks after the first exposure, pigmented spots suddenly began to appear. These pigmented spots were less than 2 mm in diameter and light brown in color. The length of the latent period until appearance and the extent of development of these spots were dependent on the exposure period. Histological examination revealed increased numbers of active melanocytes and melanin granules in the affected epidermis. These pigmented spots closely resemble solar lentigines in humans, and the mice should be useful as an animal model of solar lengtigines. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pigmentation; Pigmented spots; Melanin; Solar lentigo; UVB; Mouse
1. Introduction Wrinkles, sagging and pigmented spots are representative symptoms of photoaging in human skin [1]. In experimental studies, wrinkles, sagging and cancers have been elicited on animal skin [2 – 4], mostly in albino hairless mice. Further, * Corresponding author. Tel.: +81-3-34908451; fax: + 813-34906950.
long-term ultraviolet exposure of the skin of Skh:HR-1 albino hairless mice results in skin injury resembling solar elastosis of human skin, as well as skin cancer [5]. Using this mouse model, changes of matrix components induced by UV exposure have been investigated [6,7] and several chemicals were found to protect hairless mice skin from photoaging [8,9]. However, pigmented spots such as solar lentigines or senile lentigines could not be studied since these mice have no active melanocytes.
0923-1811/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0923-1811(00)00098-0
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M. Naganumaa et al. / Journal of Dermatological Science 25 (2001) 29–35
There have been many experimental studies using pigmented hairy or hairless mice. The effects of UV irradiation on the pigmentary system [10 – 12] and the effects of various agents upon acute pigmentation induced by UV exposure [13,14] have been reported. Attempts to cause melanoma by repeated UV exposure alone were unsuccessful [15], although UVA irradiation in the presence of psoralen was reported to induce pigmented melanocytic tumors [16]. However, there has been no report describing the experimental induction of delayed pigmented spots such as solar lentigines; these are quite distinct from the acute pigmentation uniformly induced on the irradiated area during UV exposure. Solar lentigines [17,18] and PUVA lentigines [19] are known to be formed during skin aging or long-term PUVA therapy. These pigmentations are induced in patches, only on the irradiated area, long after exposure. These facts suggested that they are caused, or at least accelerated, by UV irradiation. We therefore investigated delayed pigmentation induced on pigmented hairless mouse skin by UVB irradiation with the aim of developing an animal model of solar lentigines.
2. Materials and methods
2.1. Animal F1 hairless mice of HR-1 ×HR/De, which have pigmented eyes, ears and tail, were used. The mice were 8 weeks old at the start of the experiment, and were given water and a commercial diet (Oriental Yeast Co., CRF-1, Tokyo, Japan), ad libitum. Ambient lighting was automatically regulated on a 12-h light/day cycle. The animals were divided into six groups, each of eight to ten animals.
UVB three times a week for a period of 1, 2, 4 or 8 weeks. The daily dose was 94 mJ/cm2 (high dose). Another group of mice was exposed to 38 mJ/cm2 (low dose) per day for 4 weeks. An agematched control group without UV exposure was also used.
2.3. E6aluation of pigmentation and pigmented spot During and after exposures, skin response, particularly skin pigmentation, was visually examined every week for 78 weeks. The degree of pigmented spot induction was classified into four grades with the naked eye; 0: no pigmented spots, 1: a few small pigmented spots, 2: presence of distinct pigmented spots, 3: many distinct pigmented spots over the whole back. The color of the back skin was also determined using a Minolta CR 100 colorimeter in the CIE-L*, a*, b* system. The results were expressed in term of L value (white = 100, black=0).
2.4. Histology The skin of mice irradiated for 4 weeks was taken immediately or at 32 weeks after completion of irradiation. Pieces of skin were fixed in buffered formalin, sectioned, and stained by means of Fontana–Masson technique for light microscopic observation. With other pieces of skin, the epidermis was separated from the dermis by use of 1 N NaBr solution, and subjected to dopa reaction.
2.5. Statistical e6aluation Student’s t test was used to evaluate the significance of differences in skin color and the Mann– Whitney U-test was used to analyze data on pigmented spots.
2.2. UV Radiation The UVB light source was a bank of seven Toshiba FL-20 SE fluorescent lamps, with a peak emission at 305 nm. The irradiance was 0.31 mW/cm2, as measured with a UV-Radiometer (TOPCON). Five groups of mice were exposed to
3. Results
3.1. Obser6ation of acute pigmentation Skin color of mice was determined once a week
M. Naganumaa et al. / Journal of Dermatological Science 25 (2001) 29–35
during the exposure period with a colorimeter. The results are shown in Fig. 1. Hairless mice showed no tanning at the start of irradiation. During the first exposure week, erythema was observed only on skin irradiated with the high dose. Perceptible pigmentation appeared 1 week after the first exposure only on skin irradiated with the low daily dose, without erythema. All exposed mice showed apparently uniform pigmentation on their back skin 2 weeks later and the L* value dropped rapidly from 63 to about 60 in all groups except the non-irradiated group after 2 weeks. Pigmentation (Fig. 2) persisted during irradiation and there was no significant difference in L* value between animals exposed to the low dose and high dose. Those changes well reflected the changes of skin color observed with the naked eye. While irradiation was continuing, the peak of pigmentation was always seen at the third or fourth week i.e. exposure for over 4 weeks did not produce darker pigmentation. After cessation of UV irradiation this pigmentation began gradually to disappear and after about 2 weeks the skin color became the same as that of non-irradiated skin, irrespective of the length of the irradiation
Fig. 1. Time course of skin color (L* value is reflected skin brightness). The lowest values were recorded at the third or fourth week after the first exposure. The L* value increased rapidly after the final exposure, becoming the same as that of non-irradiated mice. Non-irradiated: a, — , high dose irradiated group for 1 week; b, — —, 2 weeks; c,· · · · , 4 weeks; d, – · –, 8 weeks; e, – · · – low dose irradiated group for 4 weeks: f, – – – .
