Efficacy of Hydroquinone in the Treatment of Cutaneous Hyperpigmentation in Hairless Descendants of Mexican Hairless Dogs (Xoloitzcuintli)
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Laboratory Animal Science Vol 48, No 5 Copyright 1998 October 1998 by the American Association for Laboratory Animal Science Efficacy of Hydroquinone in the Treatment of Cutaneous Hyperpigmentation in Hairless Descendants of Mexican Hairless Dogs (Xoloitzcuintli) Tohru Kimura1* and Kunio Doi2 Abstract _ The skin of adult hairless dogs is clinically nonpigmented, clinically lightly pigmented, or clini- cally hyperpigmented (spotty pigmented). The pigment noted clinically is attributable to melanin granules in the epidermis. Spotty pigmentation in the skin of adult hairless dogs was treated by administration of the depigmenting agent (3% hydroquinone, HQ) for 1 month. Depigmenting effects were examined by use of three methods: skin color, dihydroxyphenylalanine (DOPA)-positive melanocyte count, and histologic evaluation. The treated skin of hairless dogs began to become depigmented after application of HQ for 1 week. After 1 month of treatment with HQ, depigmentation spread over a quarter of the body. The number of DOPA-posi- tive melanocytes in the HQ-treated sites decreased to less than approximately a fifth of that before treatment. In HQ-treated skin, histologic staining by use of Fontana-Masson’s (FM) method revealed complete absence of melanin pigment. These results suggested that hairless dogs should be a useful animal model for investigat- ing the effects and cutaneous toxicity of depigmenting agents. In studies of pigment cells, various methods (in vitro and fects of hydroquinone (HQ) on spotty pigmentation in the in vivo) have been used to evaluate the effects of skin of adult hairless dogs. To estimate the depigmenting depigmenting agents. The most reliable in vitro test involves effects in the skin, we examined the changes in skin color, use of B16 mouse melanoma cell strains (1). In contrast, a melanocyte count, and skin histologic features. number of animal models, such as colored guinea pigs (2–5) and grey and black moor goldfish (6), have been used for Materials and Methods evaluating the effects of various chemical agents that cause Experiment 1 depigmentation. However, few animals have a large num- Dogs: Eight male N2 hairless hybrid crosses (3 weeks ber of melanocytes, not only in pilar structures but in the old) between male N1 hairless hybrids (male MHD x fe- epidermis. Additionally, they rarely develop spontaneous male beagle) and female beagles were studied. In hairless cutaneous hyperpigmentation in the skin. Until recently, descendants of MHDs, an autosomal dominant monogenic we could not examine effects of depigmenting agents on gene (Hm; hairless, Mexican type) is responsible for hair- cutaneous hyperpigmentation. less characteristics (8). These hairless dogs were housed Kimura et al. (7) reported age-related changes in the skin with their dam (beagle) in stainless steel cages (85 x 95 x of hairless descendants of Mexican hairless dogs (MHD, 75 cm) in an animal room environmentally controlled be- Xoloitzcuintli). Their epidermis has abundant melanocytes tween 20 and 258C and 50 and 70% relative humidity, with with stout dendrites. In hairless dogs >1.5 years old, skin 10 to 15 exchanges of 100% fresh air/h and 12-h light (0700 surfaces develop spotty pigmentation similar to that of se- to 1900 h)/dark (0700 to 1900 h) cycle. The dam was fed a nile lentigines. The lesion had heavy deposition of melanin commercial dry dog food (Labo D Standard; Nihon Nosan granules in the stratum basale, spinosum, granulosum, and Kogyo Co., Ltd., Yokohama, Japan) and water ad libitum. corneum. In addition, some hairless dogs have clinically Extra heat was administered to infant pups. nonpigmented areas on the skin. The study reported here All procedures involving animals were performed in ac- was undertaken to extrapolate such canine lesions to hu- cordance with protocols approved by the Research Center man pigment disorders. Thus the purpose of this study was Institutional Animals Care and Use Committee. twofold: to investigate histologically two types of the skin Histologic examination of the skin: Tissue specimens of infant hairless pups (clinically pigmented and clinically were obtained from two (clinically pigmented and clinically nonpigmented sites), and to evaluate the depigmenting ef- nonpigmented) skin sites of each hairless pup, using a 6- mm biopsy punch; local anesthesia (0.5% procaine; Research Center, Nihon Nosan Kogyo Co., Ltd., Tsukuba, Ibaraki, Japan1 Omnicaine, Fujisawa Co., Ltd., Osaka, Japan) was applied and Department of Veterinary Pathology, Faculty of Agriculture, The Uni- in a ring surrounding the 6-mm biopsy punch site. versity of Tokyo, Bunkyo, Tokyo, Japan2 *Address correspondence to Dr. Tohru Kimura, Research Center, Nihon The skin specimens were fixed in neutral-buffered 10% Nosan Kogyo Co., Ltd., Takura 5246, Tsukuba, Ibaraki 300-2615, Japan. formalin, and 4-mm paraffin sections were stained with 469 Vol 48, No 5 Laboratory Animal