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Fig. 2. Pigmentation on hairless mouse skin immediately after UV exposure for 4 weeks. This pigmentation is dark brown in color and spread uniformly on the back skin.
period. The L* value started to increase on all irradiated mice immediately after the final UV exposure, and reached the control value 2 or 3 weeks later.
3.2. Obser6ation of pigmented spots When observation was continued after the recovery of normal skin color, the skin showed no pigmentation for a long time, though the L* values of all groups, including the non-irradiated group, increased slowly but steadily (Fig. 1). The increase of L* values was caused by the skin becoming opaque. At about 28 weeks small pigmented spots began to develop, at first on the dorsal skin of mice irradiated for 8 weeks. Subsequently, the size and number of the spots increased on the irradiated area. The spots had a diameter of less than 2 mm and were light brown (Fig. 3). The L* value of mice having many pigmented spots was not different from that of non-exposed mice having no spots (Fig. 1). Thus, determination of skin color could not indicate the degree of formation of pigmented spots. Accordingly, we scored the spots into four grades by visual evaluation (Fig. 4). Score 0 means no pigmented spots and score 3 indicates extensive pigmented spots. The group exposed to a daily high dose of UVB for 8 weeks showed a mean score of 1.0 at the
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Fig. 3. Pigmented spots 57 weeks after cessation of a 4-week UV exposure. The pigmented spots began to appear 24 weeks after the final irradiation. And then they became gradually darker and bigger.
20th week after the first exposure and a mean score of 2.6 was recorded at the 78th week. The group exposed to a daily low dose for 4 weeks (total dose: 456 mJ/cm2) showed similar changes to those of the group exposed to a daily high dose for 4 weeks (total dose: 1128 mJ/cm2), which did
Fig. 4. Time course of the appearance of pigmented spots. The first appearance of them and the degree of them were dependent on irradiated dose. Non-irradiated: a ——, high dose irradiated group for 1 week: b, —; 2 weeks: c · · · · ; 4 weeks: d – · – ; 8 weeks: e – · · –; low dose irradiated group for 4 weeks: f, – – – . Pigmented spots began to appear at the 28th week of the experimental period on skin of mice irradiated for 8 weeks. The Mann – Whitney U test was used for statistical evaluation at the 45th experimental week. High dose group: 0 week versus 4 weeks, 0 versus 8, 1 versus 4, 1 versus 8, 2 versus 4, PB 0.001. High dose group: 2 weeks versus 8 weeks, P B0.01; 2 weeks versus 4 weeks, PB 0.05. The low dose group is not significantly different from the high dose group at 4 weeks.
not produce erythema. In addition, the former group received 456 mJ/cm2 for 4 weeks showed a higher mean score than that of the group exposed to a daily high dose for 2 weeks (total dose: 564 mJ/cm2). Unexposed mice never developed such pigmented spots during the experimental period (78 weeks). The time lag until appearance of pigmented spots and the degree of induction of pigmented spots were dependent on the exposure period, not on the total amount of UV exposure: the longer exposure period, the shorter the latent period and the greater the degree of induction of pigmented spots. Exposure even for just 1 week could induce the spots.
4. Histology of exposed skin Epidermal hyperplasia and accumulation of melanin granules in the epidermis were noted on skin specimens taken immediately after final exposure of a mouse exposed for 4 weeks (Fig. 5a). Melanins were stained strongly by Fontana–Massons stain in all layers of the epidermis except hair follicles. Many active melanocytes were visualized by dopa stain (Fig. 6a) and they were very dendritic. They overspread on the epidermis, showing pigmentation except around hair follicles. On the other hand, examination of skin specimens taken from exposed mice 32 weeks after final exposure revealed that the thickness of the epidermis was not significantly different from that of non-irradiated epidermis, and there were melanin granules only in those parts of the epidermis having pigmented spots (Fig. 5b). Active melanocytes similar to those observed immediately after final exposure were present only in areas showing pigmented spots (Fig. 6b), though they were very dendritic. These results showed that the pigmented spots were due to melanin synthesized by melanocytes in the epidermis, not in the dermis.
5. Discussion We previously used HR-1 mice (aabbccDDPPhrhr) to investigate the changes of skin upon
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repeated UVB irradiation and reported that the number of melanocytes began to increase 2 days after the first exposure and reached almost saturation at about 14 days. This increase of melanocyte number was partially due to mitosis. We observed the development of a similar uniform pigmentation on the back skin of hairless mice exposed to repeated UVB irradiation. Furthermore, we showed quantitatively that the decrease of L* value saturated after 3 to 4 weeks, by measurement of skin color with a colorimeter. These results are reasonable, since melanocyte density is expected to peak first, then melanin particles would be transferred to surrounding keratinocytes, which would eventually cover the epider-
Fig. 5. Skin specimens were stained using the Fontana–Masson method. (a): Skin taken immediately after a 4-week high dose UV exposure. This photo shows hyperplasia and melanin particles uniformly in all layers of the epidermis. (b): Skin taken 32 weeks after a 4-week high dose UV exposure. Melanin particles are stained only on the epidermis, not the dermis, in pigmented spots.
chronic UV exposure [20], but we could not observe the effect on melanocytes, because HR-1 mice are albino. On the other hand, HR/De mice (AABBCCDDpphrhr) do not show pigmentation upon UV exposure, although they are not genetically albino. So we crossed HR-1 with HR/De, and found that the obtained F1 mice (AaBbCcDDPphrhr) had the wild coat color and their skin darkened upon UV irradiation. Exposure of pigmented mice to UV radiation has long been known to induce pigmentation [10 – 12], and the mechanisms involved have been examined by many researchers. Sato and Kawada [10] irradiated the backs of hairless mice and observed mitotic activity of epidermal melanocytes. Rosdahl and Szabo [11] examined the ears of hairy mice, C57B1/6J, exposed to
Fig. 6. Epidermis stained with dopa reaction. (a): Skin taken immediately after a 4-week high dose UV exposure. Active and dendritic melanocytes are seen except at hair follicles. (b): Skin taken 32 weeks after a 4-week high dose UV exposure. Active and dendritic melanocytes appear only in areas showing pigmented spots. However, the morphology of melanocytes is very similar to that in (a).
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mis. The appearance of this pigmentation in mice did not require erythema. When UV irradiation was stopped, the pigmentation began to fade. Although this phenomenon is well known, extended observations after recovery of normal skin color have rarely been made. We found that after an exposure time-dependent latent period following the recovery of normal skin color, pigmented spots began to appear and the size and number of them increased gradually. We found no indication of hyperplasia of the epidermis or tumors on the skin during the late experimental period, when the pigmented spots appeared. Thus the pigmented spots seem to be the first symptoms of photoaging. Tumor or melanoma induction on mouse skin subjected to long-term UV exposure has been investigated [15,16,21], but little attention was paid to the induction of pigmentation or pigmented spots. Since the skin was monitored only during the exposure period, delayed changes would have been missed. Alcalay et al. [16] described macules elicited on the skin of mice treated with psoralen plus ultraviolet A radiation, but these macules resulted from melanin accumulation in the dermis, not the epidermis. For the first time, we have shown that the skin of pigmented hairless mice, once UV-irradiated, always develops pigmented spots after a latent period, which is inversely proportional to the exposure period. The longer the exposure period, the greater was the induction of pigmented spots. Induced pigmented spots very slowly became darker and larger even after the final observation (78 weeks after first exposure, data not shown), and never disappeared. As denaturation of matrix components was reported to be reparable, the pigmented spots are distinct from such phenomena. This means that the UV irradiation history of skin, even exposure for as short a time as one week, affected the skin condition much later. We found that the same degree of induction of pigmented spots appeared on mice of two groups irradiated with different daily doses for the same period. These groups showed the same pigmentation during the irradiation period, although mice irradiated with the low dose did not develop erythema. Kiss et al. [4] reported similar phenom-
ena in connection with wrinkle formation on chronically irradiated hairless mice i.e. equal cumulative UVB doses did not always result in identical grades of wrinkle formation. Furthermore Wulf et al. [20] found that different doses of UV were equally carcinogenic when mouse skin was subjected to repeated UV exposure from the same light source. We found that the degree of delayed pigmentation response in the epidermis appeared to be the same even when different irradiation doses were applied during the same period. It seems that a sufficient stimulus to maintain an abnormal condition for certain time, is required, even if that stimulus is small i.e. there is a threshold effect. Many kinds of pigmented spots are observed in human skin. Among them, freckles, pigmentatio pedaloides actinica and solar lentigo are related to sunlight. Freckles have a genetic basis. Pigmentatio pedaloides actinica appear on sites exposed to large amounts of UV, following severe erythema. Pigmented spots in mice were not genetically determined, and could be elicited without erythema. On the other hand, solar lentigines are induced on exposed sites in the absence of severe damage. So solar lentigines seem to be a result of chronic UV exposure. Furthermore, melanocytes were histologically reported to increase in solar lentigines and melanin was observed in the epidermis [18,22]. Histological examination of pigmented spots in mice revealed that they contained active melanocytes, had melanin in the epidermis, not in the dermis, and never showed epidermal hyperplasia. Thus, based on the reported findings and pathological examination, we consider that pigmented spots in mice resemble solar lentigines in humans. Symptoms of photoaging include wrinkles, pigmented spots, solar elastosis, and skin cancer. Wrinkles and skin cancer have been shown to be elicited in animals by long UV exposure. Animal models have been used to investigate the mechanisms of these changes and to develop protective agents. However, there is no animal model of pigmented spots. Our results indicate that UVBirradiated pigmented hairless mice could be useful as a model of human solar lentigines.