Science October 1998 Figure 1. Photomicrograph of a section of dorsal pigmented skin Figure 2. Photomicrograph of a section of dorsal pigmented skin (tan-colored area) from a 3-week-old hairless dog. The stratum (tan-colored area) from a 3-week-old hairless dog. Some melano- basale and spinosum contain many melanin granules. Some mel- cytes containing abundant melanin granules are seen in the der- anocytes are seen in the dermis (arrowheads). Fontana-Masson’s mis. FM method; bar = 10 mm. (FM) method; bar = 20 mm. hematoxylin and eosin (H&E) or toluidine blue (TB), or by (Y = luminance factor, x and y = chromaticity coordinates); use of Fontana-Masson’s (FM) method. L * a * b * systems (L * = luminance factor, a * = from Experiment 2 redness [+] to greenness [-], b * = from yellowness [+] to Dogs: Three male N1 hairless hybrid crosses (4 years blueness [-]); and LCH systems (L = luminance factor, C = old) between a male MHD and female beagle were studied chroma, H = hue). In Yxy systems, Y values express rela- to assess the depigmenting potency of the tested chemical. tive brightness (reflectance rate) of the color. The additional These animals were maintained under the aforementioned two parameters (x and y values) can detect the changes of environmental conditions. chroma and hue in the skin. In the L * a * b * systems, the Procedures: Hydroquinone (Wako Pure Chemicals Co., L *, a *, and b * values represent the white-black, red-green, Ltd., Tokyo, Japan; 3% solution in a vehicle of 20% propy- and yellow-blue components of the objects, respectively. In lene glycol, 40% ethanol, and 40% distilled water) was used LCH systems, the C values represent from “dull” to “vivid.” as a potent depigmenting agent. The H values represent the circle of colors (red → yellow → Commercial patch test plasters (2 x 2 cm) containing 3% green → blue). Spectrophotometers provide reproducible HQ (100 ml) were applied to the clinically hyperpigmented quantification of skin colors, such as pigmentation, in a skin of hairless dogs, and were reinforced with occlusive manner equivalent to perception of human eyes but with- bandage to prevent them from peeling off. This procedure out the inherent bias associated with human judgment. was repeated once a day for 1 month; 3% HQ solution was Recording was done at 1 day before beginning of the depig- prepared daily before the treatment. mentation test, and at 1 month of 3% HQ treatment. Assessment of skin colors: As reported in previous Histologic examination of the skin: Tissue specimens were studies (7, 9, 10), spectrophotometers are used to assess obtained from two (HQ-treated sites and untreated) dorsal skin skin colors in dermatologic science. A spectrophotometer sites of hairless dogs in a manner similar to that described for (CR-200; Minolta Co., Ltd., Tokyo) was used to record skin examination 1. Punch biopsy was performed the day before colors in a three-dimensional space as follows: Yxy systems beginning treatment and at 1 month after 3% HQ treatment. 470 Depigmentation of the Skin of Hairless Dogs Figure 3. Photomicrograph of a section of dorsal pigmented skin Figure 4. Photomicrograph of a section of dorsal pigmented skin (tan-colored area) from a 3-week-old hairless dog. Notice melano- (tan-colored area) of a 3-week-old hairless dog. Melanocytes con- cytes with stout dendrites in the epidermal ingrowth. FM method; taining abundant melanin granules are seen in a small number bar = 20 mm. of hair follicles in the skin of this breed. FM method; bar = 20 mm. Half the skin specimens were rinsed in 0.1 M phosphate dermis were found in some portions (Figure 1). In the epi- buffer (pH 7.4) and incubated in 2 N sodium bromide for 2 dermis of lightly pigmented (tan-colored) skin of infant h at 378C. The epidermal sheets separated from the dermis hairless pups, many melanin granules were detected in the were fixed in cold neutral-buffered 10% formalin for 30 min, stratum basale and spinosum. The dermis contained mel- washed twice with 0.1 M phosphate buffer (pH 7.4), and anocytes (Figure 2). A cluster of melanocytes with abun- incubated in 0.1% dihydroxyphenylalanine (DOPA) in 0.1 dant melanin granules was found in the epidermal in- M phosphate buffer (pH 7.4) for 5 h. The number of DOPA- growths and/or hair follicles (Figures 3 and 4). On the other positive melanocytes in each of ten 1-mm2 fields was hand, neither epidermis nor dermis in clinically nonpig- counted for each specimen under light microscopy, and mented areas had melanin granules, and the epidermal mean number was calculated. ingrowths projecting into the dermis, which appeared to The remaining skin specimens were fixed in neutral-buff- be equivalent to the rudiments of hair follicles did not con- ered 10% formalin, and 4-mm paraffin sections were stained tain pigment. with H&E or TB or by the FM method. The skin of the tan-colored sites contained a small num- Statistical analysis: All data were expressed as mean ber of pigmented hair shafts, erector pili muscles, and as- 6 SD, and statistical analysis was performed by use of the sociated sebaceous and apocrine sweat glands.