M. Naganumaa et al. / Journal of Dermatological Science 25 (2001) 29–35
References [1] Kligman LH, Kligman AM. The nature of photoaging: its prevention and repair. Photodermatology 1986;3:215–27. [2] Bissett DL, Hillebrand GG, Hannon DP. The hairless mouse as a model of skin photoaging: its use to evaluate photoprotective materials. Photodermatology 1989;6:228 – 33. [3] Kligman LH. The ultraviolet-irradiated hairless mouse: a model for photoaging. J Am Acad Dermatol 1989;21:623 – 31. [4] Kiss I, Chen S, Tramposch KM. The effect of high and low ultraviolet-B dose exposure on the degree of hairless mouse skin wrinkling. Photochem Photobiol 1991;53:109 – 12. [5] Moloney SJ, Edmonds SH, Giddens LD, Learn DB. The hairless mouse model of photoaging: evaluation of the relationship between dermal elastin, collagen, skin thickness and wrinkles. Photochem Photobiol 1992;56:505–11. [6] Takahashi Y, Ishikawa O, Okada K, Ohnishi K, Miyachi Y. Disaccharide analysis of the skin glycosaminoglycans in chronically ultraviolet light-irradiated hairless mice. J Dermatol Sci 1995;10:139–44. [7] Starcher B, Pierce R, Hinek A. UVB irradiation stimulates deposition of new elastic fibers by modified epithelial cells surrounding the hair follicles and sebaceous glands in mice. J Invest Dermatol 1999;112:450–5. [8] Bissett DL, Majeti S, Fu JJ, McBride JF, Wyder WE. Protective effect of topically applied conjugated hexadienes against ultraviolet radiation-induced chronic skin damage in the hairless mouse. Photodermatol Photoimmunol Photomed 1990;7:63–7. [9] Kochevar IE, Moran M, Lyon N, Flotte T, Siebert E, Gange RW. Effects of systemic indomethacin, meclizine, and BW755C on chronic ultraviolet B-induced effects in hairless mouse skin. J Invest Dermatol 1993;100:186–93. [10] Sato T, Kawada A. Mitotic activity of hairless mouse epidermal melanocytes: its role in the increase of melanocytes during ultraviolet radiation. J Invest Dermatol 1972;58:329 – 95. [11] Rosdahl IK, Szabo G. Mitotic activity of epidermal melanocytes in UV-irradiated mouse skin. J Invest Dermatol 1978;70:143 – 8. [12] Uesugi T. Electron microscopic characterization in the processes of activation and differentiation of dormant
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
.
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melanocytes in epidermis of C57 black mouse skin after UV-irradiation (UV-A). Jpn J Dermatol 1978;88:686 – 700. Warren R. Sensitivity of mouse Skh: HR-2 to ultraviolet light: mouse pigmentation model. Photochem Photobiol 1986;43:41 – 7. Hoshino S, Nishimura M, Fukuyama K, Gellin GA, Epstein JH. Effects of 4-tertiary butyl catechol on melanocytes of hairless mice. J Invest Dermatol 1981;76:231 – 8. van Weelden H, van der Putte SCJ, Toonstra J, van der Leun JC. Ultraviolet B-induced tumors in pigmented hairless mice, with an unsuccessful attempt to induce cutaneous melanoma. Photodermatol Photoimmunol Photomed 1990;7:68 – 72. Alcalay J, Bucana C, Kripke ML. Cutaneous pigmented melanocytic tumor in a mouse treated with psoralen plus ultraviolet A radiation. Photodermatol Photoimmunol Photomed 1990;7:28 – 31. Roth DE, Hodge SJ, Callen JP. Possible ultraviolet A-induced lentigines: a side effect of chronic tanning salon usage. J Am Acad Dermatol 1989;20:950 – 4. Rhodes AR, Albert LS, Barnhill RL, Weinstock MA. Sun-induced freckles in children and young adults: a correlation of clinical and histopathologic features. Cancer 1991;67:1991 – 2001. Takashima A, Matsunami E, Yamamoto K, Kitajima S, Mizuno N. Cutaneous carcinoma and 8-methoxypsoralen and ultraviolet A (PUVA) lentigines in Japanese patients with psoriasis treated with topical PUVA: a follow-up study of 214 patients. Photodermatol Photoimmunol Photomed 1990;7:218 – 21. Maeda K, Naganuma M, Fukuda M. Effects of chronic exposure ultraviolet-A including 2% ultraviolet-B on free radical reduction systems in hairless mice. Photochem Phobobiol 1991;54:737 – 40. Wulf HC, Hansen AB, Bech-Thomsen N. Differences in narrow-band ultraviolet B and broad-spectrum ultraviolet photocarcinogenesis in lightly pigmented hairless mice. Photodermatol Photoimmunol Photomed 1994;10:192 – 7. Rhodes AR, Melski JW, Sober AJ, Harrist TJ, Mihm MC, Fitzpatrick TB. Increased intraepidermal melanocyte frequency and size in dysplastic melanocytic nevi, superficial spreading melanoma, nevocellular nevi, and solar lengigines. J Invest Dermatol 1983;80:452 – 9.
Delayed induction of pigmented spots on UVB-irradiated hairless mice Masako Naganumaa a,*, Eiichiro Yagi b, Minoru Fukuda b a
Scientific Research Di6ision, Research and De6elopment Headquaters, Shiseido Co. Ltd., 3 -9 -1 Nishigotannda Shinagawa-ku, Tokyo, 141 -0031 Japan b Shiseido Research Center, Yokohama, Japan Received 5 January 2000; received in revised form 3 April 2000; accepted 5 April 2000
Abstract Human skin exposed to solar radiation for a long time subsequently develops pigmented spots, which are named solar lentigines. Since no animal model of this process is currently available, we attempted to induce similar spots in pigmented hairless mice. The mice were irradiated at 38 or 94 mJ/cm2 three times/week for various periods of time (1–8 weeks) under an ultraviolet light source (Toshiba FL-SE; UVB). Skin pigmentation of irradiated mice was visually observed and skin color was determined with a colorimeter for 78 weeks. Uniform pigmentation was induced, but persisted only during exposure, disappearing completely within 2 weeks after cessation of exposure. At about 28 weeks after the first exposure, pigmented spots suddenly began to appear. These pigmented spots were less than 2 mm in diameter and light brown in color. The length of the latent period until appearance and the extent of development of these spots were dependent on the exposure period. Histological examination revealed increased numbers of active melanocytes and melanin granules in the affected epidermis. These pigmented spots closely resemble solar lentigines in humans, and the mice should be useful as an animal model of solar lengtigines. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pigmentation; Pigmented spots; Melanin; Solar lentigo; UVB; Mouse
1. Introduction Wrinkles, sagging and pigmented spots are representative symptoms of photoaging in human skin [1]. In experimental studies, wrinkles, sagging and cancers have been elicited on animal skin [2 – 4], mostly in albino hairless mice. Further, * Corresponding author. Tel.: +81-3-34908451; fax: + 813-34906950.
long-term ultraviolet exposure of the skin of Skh:HR-1 albino hairless mice results in skin injury resembling solar elastosis of human skin, as well as skin cancer [5]. Using this mouse model, changes of matrix components induced by UV exposure have been investigated [6,7] and several chemicals were found to protect hairless mice skin from photoaging [8,9]. However, pigmented spots such as solar lentigines or senile lentigines could not be studied since these mice have no active melanocytes.
0923-1811/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0923-1811(00)00098-0
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M. Naganumaa et al. / Journal of Dermatological Science 25 (2001) 29–35
There have been many experimental studies using pigmented hairy or hairless mice. The effects of UV irradiation on the pigmentary system [10 – 12] and the effects of various agents upon acute pigmentation induced by UV exposure [13,14] have been reported. Attempts to cause melanoma by repeated UV exposure alone were unsuccessful [15], although UVA irradiation in the presence of psoralen was reported to induce pigmented melanocytic tumors [16]. However, there has been no report describing the experimental induction of delayed pigmented spots such as solar lentigines; these are quite distinct from the acute pigmentation uniformly induced on the irradiated area during UV exposure. Solar lentigines [17,18] and PUVA lentigines [19] are known to be formed during skin aging or long-term PUVA therapy. These pigmentations are induced in patches, only on the irradiated area, long after exposure. These facts suggested that they are caused, or at least accelerated, by UV irradiation. We therefore investigated delayed pigmentation induced on pigmented hairless mouse skin by UVB irradiation with the aim of developing an animal model of solar lentigines.
2. Materials and methods
2.1. Animal F1 hairless mice of HR-1 ×HR/De, which have pigmented eyes, ears and tail, were used. The mice were 8 weeks old at the start of the experiment, and were given water and a commercial diet (Oriental Yeast Co., CRF-1, Tokyo, Japan), ad libitum. Ambient lighting was automatically regulated on a 12-h light/day cycle. The animals were divided into six groups, each of eight to ten animals.
UVB three times a week for a period of 1, 2, 4 or 8 weeks. The daily dose was 94 mJ/cm2 (high dose). Another group of mice was exposed to 38 mJ/cm2 (low dose) per day for 4 weeks. An agematched control group without UV exposure was also used.
2.3. E6aluation of pigmentation and pigmented spot During and after exposures, skin response, particularly skin pigmentation, was visually examined every week for 78 weeks. The degree of pigmented spot induction was classified into four grades with the naked eye; 0: no pigmented spots, 1: a few small pigmented spots, 2: presence of distinct pigmented spots, 3: many distinct pigmented spots over the whole back. The color of the back skin was also determined using a Minolta CR 100 colorimeter in the CIE-L*, a*, b* system. The results were expressed in term of L value (white = 100, black=0).
2.4. Histology The skin of mice irradiated for 4 weeks was taken immediately or at 32 weeks after completion of irradiation. Pieces of skin were fixed in buffered formalin, sectioned, and stained by means of Fontana–Masson technique for light microscopic observation. With other pieces of skin, the epidermis was separated from the dermis by use of 1 N NaBr solution, and subjected to dopa reaction.
2.5. Statistical e6aluation Student’s t test was used to evaluate the significance of differences in skin color and the Mann– Whitney U-test was used to analyze data on pigmented spots.
2.2. UV Radiation The UVB light source was a bank of seven Toshiba FL-20 SE fluorescent lamps, with a peak emission at 305 nm. The irradiance was 0.31 mW/cm2, as measured with a UV-Radiometer (TOPCON). Five groups of mice were exposed to
3. Results
3.1. Obser6ation of acute pigmentation Skin color of mice was determined once a week
M. Naganumaa et al. / Journal of Dermatological Science 25 (2001) 29–35
during the exposure period with a colorimeter. The results are shown in Fig. 1. Hairless mice showed no tanning at the start of irradiation. During the first exposure week, erythema was observed only on skin irradiated with the high dose. Perceptible pigmentation appeared 1 week after the first exposure only on skin irradiated with the low daily dose, without erythema. All exposed mice showed apparently uniform pigmentation on their back skin 2 weeks later and the L* value dropped rapidly from 63 to about 60 in all groups except the non-irradiated group after 2 weeks. Pigmentation (Fig. 2) persisted during irradiation and there was no significant difference in L* value between animals exposed to the low dose and high dose. Those changes well reflected the changes of skin color observed with the naked eye. While irradiation was continuing, the peak of pigmentation was always seen at the third or fourth week i.e. exposure for over 4 weeks did not produce darker pigmentation. After cessation of UV irradiation this pigmentation began gradually to disappear and after about 2 weeks the skin color became the same as that of non-irradiated skin, irrespective of the length of the irradiation
Fig. 1. Time course of skin color (L* value is reflected skin brightness). The lowest values were recorded at the third or fourth week after the first exposure. The L* value increased rapidly after the final exposure, becoming the same as that of non-irradiated mice. Non-irradiated: a, — , high dose irradiated group for 1 week; b, — —, 2 weeks; c,· · · · , 4 weeks; d, – · –, 8 weeks; e, – · · – low dose irradiated group for 4 weeks: f, – – – .
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Fig. 2. Pigmentation on hairless mouse skin immediately after UV exposure for 4 weeks. This pigmentation is dark brown in color and spread uniformly on the back skin.
period. The L* value started to increase on all irradiated mice immediately after the final UV exposure, and reached the control value 2 or 3 weeks later.
3.2. Obser6ation of pigmented spots When observation was continued after the recovery of normal skin color, the skin showed no pigmentation for a long time, though the L* values of all groups, including the non-irradiated group, increased slowly but steadily (Fig. 1). The increase of L* values was caused by the skin becoming opaque. At about 28 weeks small pigmented spots began to develop, at first on the dorsal skin of mice irradiated for 8 weeks. Subsequently, the size and number of the spots increased on the irradiated area. The spots had a diameter of less than 2 mm and were light brown (Fig. 3). The L* value of mice having many pigmented spots was not different from that of non-exposed mice having no spots (Fig. 1). Thus, determination of skin color could not indicate the degree of formation of pigmented spots. Accordingly, we scored the spots into four grades by visual evaluation (Fig. 4). Score 0 means no pigmented spots and score 3 indicates extensive pigmented spots. The group exposed to a daily high dose of UVB for 8 weeks showed a mean score of 1.0 at the
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Fig. 3. Pigmented spots 57 weeks after cessation of a 4-week UV exposure. The pigmented spots began to appear 24 weeks after the final irradiation. And then they became gradually darker and bigger.
20th week after the first exposure and a mean score of 2.6 was recorded at the 78th week. The group exposed to a daily low dose for 4 weeks (total dose: 456 mJ/cm2) showed similar changes to those of the group exposed to a daily high dose for 4 weeks (total dose: 1128 mJ/cm2), which did
Fig. 4. Time course of the appearance of pigmented spots. The first appearance of them and the degree of them were dependent on irradiated dose. Non-irradiated: a ——, high dose irradiated group for 1 week: b, —; 2 weeks: c · · · · ; 4 weeks: d – · – ; 8 weeks: e – · · –; low dose irradiated group for 4 weeks: f, – – – . Pigmented spots began to appear at the 28th week of the experimental period on skin of mice irradiated for 8 weeks. The Mann – Whitney U test was used for statistical evaluation at the 45th experimental week. High dose group: 0 week versus 4 weeks, 0 versus 8, 1 versus 4, 1 versus 8, 2 versus 4, PB 0.001. High dose group: 2 weeks versus 8 weeks, P B0.01; 2 weeks versus 4 weeks, PB 0.05. The low dose group is not significantly different from the high dose group at 4 weeks.
not produce erythema. In addition, the former group received 456 mJ/cm2 for 4 weeks showed a higher mean score than that of the group exposed to a daily high dose for 2 weeks (total dose: 564 mJ/cm2). Unexposed mice never developed such pigmented spots during the experimental period (78 weeks). The time lag until appearance of pigmented spots and the degree of induction of pigmented spots were dependent on the exposure period, not on the total amount of UV exposure: the longer exposure period, the shorter the latent period and the greater the degree of induction of pigmented spots. Exposure even for just 1 week could induce the spots.
4. Histology of exposed skin Epidermal hyperplasia and accumulation of melanin granules in the epidermis were noted on skin specimens taken immediately after final exposure of a mouse exposed for 4 weeks (Fig. 5a). Melanins were stained strongly by Fontana–Massons stain in all layers of the epidermis except hair follicles. Many active melanocytes were visualized by dopa stain (Fig. 6a) and they were very dendritic. They overspread on the epidermis, showing pigmentation except around hair follicles. On the other hand, examination of skin specimens taken from exposed mice 32 weeks after final exposure revealed that the thickness of the epidermis was not significantly different from that of non-irradiated epidermis, and there were melanin granules only in those parts of the epidermis having pigmented spots (Fig. 5b). Active melanocytes similar to those observed immediately after final exposure were present only in areas showing pigmented spots (Fig. 6b), though they were very dendritic. These results showed that the pigmented spots were due to melanin synthesized by melanocytes in the epidermis, not in the dermis.
5. Discussion We previously used HR-1 mice (aabbccDDPPhrhr) to investigate the changes of skin upon
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repeated UVB irradiation and reported that the number of melanocytes began to increase 2 days after the first exposure and reached almost saturation at about 14 days. This increase of melanocyte number was partially due to mitosis. We observed the development of a similar uniform pigmentation on the back skin of hairless mice exposed to repeated UVB irradiation. Furthermore, we showed quantitatively that the decrease of L* value saturated after 3 to 4 weeks, by measurement of skin color with a colorimeter. These results are reasonable, since melanocyte density is expected to peak first, then melanin particles would be transferred to surrounding keratinocytes, which would eventually cover the epider-
Fig. 5. Skin specimens were stained using the Fontana–Masson method. (a): Skin taken immediately after a 4-week high dose UV exposure. This photo shows hyperplasia and melanin particles uniformly in all layers of the epidermis. (b): Skin taken 32 weeks after a 4-week high dose UV exposure. Melanin particles are stained only on the epidermis, not the dermis, in pigmented spots.
chronic UV exposure [20], but we could not observe the effect on melanocytes, because HR-1 mice are albino. On the other hand, HR/De mice (AABBCCDDpphrhr) do not show pigmentation upon UV exposure, although they are not genetically albino. So we crossed HR-1 with HR/De, and found that the obtained F1 mice (AaBbCcDDPphrhr) had the wild coat color and their skin darkened upon UV irradiation. Exposure of pigmented mice to UV radiation has long been known to induce pigmentation [10 – 12], and the mechanisms involved have been examined by many researchers. Sato and Kawada [10] irradiated the backs of hairless mice and observed mitotic activity of epidermal melanocytes. Rosdahl and Szabo [11] examined the ears of hairy mice, C57B1/6J, exposed to
Fig. 6. Epidermis stained with dopa reaction. (a): Skin taken immediately after a 4-week high dose UV exposure. Active and dendritic melanocytes are seen except at hair follicles. (b): Skin taken 32 weeks after a 4-week high dose UV exposure. Active and dendritic melanocytes appear only in areas showing pigmented spots. However, the morphology of melanocytes is very similar to that in (a).
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mis. The appearance of this pigmentation in mice did not require erythema. When UV irradiation was stopped, the pigmentation began to fade. Although this phenomenon is well known, extended observations after recovery of normal skin color have rarely been made. We found that after an exposure time-dependent latent period following the recovery of normal skin color, pigmented spots began to appear and the size and number of them increased gradually. We found no indication of hyperplasia of the epidermis or tumors on the skin during the late experimental period, when the pigmented spots appeared. Thus the pigmented spots seem to be the first symptoms of photoaging. Tumor or melanoma induction on mouse skin subjected to long-term UV exposure has been investigated [15,16,21], but little attention was paid to the induction of pigmentation or pigmented spots. Since the skin was monitored only during the exposure period, delayed changes would have been missed. Alcalay et al. [16] described macules elicited on the skin of mice treated with psoralen plus ultraviolet A radiation, but these macules resulted from melanin accumulation in the dermis, not the epidermis. For the first time, we have shown that the skin of pigmented hairless mice, once UV-irradiated, always develops pigmented spots after a latent period, which is inversely proportional to the exposure period. The longer the exposure period, the greater was the induction of pigmented spots. Induced pigmented spots very slowly became darker and larger even after the final observation (78 weeks after first exposure, data not shown), and never disappeared. As denaturation of matrix components was reported to be reparable, the pigmented spots are distinct from such phenomena. This means that the UV irradiation history of skin, even exposure for as short a time as one week, affected the skin condition much later. We found that the same degree of induction of pigmented spots appeared on mice of two groups irradiated with different daily doses for the same period. These groups showed the same pigmentation during the irradiation period, although mice irradiated with the low dose did not develop erythema. Kiss et al. [4] reported similar phenom-
ena in connection with wrinkle formation on chronically irradiated hairless mice i.e. equal cumulative UVB doses did not always result in identical grades of wrinkle formation. Furthermore Wulf et al. [20] found that different doses of UV were equally carcinogenic when mouse skin was subjected to repeated UV exposure from the same light source. We found that the degree of delayed pigmentation response in the epidermis appeared to be the same even when different irradiation doses were applied during the same period. It seems that a sufficient stimulus to maintain an abnormal condition for certain time, is required, even if that stimulus is small i.e. there is a threshold effect. Many kinds of pigmented spots are observed in human skin. Among them, freckles, pigmentatio pedaloides actinica and solar lentigo are related to sunlight. Freckles have a genetic basis. Pigmentatio pedaloides actinica appear on sites exposed to large amounts of UV, following severe erythema. Pigmented spots in mice were not genetically determined, and could be elicited without erythema. On the other hand, solar lentigines are induced on exposed sites in the absence of severe damage. So solar lentigines seem to be a result of chronic UV exposure. Furthermore, melanocytes were histologically reported to increase in solar lentigines and melanin was observed in the epidermis [18,22]. Histological examination of pigmented spots in mice revealed that they contained active melanocytes, had melanin in the epidermis, not in the dermis, and never showed epidermal hyperplasia. Thus, based on the reported findings and pathological examination, we consider that pigmented spots in mice resemble solar lentigines in humans. Symptoms of photoaging include wrinkles, pigmented spots, solar elastosis, and skin cancer. Wrinkles and skin cancer have been shown to be elicited in animals by long UV exposure. Animal models have been used to investigate the mechanisms of these changes and to develop protective agents. However, there is no animal model of pigmented spots. Our results indicate that UVBirradiated pigmented hairless mice could be useful as a model of human solar lentigines.
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References [1] Kligman LH, Kligman AM. The nature of photoaging: its prevention and repair. Photodermatology 1986;3:215–27. [2] Bissett DL, Hillebrand GG, Hannon DP. The hairless mouse as a model of skin photoaging: its use to evaluate photoprotective materials. Photodermatology 1989;6:228 – 33. [3] Kligman LH. The ultraviolet-irradiated hairless mouse: a model for photoaging. J Am Acad Dermatol 1989;21:623 – 31. [4] Kiss I, Chen S, Tramposch KM. The effect of high and low ultraviolet-B dose exposure on the degree of hairless mouse skin wrinkling. Photochem Photobiol 1991;53:109 – 12. [5] Moloney SJ, Edmonds SH, Giddens LD, Learn DB. The hairless mouse model of photoaging: evaluation of the relationship between dermal elastin, collagen, skin thickness and wrinkles. Photochem Photobiol 1992;56:505–11. [6] Takahashi Y, Ishikawa O, Okada K, Ohnishi K, Miyachi Y. Disaccharide analysis of the skin glycosaminoglycans in chronically ultraviolet light-irradiated hairless mice. J Dermatol Sci 1995;10:139–44. [7] Starcher B, Pierce R, Hinek A. UVB irradiation stimulates deposition of new elastic fibers by modified epithelial cells surrounding the hair follicles and sebaceous glands in mice. J Invest Dermatol 1999;112:450–5. [8] Bissett DL, Majeti S, Fu JJ, McBride JF, Wyder WE. Protective effect of topically applied conjugated hexadienes against ultraviolet radiation-induced chronic skin damage in the hairless mouse. Photodermatol Photoimmunol Photomed 1990;7:63–7. [9] Kochevar IE, Moran M, Lyon N, Flotte T, Siebert E, Gange RW. Effects of systemic indomethacin, meclizine, and BW755C on chronic ultraviolet B-induced effects in hairless mouse skin. J Invest Dermatol 1993;100:186–93. [10] Sato T, Kawada A. Mitotic activity of hairless mouse epidermal melanocytes: its role in the increase of melanocytes during ultraviolet radiation. J Invest Dermatol 1972;58:329 – 95. [11] Rosdahl IK, Szabo G. Mitotic activity of epidermal melanocytes in UV-irradiated mouse skin. J Invest Dermatol 1978;70:143 – 8. [12] Uesugi T. Electron microscopic characterization in the processes of activation and differentiation of dormant
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
.
35
melanocytes in epidermis of C57 black mouse skin after UV-irradiation (UV-A). Jpn J Dermatol 1978;88:686 – 700. Warren R. Sensitivity of mouse Skh: HR-2 to ultraviolet light: mouse pigmentation model. Photochem Photobiol 1986;43:41 – 7. Hoshino S, Nishimura M, Fukuyama K, Gellin GA, Epstein JH. Effects of 4-tertiary butyl catechol on melanocytes of hairless mice. J Invest Dermatol 1981;76:231 – 8. van Weelden H, van der Putte SCJ, Toonstra J, van der Leun JC. Ultraviolet B-induced tumors in pigmented hairless mice, with an unsuccessful attempt to induce cutaneous melanoma. Photodermatol Photoimmunol Photomed 1990;7:68 – 72. Alcalay J, Bucana C, Kripke ML. Cutaneous pigmented melanocytic tumor in a mouse treated with psoralen plus ultraviolet A radiation. Photodermatol Photoimmunol Photomed 1990;7:28 – 31. Roth DE, Hodge SJ, Callen JP. Possible ultraviolet A-induced lentigines: a side effect of chronic tanning salon usage. J Am Acad Dermatol 1989;20:950 – 4. Rhodes AR, Albert LS, Barnhill RL, Weinstock MA. Sun-induced freckles in children and young adults: a correlation of clinical and histopathologic features. Cancer 1991;67:1991 – 2001. Takashima A, Matsunami E, Yamamoto K, Kitajima S, Mizuno N. Cutaneous carcinoma and 8-methoxypsoralen and ultraviolet A (PUVA) lentigines in Japanese patients with psoriasis treated with topical PUVA: a follow-up study of 214 patients. Photodermatol Photoimmunol Photomed 1990;7:218 – 21. Maeda K, Naganuma M, Fukuda M. Effects of chronic exposure ultraviolet-A including 2% ultraviolet-B on free radical reduction systems in hairless mice. Photochem Phobobiol 1991;54:737 – 40. Wulf HC, Hansen AB, Bech-Thomsen N. Differences in narrow-band ultraviolet B and broad-spectrum ultraviolet photocarcinogenesis in lightly pigmented hairless mice. Photodermatol Photoimmunol Photomed 1994;10:192 – 7. Rhodes AR, Melski JW, Sober AJ, Harrist TJ, Mihm MC, Fitzpatrick TB. Increased intraepidermal melanocyte frequency and size in dysplastic melanocytic nevi, superficial spreading melanoma, nevocellular nevi, and solar lengigines. J Invest Dermatol 1983;80:452 – 9.